Distal Enhancer – Gene Interactions at the Lmo2 locus in Mouse Erythroid cells

by

Anandi Bhattacharya

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Cell and Systems Biology University of Toronto

© Copyright by Anandi Bhattacharya 2012

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Distal Enhancer – Gene Interactions at the Lmo2 locus in Mouse

Erythroid cells

Anandi Bhattacharya

Master of Science

Department of Cell and Systems Biology University of Toronto

2012

Abstract

Distal regulatory elements (DREs) have been identified upstream of the hematopoietic

regulator Lim domain only 2 (Lmo2) gene in the human and mouse genomes. In this thesis I

have investigated how these elements regulate Lmo2 transcription in erythroid cells. My

results show that strong chromatin-chromatin interactions exist between the DREs and the

Lmo2 gene promoter in erythroid cells. These interactions are absent from kidney cells that

do not express Lmo2. Within the distal chromatin interaction cluster encompassing three of

the DREs increased DNase I sensitivity, presence of high levels of H3K4me1, and binding of

multiple transcription factors, p300, cohesin (RAD21) and CTCF are observed. CTCF bound

regions are located between the farthest DRE and the neighboring Caprin1 promoter

suggesting that CTCF insulates Caprin1 from the DREs. Hence, my data suggests that these

DREs function through a chromatin looping mechanism supported by cohesin associated

with CTCF and transcription factor bound regions.

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¨ The purpose of learning is growth, and our minds, unlike our bodies,

can continue growing as we continue to live. ¨

Mortimer Adler

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Acknowledgments

I would like to take this opportunity to thank all those people without whom this thesis would

not have been possible. At the very beginning I would like to thank my supervisor Prof.

Jennifer Mitchell, for her guidance, unwavering support and inspiration through the last 2.5

years. In spite of her extremely busy schedule, she always had time for me, and each and

every discussion with her was illuminating, to say the least. I have learnt a lot from her and I

am deeply indebted to her. Because of her I was introduced not only to the world of

genomics but also to this project which to say the least has been a life changing experience. I

would also like to express my sincere gratitude to Prof. Dorothea Godt and Prof. Vincent

Tropepe for their invaluable advice and encouragement and all the help that they have

provided me for completing my graduate study. I would especially like to thank Prof. Godt for

her invaluable support throughout the entire graduate program. The last two and half years

have been a very humbling experience for me and I consider myself privileged to have got

this opportunity to study at the University of Toronto.

I would also like to thank all those people who have played a very important role in bringing

me to the place where I stand today; my parents Sankarlal and Indrani Bhattacharya, for

believing in me, Prof. Sudeep Banerjee for inspiring me to be a researcher, my friends Conny

Bartholmes, Julie Chen, Kamelia Miri, Mike Schwartz and Huda Abdel-Aleem for their

invaluable companionship, Ian Buglass for answering all the innumerable questions that I had

throughout the entire duration of my study here at the department of Cell and systems

biology. I would also like to thank Dr. Neil Macpherson for providing me with invaluable

inputs both on my project as well as on my presentations thereby helping me immensely and

also Dr. Scott Davidson for the discussions on biological and bioinformatics research. I

would also like to thank my labmate Harry Zhou for helping me a lot during my gel running

sessions and Pooya Dibajnia for helping me in my experiments. I would especially like to

thank Julie Chen for working along with me on my project. without Julie`s expertise in

bioinformatic analyses this thesis would not have been possible. Another person who has

contributed a lot to my project is Sara Ho, I would really like to thank her as well. Finally, I

would like to thank my husband and best friend Kunal Dasgupta for his understanding and

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unwavering support, if it had been not for him I would not have been a part of University of

Toronto.

At the very end I would also like to thank the department of Economics and department of

Cell and Systems Biology for funding my education here at the University of Toronto.

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Table of contents

Acknowledgments .......................................................................................................................... iv

Table of contents............................................................................................................................. vi

List of Tables .................................................................................................................................. ix

List of Figures .................................................................................................................................. x

List of Appendices .......................................................................................................................... xi

Chapter 1: Introduction .................................................................................................................. 1

1.1 General Introduction ............................................................................................................ 2

1.2 Mechanisms of regulating gene transcription ...................................................................... 3

1.2.1 First Level of regulation of gene transcription: DNA sequences ............................ 4

1.2.1.1 Core Promoter and Proximal Promoter ..................................................... 5

1.2.1.2 Cis regulatory elements ............................................................................. 6

1.2.1.2.1 Distal Regulatory Elements: Enhancers and Insulators ............................ 7

1.2.2 Second Level of regulation of gene transcription: Epigenetic modifications.......... 8

1.2.2.1 Epigenetic signatures, tissue specificity and enhancers ............................ 9

1.2.2.1.1 Low nucleosome occupancy and DNaseI sensitivity ................................ 9

1.2.2.1.2 p300 binding ............................................................................................ 10

1.2.2.1.3 Cobound by multiple TFs ........................................................................ 10

1.2.3 Third Level of regulation of gene transcription: Spatial organization of the chromatin ............................................................................................................... 11

1.2.3.1 Relation between transcription and organization of chromatin structure ................................................................................................... 11

1.2.3.2 Chromatin looping and regulation of gene transcription ........................ 12

1.2.3.2.1 Enhancers and chromatin looping .......................................................... 12

1.2.3.2.2 Active chromatin hub .............................................................................. 13

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1.2.3.2.3 Insulators and chromatin looping ............................................................ 14

1.2.3.3 Role of protein complexes in maintaining higher-order chromatin conformation ........................................................................................... 14

1.3 Lmo2: A candidate for investigating tissue specific chromatin conformation ................. 16

Chapter 2: Methods........................................................................................................................ 18

2.1 Cell Isolation ...................................................................................................................... 19

2.2 Chromosome Conformation Capture (3C) ........................................................................ 19

2.3 RNA isolation and real-time RT-qPCR ............................................................................. 21

2.4 Statistical analysis .............................................................................................................. 22

2.5 Genome Mapping and Peak Identification of ChIP-seq datasets in erythroid cells .......... 22

Chapter 3: Distal regulatory elements located upstream of Lmo2 are associated with tissue-specific chromatin features ............................................................................................. 24

3.1 Introduction........................................................................................................................ 25

3.2 Results ............................................................................................................................... 26

3.2.1 Identification and mapping of the enhancer elements on the mouse genome ................................................................................................................... 26

3.2.2 TFs bind to the distal regulatory elements ............................................................. 28

3.2.2.1 Multiple TFs binding to DREs in mouse erythroid cells ........................ 28

3.2.3 The DREs have erythroid cell-specific epigenetic features ................................... 32

3.2.4 Intergenic transcription occurs at the distal regulatory elements .......................... 34

3.2.5 CTCF and RAD21 bind to multiple regions across the Lmo2/Caprin1 locus ....................................................................................................................... 36

3.3 Discussion .......................................................................................................................... 39

3.3.1 TFs bind to the distal regulatory elements ............................................................. 39

3.3.2 The DREs have erythroid cell-specific epigenetic features ................................... 41

3.3.3 Intergenic transcription occurs at the distal regulatory elements .......................... 42

3.3.4 CTCF and RAD21 bind to multiple regions across the Lmo2/Caprin1 locus ....................................................................................................................... 42

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3.3.5 The big picture ....................................................................................................... 42

3.4 Future work ........................................................................................................................ 43

Chapter 4:Chromatin-Chromatin interactions at the Lmo2 locus .................................................. 44

4.1 Introduction........................................................................................................................ 45

4.2 Results ............................................................................................................................... 46

4.2.1 The 75 distal regulatory element contacts the Lmo2 proximal promoter .............. 46

4.2.2 Several upstream distal regulatory elements contact the Lmo2 promoter ............. 50

4.2.3 The Caprin1 promoter does not interact with the identified distal regulatory elements ................................................................................................ 52

4.3 Discussion .......................................................................................................................... 54

4.3.1 The 75 distal regulatory element and several other upstream distal regulatory elements contact the Lmo2 proximal promoter .................................... 54

4.4 Future work ........................................................................................................................ 56

Chapter 5: General Discussion ...................................................................................................... 57

5.1 Discussion .......................................................................................................................... 58

5.2 Summary ............................................................................................................................ 61

References...................................................................................................................................... 63

Appendices .................................................................................................................................... 75

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List of Tables

Table 2-1. Chromatin immunoprecipitation sequencing data........................................................ 23

x

List of Figures

Figure 2-1.Chromosome Conformation Capture (3C)................................................................... 21

Figure 3-1. Genomic map of the Lmo2/Caprin1 locus showing the identified DREs .................. 27

Figure 3-2. Distal regulatory elements upstream of Lmo2 overlap transcription factor bound

regions in erythroid cells ............................................................................................................... 29

Figure 3-3. Distal regulatory elements upstream of Lmo2 overlap transcription factor bound

regions in HPC7 hematopoietic progenitor cells ........................................................................... 31

Figure 3-4. Distal regulatory elements upstream of Lmo2 overlap transcription factor bound

regions and have different epigenetic marks in erythroid cells ..................................................... 33

Figure 3-5. Intergenic transcription occurs in anaemic spleen erythroid cells. ............................. 35

Figure 3-6. CTCF and RAD21 are bound within the Lmo2-Caprin1 region ................................ 37

Figure 3-7. CTCF bound upstream region of Lmo2 in different cell types. .................................. 38

Figure 4-1.Lmo2 primary transcripts are abundant in anaemic spleen erythroid cells .................. 47

Figure 4-2. The Lmo2/Caprin1 region on mouse chromosome 2 ................................................. 48

Figure 4-3. The 75DRE interacts with the Lmo2 proximal promoter in anaemic spleen

erythroid cells ................................................................................................................................ 49

Figure 4-4. Distal regulatory elements interact with the Lmo2 proximal promoter in anaemic

spleen erythroid cells ..................................................................................................................... 51

Figure 4-5. The distal regulatory elements upstream of Lmo2 do not interact with the Caprin1

promoter ......................................................................................................................................... 53

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List of Appendices

Appendix 1. Coordinates of distal regulatory elements located upstream of the Lmo2

promoter in the mouse genome...................................................................................................... 75

Appendix 2. Coordinates of the Lmo2 proximal and distal promoters in the mouse genome ....... 77

Appendix 3. Primers used in quantitative chromosome conformation capture and RT-qPCR ..... 78

Appendix 4. Restriction digestion efficiency in chromosome conformation capture ................... 85

Appendix 5. Transcription factor bound regions at the 75 and 12DREs ....................................... 86

Appendix 6. QPCR products of gene expression profile run on agarose gel ................................ 87

Appendix 7. QPCR products of intergenic transripts at 90,75, and12 DREs run on agarose gel . 87

Appendix 8.QPCR products of the intergenic transcripts at 25,35,40,43,47,58,64, and 70

DREs run on agarose gel ............................................................................................................... 88

Appendix 9. QPCR products of the intergenic transcripts located between the 47and 58DREs,

64 and 58DREs, and between the 90 and 75DREs run on agarose gel ......................................... 88

Appendix 10. Chromosome conformation capture (3C) products run on agarose gel (75E as

the anchor fragment) ...................................................................................................................... 89

Appendix 11. Chromosome conformation capture (3C) products with Lmo2 proximal

promoter as anchor fragment run on agarose gel........................................................................... 89

Appendix 12. Chromosome conformation capture (3C) products with Caprin1 promoter as

anchor fragment run on agarose gel............................................................................................... 90

Appendix 13. Primary intergenic transcript levels in adult mouse anaemic spleen and kidney

cells at the Lmo2/Caprin1 locus .................................................................................................... 90

1

Chapter 1: Introduction

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1.1 General Introduction

The completion of the human genome project in 2003 launched the genomics revolution in

molecular biology research (Lander et al., 2001; Venter et al., 2001). Even though science has

since experienced a succession of rapid advances, there are several fundamental gaps in our

current knowledge. For example we know that every cell in our body contains the same genetic

material; however genes expressed in the neurons of our brain are different from the genes

expressed in our blood cells. So how is this tissue specific gene expression pattern generated?

Classical factors that allow cells to exhibit differential gene expression patterns without

changing genome sequences are chemical modifications to DNA sequences and histones and the

presence of various cell type specific proteins that interact with DNA (Jaenisch and Bird, 2003).

Recently the manner in which the chromatin is organized at a specific gene locus has emerged

as a new paradigm in gene expression regulation and new techniques have allowed the

investigation of the chromatin organization at a gene locus within a cell type in fine detail

(Dekker et al., 2002; Ethier et al., 2012; Palstra et al., 2003; Tolhuis et al., 2002). It has become

clear that to understand the tissue-specific regulation of genes all of these factors that contribute

to the cell-type specific epigenome need to be considered. For example a study has shown that a

2Mb region encompassing the human β-globin locus along with the flanking olfactory receptor

genes is organized in cell type specific manner in the human erythroid K562 cells and the

formation of this cell type-specific conformation is mediated by two proteins namely CTCF and

cohesin (Hou et al., 2010). Furthermore, the results also suggest that there is a correlation

between β-globin gene transcription and the histone methylation pattern of the locus (Hou et al.,

2010). Therefore, in order to understand tissue specific regulation of gene transcription a

combination of different biological data is required to explain how different biological factors

work synergistically to execute a specific transcriptional program.

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1.2 Mechanisms of regulating gene transcription

Regulation of gene transcription within the nuclear space is a multistep process. As multiple

events occur before and after the actual initiation of transcript (RNA) synthesis, hence gene

transcription can be regulated not only at different steps but these steps can be part of different

levels of regulation (Fuda et al., 2009). The first step that hails the onset of transcription is the

decondensation of the locus followed by remodeling of the nucleosomes so that RNA

Polymearse II (RNAPII) can gain access to the gene promoter (Fuda et al., 2009; Smale and

Kadonaga, 2003). In the next step the pre-initiation complex is assembled at the gene promoter

following which the DNA is unwound and RNAPII initiates transcription (Fuda et al., 2009).

After the actual initiation of RNA synthesis, the RNAPII pauses so as to undergo promoter

clearance before engaging in productive elongation (Sims et al., 2004). For promoter clearance

the C-terminal domain (CTD) of RBP1, the largest subunit of RNAPII has to be phosphorylated

(Komarnitsky et al., 2000; Sims et al., 2004). In this context it should be mentioned that the

CTD of RNAPII comprises of multiple repeats of the heptapeptide sequence (YSPTSPS) that

can be phosphorylated at the serine 2, 5 and 7 positions in each of the repeats (Chapman et al.,

2007; Corden, 1993; Sims et al., 2004). Whereas phosphorylation of serine 2 of the RNAPII

CTD occurs at the transition to productive elongation, phosphorylation of serine 5 of RNAPII

CTD is required for promoter clearance of the paused RNAPII (Komarnitsky et al., 2000;

Marshall et al., 1996; Sims et al., 2004). It is only after the release of the paused polymerase that

the gene is transcribed throughout its entire length (Fuda et al., 2009; Sims et al., 2004). After

this the last step of transcription is performed by the RNAPII that is `termination` following

which RNAPII can initiate a new round of transcription (Fuda et al., 2009). Therefore gene

transcription can be regulated during different stages of transcription namely before, during or

after the initiation of RNA synthesis (Fuda et al., 2009). Although there are multiple

mechanisms by which gene transcription can be regulated after the initiation of RNA synthesis,

in this thesis I will focus on the regulatory events that occur before the process of initiation of

RNA synthesis.

In eukaryotes, mechanisms that are involved in regulating gene transcription before the process

of initiation of RNA synthesis can be arranged into a series of different levels (Dillon, 2006).

The most basic level of regulation lies at the DNA sequence level (Dillon, 2006). Studies have

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shown that binding of transcription factors (TFs) to specific sequences in the gene promoter and

more distally located DNA elements helps in the assembly of the transcription pre initiation

complex at the promoter, the first step in productive transcription (Dillon, 2006). The next level

of regulation of gene transcription involves interactions between chromatin-associated proteins

and the DNA sequences so as to cause chemical modifications of DNA sequences as well as the

histone proteins (Li et al., 2007). These modifications are essential because in spite of the DNA

sequences having all the information required for regulation gene transcription, these DNA

sequences are often not accessible to the regulatory proteins in different cell types due to the

packaging of the DNA sequences around nucleosomes (Li et al., 2007). Therefore epigenetic

modifications of the DNA sequences and associated proteins that impact regulation of gene

transcription constitute the second level of regulation of gene transcription. Finally a

combination of the presence of cell type-specific regulatory proteins and the epigenetic

modifications along with chromatin de-condensation and looping helps in organizing the

chromatin in a tissue specific manner (Chambeyron and Bickmore, 2004; de Laat and Grosveld,

2003; Dillon, 2006). Formation of this tissue-specific conformation of the chromatin in turn

plays a vital role in the regulation of gene transcription by bringing distal regulatory sequences

into physical proximity to gene promoters (de Laat and Grosveld, 2003; Palstra et al., 2003;

Spilianakis and Flavell, 2004; Tolhuis et al., 2002). This kind of regulation of gene transcription

constitutes the third level of regulation of gene transcription. All three levels of regulation of

gene transcription together coordinate tissue specific transcriptional programs thereby

conferring a tissue specific gene expression pattern.

