genome-wide transcription factor localization and function in … · 2020. 1. 6. · genome-wide...

Genome-wide transcription factorlocalization and function in stem

cellsWai-Leong Tam1 and Bing Lim1,2, 1Genome Institute of Singapore,Singapore and 2Harvard Institutes of Medicine, Harvard Medical School,Boston, MA 02115, USA

Table of Contents1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.1. Regulation of gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2. A stem cell window into transcription factor function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3. Interrogation of transcription factor binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2. Key regulators of pluripotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53. The transcriptional landscape in ESCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3.1. Auto-regulation and feedback loops maintain gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2. Feed-forward regulation of common target genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.3. Fine-tuning the transcription output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.4. Transcription factor pleiotrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.5. Functional validation of transcriptional targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4. The transcriptional landscape in somatic stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114.1. Distinct transcription factors direct a common program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114.2. Spatial and temporal expression of transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114.3. A transcriptional link between hematopoiesis and leukemia? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.4. Co-factors and post-translation modifications impart transcriptional specificity . . . . . . . . . . . . . . 124.5. Surrogates to somatic stem cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

5. Reactivating pluripotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136. Cell type-dependent regulation of distinct circuitries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

6.1. Lineage-specific recruitment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.2. Cofactor-mediated differential recruitment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

7. The integration of microRNAs in transcription circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1810. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

*Edited by Bradley E. Bernstein. Last revised July 28, 2008. Published September 15, 2008. This chapter should be cited as: Tam, W.-L. andLim, B., Genome-wide transcription factor localization and function in stem cells (September 15, 2008), StemBook, ed. The Stem Cell ResearchCommunity, StemBook, doi/10.3824/stembook.1.19.1, http://www.stembook.org.

Copyright: C© 2008 Wai-Leong Tam and Bing Lim. This is an open-access article distributed under the terms of the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.§To whom correspondence should be addressed. E-mail: [email protected] or [email protected].

1

stembook.org

http://www.stembook.org

Genome-wide transcription factor localization and function in stem cells

Abstract

Stem cells are biologically and clinically important cells characterized by their ability to self-renewor differentiate into many cell types. The decision between self-renewal and differentiation is governed byextracellular signals, coupled to intracellular signaling cascades which activate transcription programs. Hence,transcription regulation plays a determining role in conferring cellular identity and function. As intrinsicdeterminants, transcription factors provide an entry point for uncovering how stem cells attain their phenotype,and how lineage-specific differentiation is initiated. The step-wise maturation of stem cells into terminallydifferentiated cell types requires the timely activation of a cascade of transcription programs governed bylineage-specifying transcription factors. The identification of key regulators in the hierarchy and their targetscan provide clues into the manipulation of stem cells. This review examines the role of key transcription factorsin various stem cell types, and emphasizes on their control of downstream gene expression, which ultimatelycontributes to cellular function.

1. Introduction

1.1. Regulation of gene expression

Transcription regulation constitutes a major component of biological activities in the cell. A key function oftranscriptional activity is the control of gene expression necessary for the maintenance of a cellular state. In metazoan,all cell types, with the exception of mature germ cells, contain the same genetic information within the species.Yet, each cell type expresses a unique subset of all genes encoded. Differential gene expression can be controlledat many levels. One of the foremost steps occurs at the first stage of expression: transcription. Coding and non-coding genes contain promoter and enhancer sequences that are bound by transcription activators and repressors –transcription factors (TFs; Struhl, 1995). A large proportion of TFs are DNA-binding proteins that localize to cis-regulatory DNA elements located upstream or downstream of their target genes. The levels and activities of TFsdetermine to a large part the extent of transcription. Hence, the active quantity of these regulators in turn must betightly regulated. The regulatory mechanisms of TFs are diversified by the combinatorial action of upstream regulatorygenes, post-translational modifications (Hill and Treisman, 1995), ligand-dependant activation for nuclear hormonereceptors (Beato et al., 1995; Mangelsdorf et al., 1995), and protein-protein interactions (Swillens and Pirson, 1994;Tsai and O’Malley, 1994). Two classes of TFs can be distinguished in the eukaryotes. General transcription factors(GTFs) bind to a core promoter usually close to the transcription initiation site, together with the assembly of thepre-initiation complex, while sequence-specific TFs bind regulatory DNA elements at varying distances away fromthe transcription start site. The occupancy of transcription activators on a promoter recruits the transcription initiationmachinery which consists of RNA polymerases and more than 50 other components (Green, 2000; Johnson et al.,2001; Myer and Young, 1998; Roeder, 1996). This is followed by promoter clearance, elongation, and the terminationof transcription. Other components of the post-transcription machinery are also involved in augmenting the overalllevels of gene transcripts in the cell. These include regulated mRNA decay which is achieved through the interactionsbetween trans-acting factors with the mRNA’s structural components, the sequence dependent mRNA stability, andthe association of mRNA with general or mRNA-specific RNA-binding proteins (Guhaniyogi and Brewer, 2001).Therefore, transcription regulation, coupled with post-transcriptional modifications, help determine the base-line ofdifferential gene expression profile that confers cellular identity.

1.2. A stem cell window into transcription factor function

Stem cells are well-suited for the study of transcription regulation and TF function. As these remarkable cells arecharacterized by their ability to self-renew as well as differentiate into functional cell types, they respond to externalstimuli and intracellular perturbations that are observable and detectable. During the course of animal development,cells transit from a pluripotent state to one of many committed lineages. Several developmental stage-specific stemor progenitor cells have been detected and isolated from the mammalian embryo, as well as from post-natal andadult tissues. These include lineage-committed multipotent stem or progenitor cells such as hematopoietic stem cells(HSCs), neural progenitor cells (NPCs), epithelial stem cells and mesenchymal stem cells. Of particular interest arecells of the embryonic inner cell mass (ICM) and their derivatives , the embryonic stem cells (ESCs), which are capableof giving rise to all subsequent cell types of the fetus. This makes ESCs highly promising in regenerative medicine.An important requirement for directing differentiation of stem cells is to first understand how the undifferentiated

2

stembook.org


‘stemness’ state is established and maintained. This would give us clues on how to isolate and expand stem cellpopulation, as well as control their cell fate specification either chemically or genetically.