1.2.1 First Level of regulation of gene transcription: DNA sequences

The most basic level of regulation is at the DNA sequence level (Dillon, 2006). Studies have

shown that sequences both proximal and distal to a gene can regulate the transcription of that

gene (Lettice et al., 2003; Maston et al., 2006; Van der Ploeg et al., 1980). As all the regulatory

information required by a cell is encoded within its genome in the form of the DNA sequence,

regulatory elements such as promoters, silencers, insulators, enhancers and other elements (such

as locus control regions) play an important role in the regulation of gene transcription (Maston

et al., 2006). Not only do the DNA sequences serve as the docking sites for the different kinds

of regulatory and chromatin-associated proteins but are also the target of different kinds of

modification, all executed in order to regulate gene transcription within the cell (Maston et al.,

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2006). Therefore we can consider the DNA sequence to be the first basic level of regulation of

gene transcription in any living organism.

1.2.1.1 Core Promoter and Proximal Promoter

The transcription start site (TSS) along with the DNA sequences located immediately around the

TSS (~ 35bp upstream and/or downstream of the TSS) is defined as the core promoter of a gene

(Smale and Kadonaga, 2003). The core promoter consists of different functional DNA motifs

known as core promoter elements that help in the assembly and initiation of the RNAPII

transcription machinery and also directly interact with the components of the basal transcription

machinery (Smale and Kadonaga, 2003). The first core promoter element to be described was

the TATA box that serves as the binding site of the TFIID subunit of the TATA binding proteins

(Maston et al., 2006; Smale and Kadonaga, 2003). In addition to the TATA box other types of

core promoter elements have been identified in metazoan core promoters such as the initiator

element, downstream core element, downstream promoter element, motif ten element and

TFIIB-recognition element (Maston et al., 2006; Smale and Kadonaga, 2003). Furthermore,

statistical sequence analysis of about 10,000 human RNAPII core promoter elements have

shown that whereas the downstream promoter element and the TFIIB-recognition element were

each present in roughly 25% of the promoters, only 12.5% of the core promoter elements

contained the TATA box motif (Gershenzon and Ioshikhes, 2005). Interestingly, whereas the

TATA box motifs have been only found in strong tissue-specific core promoters, the core

promoters of housekeeping genes have been found to be associated with CpG islands (Maston et

al., 2006). For example studies have shown that a correlation exists between the presence of

CpG islands and the presence of some core promoter elements (Gershenzon and Ioshikhes,

2005). TATA boxes are usually present in core promoters that do not have any CpG islands

located nearby, whereas TFIIB-recognition elements are found in core promoters that have a

CpG island located nearby (Gershenzon and Ioshikhes, 2005). In this context it should be

mentioned that CpG islands are regions of the genome that have a higher concentration of CpG

(Cytosine-phosphate-Guanine) sites, methylation of these CpG sites within the promoters of

genes can lead to gene silencing by blocking the binding of TFs to their recognition sequences

(Gardiner-Garden and Frommer, 1987; Gonzalez-Zulueta et al., 1995; Jones and Baylin, 2002;

Maston et al., 2006).

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In addition to the core promoter, proximal gene promoters are also present in higher eukaryotic

genomes (Maston et al., 2006). Proximal promoter (pP) of a gene denotes the region located

immediately upstream of the core promoter that can range from fifty to a few hundred base pairs

and contains multiple binding sites for proteins that act synergistically to either activate or

repress transcription of the linked gene (Maston et al., 2006).

1.2.1.2 Cis regulatory elements

Transcription is not regulated only by the gene promoters but in many cases also by other DNA

elements (Maston et al., 2006). For many years promoters were considered to be the only gene

regulatory elements as a result of which a contiguous stretch of DNA sequence located upstream

of the TSS were cloned and analysed for regulatory activity. However in 1980 it was identified

for the first time that a deletion outside the intact human ß-globin gene had caused thalassaemia

indicating that sequences apart from the gene promoter can play a vital role in regulation of gene

transcription (Van der Ploeg et al., 1980). Moreover, studies over the past three decades have

indicated that transcription is regulated not only by the sequences located immediately upstream

of the gene TSS but in many cases by DNA elements which can be located up or downstream of

the genes they regulate (Mills et al., 1997; Tuan et al., 1989). A classical example of this being

alteration in the DNA sequences located as far as 1Mb from the Sonic Hedge Hog (SHH) gene

causes malformation of limbs in patients due to a condition known as preaxial polydactyly

(Lettice et al., 2003). Interestingly in some cases these DNA regulatory elements have also been

found to be located in a different chromosome altogether (Lomvardas et al., 2006). For example

the enhancer element H for the olfactory receptor genes is able to regulate transcription of

olfactory receptor genes found on multiple chromosomes (Lettice et al., 2003; Lomvardas et al.,

2006). These distal regulatory DNA sequences in many cases are bound by specific proteins that

regulate transcription (Drissen et al., 2004; Sawado et al., 2001; Song et al., 2007). Chromatin

immunoprecipitation sequencing (ChIP-Seq) studies can be used to investigate the binding of

TFs and the presence of distinct epigenetic marks at specific locations of the genome

(Kharchenko et al., 2008). Such a genome wide ChIP-Seq study for several TFs has revealed

that a significant proportion (40-60%) of transcription factor bound regions are located in the

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intergenic regions of the genome ≥ 10 kb from a gene TSS (Chen et al., 2008; Fullwood et al.,

2009; Yu et al., 2009). Taken together these data suggests that in addition to the gene

promoters, DNA sequences located > 10 kb from gene promoters play a vital role in regulating

transcription on a genome-wide scale. Nevertheless, in this context it should be mentioned that

these elements could not only regulate transcription of protein coding target genes but also of

different types of non coding RNAs which remain largely un-annotated in mammalian genomes

and whose roles are not completely defined though many are themselves involved in regulating

gene expression.

1.2.1.2.1 Distal Regulatory Elements: Enhancers and Insulators

Distal regulatory elements (DREs) regulate transcription by enhancing (enhancers), repressing

(repressors) or by insulating (insulators) transcription of their target genes (Gowri et al., 2003;

Maston et al., 2006; Tuan et al., 1989). In fact in some cases these DREs can act as an enhancer

in one context and a repressor in another (Murayama et al., 2004; Noonan and McCallion, 2010;

Perissi et al., 2004). Hence whether or not a specific DRE will enhance or repress transcription

depends upon its genomic environment and the protein complex that associates with the DRE

(Murayama et al., 2004; Noonan and McCallion, 2010; Perissi et al., 2004). We have defined

DREs as DNA sequences located either upstream or downstream at > 10 kb from a potential

target gene (Chen et al., 2012).

Enhancers

Enhancers are DNA sequences that are capable of activating gene transcription irrespective of

their orientation, distance and location relative to the target gene (Banerji et al., 1981;

Blackwood and Kadonaga, 1998; Dillon and Sabbattini, 2000; Jin et al., 2011; Ptashne, 1986).

Enhancers are functionally comprised of multiple protein binding sites as a result of which

different TFs can bind to them thereby regulating gene transcription (Jin et al., 2011; Maston et

al., 2006). A tandem SV40 repeat comprising of two identical 72bp elements were the first

characterised enhancers which were located 200bp upstream of the TSS (Moreau et al., 1981).

Insulators

Insulators are DNA elements that have the ability to prevent a gene from getting inappropriately

8

either activated or silenced due to outside or surrounding influences (Wallace and Felsenfeld,

2007). There are two classes of insulators based on their function namely the enhancer blocking

(EB) Insulators and the barrier insulators (Wallace and Felsenfeld, 2007). Whereas, the barrier

insulator prevents the inappropriate silencing of a gene by spreading of heterochromatin through

the gene, the EB insulators when placed between an enhancer and its target gene promoter

prevent the enhancer from activating the gene promoter (Wallace and Felsenfeld, 2007). A

classical example of the EB insulator is the insulator element that is bound by the CTCF protein

at the mouse Igf2/H19 imprinted locus and at the ß-globin locus (Bell and Felsenfeld, 2000;

Chung et al., 1993).

1.2.2 Second Level of regulation of gene transcription: Epigenetic

modifications

Although the DNA template contains all the information required for the execution of a

transcriptional program, an important factor that can regulate the process of gene transcription is

the accessibility of the DNA sequences that can be modulated by epigenetically modifying the

sequence itself or modifying nucleosomes that associate with DNA (Ong and Corces, 2011; Xi

et al., 2007). Packaging of DNA sequences around nucleosomes restricts access of regulatory

proteins to the DNA sequence and in turn regulates all DNA-based processes including gene

transcription (Ong and Corces, 2011). The modulation of DNA accessibility which in turn

affects gene transcription in fact involves multiple mechanisms such as temporary removal of

the core histone octamer from the DNA or epigenetic modifications to the histone proteins

(histone acetylation, methylation, phosphorylation, ubiquitination) as well as chemical

modifications to DNA sequences such as DNA methylation (Xi et al., 2007). For example

studies by Cui et al. have shown that modification of histone proteins plays a crucial role in the

maintenance and differentiation of hematopoietic stem cells (HSCs) (Cui et al., 2009). The

authors have shown that as the HSCs undergo differentiation to give rise to the different

lineages of blood cells there is a change in the gene expression pattern, and their results suggest

that there is a correlation between the histone marks at the HSC stage and the subsequent

changes in the gene expression pattern (Cui et al., 2009). Furthermore, there is a correlation

between epigenetic modifications at distal enhancers and tissue specific transcription of nearby

genes (Chen et al., 2012; Heintzman et al., 2009).

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1.2.2.1 Epigenetic signatures, tissue specificity and enhancers

Genome wide studies have shown that enhancers have a distinct epigenetic signature (Blow et

al., 2010; Heintzman et al., 2009; Heintzman et al., 2007; Visel et al., 2009). Enhancers in many

cases are marked with increased levels of histone H3 lysine 4 monomethylation (H3K4me1) and

with reduced marks of histone H3 trimethylation of lysine 4 (H3K4me3) (Chen et al., 2008;

Gross and Garrard, 1988; Ren et al., 2007; Visel et al., 2009; Visel et al., 2010; Wu, 1980).

Furthermore, studies conducted by Heintzmann et al. have also shown that enhancers are

marked by highly cell type-specific histone modification patterns which correspond to their

functional status within the specific cell types (Heintzman et al., 2009). However, gene

promoters have been shown to have a more uniform histone modification pattern in different

cell types suggesting that distal enhancers more functionally relevant in regulating tissue

specific gene expression (Heintzman et al., 2009; Visel et al., 2009). Tissue specific chromatin

signatures at enhancers include an increased sensitivity to DNaseI, binding of multiple TFs, the

histone acetyl transferase protein p300, H3K4me1 and H3K27ac (Blow et al., 2010; Heintzman

et al., 2009; Heintzman et al., 2007; Visel et al., 2009; Xi et al., 2007). For example the

regulatory elements of the ß-globin (Hbb) locus control region (LCR) consists of a series of

transcription factor bound DNaseI hypersensitive sites 50 kb upstream of the Hbb-b1 gene

(Forrester et al., 1990; Tuan et al., 1989). The distinct chromatin features of DREs/Enhancers

combined with ChIP-Seq experiments have begun to allow for regulatory element identification

on a genome-wide scale increasing our understanding of gene regulation for mammalian

genomes (Blow et al., 2010; Heintzman et al., 2009; Visel et al., 2009).

1.2.2.1.1 Low nucleosome occupancy and DNaseI sensitivity

Genome wide studies have shown that nucleosome occupancy tends to be low at transcription

start sites and at the boundaries of the cis-regulatory elements indicating that the changes in the

nucleosome dynamics can play a critical role in regulating gene transcription (Mito et al., 2007;

Schones et al., 2008). As a result of low nucleosome occupancy and due to the presence of

``open`` chromatin genomic regions that are rich in DREs tend to exhibit increased sensitivity to

DNaseI treatment in a tissue specific manner (Gross and Garrard, 1988). Moreover, studies have

10

also shown that TFs can induce displacement of nucleosomes thereby inducing changes in the

chromatin structure (He et al., 2010; Li et al., 2007). Studies in prostate cancer cells where

androgen receptor binds to enhancers have shown that in response to stimulation with androgen,

the central H2A.Z containing nucleosome present at androgen receptor binding sites that is

flanked by a pair of marked nucleosomes disappears (He et al., 2010). Hence, this study

suggests that TFs have the ability to displace nucleosomes at their binding sites thereby

changing the chromatin structure.

1.2.2.1.2 p300 binding

A study conducted in the human genome over a region of 30Mb has shown that there is a

correlation between enhancer function and binding of the co-activator protein p300 (Birney et

al., 2007; Heintzman et al., 2007). Furthermore ChIP-Seq studies performed to map thousands

of p300 binding sites across the mouse genome have been able to accurately predict the

presence of novel enhancers in mouse brain, limbs and heart suggesting that there is a

correlation between p300 binding and enhancer function (Blow et al., 2010; Visel et al., 2009).

1.2.2.1.3 Cobound by multiple TFs

DREs are bound by multiple TFs (Chen et al., 2008; Heintzman and Ren, 2009; Jin et al.,

2011). The well characterized regulatory elements of the ß-globin gene are bound by multiple

TFs in the adult erythroid cells (Cho et al., 2008; Song et al., 2007; Wijgerde et al., 1996). TFs

such as GATA1, EKFL1 have been found to bind to the regulatory elements of the ß-globin

gene in a tissue specific manner and have been shown to play an active role in regulation of the

ß-globin gene transcription (Cho et al., 2008; Drissen et al., 2004; Kim et al., 2007; Vakoc et al.,

2005; Wijgerde et al., 1996). As mentioned above TFs have the ability to change the chromatin

structure by displacing nucleosomes at their binding sites thereby regulating gene expression

(He et al., 2010).

11

1.2.3 Third Level of regulation of gene transcription: Spatial organization of

the chromatin

The organization of chromatin in the three dimensional nuclear space plays a vital role in the

regulation of transcription of genes (de Laat and Grosveld, 2003; Palstra et al., 2003). To

understand how chromatin regulates transcription of genes it is important to understand the role

of both chromatin decondensation and chromatin looping in regulating gene transcription.

Studies with the murine ß-globin locus and the cytokine gene cluster have suggested that

formation of higher order chromatin structure not only seems to be an important parameter in

regulation of gene transcription but also correlates with transcription of genes (Palstra et al.,

2003; Spilianakis and Flavell, 2004). Furthermore different proteins such as CTCF, RAD21, and

SATB1 appear to mediate the formation of these higher order structures thereby playing an

active role in regulating gene transcription (Cai et al., 2006; Handoko et al., 2011; Kagey et al.,

2010). Moreover various techniques have been developed over the last decade that are routinely

used to investigate the three dimensional organization of different gene loci to understand how

chromatin conformation can correlate with specific transcriptional states of genes (Dekker et al.,

2002; Dostie et al., 2006; Ethier et al., 2012; Tolhuis et al., 2002; Vassetzky et al., 2009).

Hence, the third and the highest level of regulation of gene transcription lies within the three

dimensional spatial organization of different genetic loci.

1.2.3.1 Relation between transcription and organization of chromatin structure

Studies have shown that there is a strong correlation between transcription and the organization

of chromosomes (Mahy et al., 2002). In higher eukaryotes individual chromosomes occupy a

discrete location within the nucleus known as a chromosome territory (CT) (Cremer and

Cremer, 2001). Within a CT the DNA is further organized, wherein the gene rich regions are

mostly kept separate from gene poor regions (Cremer and Cremer, 2001; Croft et al., 1999;

Lieberman-Aiden et al., 2009). Studies also show that active genes are mostly located at the

periphery of the CT, whereas the inactive genes are concentrated towards the interior of the CT

(Harnicarova et al., 2006). Furthermore, regions with high density of active genes can be located

in loops that extend outside the CTs only in the cells that express those genes but not otherwise

(Volpi et al., 2000; Williams et al., 2002). Studies by Mahy et al. have also shown that genomic

regions comprising of a large number of expressed genes localize outside their CTs (Mahy et al.,

12

2002). Furthermore, it has been shown that chromatin decondensation correlates with the

transcriptionally active state of a genomic region (Chambeyron and Bickmore, 2004). For

example in response to induction by retinoic acid the HoxB gene cluster de-condenses and loops

out of its chromosome territory in order to express the HoxB gene (Chambeyron and Bickmore,

2004). This looping out of the HoxB genes was also accompanied by an overall change in the

histone modification state of the entire locus (Chambeyron and Bickmore, 2004). Hence all

these studies together suggest that not only high levels of transcriptional activity correlates with

localization of genes outside their CTs but also that chromatin structure could probably undergo

a complete reorganization in a tissue specific manner in order to execute the transcriptional

program of a set of genes.

1.2.3.2 Chromatin looping and regulation of gene transcription

Several studies have shown that there is a correlation between formation of tissue specific

chromatin loops and the regulation of gene transcription (Palstra, 2009; Palstra et al., 2003;

Spilianakis and Flavell, 2004; Tolhuis et al., 2002). Chromatin looping can help in bringing not

only DREs close to their target genes but also in sequestering regulatory elements together

thereby forming a tissue specific spatial organization of the chromatin in order to regulate gene

transcription (de Laat and Grosveld, 2003; Palstra et al., 2003).

1.2.3.2.1 Enhancers and chromatin looping

Enhancers have been shown to regulate transcription of their target genes through the chromatin

looping mechanism (Palstra, 2009; Tolhuis et al., 2002). As enhancers can be located far from

their target genes in the DNA sequence they have been shown to come in physical contact with

the target gene promoter in order to regulate gene transcription while the intervening DNA

sequence is looped out (Carter et al., 2002; Palstra et al., 2003; Tolhuis et al., 2002). This

mechanism in known as the chromatin looping mechanism and the process as a whole is known

as the long-range regulation of gene transcription. For example in adult erythroid cells, the

regulatory elements of the Hbb LCR located at a distance of 50 kb from the Hbb-b1 gene is

found in close proximity to the active Hbb genes while the intervening 50 kb of DNA sequence

containing the embryonic erythroid cell expressed genes is looped out (Carter et al., 2002;

Palstra et al., 2003; Tolhuis et al., 2002). As a result of this chromatin looping, the ß-globin

13

locus adopts a tissue specific conformation in adult erythroid cells wherein it is highly

transcribed (Palstra et al., 2003; Tolhuis et al., 2002). On the other hand the locus adopts a more

random conformation in other cell types such as in erythroid progenitor cells and brain cells

where Hbb-b1 is not expressed (Tolhuis et al., 2002). Hence it has been suggested that

conformation of the locus is associated with its active state as well its gene expression pattern

(Palstra et al., 2003).

Currently interactions between different genomic regions are detected by using the

Chromosome Conformation Capture (3C) technique (Dekker et al., 2002). The technique was

initially developed to investigate the three dimensional organization of yeast chromosome 3,

however later it was quickly adapted to understand the regulatory relationships between various

mammalian genes and their DREs (Dekker et al., 2002; Ethier et al., 2012; Spilianakis and

Flavell, 2004; Tolhuis et al., 2002). The 3C technique showed for the first time that long-range

regulation of the β-globin genes is mediated by actual physical interactions between genomic

fragments containing the regulatory elements and the gene promoters (Tolhuis et al., 2002). 3C

since has been used to detect chromatin-chromatin interactions between DREs and several other

genes including; Hba, Shh, TH2, HoxB1 and olfactory receptor genes (Amano et al., 2009;

Lomvardas et al., 2006; Spilianakis and Flavell, 2004; Vernimmen et al., 2007; Wurtele and

Chartrand, 2006). Based on the basic principle of the 3C technique various other techniques

have been developed such as 4C, e4C, 5C, Hi-C that can identify genome wide chromatin-

chromatin interactions and can also detect the conformation of a genome as a whole (Dostie et

al., 2006; Dostie et al., 2007; Ethier et al., 2012; Lieberman-Aiden et al., 2009; Schoenfelder et

al., 2010; Zhao et al., 2006). For example recent Hi-C studies have been able to describe the

conformation of the whole Drosophila melanogaster genome (Sexton et al., 2012).

1.2.3.2.2 Active chromatin hub

At some genetic loci the spatial organization of the chromatin is such that all the regulatory

elements are sequestered to form a tissue specific chromatin structure involved in activating

target gene transcription (de Laat and Grosveld, 2003). For example at the ß-globin locus all the

regulatory elements are sequestered together in order to form the active chromatin hub (ACH)

(de Laat and Grosveld, 2003). ACH formation and spatial clustering of enhancer elements has

been suggested to maintain a high local concentration of tissue specific TFs required for

14

efficient transcription of the target genes. Studies have shown that the formation of the ACH

requires the presence of protein factors bound to the regulatory DNA elements (de Laat and

Grosveld, 2003; Drissen et al., 2004; Vakoc et al., 2005). For example the TFs EKLF and

GATA-1 are required for the formation of the ß-globin ACH (Drissen et al., 2004; Vakoc et al.,

2005).

1.2.3.2.3 Insulators and chromatin looping

The insulator protein CTCF has been shown to participate in intra- and inter-chromosomal

looping from individual gene loci including Hbb, Igf2/H19 and HoxA (Ferraiuolo et al., 2010;

Kooren et al., 2007; Kurukuti et al., 2006; Yang and Corces, 2011). A genome-wide analysis of

chromatin-chromatin interactions at CTCF bound regions identified four distinct classes of

interactions occurring between CTCF bound regions, based on histone modifications: active

domain interactions, repressive domain interactions, enhancer-gene promoter interactions and

loops at the border of opposite chromatin states where CTCF acts as a boundary element

(Handoko et al., 2011).

1.2.3.3 Role of protein complexes in maintaining higher-order chromatin

conformation

CTCF and other proteins like cohesin play an important role in maintaining higher-order

chromatin conformation (Degner et al., 2011). Genome wide studies have shown that CTCF and

cohesin, a protein complex that mediates sister chromatid cohesion, localise to the same regions

of the genome (Parelho et al., 2008; Rubio et al., 2008). Furthermore, at the imprinted IGF2-

H19 locus both cohesin and CTCF are required for maintaining higher-order chromatin

conformation (Merkenschlager et al., 2009; Nativio et al., 2009). Studies with the human

Hep3B cells have shown that the genomic region containing the apolipoprotein gene cluster

APO A1/C3/A4/A5 has overlapping CTCF and cohesin (Rad21) binding sites (Mishiro et al.,

2009). The 3C studies of the locus showed the presence of two chromatin loops, whereas in one

loop the APOA1 promoter is present in the other loops the APO C3/A4/A5 promoters and the C3

enhancer is present (Mishiro et al., 2009). Furthermore, reduction in the levels of either CTCF

or Rad21 disrupts the chromatin loops, thereby causing not only a significant change in the

expression of the APO genes but also a reduction in the localization of the transcription factor

15

HNF-4a and RNAPII-Ser5P specifically at the gene promoter of APO3 (Mishiro et al., 2009).

Interestingly whereas the CTCF bound regions of the genome show limited differences between

cell types, CTCF/cohesin bound regions form tissue specific chromatin loops (Cuddapah et al.,

2009; Hou et al., 2010). Cohesin mediated chromatin loops have also been found at other

genetic loci such as at the IGF2/H19 locus and at the ß-globin locus (Hou et al., 2010). Hence

all these studies together suggest that CTCF-cohesin mediated chromatin looping plays a critical

role in mediating tissue specific regulation of gene transcription.

Members of the cohesin complex also interact with mediator, a complex recruited by TFs which

acts as a bridge to the RNA polymerase II preinitiation complex (Conaway and Conaway, 2011;

Kagey et al., 2010). ChIP-Seq analyses in mouse embryonic stem cells (ESC) showed that

mediator, cohesin and the cohesin loading factor NIPBL proteins co-localise at thousands of

sites across the ESC genome (Kagey et al., 2010). Furthermore knockdown of mediator, NIPBL

or cohesin changed the expression pattern of multiple genes whose enhancers were co-bound by

mediator and cohesin proteins (Kagey et al., 2010). The study also showed that both mediator

and cohesin proteins are bound at enhancers which form ES-cell specific chromatin loops with a

nearby gene promoter thereby regulating gene transcription in a tissue specific manner (Kagey

et al., 2010). Hence this study suggests that cohesin is capable of stabilizing higher-order

chromatin conformation across the genome and necessary for mediating enhancer-gene

interactions.

16

1.3 Lmo2: A candidate for investigating tissue specific chromatin conformation

Lim domain only 2 (LMO2) is a critical transcriptional regulator of hematopoiesis. Gene

targeting experiments conducted to introduce null mutations in the mouse Lmo2 gene, have

shown that Lmo2 is necessary for embryonic yolk sac erythropoiesis (Warren et al., 1994).

During differentiation of hematopoietic progenitor cells, Lmo2 expression is maintained in

erythroid cells but down regulated in the T-cell lineage (Boehm et al., 1991; Foroni et al., 1992;

Royer-Pokora et al., 1991; Warren et al., 1994). Aberrant expression of Lmo2 results in the

development of various T-cell related diseases; indeed Lmo2 is located at a recurrent site of T-

cell acute lymphoblastic leukemia (T-ALL) specific translocation (Fisch et al., 1992; Fitzgerald

et al., 1992; Foroni et al., 1992; Garcia et al., 1991; Larson et al., 1994; Royer-Pokora et al.,

1991). In addition, patients undergoing gene therapy for X-linked severe combined

immunodeficiency developed clonal T-cell proliferation as a result of aberrant transcriptional

activation of Lmo2 when the gene therapy vector integrated near Lmo2 (Hacein-Bey-Abina et

al., 2003; McCormack and Rabbitts, 2004).

Previous studies have shown that in erythroid cells LMO2 is usually present as part of a

complex with the transcriptional regulators, TAL1, E47, LDB1, and GATA1(Osada et al., 1995;

Valge-Archer et al., 1994; Wadman et al., 1997). This protein complex binds DNA by

recognizing a bipartite DNA sequence comprising of an E box and a GATA site (Osada et al.,

1995; Wadman et al., 1997). These LMO2 containing oligomeric complexes along with other

factors in hematopoietic cells have been found on the regulatory regions of various other genes

including, β-globin (Hbb), α-globin (Hba), retinaldehyde dehydrogenase 2, c-kit and erythroid

Kruppel-like factor (Eklf) (Anderson et al., 1998; Anguita et al., 2004; Lecuyer et al., 2002; Ono

et al., 1998; Song et al., 2007; Song et al., 2010).

The proximal promoter of the Lmo2 gene has multiple functional motifs that affect its promoter

activity (Landry et al., 2005). Lmo2 was initially believed to have two transcriptional promoters;

however, recent studies have indicated that the gene actually has three promoters, termed the

17

distal promoter, proximal promoter and intermediate promoter (Oram et al., 2010). Studies have

shown that the transcriptional activity of the proximal promoter (pP) is largely dependent on the

three Ets sites that are present within the conserved region of the pP (Landry et al., 2005).

Mutation of the first Ets site only marginally affected promoter activity of pP, however any

alterations within the second, or third site or both strongly hampered the promoter activity of pP

(Landry et al., 2005). Motif analysis has also shown that the proximal promoter (pP) contains an

E-box, which strongly influences the activity of the Lmo2 pP (Landry et al., 2005). Mutation of

the E-box element results in a small decrease in promoter activity of pP (Landry et al., 2005).

Furthermore, studies have also shown that the TFs FLI1, ELF1, and ETS1 regulate the activity

of the pP in the endothelial cells (Landry et al., 2005). In this context it should be mentioned

that though the pP of Lmo2 is sufficient to drive expression of a reporter gene in endothelial

cells in vivo, the expression levels are weak, and no expression in any other tissue has been

observed (Landry et al., 2005).

Robust expression of Lmo2 in hematopoietic cells requires the presence of multiple regulatory

elements (Landry et al., 2009). As mentioned before, though the proximal promoter of Lmo2 is

sufficient to drive its expression in endothelial cells in vivo, the expression levels are weak, and

no expression in any other tissue has been observed, hence that made researchers look into the

possibility of other regulatory elements that modulate the expression of Lmo2 in erythroid cells.

In fact, recent studies have identified eight distal regulatory elements (DREs) located upstream

of the LMO2 gene in the human genome (Landry et al., 2009). These DREs are capable of

enhancing reporter gene expression in erythroid tissues (Landry et al., 2009). Transgenic

analysis suggests that strong expression of Lmo2 in hematopoietic cells requires the combined

action of these cell-type specific distal regulatory elements (DREs) and the Lmo2 pP (Landry et

al., 2009). Interestingly though these DREs have been identified and it is known that the gene

Lmo2 is highly transcribed in erythroid cells, studies have not been conducted to investigate

how these multiple DREs regulate transcription of Lmo2 in erythroid cells and whether or not

they function cooperatively in the endogenous context.

18

2 Chapter 2: Methods

Analyses of Chip-Seq data from erythroid cells mentioned in this chapter has been

performed by Julie-Chih-Yu Chen.

19

2.1 Cell Isolation

C57/Blk6 mouse was used as the model system in this study. Adult erythroid cells were isolated

in large numbers (>1x 108) from the spleen of mice treated with phenyl hydrazine. This

treatment induces haemolytic anaemia in the mice, as a result of which the spleen becomes the

major site of red blood cell production (Dickerman et al., 1976). Three I.P. injections of 1%

phenylhydrazine solution (1 ml 10% PHZ in DMSO + 9 ml 1x PBS) are prepared and

administered at twelve-hourly intervals with 0.1 ml PHZ per 25 g body weight. Mice are

anaemic four days after first injection.

2.2 Chromosome Conformation Capture (3C)

Chromosome Conformation Capture (3C) experiments were performed as developed by Dekker

et al. 2002 with some minor modifications (Dekker et al., 2002).

3C fixation and digestion of nuclei: Anaemic spleen and kidney dissected from mouse were

strained through 70 µm strainer into chilled petridishes in cold D-MEM + 10% FBS. Cells

(suspended in room temperature medium) were next fixed for 10 min with 2% Formaldehyde.

The reaction was quenched with cold 1 M Glycine, followed by centrifugation at 1300 rpm,

4°C. The cell pellets were next washed with cold PBS and centrifuged at 1300 rpm, 4°C. In the

next step the cells were suspended in cold lysis buffer (10 mM Tris-HCl, pH 8, 10 mM NaCl,

0.2% NP-40, and complete protease inhibitors), and incubated on ice for 30 min with occasional

mixing followed by centrifugation at 1800 rpm. Next, the nuclei were resuspended in a trace of

liquid and counted using a haemocytomoter. 1x107 nuclei aliquots were next resuspended in

500 ul of 1.2x NEB2 Buffer containing 20% SDS and incubated for 1 hr at 37°C.Next 20%

Triton-X100 was added to the reaction and incubated for 1 hr at 37°C. Finally, 1500U of

HindIII were added to the tube and incubated overnight at 37°C.

3C ligation: 20% SDS was added to the tubes and incubated for 25 min at 65°C to deactivate

the HindIII enzyme. After which the contents of the tube were added to 7 ml of 1.1x ligation

buffer. 20% Triton-X100 was next added to neutralize the SDS and incubated for 1 hr at 37°C,

mixing occasionally. Next, 800U of T4 DNA ligase was added to the reaction and incubated

20

overnight in a 16°C water bath, then for 30 min at room temp. Finally 900 µg Proteinase K was

added to the ligation reaction and incubated overnight at 65°C.

3C DNA purification: The reactions were cooled to room temperatre before adding 300 µg of

RNase A and incubated at 37°C for 1 hr following which the DNA is purified using the Phenol-

Chloroform extraction and the pellet is resuspended in water.

3C validation and control template preparation: The DNA was quantified using the PicoGreen

assay. The 3C control template was prepared by mixing equimolar amounts of the BAC clone of

the entire Lmo2-Caprin1 locus (RP23-76D2) with the Alpha Aortic Actin 2 BAC clone (RP23-

2N15) followed by digestion with HindIII. The digested DNA was then ligated, and purified

using phenol extraction and ethanol precipitation. HindIII restriction enzyme digestion

efficiency was confirmed to be between 85 and 95% efficient at several genomic fragments in

anaemic spleen and kidney cells (Appendix 4). The linear range of amplification was

determined for erythroid and kidney samples by serial dilution. An appropriate amount of the

DNA within the linear range (typically 40 ng of DNA) was subsequently used for quantification.