At the heart of cellular differentiation programs lies a plethora of highly regulated TFs that enable the alteration ofcell states in response to extracellular signaling cues. For many decades, the study of TFs as regulators of downstreameffectors has been limited in scope and comprehensiveness. Deciphering the sequential order in which top-tier regulatorsare activated and identifying what are the extensive repertoire of target genes has been difficult. Different approacheshave been employed to address these issues. During cell fate specification, the activation of a hierarchy of TFs hasbeen inferred from the analyses of mutant animals. This is best exemplified in gene knockouts of TFs involved inspecific tissue development such as myogenesis (Edmondson and Olson, 1989; Hasty et al., 1993; Rudnicki et al.,1993), hematopoiesis (Huang et al., 2008; Kim et al., 2007; Orkin and Zon, 2008; Park et al., 2003) and neurogenesis(Ma et al., 1996). Downstream perturbations arising from the loss of a key TF regulator have provided clues forthe identity of regulated target genes. In recent years, the development of microarray technology and sequencing ofmany vertebrate genomes has facilitated in capturing gene expression profiles on a global scale and the unravelingof transcription regulation hierarchy. One strategy involves perturbing the TF of interest by either over-expressionor depletion, followed by analysis of the resultant changes in gene expression profiles (Menssen and Hermeking,2002; Stein and Liang, 2002; Werner, 2001a; Werner, 2001b), as exemplified in the attempt to identify target genesof the c-Myc proto-oncogene (Menssen and Hermeking, 2002). However, whether differentially expressed genes aredirectly bound and regulated by the TF, or indirectly affected through the perturbation of intermediate genes, cannotbe distinguished. Therefore, alternative approaches are required to map TF binding sites in the genome accurately andefficiently.

1.3. Interrogation of transcription factor binding sites

Chromatin immunoprecipitation (ChIP) allows for the identification of physical interactions between proteinsand DNA in the context of chromatin (Orlando and Paro, 1993; Solomon et al., 1988). An overview of the experimentalprocedure is depicted in Figure 1. ChIP involves chemical cross-linking of DNA-protein interactions in living cellsto capture TFs to their cognate binding sites. Cross-linked chromatin is fragmented and protein-DNA complexescan be enriched by immunoaffinity pull-down of the desired protein. DNA-protein cross-links are then reversed, andthe enriched DNA can be purified for analyses. When combined with high-throughput detection instrumentations,ChIP can provide a comprehensive view of protein-DNA interactions. Three major strategies have been employedfor locating TF occupancy in a genome-wide manner (reviewed in (Massie and Mills, 2008)). Briefly, ChIP-chipinvolves the amplification of ChIP-DNA followed by hybridization to whole-genome promoters or customized DNAmicroarrays (Iyer et al., 2001; Ren et al., 2000). The difference in fluorescent signal intensity of ChIP-DNA overcontrol DNA reflects the relative enrichment of the TF on genomic binding sites. ChIP-PET (paired ends di-tags)represents a sequencing-based method for detecting enrichment of ChIP-DNA (Wei et al., 2006). It involves theconstruction of a concatenated DNA fragment plasmid library which is then sequenced. Mapping both ends of afragment has the advantage of accurately decoding the identity of a gene fragment, and this method is not constrainedby array-based limitations such as probe performance and genome coverage. Recently, the application of ChIP-seq(sequencing) demonstrated considerable advantages over the other methods (Johnson et al., 2007b; Robertson et al.,2007). ChIP-seq involves direct sequencing of ChIP-DNA which has been size-selected and amplified. The method issimple, requires small amounts of ChIP-DNA, has deeper coverage, and is more efficient. Therefore, the availabilityof these methods has greatly enhanced our understanding of the complexity that governs transcription regulatorynetworks in higher eukaryotes. In addition, genome-wide ChIP analysis has also been useful for the study of non-TFsthat associate with, or are components of, the chromatin. These proteins include modified histones and epigeneticregulators such as Polycomb complexes (Bernstein et al., 2006; Boyer et al., 2006; Guenther et al., 2007; Lee et al.,2006; Mikkelsen et al., 2007; Sadikovic et al., 2008).

Genome-wide TF localization analysis has provided invaluable insights into the functions of regulators involvedin cell homeostasis, cell fate transition, immune defense and cancer (Acosta-Alvear et al., 2007; Cao et al., 2006;Carroll et al., 2005; Li et al., 2003; Lim et al., 2007; Lupien et al., 2008; Marson et al., 2007; Odom et al., 2006;Odom et al., 2004; Palomero et al., 2006). We and others have employed this strategy to examine the transcriptioncircuitry of ESCs which is governed by a hierarchy of core factors. In uncovering and reconstructing the key featuresof this circuitry, we further dissected the significance and contributions of these transcriptional components to ESCfunction. The spatial and temporal specifications of stem cells in the embryo and adult somatic tissues have raisedimportant questions pertaining to their establishment and maintenance. In particular, the contrasting role of TFs eitheras regulators in stem cell maintenance or directing differentiation is of immense interest. Thus, mapping the stemcell transcription network can provide useful clues into their manipulation. Here, we discuss key findings into the

3

stembook.org


Genomic DNA

Crosslink protein toDNA and shear DNA

into fragments

Immunoprecipitation withantibody

Release and amplifyDNA

Label DNA fragments

Hybridize to microarray

2 431

Promoter

Transcription factors

0

100

200

300

Sequ

encing

Rea

ds

Gene

0

2

4

6

8

10

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

Sign

al Inten

sity

PET Den

sity

Binding SitesBinding Site

Library cloning Size fractionation

Sequencing and Mapping Sequencing and Mapping

ChIP-chip ChIP-PET ChIP-seq

Gene

Figure 1. Genome-wide transcription factor location analysis. Transcription factors binds to the cis-regulatory sequences of chromatin. To identify thegenomic loci bound, the protein is cross-linked to DNA, and the chromatin is sheared. Chromatin immunoprecipitation is used to purify protein-DNAcomplexes using an antibody of interest. The DNA is released and analyzed. The occupancy of the TF across the genome can be accessed by threehigh-throughput methods: ChIP-chip, ChIP-PET and ChIP-seq.

4

stembook.org


deconstruction of molecular circuitries governing stem cell function, with an emphasis on ESCs. Knowledge drawnfor these studies has further led to the synthesis of key concepts in helping us understand the complexity of generegulatory network in stem cells.