PCR products of the ligated fragments were quantified using real-time quantitative PCR (qPCR)

on the Bio-Rad CFX-384 cycler. All data points were generated from an average of between

three and five independent 3C experiments with the real-time quantitative PCR performed in

triplicate. Standard curves were generated by 5 fold serial dilution of the 3C control template

and run in parallel with 3C experimental samples. The primers used for real-time quantitative

PCR are listed in apppendix 3. In each individual experiment 3C data were normalized to

neighbouring fragments at the Alpha aortic actin (α-A2) locus.

21

Figure 2-1.Chromosome Conformation Capture (3C)

Digrammatic representation of the steps involved in the 3C technique. First the cells are fixed

with formaldehyde, which forms crosslinks between proteins attached to DNA segments that are

close together in the nuclear space. The cross linked chromatin next is digested with a suitable

restriction enzyme. Next the DNA ends are ligated under dilute conditions that favour intra

molecular ligation events between cross-linked DNA fragments. Finally the cross links are

reversed and the ligation events between selected pairs of restriction fragments are quantified by

real-time quantitative PCR, using primers specific for the given hybrid fragment.

2.3 RNA isolation and real-time RT-qPCR

RNA from anaemic spleen and kidney was isolated using TRIzol, according to the

manufacturer`s instructions (Invitrogen). The isolated RNA was next treated with DNaseI

(Fermentas) followed by phenol-chloroform extraction in order to remove any DNA and DNase

I contamination from the RNA. The RNA was next used for cDNA syntheisis. The iScript First

strand synthesis cDNA kit from Bio-Rad was used for preparation of random-hexamer primed

cDNA. Real-time qPCR was performed on the Bio-Rad CFX-384 cycler. The reaction mixture

contained 2X Bio-Rad iTaq SYBR green mastermix with ROX, 0.3pM of each primer, 1uL

cDNA (10 times diluted from a 20uL reverse transcription reaction). The conditions for real-

22

time PCR were as follows: 94°C for 3 min followed by 40 cycles at 94°C for 30s, 62°C for 30s.

Expression levels of Gapdh or Epn1 were used for normalization of expression levels. The

primers used for RT-qPCR are listed in Appendix 3.

2.4 Statistical analysis

The 3C data were analyzed by two-way ANOVA using Sigma Plot12. Post tests (Holm-Sidak

method) were performed to assess significant differences between anaemic spleen and kidney at

specific genomic locations.

2.5 Genome Mapping and Peak Identification of ChIP-seq datasets in

erythroid cells

ChIP-seq raw data for GATA1, KLF1, LDB1, TAL1, and MTGR1 listed in table 2-1 were

downloaded from Gene Expression Omnibus (GEO) (Barrett et al., 2011; Cheng et al., 2009;

Soler et al., 2010). ChIP-seq data were aligned to NCBI m37 mouse assembly (mm9) using

Bowtie alignment (Langmead et al., 2009) by suppressing alignments to only 1 best reportable

alignment with a maximum number of 2 mismatches within 28 nucleotides of seed length in the

high quality end. The SISSRs (Jothi et al., 2008) algorithm was subsequently used to identify

significant transcription factor peaks compared to that of the input DNA with p <0.001. To

remove amplification bias, multiple reads aligning to the same genomic coordinate is counted as

one. Parameters for the corresponding transcription factor data were set according to original

publications using applicable input data sets. Significant transcription factor peaks were

uploaded to the UCSC genome browser for visualization (Rhead et al., 2010). The HPC7 ChIP-

Seq data analysis was performed by using published peaks (Wilson et al., 2010). ChIP-Seq data

for CTCF, p300, RAD21, DNaseI hypersensitivity, H3K4me1 data were obtained from the

mouse ENCODE project (Table 2-1) (Birney et al., 2007).

23

Table 2-1. Chromatin immunoprecipitation sequencing data

Transcription factor binding sites have been obtained from three different cell types;

differentiated murine erythroleukemia cells (MEL), hematopoietic progenitor cells (HPC7), and

GIE-ER4 a GATA1-null erythroblast cell line in which GATA1 activity was restored. CTCF,

DNaseI hypersensitivity, H3K4me1, p300 and RAD21 data have been obtained from the mouse

ENCODE project, sources listed (Principal investigator, Institution).

Protein or Chromatin

Feature

Cell type References

LDB1

Differentiated

MEL

(Soler et al., 2010)

TAL1

MTGR1

GATA1

GATA1 GIE-ER4 cells

(Cheng et al., 2009)

H3K4me1 ENCODE (R Hardison, Penn State

University)

KLF1 e14.5 fetal livers Tallack et al. 2010

ERG

HPC7

Hematopoietic

Progenitor Cells

(Wilson et al., 2010)

FLI1 RUNX1 LYL1 MEIS1 PU1 GATA1 GATA2 GFI1b TAL1 LMO2

p300 MEL ENCODE

(M Snyder, Stanford University) CTCF

MEL (2% DMSO) RAD21

DNaseI hypersensitivity MEL, Kidney ENCODE

(JA Stamatoyannopoulos, University

of Washington)

CTCF Various ENCODE

(B Ren, Ludwig Inst. for Cancer

Research)

24

3 Chapter 3: Distal regulatory elements located upstream of Lmo2 are associated with tissue-specific chromatin features

Chapters 3 and 4 have been prepared as a manuscript for submission to a peer-reviewed journal

25

3.1 Introduction

Identification of distal regulatory elements is the key to understanding tissue specific regulation

of gene transcription. Genome wide studies have shown that some of the characteristic features

of the enhancers are their ability to function in a tissue specific manner as well as to exhibit cell

type-specific chromatin features (Heintzman et al., 2009). For example genome wide studies to

identify p300 binding sites in three different embryonic tissues have shown that majority of the

binding sites are occupied by the protein in only one of the tissues (Visel et al., 2009). Further

experiments showed that these p300 bound regions exhibited tissue specific enhancer activities

in vivo (Visel et al., 2009). Moreover, enhancers identified in different cell types have been

found to be associated with tissue specific histone modification patterns (Heintzman et al.,

2009). Hence, all these studies together suggest that enhancers play a key role in driving tissue

specific transcription of genes. However, identification of the enhancer elements required for the

tissue-specific expression of specific genes is difficult because these regulatory sequences lie

embedded within the non-coding part of the mammalian genome (Xi et al., 2007). At present a

combination of molecular biology based techniques and bioinformatic tools are used to identify

these regulatory elements (Blow et al., 2010; Chen et al., 2012; Chen et al., 2008; Heintzman et

al., 2009; Heintzman et al., 2007; Visel et al., 2009; Xi et al., 2007). Features that are used in

many cases to identify DREs are DNase I hypersensitivity, histone methylation patterns, binding

of multiple TFs as well as binding of the histone acetyl transferase p300 to specific DNA

sequences (Blow et al., 2010; Chen et al., 2008; Heintzman et al., 2009; Heintzman et al., 2007;

Visel et al., 2009; Xi et al., 2007). Therefore, in order to understand the tissue specific

regulation of Lmo2, I have used a combination of bioinformatic tools and ChIP-Seq data to

identify the location and the tissue specific epigenetic features of the DREs located near the

Lmo2 gene in the mouse genome. In addition, as previous studies have shown that DREs in

some cases are transcribed at very low levels in a tissue specific manner, I have also

investigated intergenic transcription to determine if DREs and other regions within the

Lmo2/Caprin1 locus are transcribed in erythroid cells (Gribnau et al., 2000; Miles et al., 2007).

In this chapter an in depth analysis of the chromatin features of the Lmo2/Caprin1 locus has

been conducted to identify the factors that might play a role in regulating Lmo2 transcription in

adult erythroid cells.

26

3.2 Results

3.2.1 Identification and mapping of the enhancer elements on the mouse

genome

Multiple DREs upstream of Lmo2 have been identified in the human genome and confirmed to

have enhancer activity in transgenic mice (Landry et al., 2009). I mapped the proximal and

distal promoters and enhancer sequences within a region of 90kb upstream and 7kb downstream

of Lmo2 in the mouse genome (Figure 3-1). To maintain the naming used in Landry et al.2009

the mapped DREs were named based on their distances upstream or downstream from the

annotated Lmo2 TSS overlapping the proximal promoter (pP) (Landry et al., 2009). Altogether

11 enhancer elements were identified in the mouse genome located upstream of Lmo2, whereas

two enhancer elements (+1 and +7) were located downstream of the Lmo2 pP. Many of the

enhancer elements were found to have multiple subparts, with homology to the corresponding

human enhancer element. For example when looking for sequence homology with the human

enhancer element 75 (75 DRE), six regions in the mouse genome were found to have sequence

homology, these sequences are separated from one another by several base pairs. The first two

homologous regions are separated by only 60 base pairs, whereas the distance between the third,

fourth, fifth, and sixth regions is about one kb. As these sequences were located close to one

another and had sequence homology to a single human Lmo2 enhancer element, all six regions

were considered to be a subpart of the single enhancer element unit 75DRE. Similarly the

70DRE has ten subparts and all the elements together are spread over a distance of about 3 kb.

On the other hand the 35DRE has two subparts which are only separated by five bp. In addition

to the DREs I also mapped the promoter elements for the gene. Lmo2 pP also comprises of 12

subparts spread over a distance of 760bp, whereas the distal promoter (dP) of the gene has nine

subparts spanning 560b.Recent studies have with the human Lmo2 gene has identified a third

promoter known as the intermediate promoter, however due to the unavailability of its

coordinates it couldnot be mapped onto the mouse genome (Oram et al., 2010).

27

Figure 3-1. Genomic map of the Lmo2/Caprin1 locus showing the identified DREs

The mouse Lmo2-Caprin1 region on chromosome 2 is depicted with chromosome coordinates

shown at the top. The two Lmo2 promoters are indicated by red boxes. Distal regulatory

element (DRE) homology regions are indicated by black boxes joined by a line to delineate the

human enhancer construct used in the generation of transgenic mice. Proximal promoter (pP),

distal promoter (dP).

28

3.2.2 TFs bind to the distal regulatory elements

DNA sequences with regulatory roles that are located in the intergenic regions of the genome in

many cases are bound by TFs in the tissues in which they are active (Chen et al., 2008;

Fullwood et al., 2009; Yu et al., 2009). For example, the regulatory elements of the well

characterized ß-globin LCR are bound by multiple TFs in erythroid cells (Johnson et al., 2002;

Kim et al., 2007; Sawado et al., 2001; Song et al., 2007). These TFs play crucial roles in the

regulation of the ß-globin genes in adult erythroid cells (Cho et al., 2008; Johnson et al., 2002;

Sawado et al., 2001; Song et al., 2007; Wijgerde et al., 1996). As the DREs in the human

genome had been confirmed to have enhancer activity in transgenic mice, I hypothesized that

some of the mapped DREs should have TFs bound in mature erythroid cells (Landry et al.,

2009). Furthermore, several TFs (LMO2, TAL1, GATA2, FLI1, and SFPI1) had already been

found to be associated with these human DREs identified by Landry et al. using a ChIP-chip

approach (Landry et al., 2009). Hence, I wanted to investigate whether these mouse DREs

overlap any TF bound regions in erythroid cells.

3.2.2.1 Multiple TFs binding to DREs in mouse erythroid cells

I retrieved available ChIP-Seq data for definitive mouse erythroid cells (KLF1, MTGR1,

GATA1, TAL1, LDB1) to more finely map the transcription factor-bound regions within each

DRE (Cheng et al., 2009; Soler et al., 2010). My analysis revealed several TFs were bound at

three of the identified enhancer elements. Whereas, four TFs MTGR1, GATA1, TAL1, LDB1

were bound at the 75 DRE only one TF (GATA1) was bound at the 40 DRE and two TFs

(GATA1, LDB1) were bound to the 12 DRE (Figure 3-2, Appendix 5). The transcription factor

KLF1 was not found to bind to any of the DREs, however it was bound to a region located

immediately upstream of the 75DRE (Figure 3-2). Interestingly, of all the four TFs that were

bound to the DREs, GATA1 was the only TF that was bound to all the three DREs, whereas

LDB1 was bound at two of the three DREs suggesting that these TFs might be playing a crucial

role in regulating expression of the Lmo2 gene (Figure 3-2).

29

Figure 3-2. Distal regulatory elements upstream of Lmo2 overlap transcription factor

bound regions in erythroid cells

The mouse Lmo2-Caprin1 region on chromosome 2 is depicted with chromosome coordinates

shown at the top. The two Lmo2 promoters are indicated by red boxes. Distal regulatory

element (DRE) homology regions are indicated by black boxes joined by a line to delineate the

human enhancer construct used in the generation of transgenic mice. Coloured boxes represent

peaks identified from transcription factor ChIP-Seq data for erythroid (MEL and GIE-ER4)

cells. Overlapping transcription factor peaks were identified at the 75 and 12 DREs. Proximal

promoter (pP), distal promoter (dP), transcription factor (TF).

30

3.2.2.2 Multiple TFs binding to the DREs in HPC7 cells

My initial analysis of transcription factor binding at the intergenic region between Caprin1 and

Lmo2 highlighted only three DREs (75, 40, and 12) bound by TFs. To investigate additional

transcription factor bound regions I analysed ChIP-Seq data available for the HPC7

hematopoietic progenitor cell line (Pinto do et al., 1998; Wilson et al., 2010) (Table 2-1). The

HPC7 cells are a model hematopoietic stem/progenitor cell line (early fetal multipotent

hematopoietic progenitor cell line), they have the potential to form any of the hematopoietic

cells, addition of necessary growth factors such as Steel factor and Erythropoietin can efficiently

induce differentiation of the HPC7 cells to form both primitive and definitive erythroid cells

(Pinto do et al., 1998). As this was the only available ChIP-Seq data set that was collected from

a cell type closest to erythroid cells (HPC7 cells are the progenitors of erythroid cells), hence I

used their ChIP-Seq for my analysis (Pinto do et al., 1998; Wilson et al., 2010). This HPC7 data

set also contains GATA1 and TAL1, as did the earlier analysis of differentiated erythroid cell

ChIP-Seq data, along with several additional TFs (ERG, FLI1, GATA2, GFI1B, LMO2, MEIS1,

PU1, RUNX1) (Wilson et al., 2010). This data revealed multiple TFs are associated with many

of the other DREs in the intergenic region upstream of the Lmo2 gene including the 75, 40 and

12 DREs in HPC7 cells (Figure 3-3). Multiple TFs were bound at several TFs including 9, 6,

and 4 bound at the 75, 40 and 12 DREs and 3 bound at each of the 90, 70, 64 and 3 DREs

(Figure 3-3). Interestingly apart from the DREs 58, 47 and 43 all the other DREs are bound by

ERG) (Figure 3-3). Furthermore, in addition to the DREs intergenic regions upstream of the

90DRE were also bound by ERG (Figure 3-3). One of these intergenic regions was co-bound by

ERG and FLI1 and this co-binding was also observed at the DREs 75, 70, 64, 40, 35, and 25

(Figure 3-3). A fact worth mentioning here is that many of the human Lmo2 enhancer elements

were bound by the transcription factor FLI1 as detected using the ChIP-chip approach

suggesting that FLI1 and ERG could be playing an important role in the regulation of the Lmo2

gene both in humans and in mouse (Landry et al., 2005).

31

Figure 3-3. Distal regulatory elements upstream of Lmo2 overlap transcription factor

bound regions in HPC7 hematopoietic progenitor cells

The mouse Lmo2-Caprin1 region. Distal regulatory element (DRE) homology regions are

indicated by black boxes joined by a line to delineate the human enhancer construct used in the

generation of transgenic mice. Coloured boxes represent peaks identified from transcription

factor ChIP-Seq data from HPC7 hematopoietic progenitor cells (Pinto do et al., 1998; Wilson et

al., 2010). Proximal promoter (pP), distal promoter (dP).

32

3.2.3 The DREs have erythroid cell-specific epigenetic features

Enhancers are not only co-bound by multiple TFs but are also associated with increased

sensitivity to DNaseI, and are bound by the histone acetyl transferase protein p300 (also known

as EP300) (Blow et al., 2010; Visel et al., 2009; Xi et al., 2007). For example the well studied

regulatory elements of the ß-globin (Hbb) locus control region (LCR) consists of a series of

transcription factor bound DNaseI hypersensitive sites 50 kb upstream of the Hbb-b1 gene

(Tuan et al., 1989). I next investigated if the DREs in the mouse genome were associated with

any of the enhancer features mentioned above. For this purpose I used available ChIP-Seq data

from the mouse ENCODE project to identify p300 binding and DNaseI hypersensitivity across

the entire Lmo2/Caprin1 locus (Table 2-1) (Birney et al., 2007). My analysis showed that indeed

many of the DREs are not only bound by the transcriptional co-activator protein p300 but are

also associated with increased sensitivity to DNaseI (Figure 3-4). Whereas, binding of p300 was

identified at the 75, 70, 40, 25 and 12 DREs, DNaseI hypersensitivity was identified only at the

75, 25 and 12 DREs (Figure 3-4). Furthermore, comparison of the ChIP-Seq data for DNase I

sensitivity between erythroid cells and the kidney cells showed that DNaseI hypersensitivity at

75, 25 and 12 was erythroid cell specific as peaks were absent from kidney cells (Figure 3-4)

(Birney et al., 2007).

In addition to DNaseI hypersensitivity and p300 binding I also investigated the histone

modification pattern of the entire Lmo2-Caprin1 locus in erythroid cells (Birney et al., 2007).

My analyses showed that the entire region is marked with high levels of histone H3 lysine 4

monomethylation (H3K4me1), which is believed to be an epigenetic mark for enhancers (Figure

3-4) (Chen et al., 2008; Gross and Garrard, 1988; Ren et al., 2007; Visel et al., 2009; Visel et al.,

2010; Wu, 1980). In summary individual DREs as well as some intergenic regions within the

Lmo2/Caprin1 locus showed increased DNaseI hypersensitivity, presence of high levels of

H3K4me1 mark and were bound by p300 in erythroid cells suggesting that the DREs as well

some other intergenic regions in the locus might be involved in regulating the Lmo2 gene in

erythroid cells.

33

Figure 3-4. Distal regulatory elements upstream of Lmo2 overlap transcription factor

bound regions and have different epigenetic marks in erythroid cells

The mouse Lmo2-Caprin1 region on chromosome 2 is depicted with chromosome coordinates

shown at the top. The two Lmo2 promoters are indicated by red boxes. Distal regulatory

element (DRE) homology regions are indicated by black boxes joined by a line to delineate the

human enhancer construct used in the generation of transgenic mice. Mouse ENCODE ChIP-

Seq data for p300 and DNaseI hypersensitivity are shown below the DREs (Birney et al., 2007).

Coloured boxes represent peaks identified from transcription factor ChIP-Seq data for erythroid

(MEL and GIE-ER4) cells. Overlapping transcription factor peaks were identified at the 75 and

12 DREs. These regions were also occupied by p300 and showed increased sensitivity to

DNaseI. The entire locus was marked with a high level of histone H3 lysine 4 monomethylation

(H3K4me1). Proximal promoter (pP), distal promoter (dP), murine erythroleukemia cells

(MEL), Transcription factors (TF).

34

3.2.4 Intergenic transcription occurs at the distal regulatory elements

Previous studies have identified intergenic transcripts at the human Hbb LCR (Gribnau et al.,

2000; Miles et al., 2007; Tuan et al., 1992). In addition enhancer RNA (eRNA) has been

identified at several neuronal enhancers (Kim et al., 2010). Furthermore, a recent study has also

shown that long non-coding RNAs can play a role in regulating the transcription of

neighbouring genes (Morris, 2009). Therefore to investigate if intergenic transcription occurs at

the DREs upstream of Lmo2, I performed RT-qPCR using primers overlapping the DREs and

both up- and down-stream of several DREs. This RT-qPCR analysis identified measurable

levels of intergenic transcription occurring at all identified DREs in erythroid cells (Figure 3-5).

My analysis also showed that transcripts were quite abundant at the 12, 58, 64, 70, 75 DREs and

immediately upstream of the 58, 64, and 70 DREs (Figure 3-5). Interestingly regions between

the DREs were also transcribed in relatively high levels in erythroid cells but not in kidney cells

(Figure 3-5). Intergenic transcription was also detected at the distal promoter (dP) of the gene

and at a distance of 150bp downstream of the dP (Figure 3-5). However in this case transcripts

were detected in both kidney and erythroid cells, though the level of transcription was

marginally higher in erythroid cells when compared to kidney (Figure 3-5). Transcripts in

erythroid cells but not in kidney cells were also detected in the region located immediately

upstream of the 90DRE and at regions located about 8kb, 15kb, and 30kb upstream of the

90DRE (Figure 3-5). As I was able to amplify intergenic transcripts at several locations both

upstream and downstream of the DREs as well as between the individual DREs it seems that the

transcripts proceed throughout a broad region located upstream of the Lmo2 gene (Figure 3-5).

My analysis suggests that the entire region encompassing the 90, 75, 70, 64, and 58 DREs are

transcribed at moderate levels (Figure 3-5). Hence at the end it can be summarized that I found

that all identified DREs as well as the regions in between the different DREs are transcribed at

low to high levels in erythroid cells but not in kidney cells.

35

Figure 3-5. Intergenic transcription occurs in anaemic spleen erythroid cells.

Primary transcript levels in adult mouse anaemic spleen and kidney (blue) for; distal regulatory

elements (DRE), regions upstream and downstream of the DREs, distal promoter (dP), proximal

promoter (pP), and Lmo2 (exon2-intron2), Caprin1 (exon3-intron2). Levels were quantitatively

assessed by RT-qPCR and expressed relative to Gapdh. Gapdh is a ubiquitously expressed

reference gene.

36

3.2.5 CTCF and RAD21 bind to multiple regions across the Lmo2/Caprin1

locus

Investigating ChIP-Seq data released by the mouse ENCODE project (Table 2-1) (Birney et al.,

2007) I identified several CTCF and cohesin (RAD21) bound regions across the entire

Lmo2/Caprin1 locus (Figures 3-6 and 3-7). My ChIP-Seq data analysis not only showed that

some regions were co-bound by CTCF and RAD21 proteins but there were also regions that

were only bound by the RAD21 protein in erythroid cells such as the 25 DRE and a region

located downstream of the 58 DRE (Figure 3-6). Furthermore, I also identified a region located

further upstream of the 90 DRE that was co-bound by CTCF and RAD21 (Figure 3-6).

However, the CTCF bound region upstream of the 90 DRE was bound by CTCF in several cell

types (Figure 3-7). Additional CTCF and cohesin bound regions were located just down-stream

of the 75 DRE and at the Lmo2 proximal promoter (Figure 3-6). Like the CTCF bound region

upstream of the 90 DRE, CTCF was bound at the Lmo2 proximal promoter in several cell types

(Figure 3-7). In contrast CTCF was bound 400 bp downstream of the 75 DRE predominantly in

erythroid cells, suggesting that the CTCF protein bound to the 75 DRE probably plays a role in

the transcription of Lmo2 in erythroid cells.

37

Figure 3-6. CTCF and RAD21 are bound within the Lmo2-Caprin1 region

A) The mouse Lmo2-Caprin1 region on chromosome 2 is depicted with chromosome

coordinates shown at the top. B) A zoomed in view of the region upstream of Lmo2 showing

the locus wide binding of RAD21 marked in pink and CTCF marked in blue. In both the two

Lmo2 promoters are indicated by red boxes. Distal regulatory element (DRE) homology regions

are indicated by black boxes joined by a line to delineate the human enhancer construct used in

the generation of transgenic mice. Mouse ENCODE ChIP-Seq data for the cohesin complex

member RAD21 and CTCF are shown below DREs (Birney et al., 2007). Proximal promoter

(pP), distal promoter (dP), murine erythroleukemia cells (MEL differentiated with 2% DMSO).

38

Figure 3-7. CTCF bound upstream region of Lmo2 in different cell types.

The mouse Lmo2 upstream region on chromosome 2 is depicted with chromosome coordinates

shown at the top. HindIII restriction sites are indicated by blue lines. The two Lmo2 promoters

are indicated by red boxes. Distal regulatory element (DRE) homology regions are indicated by

black boxes joined by a line to delineate the human enhancer construct used in the generation of

transgenic mice. Mouse ENCODE ChIP-Seq data for the CTCF are shown below DREs in

different cell types (Birney et al., 2007). Proximal promoter (pP), distal promoter (dP), murine

erythroleukemia cells (MEL differentiated with 2% DMSO), bone marrow (BM), embryonic

stem cells (ES-Bruce4), mouse embryonic fibroblasts (MEF).

39

3.3 Discussion

In this chapter, I have used bioinformatic analysis of ChIP-Seq data for mature erythroid cells

and HPC7 hematopoietic progenitor cells to identify cell type-specific enhancer elements which

are located upstream of the Lmo2 gene in the mouse genome (Birney et al., 2007; Cheng et al.,

2009; Soler et al., 2010; Wilson et al., 2010). Furthermore ChIP-Seq data to identified cell type-

specific epigenetic features at these DREs and the binding of specific chromatin associated

proteins with a possible role in regulating the expression of the Lmo2 gene in erythroid cells.

3.3.1 TFs bind to the distal regulatory elements

Multiple TFs were bound to many of the identified DREs and other intergenic regions at the

Lmo2/Caprin1 locus both in erythroid and in HPC7 cells with the highest density of

transcription factor binding at the 75 DRE (Figures 3-2 and 3-3). Previous studies in circulating

erythroid cells of transgenic mice have show that the human 75 DRE has the strongest enhancer

activity in erythroid cells and drives the expression of a reporter gene, cooperatively with the

Lmo2 proximal promoter and +1 enhancer element (Landry et al., 2009). Interestingly, one of

the TFs that is bound to the 75 DRE is the protein LMO2 itself (Figures 3-2 and 3-3). In

addition to the 75DRE, the transcription factor LMO2 was also bound to the DREs 12, 25 and

40 in indicating that LMO2 might be involved in the regulation of its own transcription (Figures

3-2 and 3-3). In addition to LMO2 two other TFs (TAL1, GATA1) were bound to the 75DRE in

erythroid cells and to the 75 and 40DREs in the HPC7 cell line (Figure 3-3). Of note is the fact

that these three TFs (TAL1, GATA1, LMO2) along with LDB1 are a part of an oligomeric

complex that controls transcription of other important haematopoietic genes such as α-globin

(Hba), β-globin (Hbb) and erythroid Kruppel-like factor (Eklf) in erythroid cells (Anderson et

al., 1998; Anguita et al., 2004; Osada et al., 1995; Song et al., 2007; Song et al., 2010; Valge-

Archer et al., 1994; Wadman et al., 1997). Furthermore, my analysis of TF binding in erythroid

cells also shows that LDB1 binds to the 75DRE along with TAL1 and GATA1 (Figure 3-2).

Hence, this oligomeric complex might be involved in the regulation of Lmo2 in mouse erythroid

cells as well. Interestingly, GATA-1 (which is one of the components of the LMO2 oligomeric

complex) is the only transcription factor amongst the five TFs (TAL1, MTGR1, KLF1, LDB1,

GATA1) whose ChIP-Seq data from erythroid cells were analysed, that was bound to the three

DREs both in erythroid and in HPC7 cells suggesting that this TF might be playing an important

40

role in the regulation of Lmo2 transcription (Figures 3-2 and 3-3). In fact studies by Wang et al.

have already shown that GATA-1 is involved in the regulation of Lmo2 by binding to its

promoter (Wang et al., 2007).

Analysis of the ChIP-Seq data from the HPC7 cell line underlined the fact that two ETS factors

ERG and FLI1 have the highest number of binding sites across the entire Lmo2/Caprin1 locus

(Figure 3-3). Both the TFs bind not only to the DREs but also to other intergenic region across

the Lmo2/Caprin1 locus in the HPC7 cell line (Figure 3-3). Hence, it seems that these two TFs

might be playing an important role in the regulation of the Lmo2 gene. In agreement with my

analyses are two studies that have shown that FLI1 and ERG both play important roles in the

regulation of the Lmo2 gene (Landry et al., 2005; Oram et al., 2010). Whereas one of the studies

in human T-ALL samples has shown that ERG regulates LMO2 transcription by binding to the

LMO2 Intermediate promoter, another study conducted by Landry et al. has shown that the

transcription factor FLI1 regulates transcription of the human LMO2 gene by binding to its

proximal promoter (Landry et al., 2005; Oram et al., 2010). Interestingly, my analyses suggests

that in addition to controlling Lmo2 transcription individually these two ETS factors might be

part of a common circuitry that regulates Lmo2 transcription in mouse erythroid cells essentially

because multiple intergenic regions are co-bound by FLI1 and ERG (Figure 3-3). In fact a study

has shown that both the ERG and FLI1 can together mediate the aberrant expression of LMO2 in

T-cells by binding to the gene promoter in a subset of T-ALL patients (Oram et al., 2010).

In addition to ERG and FLI1 another transcription factor another transcription factor that has

been implicated in the regulation of Lmo2 transcription is PU.1 (Wang et al., 2007). Analysis of

the TF binding across the entire Lmo2-Caprin1 locus has shown that indeed in the HPC7 cell

line PU.1 binds to the DREs 75, 25 and +1, and also to a region located in between the 90 and

75DREs (Figure 3-3). Hence analysis of the HPC7 ChIP-Seq data seems to suggest that PU.1

might play a role in regulating Lmo2 transcription by binding to the upstream DRE elements

thereby helping Lmo2 to be transcribed in a tissue specific manner.

At the end it can be summarized that binding of multiple erythroid and HPC7 cell specific TFs

to the DREs and other intergenic regions at the Lmo2/Caprin1 locus suggests that transcription

of Lmo2 in erythroid cells requires the coordinated action of various erythroid cell specific TFs.

This data also underlines the potential role of these upstream sequences that are co-bound by

41

multiple TFs in the regulation of Lmo2 transcription.

3.3.2 The DREs have erythroid cell-specific epigenetic features

Analysis of the available ChIP-Seq data from the mouse ENCODE project to identify p300

binding, DNaseI hypersensitivity and the histone modification pattern across the entire

Lmo2/Caprin1 locus indicated that the DREs have multiple erythroid cell specific chromatin

features (Birney et al., 2007). My analyses showed that many of the DREs were not only bound

by the transcriptional co-activator protein p300 (also known as EP300) but were also associated

with increased sensitivity to DNaseI (Figure 3-4). According to recent studies the presence of

p300 binding and increased sensitivity to DNase I treatment suggests that the regions in question

might have tissue specific enhancer activities (Blow et al., 2010; Visel et al., 2009; Xi et al.,

2007). Hence, in this case I have identified three DREs (75, 25, and 12) all of which have both

the features (Figure 3-4). In agreement with my analysis a previous study has already shown

that in circulating erythroid cells of transgenic mice the human 75 DRE has the strongest

enhancer activity in erythroid cells and drives the expression of a reporter gene, cooperatively

with the Lmo2 proximal promoter and +1 enhancer element (Landry et al., 2009). Hence it

seems that the other two DREs (12 and 25) can also function as enhancers of the Lmo2 gene.

Interestingly, comparison of the ChIP-Seq data for DNase I sensitivity between erythroid cells

and the kidney cells showed that DNaseI hypersensitivity at 75, 25 and 12 was erythroid cell

specific as peaks were absent from kidney cells (Figure 3-4). In addition to DNaseI

hypersensitivity and p300 binding the entire Lmo2/Caprin1 region was marked with high levels

of histone H3 lysine 4 monomethylation (H3K4me1), which is believed to be an epigenetic

mark for enhancers, however as this data could not be compared with any other cell type, hence

it is difficult to understand whether or not this is an erythroid cell specific feature (Figure 4)

(Chen et al., 2008; Gross and Garrard, 1988; Ren et al., 2007; Visel et al., 2009; Visel et al.,

2010; Wu, 1980). Hence, at the end it can be summarized that both the entire Lmo2/Caprin1

locus as well as the individual DREs were marked with specific epigenetic features in erythroid

cells suggesting that some of the DREs especially the DREs 75, 25, and 12 as well some other

intergenic regions in the locus might be involved in an erythroid cell specific regulatory activity.

42

3.3.3 Intergenic transcription occurs at the distal regulatory elements

Intergenic transcripts were detected at various locations across the entire Lmo2/Caprin1 locus

(Figure 3-5). My analysis suggests that the entire region encompassing the 90, 75, 70, 64, and

58 DREs are transcribed at moderate levels in erythroid cells (Figure 5). This picture seems very

similar to what happens at the Hbb LCR, wherein a large domain encompassing all the DREs

and the region in between the regulatory elements is transcribed at fairly high levels (Gribnau et

al., 2000; Miles et al., 2007; Tuan et al., 1992). Though the exact function of these transcripts

are not known however they seem to be in some way related to the regulation of Lmo2

transcription especially due to the fact that they were mostly transcribed from DREs that seem

to play an important role in enhancing Lmo2 transcription in erythroid cells.