2. Key regulators of pluripotency

During development, the formation of the ICM in the mammalian blastocyst is a major landmark that representsthe specification of pluripotent cells. ESCs have been derived from human and mouse blastocysts (Evans and Kaufman,1981; Reubinoff et al., 2000; Thomson et al., 1998), and are similar in many developmental aspects. Althoughdifferences exist in their culture and maintenance, many molecular determinants governing their undifferentiatedstates are shared. Homeodomain TFs are evolutionary conserved and participate in cell fate determination in manyorganisms (Hombria and Lovegrove, 2003). The POU (Pit/Oct/Unc) TF, Oct4, has emerged as a dominant regulatorin ESCs (Scholer et al., 1990a; Scholer et al., 1990b). It is exclusively expressed in blastomeres, pre-implantationembryos, primodial germ cells, the ICM and a narrow range of cultured cell types (Nichols et al., 1998; Pesce et al.,1998; Rosner et al., 1990; Scholer et al., 1990a; Yeom et al., 1996). As a TF, Oct4 can activate or repress geneexpression (Botquin et al., 1998; Nichols et al., 1998; Pesce and Scholer, 2001; Scholer et al., 1991), and interactwith other molecules such as the HMG-box TF, Sox2, in pluripotent cells (Scholer et al., 1991; Yuan et al., 1995).Genetic studies have confirmed the importance of Oct4 during embryonic development and in maintaining pluripotentESCs (Nichols et al., 1998; Palmieri et al., 1994). Nanog, another homeodomain-containing TF expressed abundantlyin ESCs, primodial germ cells and embryonal carcinoma cell lines, is also necessary for ESC function (Chamberset al., 2003; Mitsui et al., 2003; Ramalho-Santos et al., 2002; Wang et al., 2003). In the mouse embryo, Nanog firstappears in the compacted morula, becomes localized to the ICM, and is finally restricted to the epiblast (Chamberset al., 2003). In Nanog−/− embryos, there is a lack of epiblast and primitive ectoderm formations, indicative of theabsolute requirement for Nanog; and Nanog−/− ESCs manifest a reduction of pluripotency and re-specification tothe extra-embryonic endoderm lineages (Mitsui et al., 2003). Strikingly, a recent observation has clarified the role ofNanog further. While Nanog is necessary for inner cell mass formation and primodial germ cell specification, it appearsdispensable in ESCs since Nanog−/− ESCs persist in culture although these cells are more prone to differentiation(Chambers et al., 2007).

A host of other TFs also appear crucial for ESC function. The neuronal repressor REST is highly abundant inmouse ESCs (Ballas et al., 2005), and its depletion either through RNA interference (RNAi) or heterozygous deletionleads to a loss of self-renewal (Singh et al., 2008). REST is a major transcriptional repressor of neurogenesis, and theactivation of REST-regulated genes can convert neural progenitor cells to neuronal phenotypes (Ballas and Mandel,2005; Coulson, 2005; Su et al., 2004). But, the manner in which it regulates gene expression in ESCs is unclear. Anotherregulator, Sall4, is an important activator of Oct4, and its loss leads to cell fate re-specification into trophoblast stem cells(Elling et al., 2006; Sakaki-Yumoto et al., 2006; Zhang et al., 2006). This effect is recapitulated during pre-implantationdevelopment where down-regulation of Sall4 causes mis-expression of the trophoblast stem cell determinant, Cdx2, inthe ICM (Zhang et al., 2006). The use of integrated approaches combining transcriptome profiling, genome-wide ChIPanalyses, as well as large-scale gain- or loss-of-function has further led to the identification of an increasing numberof developmentally important TFs. In these studies, Tcl1, Tbx3 and Esrrb have been identified as novel regulators inESCs (Ivanova et al., 2006; Loh et al., 2006). However, the precise manner in which these genes exert their regulatoryeffects has not yet been elucidated.

3. The transcriptional landscape in ESCs

Initial studies into transcriptional regulation by Oct4, Sox2, Nanog and Sall4 have revealed a small number ofgenes bound by these factors (Bohm et al., 2007; Chew et al., 2005a; Nishimoto et al., 1999; Rodda et al., 2005a;Tokuzawa et al., 2003; Tomioka et al., 2002; Wu et al., 2006; Zhang et al., 2006). These limited observations aresimplistic, and do not provide a comprehensive understanding into the complex framework of transcription hierarchywhere regulators tend to exert far-reaching effects on a vast array of genes, as observed from yeast studies (Iyer et al.,2001; Lee et al., 2002; Ren et al., 2000). In animals as diverse as C. elegans and Drosophila to Xenopus, mouseand man, activating and inhibiting signals establish a dynamic cascade of coordinated gene expression in responseto extrinsic signals and intrinsic programming. The integration of these instructions occurs in the nucleus throughcombinations of signal-activated and tissue-restricted TFs binding to, and controlling, cis-regulatory modules of genes.With the complexities arising from such intricate interactions, the ESC model, which shows a remarkable sensitivityto genetic perturbations, thus represents a suitable system for probing TF function and regulatory dynamics.

5

stembook.org


Embryo-derived stem cells

Sall4ESCcircuitry

XENcircuitry

Oct4,

Sox

2, N

anog

Gata4,Gata6,

Sox7,

Sox17

Auto-regulatory loopsin ESC

Sall4

Oct4

Sox2

Nan

og

A B C

MyoD

MyoG

Myogenic program

Priming targetgenelocus

Modulatingtranscription

activity

Figure 2. Transcription programs in stem cells. Transcription factors control the molecular circuits that govern stem cell identity. A. In ESCs, keypluripotency-associated factors Oct4, Sox2, Nanog and Sall4 form auto-regulatory loops that maintain their own transcriptional activity and gene expression.The stability of this set of core factors is further enhanced through the interconnectivity of these loops. B. During myogenesis, MyoD is an upstream regulatorwhich activates the expression of MyoG. Both TFs are necessary for the myogenic program, and regulate a common subset of genes in a temporal manner.MyoD must initiate chromatin remodeling of genes for these to be subsequently bound and regulated by MyoG. C. The same TF, Sall4, have dual roles incontrolling discrete circuits in the different embryo-derived stem cell lines, ESCs and XEN cells. In the respective cell types, Sall4 regulates distinctive setsof key factors that contribute to the specific cellular identity. In ESCs, Sall4 activates Oct4, Sox2 and Nanog; all of which positively feedback onto the Sall4promoter. In XEN cells, Sall4 activates Gata4, Gata6, Sox7 and Sox17 which are necessary for the undifferentiated state. Whether these regulated factorsdirectly feedback onto the Sall4 promoter is not known.