3.3.4 CTCF and RAD21 bind to multiple regions across the Lmo2/Caprin1

locus

The proteins CTCF and RAD21 have already been implicated in regulating gene expression in a

tissue specific manner at the murine ß-globin locus and at the human cytokine (IFNG) gene

locus by mediating the formation of cell type specific higher order chromatin conformation

(Chien et al., 2011; Hadjur et al., 2009). In this chapter I have also shown the presence of both

CTCF and RAD21 at multiple locations across the entire Lmo2/Caprin1 locus. Hence, analyses

of the ChIP-Seq data suggests that these two proteins might be involved in the regulation of the

Lmo2 gene probably by mediated the formation a higher order chromatin structure that brings in

spatial proximity the DREs and the Lmo2 gene thereby ensuring a higher level of transcription

of Lmo2 in erythroid cells (Birney et al., 2007).

3.3.5 The big picture

In this chapter bioinformatic and ChIP-Seq data analyses has helped me identify the unique

features of the Lmo2/Caprin1 locus in the mouse erythroid cells (Birney et al., 2007; Cheng et

al., 2009; Soler et al., 2010; Wilson et al., 2010). I have not only identified tissue specific DREs

at this locus but have also identified erythroid cell specific chromatin features and key

epigenetic marks of the entire Lmo2/Caprin1 locus. Hence, an in depth analysis of the

transcription factor binding pattern, DNaseI hypersensitivity pattern and a close look at the

43

binding of proteins p300, CTCF, and RAD21 and identification of histone signatures has helped

me to build a big picture of how the Lmo2/Caprin1 locus can be epigenetically different

between kidney and erythroid cells, thereby helping me to understand how the Lmo2 gene might

be regulated in erythroid cells (Birney et al., 2007).

3.4 Future work

Much of the ChIP-Seq data that has been used in this study has been obtained from the HPC7

cell line (Pinto do et al., 1998; Wilson et al., 2010). The HPC7 cells are a model hematopoietic

stem/progenitor cell line (early fetal multipotent hematopoietic progenitor cell line), they have

the potential to form any of the hematopoietic cells, addition of necessary growth factors such as

Steel factor and Erythropoietin can efficiently induce differentiation of the HPC7 cells to form

both primitive and definitive erythroid cells (Pinto do et al., 1998). Hence, in future a ChIP

study performed in erythroid cells with the TFs (ERG, FLI1, GATA2, LMO2, LYL1, PU1,

RUNX1, MEIS1) whose data are currently unavailable would be extremely helpful in

identifying the key TFs that regulate Lmo2 transcription in mature erythroid cells. In addition,

ChIP-Seq studies for the histone modification H3K4me1 in kidney cells can be used to identify

if the H3K4me1 modification pattern that has been noticed at the Lmo2/Caprin1 locus in

erythroid cells is tissue specific. Furthermore, my analysis shows that the histone acteyl

transferase p300 protein binds to the 75DRE in erythroid cells; however, due to the lack of

ChIP-Seq data for p300 in other cell types, it is difficult to understand whether or not this p300

binding pattern is specific to erythroid cells. Hence inclusion of some more ChIP-qPCR data

would be extremely helpful in understanding the tissue specific regulation of Lmo2 in erythroid

cells.

44

4 Chapter 4: Chromatin-Chromatin interactions at the Lmo2 locus

45

4.1 Introduction

The three dimensional organization of a locus plays an important role in the regulation of the

transcription of the gene in a tissue specific manner (Palstra et al., 2003). For example studies

with the mouse ß-globin locus has shown when the gene is actively transcribed, all the distal

regulatory elements of the gene are located in close spatial proximity to each other within the

nuclear space, thereby forming a compartment termed the Active Chromatin Hub (de Laat and

Grosveld, 2003; Palstra et al., 2003). In this chapter I have investigated whether the Lim domain

only 2 (Lmo2) gene is regulated in a similar manner in erythroid cells as observed at the ß-

globin locus essentially because just like at the ß-globin locus, several distal regulatory elements

too have been identified upstream of the Lmo2 gene in the human and mouse genomes (Landry

et al., 2009). These elements are capable of enhancing reporter gene expression in erythroid

cells and hence may be responsible for the high level transcription of Lmo2 in erythroid cells

(Landry et al., 2009). Nevertheless, it is unclear how these elements regulate transcription of

Lmo2 and whether or not they function cooperatively in the endogenous context. In this chapter

I have investigated whether or not chromatin-chromatin interactions indeed exist between Lmo2

proximal promoter and the upstream regulatory elements. As chromatin-chromatin interations

between DREs and several genes including; Hba, Shh, TH2, HoxB1 and olfactory receptor genes

have typically been detected using the chromosome conformation capture technique, hence in

chapter I have used the same technique to identify chromatin-chromatin interactions between the

DREs and the Lmo2 proximal promoter (Amano et al., 2009; Lomvardas et al., 2006;

Spilianakis and Flavell, 2004; Vernimmen et al., 2007; Wurtele and Chartrand, 2006). Hence the

focus of this chapter is to investigate long-range regulation of Lmo2 transcription in the context

of its endogenous genomic location using the 3C technique and also to identify possible proteins

that might be involved in mediating these chromatin-chromatin interactions

46

4.2 Results

4.2.1 The 75 distal regulatory element contacts the Lmo2 proximal promoter

To investigate whether or not chromatin loops form in erythroid cells which bring the 75 DRE

into proximity with the Lmo2 promoter I performed 3C experiments in adult erythroid cells

isolated from mouse anemic spleens 5 days after the initiation of Phenylhydrazine treatment

(Dekker et al., 2002; Dickerman et al., 1976). On day five the anaemic spleen is composed of

>85% mature globin expressing erythroid cells (Osborne et al., 2004). For comparison I used

kidney as a tissue in which Lmo2 is not transcribed at robust levels. I confirmed robust

transcription of Lmo2 in isolated anaemic spleen by measuring the levels of the primary

transcript by RT-qPCR (Figure 4-2). By contrast Lmo2 primary transcript levels were more

than fifteen fold lower in kidney. I also examined the primary transcript levels of the cell cycle

associated protein Caprin1 which is located 172 Kb upstream of Lmo2. I found that Caprin1 is

transcribed at similar levels in both adult erythroid and kidney cells (Figure 4-2).

My previous analysis of the ChIP-Seq data revealed p300 binding and the highest density of

bound TFs at the 75 DRE, hence, I performed the first set of locus wide 3C experiments using

the HindIII fragment containing the 75 DRE as the anchor fragment (Figure 4-3, restriction

fragment map and primers shown in Figure 4-2). My 3C analyses revealed that indeed

significantly increased interaction exists between the 75 DRE and the Lmo2 proximal promoter

fragment in erythroid cells as compared to the interaction frequency in kidney cells. Of note the

Lmo2 proximal promoter fragment also contains the +1 enhancer element found to cooperate

with the 75 DRE for optimal expression in circulating erythrocytes of transgenic mice (Landry

et al., 2009). I also found increased interaction in erythroid cells with the two fragments

upstream of the Lmo2 proximal promoter (Figure 4-3). This region contains the 3 DRE

identified by Landry et al 2009 which appeared not to have enhancer activity in transgenic

analysis (Landry et al., 2009). These increased interactions were not detected in cells isolated

from kidney where Lmo2 is transcribed at very low levels (Figure 4-3). I did not identify

increased interaction of the 75 DRE with the fragment containing the distal promoter and the 25

DRE in either anaemic spleen or in kidney (Figure 4-3). The 75 DRE was also found to have

significantly increased interaction in anaemic spleen as compared to kidney with a fragment

overlapping the 90 DRE but not with other DREs suggesting that the 75 and 90 DREs are more

47

closely associated in erythroid cells (Figure 4-3). The fragment upstream of the 90 DRE also

showed increased interaction in anaemic spleen compared to kidney. I found no increased

interaction between the 75 DRE and the transcribed Caprin1 gene in either tissue type (Figure

4-3). Hence, my results suggest that chromatin-chromatin interactions between the 75 DRE and

the Lmo2 proximal promoter and +1 enhancer occur when Lmo2 is transcribed in erythroid cells

but not in kidney where the gene is transcribed only at very low levels (Figure 4-3).

Figure 4-1.Lmo2 primary transcripts are abundant in anaemic spleen erythroid cells

Primary transcript levels in adult mouse anaemic spleen (red) and kidney (blue) cells for: Lmo2

(exon2-intron2), Caprin1 (exon3-intron2), Slc4a1 (exon1-intron1), Pkd2 (intron2-exon3),

Epn1(exon1-intron1), Gapdh (exon1-intron1) and Vh16 (genic). Levels were quantitatively

assessed by RT-qPCR and expressed relative to Gapdh. Epn1 and Gapdh are ubiquitously

expressed reference genes, Slc4a1 is an erythroid cell specific transcript, Pkd2 is a kidney

specific transcript, Vh16 is not expressed in either tissue.

48

Figure 4-2. The Lmo2/Caprin1 region on mouse chromosome 2

Primers used in chromosome conformation capture (3C) and HindIII restriction sites are shown

across the Lmo2/Caprin1 region of mouse chromosome 2. Promoters and distal regulatory

elements (DREs) are depicted in red and black respectively. Anchor fragments used in the

Caprin1, 75 DRE and Lmo2 3C experiments are marked with an asterisk. Distal promoter (pP),

proximal promoter (pP).

49

Figure 4-3. The 75DRE interacts with the Lmo2 proximal promoter in anaemic spleen

erythroid cells

Quantitative chromosome conformation capture (3C) was performed to detect chromatin-

chromatin interactions between the 75 DRE (distal regulatory element) upstream of Lmo2 and

the rest of the Lmo2-Caprin1 region of mouse chromosome 2. The profile of interactions

identified in anaemic spleen (red) and kidney (blue) is displayed. Black box indicates the

anchor fragment at the 75 DRE and alternating intensities of grey boxes indicate the fragments

investigated for interactions. Significantly increased interaction in anaemic spleen compared to

kidney was detected at the Lmo2 proximal promoter (pP) and at a region upstream of the 75

DRE. Data points are an average of three independent biological replicates. Error bars depict

the SEM, ** p<0.01, and *** p<0.001.

50

4.2.2 Several upstream distal regulatory elements contact the Lmo2 promoter

After detecting chromatin-chromatin interactions between the 75 DRE and the Lmo2 pP, I

wanted to investigate if any of the other DREs also contact the Lmo2 pP in a tissue specific

manner. I performed a locus-wide 3C using the HindIII fragment containing the Lmo2 proximal

promoter as the anchor fragment. 3C analyses detected significantly increased interaction

between the Lmo2 pP and multiple HindIII fragments across the locus in erythroid cells

compared to kidney cells (Figure 4-4). Interestingly, the HindIII fragments that I found to

contact the Lmo2 pP in erythroid cells contained the different DREs that I had already identified,

namely the 12, 25, 70, 75 and 90 DREs. I also identified two broad domains interacting with the

Lmo2 pP in a tissue specific manner. The first domain is located close to the Lmo2 pP and

contains the distal promoter as well as the 12 and 25 DREs. Of note is the observation that

another promoter, termed the intermediate promoter, has been identified immediately upstream

of the 12 DRE (Oram et al., 2010). The second interaction domain located further upstream of

Lmo2 contains the 70, 75 and 90 DREs as well as the HindIII fragment upstream of the 90 DRE

(Figure 4-4). Interestingly, the HindIII fragment upstream of the 90DRE interacted with the

Lmo2 pP in a very high frequency in erythroid cells when compared to all the other HindIII

fragments belonging to the second domain (HindIII fragments containing the DREs 90, 70, 75,

and 64) that were found to interact with the Lmo2 pP (Figure 4-4). This observation is especially

striking as the interaction frequency between the HindIII fragments should become

progressively lesser as the distance between the fragments become fairly large. In this case the

distance between the fragments is more than 90kb, hence the fact that the interaction frequency

in erythroid cells between the HindIII fragment located upstream of 90 DRE and the fragment

containing the Lmo2 pP is high is definitely noteworthy. I also detected a peak in the relative

interaction frequency at the 35 DRE; however this interaction was detected in both kidney and

erythroid cells (Figure 4-4).

51

Figure 4-4. Distal regulatory elements interact with the Lmo2 proximal promoter in

anaemic spleen erythroid cells

Quantitative chromosome conformation capture (3C) was performed to detect chromatin-

chromatin interactions between the Lmo2 proximal promoter (pP) and distal regulatory elements

(DREs). The profile of interactions identified in anaemic spleen (red) and kidney (blue) is

displayed. Black box indicates the anchor fragment at the Lmo2 pP and alternating intensities of

grey boxes indicate the fragments investigated for interactions. Significantly increased

interaction in anaemic spleen compared to kidney was detected at the 12, 25, 70, 75 and 90

DREs. Data points are an average of five independent biological replicates. Error bars depict

the SEM, * p<0.05, ** p<0.01, and *** p<0.001.

52

4.2.3 The Caprin1 promoter does not interact with the identified distal

regulatory elements

Caprin1 is a ubiquitously expressed gene located 172kb upstream of Lmo2 and transcribed from

the opposite strand. As Caprin1 is transcribed in erythroid cells I was interested to investigate

whether or not the Caprin1 promoter physically interacts with the DREs located between

Caprin1 and Lmo2. My initial 3C experiments performed with the 75 DRE as the anchor

fragment did not show any increase in the relative interaction frequency between the Caprin1

promoter and the 75 DRE in erythroid cells as compared to kidney cells (Figure 4-3), however

as Caprin1 could physically interact with other DREs located between Caprin1 and Lmo2 I

performed locus wide 3C experiments using the HindIII fragment containing the Caprin1

promoter as the anchor fragment (Figure 4-5). My results did not show any significant peaks in

the relative interaction frequency with the Caprin1 promoter in the entire region between the

Caprin1 and Lmo2 genes (Figure 4-5). Furthermore I did not identify any significant

differences between the relative interaction frequency of any HindIII fragments with the

Caprin1 promoter for cells isolated from anaemic spleen and kidney (Figure 4-5). Hence, my

locus wide 3C experiments with the Caprin1 promoter showed that in erythroid cells the

Caprin1 promoter does not interact with any of the identified DREs located upstream of the

Lmo2 gene.

53

Figure 4-5. The distal regulatory elements upstream of Lmo2 do not interact with the

Caprin1 promoter

Quantitative chromosome conformation capture (3C) was performed to detect chromatin-

chromatin interactions between the Caprin1 promoter and distal regulatory elements (DREs).

The profile of interactions identified in anaemic spleen (red) and kidney (blue) is displayed.

Black box indicates the anchor fragment at Caprin1 and alternating intensities of grey boxes

indicate the fragments investigated for interactions. Data points are an average of three

independent biological replicates. Error bars depict the SEM, no significant differences were

identified throughout this region.

54

4.3 Discussion

In this chapter I investigated the presence of chromatin-chromatin interactions throughout the

mouse genomic region containing Lmo2 and Caprin1 genes. My 3C experiments identified

several erythroid cell specific interactions between the Lmo2 proximal promoter and upstream

DREs, specifically a cluster of three transcription factor bound DREs 70-90 kb upstream interact

with the Lmo2 promoter as do the more proximal 12 and 25 DREs each of which are close to an

alternate promoter (intermediate and distal respectively). My 3C experiments revealed no

significant interactions between the Caprin1 promoter and the DREs suggesting these elements

are specific to Lmo2.

4.3.1 The 75 distal regulatory element and several other upstream distal

regulatory elements contact the Lmo2 proximal promoter

My 3C data confirmed specific interaction of the 75 DRE with the Lmo2 proximal promoter and

interaction of the Lmo2 proximal promoter with a broad region containing the 75 DRE as well

as the 90 and 70 DREs. Previous studies in circulating erythroid cells of transgenic mice have

shown that the 75 DRE has the strongest enhancer activity in erythroid cells and drives the

expression of a reporter gene, cooperatively with the Lmo2 proximal promoter and +1 enhancer

element (Landry et al., 2009). I have shown that the 75 DRE functions via contacting the Lmo2

proximal promoter/+1 DRE region to form an erythroid cell specific chromatin loop.

Furthermore, I identified a broad region of interaction with the Lmo2 proximal promoter region

containing the 70, 75, 90 DREs and a region located upstream of the 90 DRE. This suggests

that in the endogenous context the three DREs coordinately regulate Lmo2 transcription in

erythroid cells.

I also identified increased interaction between fragments containing the 25 and 12 DREs and the

Lmo2 proximal promoter in erythroid cells compared to kidney cells. This suggests that this

more proximal region also contributes to transcriptional regulation of endogenous Lmo2 through

increased interaction with the Lmo2 proximal promoter region. All three of these regions

contain a promoter (distal, intermediate and proximal) close to the identified DRE (25, 12 and

+1), therefore the increased interaction I identified could be mediated by the DREs, promoters

55

or both. However, in chapter 3 I have shown that the 25 and 12 DREs overlap with multiple

transcription factor peaks in HPC7 cells and are bound by p300 in erythroid cells. Hence, this

piece of data seems to suggest that the 12 and 25DREs may have a regulatory role.