3.1. Auto-regulation and feedback loops maintain gene expression

One emergent theme arising from transcription regulation in ESCs is the revelation that a core set of factorsact together in a tightly regulated framework to control similar as well as divergent downstream genes (see Table 1).Auto-regulation by top-tier TFs is a key feature. The pluripotent molecules Oct4, Sox2, Nanog and Sall4 auto-regulatethrough binding to their own promoters (Boyer et al., 2005; Chew et al., 2005b; Loh et al., 2006; Rodda et al., 2005b;Tomioka et al., 2002; see Figure 2A). On its own, each factor maintains its expression that is individually requiredfor ESC function. Collectively, all four factors regulate one another through the interconnectivity of positive feedbackloops (see Table 1). The generation of these feedback circuits reinforces the transcription activity of each factor, andmaintains appropriate levels of the necessary factors.

Recently, the transcription landscape has been extended through the analyses of a greater number of factorsassociated with ESC pluripotency, as well as those implicated in the reprogramming of somatic cells to induced-pluripotent stem (iPS) cells. The extended repertoire examined reinforces previous observations that auto-regulatoryand feedback loops are prevalent features of ESC transcriptional landscape. In one study, the interconnectivity ofnine TFs (Oct4, Sox2, Nanog, Dax1, Nac1, Klf4, Zfp281, Rex1 and c-Myc) has been analyzed. Of these regulators,the promoters of Oct4, Sox2, Nanog and Dax1 are bound by at least four factors including itself (Kim et al., 2008),suggestive of a robust positive feedback network. Strikingly, new regulatory foci that include Sall4, Rif1, REST andDax1, bound by multiple TFs, have also been uncovered (Kim et al., 2008), integrating these hubs to the core circuitry.

Auto-regulation and feedback loops appear to confer several advantages. A core set of factors that maintainsits own expression, as well as that of others in the vicinity, enhances the stability of gene expression (Boyer et al.,2005; McAdams and Arkin, 1997). This is especially vital to ESCs since the prolonged loss of any key factor issufficient to cause irreversible differentiation. Based on empirical evidence that have emerged from biological studies,the computational notion of a ‘bistable’ switch arising from the positive feedback loops comprised of Oct4, Sox2 andNanog has been proposed (Chickarmane et al., 2006). It is thought that such a mechanism stabilizes the expressionlevels of all three factors, which through their regulatory control of downstream targets, appears extremely tolerant toparameter change and could counter input noise (Chickarmane et al., 2006; Rao et al., 2002).

3.2. Feed-forward regulation of common target genes

The second observation is the regulation of common target genes by ESC regulators. Oct4, Sox2 and Nanogform feed-forward loops (see Table 1) that involve over 350 protein-coding genes in human ESCs (Boyer et al.,2005). In the mouse counterpart, Oct4 and Nanog occupy approximately 1,000 and 3,000 genomic binding sites

6

stembook.org


Table 1. Models of transcription regulatory network

Key Concepts Remarks Model

Auto-regulatory andfeedback loop

� Transcription factors (X and Y) auto-regulate by binding to their own promoters.� X and Y also bind the promoters of each other in a feedback loop.� Auto-regulation and feedback loops are features of ‘master’ regulators in the

transcription network of eukaryotes.� Provides enhanced stability of gene expression that maintains high levels of both

factors.� Examples: Oct4, Sox2, Nanog and Sall4 in ESCs; HNF1α, HNF4α and HNF6 in

hepatocytes.

X Y

Feed-forwardloop

� Transcription factor (X) controls another regulator (Y), and both of them regulatedownstream target gene (Z).

� Multiple positive inputs provide consistent activity in regulating a commontarget, rendering it insensitive to transient changes in individual input.

� Particularly important in stem cells to maintain either a stable self-renewing ordifferentiated state.

� Examples: In ESCs, Nanog regulates Oct4 and Sall4, where Oct4 also controlsSall4; upon differentiation, Sall4 is down-regulated partly owing to the loss ofNanog and Oct4.

X

Y

Z

Onetranscriptionfactor →Multiple targetgenes

� A regulator (X) controls the expression of many target genes.� Dominant key factors control a wide repertoire of genes in a positive or negative

manner, which may require the activity of co-factors.� The sum of activity of all downstream targets represents the cellular state.� Example: REST represses extensive neuronal lineage-associated targets in

non-neuronal cells; HNF1α controls hepatocyte biochemistry-related genes inliver; Oct4 activates ESC-associated genes and repressesdifferentiation-associated genes in ESCs.

X

a

d e

b c

Core set oftranscriptionfactors →Common targetgenes

� Transcription factors (X, Y and Z) forms a central regulatory hub.� All three factors tend to bind common target genes, in addition to other targets.� Core factors are known to regulate a set of common downstream regulators which

themselves are crucial for the maintenance of a particular cellular state.� Examples: Oct4, Sox2, Nanog regulates common genes in undifferentiated ESCs;

MyoD and MyoG maintains the expression of common genes during myogenesisin a temporal manner.

X Y Z

a b c d e f

Combinat-orial inputs →Transcriptionoutput

� Transcription factors comprising of activator (A) and repressor (R) bind to acommon target gene.

� The combinatorial input of activators and repressors determine the transcriptionoutput of Y.

� Output may be binary (On/Off) or graded (High/Low).� Repressors may behave as ‘transistors’ which moderate gene expression levels.� Example: Regulation of the Oct4 gene activity by the activator Nanog and the

repressor Tcf3 in ESCs.

A A R

Y

ON / OFF

HIGH / LOW OUTPUT

Onetranscriptionfactor →Distinctcircuitries

� The transcription factor (X) may control different and/or overlapping sets oftarget genes in a cell-type dependent manner.

� The regulation of dissimilar gene sets is responsible for a varied role in differentcell types or cellular contexts.

� The control of a similar set of genes may suggest a common function for thefactor in different cell types.