Interestingly, I did not identify interactions of the 75 DRE with the fragments containing the 25

or 12 DREs. Transgenic analysis by Landry et al 2009 of these elements did reveal that the 25

and 12 DREs conferred expression in the fetal liver whereas only the 75 DRE conferred

expression in fetal liver as well as in circulating blood cells suggesting that these elements have

different functional roles in regulating Lmo2 expression (Landry et al., 2009). The fact that I

did not identify specific interactions between the 75 DRE and the fragment containing the 25 or

12 DREs suggests that the interactions of these two regions with the proximal promoter are

mutually exclusive. These mutually exclusive interactions could occur in different sub-

populations of cells within the anaemic spleen or the interactions could be dynamic within

individual cells with the proximal promoter region alternately contacting the 25-12 region and

the 70-90 region.

I did identify one distal chromatin-chromatin interaction with the Lmo2 proximal promoter in

both erythroid cells and kidney cells. This interaction occurred at the 35 DRE that also showed

comparable, though low, levels of intergenic transcription in both tissues as noted in chapter 3.

In transgenic mice a construct containing the 35 DRE and the proximal promoter showed similar

levels of LacZ reporter gene expression in endothelial cells when compared with the proximal

promoter alone (Landry et al., 2009). The chromatin-chromatin interactions that I identified

between the 35 DRE and the Lmo2 proximal promoter in both erythroid and kidney cells

suggests this region has a subtle role in regulating low level transcription of Lmo2 in endothelial

cells.

56

4.4 Future work

In this chapter I have shown that Lmo2/Caprin1 locus adopts an erythroid cell specific

conformation that correlates with the enhanced level of transcription of the gene in erythroid

cells. An interesting study that can be performed in future is to investigate how the locus is

organized in a cell type where the gene is not expressed for example in mouse embryonic stem

cells (ESCs). An important aspect that can be investigated is the manner in which the chromatin

loops are acquired as ESCs differentiate to hematopoietic progenitor cells and mature erythroid

cells wherein the Lmo2 gene is transcribed at high levels. Capturing the spatial and temporal

changes that occur as the Lmo2 gene goes from a non expressed state to a highly transcribed

state would provide invaluable information about how changes in chromatin conformation

correlates with the regulation of Lmo2 transcription.

An important conclusion that I derived from my combines analyses from this chapter and

chapter 3 is that the distal regulatory elements located upstream of the Lmo2 gene function

through a chromatin looping mechanism. Analysis of ChIP-seq data from chapter 3 suggests that

these chromatin loops are supported by cohesin associated with CTCF and transcription factor

bound regions. Hence, an important experiment that can be conducted in the future is to

knockdown the CTCF and Cohesin proteins in erythroid cells to identify what are the changes

that happen not only to the conformation of the Lmo2/Caprin1 locus but also to the levels of

transcription of both the genes. According to my model the gene Caprin1 does not interact with

any of the DREs possibly because of the presence of the insulator protein CTCF at three

different regions located between the 90DRE and the Caprin1 gene promoter. A depletion of the

CTCF protein may allow the DREs to interact with Caprin1 potentially enhancing transcription

of the gene. Furthermore, my model also suggests that the cohesin subunit RAD21 mediates the

formation of the chromatin loops between the DREs and the Lmo2 proximal promoter, therefore

a depletion in the levels of the cohesin protein complex should result in the loss of these

chromatin loops thereby decreasing the level of Lmo2 transcription in erythroid cells. Therefore

a knockdown of the Cohesin and CTCF proteins would provide concrete evidence to back up

my model indicating that the DREs indeed function through a chromatin looping mechanisms

and the formation of these loops are supported by the presence of the Cohesin and CTCF protein

bound regions.

57

5 Chapter 5: General Discussion

58

5.1 Discussion

In the post genome era, the availability of multiple low-cost, high-throughput technologies has

helped in sequencing genomes of several organisms, and the functions of the distal regulatory

elements that lie embedded within the non-coding part of these sequenced genomes are at last

beginning to be unveiled. Previous studies have shown that distal regulatory elements can

regulate gene transcription in a tissue specific manner and that specific chromatin features such

as histone modification patterns can help in identifying these tissue specific DREs (de Laat and

Grosveld, 2003; Heintzman et al., 2009; Heintzman et al., 2007; Palstra et al., 2003; Tolhuis et

al., 2002). The purpose of the work presented in this thesis is to integrate various biological data

together to understand the role of the DREs and other factors in the tissue specific regulation of

Lmo2.

In this study using a combination of ChIP-Seq data for mature erythroid cells and HPC7

hematopoietic progenitor cells I identified binding of multiple TFs and other chromatin

associated proteins to many of the DREs that have been identified upstream of the Lmo2 gene

(Birney et al., 2007; Cheng et al., 2009; Soler et al., 2010; Wilson et al., 2010). Furthermore my

3C data also confirmed the presence of increased interactions between Lmo2 proximal promoter

with a broad region containing the 75 DRE as well as the 90 and 70 DREs in erythroid cells

when compared with kidney cells.

Although I identified erythroid-cell specific chromatin loops between upstream DREs and the

Lmo2 proximal promoter; however the question remains as to which factors are mediating these

looping interactions. Possible candidates that could be mediating the formation of these loops

are the Cohesin protein complex subunit RAD21, CTCF, the TFs LMO2, LDB1, GATA1, and

KFL1. Both CTCF and RAD21 have been shown to mediate the formation of chromatin loops at

other genomic locations (Chien et al., 2011; Hadjur et al., 2009). Analyses of the ChIP-Seq data

revealed the presence of several CTCF and cohesin (RAD21) bound regions across the entire

Lmo2/Caprin1 locus indicating that CTCF and RAD21 could be mediating the formation of

chromatin loops even at the Lmo2/Caprin1 locus (Figures 3-6 and 3-7) (Birney et al., 2007).

Cohesin (RAD21) is bound within both the distal and proximal interacting domains, specifically

at the 75, 25 DREs as well as at the proximal promoter suggesting cohesin bound at the

upstream DREs supports erythroid cell specific looping interactions with the Lmo2 proximal

59

promoter. Cohesin is also recruited to CTCF occupied regions and identified several

CTCF/RAD21 bound regions throughout the Lmo2 upstream region, all of which occur within

erythroid cell specific interacting domains, though the majority of the CTCF sites were not

specific to erythroid cells (Parelho et al., 2008; Rubio et al., 2008). This is similar to the

findings at the Hbb locus where CTCF bound regions, invariant between cell types, formed cell

type specific chromatin loops (Hou et al., 2010). CTCF is often associated with insulator

activity; the CTCF/RAD21 occupied region upstream of the 90 DRE could have an important

role in preventing Caprin1 from interacting with the DREs that enhance Lmo2 transcription

(Phillips and Corces, 2009). As overexpression of Caprin1 causes inhibition of cell division it is

critical to prevent its aberrant up-regulation in rapidly dividing erythroid cells (Grill et al.,

2004). I did identify one CTCF/RAD21 bound region, downstream of the 75 DRE, which was

enriched predominantly in erythroid cells and may be critical in generating the tissue specific

looping pattern that I identified.

My results suggest that chromatin-chromatin interactions throughout the Lmo2 locus are being

supported by cohesin recruited both to CTCF bound regions (upstream of 90, downstream of 75)

as well as at transcription factor and p300 bound DREs not associated with CTCF (90, 70, 25,

12). The results also suggest that in the endogenous context the three DREs coordinately

regulate Lmo2 transcription in erythroid cells. I also identified increased chromatin-chromatin

interactions between fragments containing the 25 and 12 DREs and the Lmo2 proximal

promoter in erythroid cells compared to kidney cells. This suggests that this more proximal

region also contributes to transcriptional regulation of endogenous Lmo2 through increased

interaction with the Lmo2 proximal promoter region. As all three of these regions contain a

promoter (distal, intermediate and proximal) close to the identified DRE (25, 12 and +1),

therefore the increased interaction that I identified could be mediated by the DREs, promoters or

both. However, the 25 and 12 DREs were found to overlap multiple transcription factor peaks

in HPC7 cells and are bound by p300 in erythroid cells suggesting they have a regulatory role.

In addition to CTCF and RAD21, proteins other proteins that might mediate the formation of the

chromatin loops are the TFs LMO2, LDB1, GATA1, AND KLF1. As LMO2 itself was bound to

the upstream DREs, specifically at 75, 25 and 12, all of which showed increased interaction with

the Lmo2 promoter, hence my results suggest that the LMO2 oligomeric complex can play an

60

important role in mediating the formation of chromatin loops. In support of this, a recent study

found LDB1 (a member of the LMO2 complex) at regions of chromatin interaction with the

LDB1 bound Hbb-b1 promoter (Soler et al., 2010). GATA1 and KLF1 are also bound within

the distal interacting region, these TFs have been shown to be required, though not sufficient for

chromatin looping between Hbb-b1 and the LCR (Drissen et al., 2004; Kooren et al., 2007;

Vakoc et al., 2005) and may have a similar role in regulating looping within the Lmo2 locus.

At the end it can be summarized that integration of a wide range of biological data obtained

from ChIP-Seq, 3C, and RT-qPCR experiments has helped me in identifying the long range

chromatin-chromatin interactions that occur between the Lmo2 proximal promoter and two

broad regions, 3-31 and 66-105 kb upstream of Lmo2 containing transcription factor bound

regulatory elements suggesting that these elements cooperate in regulating high level

transcription of Lmo2 in erythroid cells. Furthermore my data also supports a model in which

these distal regulatory elements function through a chromatin looping mechanism supported by

cohesin associated with CTCF and transcription factor bound regions thereby enhancing the

transcription of Lmo2 in mouse erythroid cells.

61

5.2 Summary

The contributions of the work presented in this thesis include highlighting the role of chromatin

organization in regulation of Lim domain only 2 (Lmo2) gene transcription and identifying

various other biological factors that help in the formation of an erythroid cell specific

conformation of the entire Lmo2/Caprin1 locus. The ChIP-Seq data sets that have been used in

this study identified the wide range of biological factors (such as presence of TFs and other

chromatin associated proteins, histone modification patterns, genomic features, presence of

intergenic transcripts) that help to regulate the transcription of Lmo2 in erythroid cells (Birney et

al., 2007; Cheng et al., 2009; Soler et al., 2010; Wilson et al., 2010)

The significance of the study lies in explaining how multiple distal regulatory elements regulate

transcription of Lmo2 in erythroid cells and whether or not they function cooperatively in the

endogenous context. My study not only shows that strong interactions exist between upstream

regulatory elements and the Lmo2 gene promoter in erythroid cells, but also identifies the

specific regulatory elements (i.e. the 12, 25, 70, 75 and 90 DREs) that might be playing a more

important role in regulating of the Lmo2 gene in erythroid cells. I also found that the Lmo2-

Caprin1 locus adopts a tissue-specific conformation in erythroid cells. This tissue specific

organization of the locus brings several but not all of the DREs into proximity with the Lmo2

proximal promoter while excluding the Caprin1 promoter. A distal region covering 39 kb and

containing three DREs (90, 75 and 70) and forms a strong interaction with the Lmo2 proximal

promoter in erythroid cells. In addition a more proximal region containing the 12 and 25 DREs

as well as the distal and intermediate promoters interacts with the Lmo2 proximal promoter in

erythroid cells. Furthermore, a CTCF bound region upstream of the 90 DRE interacts with the

Lmo2 proximal promoter and may function as an insulator preventing the interaction of the

Caprin1 promoter with the erythroid cell specific DREs. In addition, multiple locations within

the entire Lmo2/Caprin1 locus has erythroid cell specific epigenetic signatures such as enriched

histone modification patterns, and DNaseI sensitivity which is in agreement with previous

studies that have shown the correlation between tissue specific epigenetic marks, chromatin

organization, and tissue specific regulation of specific genes. Hence, it can be concluded that a

combination of ChIP-Seq data, bioinformatics and 3C analyses has not only helped in

identifying distal regulatory elements that are located upstream of the Lmo2 gene in the mouse

62

genome but has also helped me in understanding how these enhancer-gene interactions are

mediated within the erythroid cells so as to regulate transcription of Lmo2 in a tissue specific

manner.

63

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Zhao, Z., Tavoosidana, G., Sjolinder, M., Gondor, A., Mariano, P., Wang, S., Kanduri, C., Lezcano, M., Sandhu, K.S., Singh, U., et al. (2006). Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet 38, 1341-1347.

75

Appendices

Appendix 1. Coordinates of distal regulatory elements located upstream of the Lmo2

promoter in the mouse genome

Distal regulatory elements are named acording to their distance upstream of the annotated Lmo2

transcription start site overlapping the proximal promoter. Coordinates are given for homology

regions identified by BLAT. All fragments were mapped in NCBI m37 mouse assembly

(mm9).

Distal Regulatory

Element

Chromosome Starts at Ends at

90 Chr2 103720746

103720876

Chr2 103720495

103720607

Chr2 103720233

103720316

Chr2 103720614

103720670

Chr2 103720684

103720729

Chr2 103720909

103720959

75 Chr2 103736385

103736662

Chr2 103733907

103733995

Chr2 103734073 103734138

Chr2 103741698

103741839

Chr2 103740691

103740810

70 Chr2 103743108

103743214

Chr2 103743323

103743439

Chr2 103740817

103740859

Chr2 103743246

103743284

Chr2 103742632

103742668

Chr2 103743288

103743312

Chr2 103741882

103741906

Chr2 103741847

103741854

64 Chr2 103746643 103746795

Chr2 103746829 103746975

Chr2 103746393 103746425

76

Chr2 103746616 103746634

Chr2 103746616 103746634

58 Chr2 103753051 103753386

Chr2 103753006 103753038

47 Chr2 103764161 103764463

43 Chr2 103768003 103768252

40 Chr2 103770972

103771182

Chr2 103770692

103770883

Chr2 103770897

103770953

35 Chr2 103776285 103776669

Chr2 103776677 103776702

25 Chr2 103786638

103786800

Chr2 103786084

103786206

Chr2 103785920 103785949

Chr2 103786575

103786598

Chr2 103786541

103786557

Chr2 103786363

103786383

Chr2 103785810

103785823

Chr2 103786233

103786243

Chr2 103786568

103786573

12 Chr2 103798740 103798811

Chr2 103797914 103797994

Chr2 103798059 103798101

3 Chr2 103807666 103807799

Chr2 103807840 103807879

Chr2 103807626 103807654

Chr2 103807906 103807921

+1 Chr2 103811961 103812138

Chr2 103811728 103811830

Chr2 103811901 103811939

Chr2 103811837 103811880

77

+7 Chr2 103818142 103818313

Chr2 103818037 103818138

Chr2 103817938 103817999

Chr2 103818354 103818374

Chr2 103818324 103818338

Chr2 103818018 103818031

Appendix 2. Coordinates of the Lmo2 proximal and distal promoters in the mouse genome

The coordintes of proximal promoters and distal promoters for the Lmo2 gene in the mouse

genome are listed in the table. Coordinates are given for homology regions identified by BLAT.

All fragments were mapped in NCBI m37 mouse assembly (mm9).

Promoter Element Chromosome Starts at Ends at

Distal Promoter (dp)

Chr2 103788235

103788347

Chr2 103788107

103788162

Chr2 103788392

103788432

Chr2 103788641

103788683

Chr2 103788499

103788515

Chr2 103788081

103788091

Chr2 103788372

103788379

Chr2 103788202

103788208

Chr2 103788222

103788228

Chr2 103788168

103788172

Proximal promoter

(pP)

Chr2 103810212

103810400

Chr2 103810495

103810533

Chr2 103810398

103810445

Chr2 103810212

103810400

78

Proximal promoter

extended (pPex)

Chr2 103810620

103810724

Chr2 103810734

103810812

Chr2 103809864

103809907

Chr2 103810495

103810545

Chr2 103809630

103809688

Chr2 103810398

103810445

Chr2 103810140

103810170

Appendix 3. Primers used in quantitative chromosome conformation capture and RT-

qPCR

Specific primers are listed for the chromosome conformation capture (3C) and RT-qPCR analyses. Left primer (L), right primer (R), primers used to test HindIII restriction digestion efficiency are marked as REX.