� Example: Sall4 maintains lineage-distinct ESCs and XEN cells by binding todifferent sets of genes; c-Myc regulates similar proliferation-associated genes inESCs and cancer cells.

XType Acell

Type Bcell

Co-factordependentrecruitment totargets

� The activity and target gene occupancy profile of a transcription factor (X) isdependent on its association with specific co-factors (α or β).

� Co-factors can mediate recruitment of X to specific target genes that may bemotif (DNA sequence) dependent.

� Example: FoxA1 recruitment to differential targets in breast and prostate cancercells is mediated by the ERα and AR, respectively.

X X

a

b

c

d

e

f

Condition-dependentrecruitment totargets

� The transcription factor (X) regulates a set of target genes in the native state ofthe cell.

� Upon stimuli, the binding dynamics of X may become expanded or altered toinclude either a larger repertoire of targets relevant for a specific response, or anentirely different set of genes.

� The expression of co-factors induced by stimulation may be necessary forrecruitment to targets.

� Example: Stimulation of monocytes by LPS expands the binding profile ofNF-κB which is partly dependent upon E2F1.

X

Inactive

Active

7

stembook.org


Oct4

Sox2Nanog

Dax1

Nac1Klf4

At least 5 out of 6 factors

Gbx2

Sox2Rest

Sall4Tbx3 Zfp206

Zfp42

Tp53bp1

Oct4Nanog

Phc1

Klf2

Msh6

Mybl2

0

200 NanogOct4Sox2Klf4E2f1Esrrbn-Mycc-MycSmad1STAT3Tcfcp2l1Zfx

Multiple Transcriptionfactor binding Loci

(MTL)or

“Enhanceosome”

Oct4 locus

A BESC regulators

Activation of transcription targets by co-occupancy of multiple factors at target

gene promoter

Input Synergism

Multiple transcription factor occupancy atenhancers

Figure 3. Analysis of multiple transcription factor occupancy. Several TFs may bind to common gene promoters, or at the same genomic loci. A. InESCs, different combinations of pluripotency-associated factors, Oct4, Sox2, Nanog, Klf4, Nac1 and Dax1, occupy the promoters of common genes. In thismultiple co-occupancy where at least five factors are bound, it typically leads to transcription activation of the target genes, highlighting a synergistic effectthat is mediated by a combined positive transcriptional input. B. Multiple TFs are also found to be located on enhancers where these dense ‘hotspots’ arereferred as enhanceosomes. As many as 12 factors are found to occupy the enhancer of Oct4, which is a key ESC regulator. Several of these TFs have beenshown to biochemically co-purify as part of a larger complex, supporting the synergistic activation model.

respectively (Loh et al., 2006). Many of these Oct4-bound loci also contain Sox2 sites, confirming both factors act intandem. Significantly, Oct4 and Nanog positively control downstream genes important for maintaining pluripotencyand inhibiting differentiation. These included Foxd3, Setdb1, Rif1, Esrrb and n-Myc which help maintain pluripotency(Adams and McLaren, 2004; Cartwright et al., 2005; Dodge et al., 2004; Guo et al., 2002; Hanna et al., 2002; Luo et al.,1997; Mitsunaga et al., 2004). A cascade of other pathways that are connected to self-renewal, genome surveillanceand cell fate determination are also implicated (Loh et al., 2006). In addition to their role as activators, Oct4 and Nanogcan repress differentiation-associated genes. One key example repressed by Oct4 is Cdx2, necessary for trophectodermformation (Niwa et al., 2005). Additionally, integrated analysis of a larger set of TFs revealed that target genesco-occupied simultaneously by multiple other factors (Oct4, Sox2, Nanog, Dax1, Nac1 or Klf4), are found to begenerally active in ESCs, and repressed upon differentiation; whereas those bound by a single factor alone tend to beinactive or repressed (Kim et al., 2008; see Figure 3A). This suggests multiple inputs may be necessary for a concertedactivation signal, possibly through the assembly of activation complexes. For single inputs, these could be associatedwith co-repression complexes, as revealed by protein network analysis where there exist multiple connections betweencore factors and repressive chromatin complexes such as NuRD and Polycomb (Wang et al., 2006).

Interestingly, discrete feed-forward loops comprised of divergent target sets bound by different subsets of TFscan be discerned. In a separate study involving the genome-wide mapping of 13 TFs (Oct4, Sox2, Nanog, Klf4, c-Myc,n-Myc, Esrrb, Zfx, Tcfcp2l1, Stat3, Smad1, E2f1 and CTCF), it is striking that whereas targets of Oct4, Sox2, Nanog,Stat3 and Smad1 appear more closely associated based on hierarchical clustering, targets of c-Myc, n-Myc and Zfxfall into a separate grouping (Chen et al., 2008; Kim et al., 2008) .The interrogation of chromatin occupancy for a largeset of TFs, not limited to promoters, has surprisingly revealed genomic regions, or hotspots, which are co-occupied. Ifonly a few factors were examined, the existence of these hotspots would not have been evident. These nucleoproteincomplexes comprised of a combination of TFs binding to enhancer DNA are referred to as enhanceosomes (Thanos andManiatis, 1995). It is furthermore striking that the densest binding loci occupied by 12 of 13 factors is the previouslycharacterized distal enhancer of Oct4 (Chen et al., 2008; Chew et al., 2005a; see Figure 3B). Several factors in this

8

stembook.org


complex have already been reported to interact with one another (Chew et al., 2005a; Okumura-Nakanishi et al., 2005;Suzuki et al., 2006; Wang and Orkin, 2008). Given the limited understanding of enhancer functions, in-depth analysesof enhanceosomes, or common target loci, could provide valuable insights into key regulatory regions unique to ESCs.

The reliance on feed-forward loops has several advantages. When more than one positive regulator control acommon gene, either directly or indirectly through intermediates, the multiple inputs provide consistent activity thatrender it relatively insensitive to transient changes in individual input strength (Mangan and Alon, 2003; Manganet al., 2003; see Table 1). When these regulators have either a positive or negative function, the feed-forward loopthen behaves as a sensor that can respond rapidly to the balance of the inputs. In that sense, the upstream geneticcis-regulatory elements can be viewed as a ‘transistor’ for receiving and moderating the input signals of the TFs whichmay carry either an activating or repressive function. This concept of feed-forward loops appears particularly vital tostem cells which must react appropriately to either self-renewal or differentiation cues (Boyer et al., 2005).

Much less is understood about the consequences of having elevated levels of pluripotency factors. As exemplifiedby Oct4 and Nanog, their elevation at non-physiological levels dramatically alters ESCs. An up-regulation of Oct4causes ESCs differentiation towards the mesendoderm lineage while an increase in Nanog limits their ability todifferentiate (Chambers et al., 2003; Mitsui et al., 2003; Niwa et al., 2000; Tam et al., 2008). Furthermore, the ectopicmis-expression of Oct4 in somatic tissues of adult mice can lead to dysplastic growth in epithelial tissues and blockprogenitor cell differentiation (Hochedlinger et al., 2005). Thus, it is imperative to understand the fine-tuning ofappropriate transcription output by repressors.

3.3. Fine-tuning the transcription output

The negative regulation of pluripotency-associated genes is poorly understood and largely under-appreciated.The tumor suppressor p53, for example, suppresses transcription of Nanog and induces differentiation in response toDNA damage, suggesting its role in maintaining genetic stability (Lin et al., 2005); and, the orphan nuclear receptorGCNF, was reported to repress Oct4 and Nanog during retinoic acid-induced differentiation of ESCs (Gu et al., 2005).Recent studies have further highlighted the role of the Wnt TF, Tcf3, as a potent repressor of Oct4 and Nanog (Pereiraet al., 2006; Tam et al., 2008; Yi et al., 2008a). The Wnt signaling cascade is frequently implicated in the establishmentof stem cell identity, as well as directing cell fate commitment (Hirabayashi et al., 2004; Huelsken et al., 2001; Lagutinet al., 2003; Merrill et al., 2004; Nguyen et al., 2006; Sato et al., 2004). But, the precise mechanism in which itsdownstream effectors, Tcf/Lef factors, mediate these decisions is not known. Interestingly, the depletion of Tcf3 limitsthe ability of ESCs to differentiate (Pereira et al., 2006; Tam et al., 2008). Part of this is attributed to the relief ofrepression on Oct4 and Nanog, whereby their levels become induced. Comprehensive analyses of Tcf3 binding sitesacross the genome surprisingly revealed that this repressive factor share many common targets with Oct4, Sox2 andNanog (Cole et al., 2008; Tam et al., 2008; see Figure 4A). The close proximity of these factors on co-occupied genesfurther suggests an integrative regulatory function, and many of their common targets have roles in ESC self-renewal.It appears conceivable that Tcf3 counters the effects of positive regulators on these genes as Tcf3 depletion inducesthe up-regulation of these targets (Tam et al., 2008). The conglomeration of data indicates that fine-tuning the dosageof ESC factors is a crucial component in transcription regulation (see Figure 4B). Currently, the interaction betweenactivators and repressors on the same gene promoter is not well-understood, much less their complex dynamics on aglobal scale. The delicate balance between positive and negative inputs necessary to dictate the transcriptional outputneeds to be better established before quantitative evaluation and predictions of the transcription circuitry are possible(see Table 1). The recruitment of co-repressors, such as Grouchos/TLEs or activators such as β-catenin in the case ofTcf/Lef factors (Chen and Courey, 2000; Eastman and Grosschedl, 1999; Roose and Clevers, 1999), further confoundsour understanding on how TFs act as molecular switches in a combinatorial manner. In-depth studies of interactionsamong transcription regulators and the global mapping of an increasing number of ESC factors are pertinent in thesystems approach to dissecting the stem cell identity, as exemplified in a computational study model (Zhou et al.,2007).

3.4. Transcription factor pleiotrophy

Surprisingly, the role of Tcf3 extends beyond its control of pluripotency-associated genes. It also binds thepromoters of homeodomain genes which include members of the Fox, Tbx and Sox gene families known to regulateearly developmental programs (Buckingham et al., 2003; Kiefer, 2007; Plageman and Yutzey, 2005; Tam et al., 2008;Wijchers et al., 2006). Tcf3 appears necessary to prevent the premature activation of differentiation programs. Whereaspolycomb group (PcG) proteins act as general transcription repressors that blocks the expression of a large cohort ofdevelopmental regulators (Boyer et al., 2006), Tcf3 binds to two categorically discrete sets of regulators in the same

9

stembook.org


Oct4

Nanog

Tcf3

Rif1

Tbx3

Dppa5

Phc1

Jarid2

Oct4

Nanog

N-myc

Fzd5

Tle1

Tcf3

Trp53bp1

ESC Regulators

Wnt signaling

Epigenetic Regulators

Tumor suppressor

Telomere-associated

RNA binding protein

Pluripotency Differentiation

Oct4 Oct4

Oct4

NanogSox2

Sall4LRH1

Tcf3

GCNF

A B

Figure 4. Repressors modulate transcription output. A. Oct4, Nanog and Tcf3 co-occupy a common set of pluripotency-associated factors in ESCs.Whereas Oct4 and Nanog may activate genes required for pluripotency, Tcf3 behaves as a repressor that also binds these common genes, thereby controllingthe overall gene expression levels. B. At the single gene level, the transcription output is partly determined by the combination of factors that activate orrepress transcription. The well-characterized Oct4 gene cis-regulatory regulatory region is bound by several TFs that act as either activator or repressor. Inthe undifferentiated state, Oct4, Sox2, Nanog, Sall4 and LRH1 activate the promoter while Tcf3 and GCNF repress it. The balance of molecules acting onthis critical gene controls the cell fate of either self-renewal or differentiation.

stage-specific context, i.e. undifferentiated ESCs. Although both sets of targets become de-repressed upon the loss ofTcf3, it remains unclear why Tcf3-deficient cells tend to self-renew rather than differentiate. A plausible explanationmay be attributed to the functional dominance of ESC stemness-associated over differentiation-associated genes,as observed during somatic cell reprogramming by fusion with ESCs (Cowan et al., 2005; Tada et al., 2001). Thispleiotropic control by a TF in a genome-wide manner is puzzling, and implies critical roles for repressors. Interestingly,it has also been demonstrated that Wnt signal activation can induce ESC-associated targets of Tcf3, possibly due tothe replacement of co-repressors with the co-activator β-catenin which associates with Tcf3 (Cole et al., 2008). It,however, remains unresolved whether there is indeed a direct protein-protein interaction between Tcf3 and β-cateninupon Wnt stimulation, or that β-catenin may interact with other Tcf/Lef factors to activate common genes.