3C primers Sequence 5`-3`

LMO2-3C-pP-L AGGAGAGAAACAACAACCCTTT

LMO2-3C-upstream pP-L GGGGACCTAGGTTTTTCTCCT

LMO2-3C-downstream 12E-L TTCAGACTTCTGACATCCTTATTTC

LMO2-3C-12E-L CTGCCTTACCTTGAGCTTGG

LMO2-3C-25E-L ACCCTTGGCAATTAACGTGT

LMO2-3C-downstream 35E-L TGTGGAACACCAACTTTTCCT

LMO2-3C-35E-L CTGGGCCAAGGGGTATAGAG

LMO2-3C-47E-L CTACTCCCGCTCAAAACTGC

LMO2-3C-downstream 58E-R CTTGGTACCCAGGAACTAGCA

LMO2-3C-64E-R CTCCCCTCCCTCAAACATTA

79

LMO2-3C-70E-L GGACTACGGAGCTGAAACCA

LMO2-3C-75E-R CCCTACAACATGCATCTCCA

LMO2-3C-upstream 75E-R TGCTTGATCATGGTTACAGGTC

LMO2-3C-downstream 90E-R TTGGGGTCATTATCTCTTTGCT

LMO2-3C-90E-L GGCCCTTATAATTTGGCACA

LMO2-3C-blankregion1-L TGGCACACATCTACAAGAGCA

LMO2-3C-blankregion2-L TCTCTGAACTGTTCCCTGGAG

LMO2-3C-upstream Caprinpr-L TTTCATCAAGTGCATCTTTGC

LMO2-3C-Caprinpr-L CCAGAGAGGCTGTTGGTTACT

LMO2-3C-downstream Caprinpr-L CATTGGTATGTTCATTACCTAGACA

Alpha aortic actinHIII-4-3C-L CCCTAGTCAGCCATCTCCTCT

Alpha aortic actinHIII-5-3C-L TGCAGTTATGTTCCACAGCAG

RT-qPCR Primers Sequence 5`-3`

CTCF peak1-L ATGCTGGTTTGTCATCTCCTGA

CTCF peak1-R AGTGCATGAGGATGTGCAATTA

CTCF peak 2-L ACTCACAGATTTGCTGGAGAGAC

CTCF peak 2-R TGGTGATTAACCAACTTCAGACA

CTCF peak 3-L GGTTGCTATGGTTGCAGATAGAG

CTCF peak3-R CAGGAGTTGTGTAGACCGAGAAT

u90-L TCATCAGCACTTACAGCCTCA

u90-R ATATGGCTGCAACAATTTCTGA

90DRE-L ATTCTTGTTTATGTAGGGGTGATGT

80

90DRE-R GATACCATAAAATCAGAGGCAGGTA

d90-L CCCCCTTTAGAGTACTGCACTG

d90-R CCAGATACGATGCCTGTGATAG

d90A-L TCTTCCAGTTCAATATGCTCCTAAC

d90A-R GGAGAGGTCTGATACAGTCGTTTTA

d90BL ATATTTAGAAAGGCCAGAATTTTGCT

d90B-R GTTTGGGAATTATAGCCCTACGATA

90-75A-L ACTGAGGAAGTGCAGCAGATTAAC

90-75A-R AAGAATTTCAGCGAACTCTAAGGA

u75A-L GCAGGTGGTATTGTTTAGTGAGGTA

u75A-R AGAAGCATGGGGTAGTGGATT

u75B-L ACCAAGCGGAGGCTGTATTA

u75B-R TTAGCTGCCTCAGAAGATAATGG

75DRE-L CAGCTAACTGTTACAGGAGAAGGAG

75DRE-R TGGGATCTGGGAGAGTATACTACAG

d75-L GGAAGTAAGGGAGACCCATTG

d75-R TTTGCTAGAAATCCCAACGTG

u70-L AACTATGGGGAGCATAAGCAAA

u70-R CAGGCAAATATCTAGGGGAAAA

70DRE-L AAAGGGGCCAGCTAGGAG

70DRE-R CTCAACCTGTTGGCGTATCC

47DRE-R GTCCGAACCTTTCAGTGTTCTC

81

d70-R TACTGGGTTAAAGAAGGGGTGA

u64A-L ACCAAGTTGGCTAAGGGTAGTTTT

u64B-L GAGGAGCCAGAGTTAAACCAAGT

u64AB-R GGACACCTAATAACGTGTTAGGATTAG

u64-L GGCTAAGGGTAGTTTTGCAGAG

u64-R CGATCAGACTGAGTGTGTGAGA

64DRE-L AAGGATCAGTGTGGAACTTGC

64DRE-R2 TGGCGACAGCACAGAAATAG

d64-L CCTGCTGTTTATGCAACACTTC

d64-R GCCTAACAAACTGGGATTCACT

64-58ER GGTAGCAATCTGGATATCTTGGAG

64-58EL ATGTATCCTTCAGAGGAGGCATAG

64-58CR TCTGCTTAATTGTTGGGCCTCT

64-58CL TAAGTCAACCTGCCGTTAATTGTA

u58-L AATAAGAGAGGAGAACGCAGTATGA

u58-R CATGTTTAGAAACAGAGGGTTATGC

58DRE-L TTCAGAACTCCCCGAAGAGA

58DRE-R CTCAGTTCCAAACCGCTCAG

d58-L TGGCATTGATTTTCCCTATTTT

d58-R GCCCTGTACCTACCTCAAGATG

58-47AR TCCTTTTACGGAACATGATGAACT

58-47AL AGAGTGACTTCAATTTGGACCATT

82

58-47BR CATTTTAGCTTCCCAAATGGTTAT

58-47BL AGATACGTGACCTAAACAGCATTC

58-47CL GAAGACTGCCTCGGTTTATTCTTA

58-47CR ATTTACACCTTGTCCTGATTTCGT

58-47DL GTCTATAACACAGATGACCCATGC

58-47DR AGTTTGGGAACAATCAGAAGCTAT

47DRE-L CAGTGCATGGAGTTAATGGAAA

47DRE-R ACTACAACTTGGTGCTGGCAAT

43DRE-L GTGGGCCAATTAGTGTCTGG

43DRE-R CCCCAGGCTTTGTTCTACATT

40DRE-L GAGGGAGGGAGTTCGTAACA

40DRE-R AATAATGAATGCGCGTCTCC

35DRE-L GGCATGATCGATACAAGACAGA

35DRE-R GCACTTAAATGGAACTCCCAAC

d35-L GCCACATACCATCTAAACAGCA

d35-R CTACTGGTGCCCTGTCCTACTC

u25-L ATGACTGGATTCACCACCTTG

u25-R GCTAACCACATCAAACCAACC

25DRE-L GGGGATGAATGCATGATAGACT

25DRE-R GGCTGAAGGGAAACTGTGTAAC

d25-L AGAACAGCCAGGTGAGATGAA

d25-R AGGCATCATCCTAACCAGTGA

83

u12-L1 CTTTTCAACTCCCGGAGGAT

u12-R1 GGGAGAGGTACCTTCTTCAAGC

u12-L2 GAAGTACTGCGGTCCTTGATATG

u12-R2 TATTCTTATACAAGCATGGGCATC

12DRE-L GCAAAAAGTTGCCAGATAAAAGATA

12DRE-R ACATTGTAAGTCTTCGAGGTAGGTG

d12-L GGGATGTTAAAAGGGATCCTG

d12-R CATGAGCGAGCAGAATTTGAC

dP_Int_Lmo2-L ACTTTGCTGACTTCCACAAGGAC

dP_Int_Lmo2-R GATGTAATCCCTGTGACTCCTGAT

d-dPL1 CTAAAGTCACGAGAAGGACCAAA

d-dPR1 CCAAAGACTCCTTACTTGCTCAG

d-dPL2 CTGCACCCTAGATGAATAACACC

d-dPR2 ACTGTTTGGGTATGCTACACTCG

d-dPL3 AAGGACTTGGAATAACCTTGCTAGT

d-dPR3 TGGTAGTAGGAACACTCTCTCGTCT

pP_Int_Lmo2-L GATGGAAGGTTAAGTCCTGAGCA

pP_Int_Lmo2-R AAAGAGAGAGAGCGAATCATCCAG

Lmo2 Exon2-L2 ATCGAAAGGAAGAGCCTGGAC

Lmo2 Intron2 -R2 GGTCGATCCCAGTTACAGCTTC

Pkd2_In2-L GGAGGGAAAGAGCTGACCTTA

Pkd2_EX3-R AGCTCATCATGCCGTAGGTC

84

Vh16 genic-L GGAGGGTCCACTAAACTCTCTTG

Vh16 genic-R GCATAGCCTTTTCCACTCTCATC

GapdhE1I1-L CTTCTTGTGCAGTGCCAGGTG A

GapdhE1I1-R CGCACCAGCATCCCTAGACC

Slc4a1E1I1-L TGGGAGCTCAGCCAGTCACA

Slc4a1E1I1-R CGGGACAGATGCCAA AGGAC

Caprin1E3-L2 CCTTTCCCCTTTATTCATTCG

Caprin1I2-R2 AGCAATGGTCAGTGTTTCAAGTT

EpnE1I1-L CTGGAAGCCCGGTATAAGC

EpnE1I1-R GTACAAAAGCAGCCACAAGC

LMO2 pP REX -L AGGAGAGAAACAACAACCCTTT

LMO2 pP REX -R TGCCTCCCCAACTGTGTAAT

ULmo2 pPREX-L GGGGACCTAGGTTTTTCTCCT

ULmo2 pPREX-R GGAAGTTCCTTCCCGATAAAA

25E REX -R TTTGGCTGATGCAGAGAATG

25E REX -L ACCCTTGGCAATTAACGTGT

70E REX -L GGACTACGGAGCTGAAACCA

70E REX -R CTCCCCTCCCTCAAACATTA

75E REX -R AGCCAGGCACAAATTACCTC

75E REX -L GTGGCACTCTCTGCTGACC

UCaprin1 REX -R TCCCTGTCAAACTGATGCAC

UCaprin1 REX -L TTTCATCAAGTGCATCTTTGC

85

Caprin1pr REX -R TTTCCCAAGTAGGTCCCTGA

Caprin1pr REX -L CCAGAGAGGCTGTTGGTTACT

Appendix 4. Restriction digestion efficiency in chromosome conformation capture

Restriction digest efficiency was between 85 and 95% at several HindIII restriction sites. Lmo2

proximal promoter (pP), Distal regulatory element (DRE), Caprin1 promoter (Caprin1P). “U”

denotes a restriction fragment upstream of the indicated element.

86

Appendix 5. Transcription factor bound regions at the 75 and 12DREs

A) The 75 distal regulatory element (DRE) overlaps multiple transcription factor peaks from

erythroid (MEL and GIE-ER4) cell ChIP-Seq data. B) Similarly the 12 DRE overlaps GATA1

and LDB1 peaks from erythroid (MEL and GIE-ER4) cell ChIP-Seq data. In both DRE

homology regions are indicated by black boxes joined by a line to delineate the human enhancer

construct used in the generation of transgenic mice. Overlapping peaks obtained from p300

mouse ENCODE ChIP-Seq data are also depicted at the 75 and 12 DREs.

87

Appendix 6. QPCR products of gene expression profile run on agarose gel

The QPCR products of the gene expression profile (A-Vh16, B- Gapdh, C-Epn1, D-Slc4a1, E-

Pkd2, F-Caprin1, G-Lmo2, H-Lmo2 proximal promoter, I-Lmo2 distal promoter, J-downstream

of Lmo2 distal promoter) were run on a 2% agarose gel to check the presence of a single

amplicon. bp-base pairs.

Appendix 7. QPCR products of intergenic transripts at 90,75, and12 DREs run on agarose

gel

The QPCR products of the amplified intergenic transcripts were run on a 2% agarose gel to

check for the presence of a single amplicon. (A-d90EA, B-d90EB, C-u90, D-90DRE, E-d90EC,

F-CTCF Peak1, G-CTCF Peak2, H-CTCF Peak3, I-u75E, J-75TFBS, K-75EA, L-75EB, M-

77MID, N-d75E, O-u75, P-u12E, Q-12DRE, R-d12E), bp-base pairs, u-upstream, d-

downstream.

88

Appendix 8.QPCR products of the intergenic transcripts at 25,35,40,43,47,58,64, and 70

DREs run on agarose gel

The QPCR products of the amplified intergenic transcripts were run on a 2% agarose gel to

check for the presence of a single amplicon. (A-35DRE, B-d35E, C-58DRE, E-u58E, F-u64EA,

G-u64EB, H-u64EE, I-64DRE, J-d64E, K-u70E, L-70DRE, M-d70E, N-40DRE, O-43DRE, P-

47DRE, Q-d25E, R-25DRE), bp-base pairs,u-upstream, d-downstream.

Appendix 9. QPCR products of the intergenic transcripts located between the 47and

58DREs, 64 and 58DREs, and between the 90 and 75DREs run on agarose gel

The QPCR products of the amplified intergenic transcripts were run on a 2% agarose gel to

check for the presence of a single amplicon. 90-75: located between the 90 and 75DREs, 64-58:

located between 64 and 58DREs, 58-47: located between 58 and 47DREs.

89

Appendix 10. Chromosome conformation capture (3C) products run on agarose gel (75E

as the anchor fragment)

All the 3C products with 75E as the anchor fragment run on a 2% agarose gel.pP-Lmo2

proximal promoter, b-blank region, d-downstream, u –upstream, bp-base pairs.

Appendix 11. Chromosome conformation capture (3C) products with Lmo2 proximal

promoter as anchor fragment run on agarose gel

All the 3C products with Lmo2 proximal promoter (pP) as the anchor fragment run on a 2%

agarose gel. Cp-Caprin1 promoter, b-blank region, d-downstream, u –upstream, bp-base pairs.

90

Appendix 12. Chromosome conformation capture (3C) products with Caprin1 promoter as

anchor fragment run on agarose gel

All the 3C products with Caprin1 promoter (Cp) as the anchor fragment run on a 2% agarose

gel.pP-Lmo2 proximal promoter, b-blank region, d-downstream, u –upstream, bp-base pairs.

Appendix 13. Primary intergenic transcript levels in adult mouse anaemic spleen and

kidney cells at the Lmo2/Caprin1 locus

Primary intergenic transcript levels as observed in adult mouse anaemic spleen and kidney.

Levels were quantitatively assessed by RT-qPCR and expressed relative to Gapdh. Gapdh is a

ubiquitously expressed reference gene. Chromosome Starts at Ends at Erythroid Kidney

chr2 103719773 103719855 0.07 0.00

chr2 103720768 103720926 0.19 0.00

chr2 103721765 103721936 0.52 0.00

chr2 103733981 103734083 0.00 0.00

chr2 103734036 103734162 0.20 0.00

chr2 103736359 103736503 0.76 0.00

chr2 103733874 103733987 0.70 0.02

91

chr2 103735453 103735544 1.83 0.00

chr2 103737677 103737813 0.72 0.00

chr2 103741899 103741916 1.74 0.00

chr2 103743566 103743701 1.67 0.02

chr2 103740379 103740594 0.98 0.01

chr2 103744683 103744817 1.40 0.02

chr2 103747146 103747258 0.83 0.00

chr2 103753011 103753170 5.77 0.04

chr2 103753441 103753558 4.69 0.03

chr2 103764283 103764406 0.32 0.03

chr2 103768070 103768257 0.32 0.00

chr2 103770958 103771051 0.24 0.01

chr2 103776571 103776653 0.25 0.14

chr2 103776976 103777175 0.25 0.04

chr2 103785900 103786097 0.09 0.01

chr2 103787110 103787196 0.03 0.00

chr2 103785656 103785825 0.16 0.00

chr2 103796380 103796571 2.50 0.02

chr2 103797876 103798028 0.55 0.00

chr2 103799435 103799515 21.57 0.07

chr2 103788221 103788401 0.14 0.02

chr2 103788891 103788984 0.06 0.01

chr2 103810515 103810638 5.89 0.20

chr2 103746604 103746751 2.95 0.01

chr2 103798226 103798325 0.55 0.00

chr2 103712293 103712439 0.15 0.00

chr2 103705781 103705877 0.15 0.00

92

chr2 103688916 103689025 0.04 0.01

chr2 103810841 103811055 15.07 1.28

chr2 103623318 103623538 1.00 0.43

chr2 103746245 103746326 4.73 0.03

chr2 103722341 103722435 0.84 0.00

chr2 103725075 103725176 0.9 0.00

chr2 103731605 103731706 0.18 0.00

chr2 103752577 103752695 3.2 0.00

chr2 103792261 103792371 0.00 0.00

chr2 103794182 103794283 0.00 0.00

chr2 103729128 103729266 0.41 0.00

chr2 103749894 103749988 0.42 0.00

chr2 103750643 103750736 1.71 0.03

chr2 103754295 103754405 0.83 0.00

chr2 103755261 103755406 0.69 0.00

chr2 103759636 103759735 0.43 0.03

chr2 103753751 103753850 2.26 0.00