3.5. Functional validation of transcriptional targets

A major limitation of genome-wide studies is the generation of large interactome datasets that are not functionallytested in a biologically relevant manner. Technically, it is demanding to prove a causative relationship between geneoccupancy and transcription activity one gene at a time. Although an alternative method is to correlate global transcriptchanges with factor activity for implying a direct transcription control, many studies surprisingly revealed that a largeproportion of these gene targets do not appear directly regulated. Transcription factor over-expression or depletiondoes not necessarily alter the transcript levels of target genes. This, therefore, raises an important question of whetherTF occupancy alone is sufficient to infer function in an overall network, or is the association with co-factors and proteincomplexes really the critical determinant. Furthermore, the epigenetic state of chromatin in altering permissivenessto transcription activity is another major consideration not discussed here (Azuara et al., 2006; Bernstein and Kellis,2005; Bernstein et al., 2006; Guenther et al., 2007; Mikkelsen et al., 2007). One approach which clarifies the roleof transcription targets is through gain- or loss-of-function studies to test if putative candidates are physiologicallyrelevant. The availability of extensive gene expression libraries has made this type of validation possible (Chang et al.,2006; Paddison et al., 2004; Silva et al., 2005; Silva et al., 2004). It is now feasible to establish high-throughput

10

stembook.org


platforms for introducing a library of over-expression or RNAi plasmid constructs into stem cells, accompanied bya suitable assay method for assessing gene function. For genetic studies in ESCs and gene function during animaldevelopment, the accessibility of gene-trap mouse ESC lines has made it easier to analyze gene functions in the animal(Hansen et al., 2003; Nord et al., 2006; Stryke et al., 2003; To et al., 2004). Hence, genomic discoveries create aframework which supports hypothesis-driven candidate gene function analysis.

4. The transcriptional landscape in somatic stem cells

4.1. Distinct transcription factors direct a common program

The global transcription landscape in many somatic stem cells is poorly understood. Part of this limitation isthe inherent difficulty of obtaining a large number of homogenous bona fide somatic stem cells in vitro for analyses.Most somatic stem cells, unlike ESCs, arrest at the interphase of cell cycle and do not proliferate rapidly unlessinduced. Limited knowledge of the culture condition has prevented efficient expansion. Notwithstanding, unipotentand multipotent stem or progenitor cells of several tissues can be isolated and cultured. During myogenic differentiation,the convergence of lineage-defining TFs on a common set of targets amplifies the recurrent theme of a core transcriptionnetwork. Precursor myoblast cells proceed through irreversible cell cycle arrest, followed by an increase in musclegenes expression. This process is orchestrated through a series of transcription networks governed by distinct myogenicTFs. Several basic helix-loop-helix (bHLH) family members play discrete roles. MyoD and Myf5 are necessary tospecify the initial skeletal muscle lineage (Rudnicki et al., 1993), where the ectopic expression of MyoD aloneis sufficient to force non-muscle cells to complete the myogenic program (Tapscott et al., 1988). The subsequentexpression of MyoG causes the terminal differentiation of specified muscle cells (Hasty et al., 1993; Nabeshima et al.,1993), and Mrf4 appears to act as a specification as well as differentiation factor (Kassar-Duchossoy et al., 2004).In the skeletal muscle program, genome-wide identification of MyoD and MyoG binding sites revealed that bothfactors have distinct and sequential regulatory roles on a similar set of genes promoters (Cao et al., 2006; see Figure2B). MyoD initiates chromatin remodeling but is insufficient for gene expression, and MyoG can only bind genespreviously initiated by MyoD. The shared consensus DNA binding sequences of MyoD and MyoG further confirmstheir convergence (Berkes et al., 2004). This sustained temporal activation of similar targets by two stage-specificfactors is surprising because previous evidence suggest that MyoD and MyoG act on separate sets of myogenic genepromoters thereby promoting a step-wise maturation (Asakura et al., 1993; Yutzey et al., 1990). The convergence ofboth factors on common targets could not have been revealed in limited candidate gene analyses, as a comprehensiveapproach to mapping the transcription landscape during myogenesis is necessary to resolve these differences.

4.2. Spatial and temporal expression of transcription factors

The specification of the vertebrate central nervous system is defined by neural progenitor/stem cells (NPCs/NSCs)which proliferate, self-renew, and give rise to neurogenic and gliogenic lineages. A few pro-neural genes which encodethe bHLH TFs are necessary and sufficient to initiate the formation of neural lineages, and also to promote the generationof progenitors that are committed to differentiation (Guillemot, 1999). Neurogenins (Ngn1/2/3) and NeuroD are keyregulators of vertebrate neurogenesis. Ngn1 or Ngn2 mutant mice lack cranial and spinal sensory ganglia and ventralspinal cord neurons (Fode et al., 1998; Ma et al., 1998; Ma et al., 1999), while NeuroD mutants manifest the loss ofcerebellar and hippocampal granule cells, inner ear sensory neurons, and retinal photoreceptor cells (Kim et al., 2001;Miyata et al., 1999; Pennesi et al., 2003). The expression of pro-neural genes in NPCs is transient as these must bedown-regulated before progenitors exit the proliferative zone of the neural tube and differentiate (Fode et al., 2000).Hence, pro-neural genes appear to promote complete neuronal differentiation through the induction of downstreamregulators. The direct transcriptional targets of pro-neural genes has not been systematically defined, but indirectevidence in Xenopus suggests that Ngn1/2 and NeuroD may regulate many genes such as Nhlh1, NeuroD4 and Hes,which are related to neurogenesis (Seo et al., 2007). In pancreatic islet cells, NeuroD also appears to be a downstreammediator of Ngn activities (Huang et al., 2000).

Unlike ESC regulators which must remain constitutively elevated, the control of NPC fate by pro-neural genesoccurs in two phases (Bertrand et al., 2002). The first involves a reversible selection of progenitors which are notcommitted to differentiation when pro-neural genes are expressed at a low basal level. This is followed by a secondphase whereby elevated levels of pro-neural genes force an irreversible commitment of progenitors to differentiate.Although signaling pathways that include Notch, sonic hedgehog (Shh) and bone morphogenic protein (BMP) provideextrinsic cues for the initiation of pro-neural genes, it is likely that other positive feedback mechanisms mediated bydownstream transcription targets help increase or maintain their appropriate expression in the selected progenitors.Subsequently, upon the induction of differentiation, lineage-restricted genes become elevated and direct the progenitors

11

stembook.org


to adopt their committed fate. Even though pro-neural and neuronal-differentiation genes perform distinct functions,the structural similarities between them have raised the question of whether their disparate functions may be due tothe consequences of stage-specific and spatial-dependent expression in the neural lineages, or intrinsic biochemicaldifferences such as protein activity and/or stability (Bertrand et al., 2002). In the spinal cord, the spatial expressionof neural bHLH proteins Math1, Ngn1 and Mash1 in distinct dorso-ventral progenitor domains is responsible forcontrolling the specification of interneuron subtypes (Gowan et al., 2001). Observations from myogenic differentiationappear to support the view that temporal expression of structurally related TFs plays a major role in defining thetranscription program of either self-renewal or differentiation. Therefore, the expression specificity in a spatial andtemporal context, rather than unique biochemical properties, of bHLH pro-neural genes may determine the control ofdifferential target genes.

Several members of the Sox family of TFs also mark neural progenitor cells throughout the central nervoussystem, as well as maintaining self-renewing NPCs in vitro. The constitutive expression of Sox2 inhibits neuronaldifferentiation and results in the maintenance of progenitor characteristics, whereas its loss results in NPCs exitingcell cycle and the onset of neuronal differentiation (Graham et al., 2003). The multipotent NSCs obtained from mouseembryonic neuroepithelial cells express Sox2, along with the neural cell marker Nestin. Strikingly, Sox2 is also adeterminant of ESC function. This underlying importance of the same factor in two stem cell types could suggest thateither the temporal and lineage-specific expression of a TF determines their cell fate, or Sox2 could be a ‘stemness’gene common to certain stem cells. Nevertheless, the lack of information on Sox2 transcription targets in NSCshampers a direct comparison of the mechanisms which it may utilize in different stem cells.

4.3. A transcriptional link between hematopoiesis and leukemia?

During hematopoiesis, the establishment and maintenance of the entire blood system depends on rare hematopoi-etic stem cells (HSCs) residing in the bone marrow. The balance between self-renewal, and the differentiation of HSCsinto lineage-restricted progenitors, and ultimately into diverse blood cell types, is intricately controlled through ex-tracellular and intracellular signaling cues. These feed into the activation of regulators, which establish complextranscription networks. Briefly, HSCs can give rise to two major lineage-restricted progenitor cell types: the commonmyeloid progenitor which diversifies into myeloid cells comprised of monocytes, neutrophils, eosinophils, erythroids,mast cells and megakaryocytes; and the common lymphoid progenitor which produces the B and T lymphocytes (re-viewed in (Orkin, 2000)). Several TFs maintain HSC self-renewal, whereas others specify differentiated hematopoieticlineages. The bHLH factor SCL, the SET-domain containing histone methyltransferase MLL and runt-domain Runx1factors, are essential for production, survival and self-renewal of HSCs (Orkin, 2000). MLL and Runx1 are reportedto regulate HoxB4 and Pu.1, respectively, whereby the loss of either factor blocks self-renewal (Diehl et al., 2007;Huang et al., 2008). Presumably, other yet unidentified downstream regulators may also be important. One lessondrawn from the identification HSC regulators is that many of these are concurrently mis-expressed in various humanleukemias (Orkin and Zon, 2008). For instance, SCL is associated with T-cell acute leukemia, and MLL and Runx1is associated with myeloid and lymphoid leukemias. The transcriptional control of genetic targets by these regulatorsduring hematopoiesis and oncogenesis has not been forthcoming. It remains uncertain if a common factor does indeedcontrols similar self-renewal genes which is regulated in one cell type but deregulated in the other.

4.4. Co-factors and post-translation modifications impart transcriptional specificity

Important principles underlying the transcriptional control of erythropoiesis have emerged from mechanisticstudies on the synergism and antagonism of factors involved in lineage transition. Gata1, the founding member of theGATA TF family is a determinant for common myeloid progenitor specification. Analyses of chromatin occupancyrevealed that Gata1 occupies only a small proportion of high-affinity GATA motifs in the genome (Grass et al.,2006; Johnson et al., 2007a). Interestingly, Gata2, which contains a nearly identical dual zinc finger DNA bindingdomain, function uniquely to control distinct aspects of hematopoiesis (Lurie et al., 2008). Gata2 is expressed inHSCs to promote their proliferation, survival and function. Intriguingly, during erythropoiesis, Gata1 and Gata2 showreciprocal expression, with the levels of Gata1 increasing and Gata2 declining. Since both Gata1 and Gata2 couldoccupy similar target genes containing the GATA motif, how does the subsequent expression of Gata1 determine theactivation of genes necessary for erythropoiesis? This selectivity appears to be partially imparted by the cell-specificco-regulator Friend of Gata1 (FOG-1) which mediates certain functions of Gata1 (Letting et al., 2004). Co-regulatorproteins can be recruited to chromatin by sequence-specific TFs and catalyze chromatin modifications that controlDNA accessibility and binding of the transcription machinery (Pal et al., 2004). However, the logic which dictatesspecific recognition of cognate Gata1 versus Gata2 targets, as well as their unique transcription output remains unclear.The question of how related factors are recruited to common versus distinct chromatin target sites needs to be resolved.

12

stembook.org


While the intrinsic differences in biochemical properties between related TFs are often under-appreciated, itcould explain the phasic expression of related members which may perform unique and/or redundant functions. Forexample, the considerable protein stability of Gata1 compared with Gata2, conferred by the C-termini, provides analternative explanation for selective target gene occupancy (Lurie et al., 2008). Gata1 is subjected to phosphorylationand acetylation, but it is unclear how these modifications affect Gata1 activity or function. The relevance of post-translation modifications to stem cell factors in defining genome-wide localization has not been examined. Althoughphosphorylation of the downstream effectors, Stat3 and Smad, of the Jak and BMP signaling pathways are essentialfor nuclear localization and DNA binding properties in ESCs, it is unclear how such modifications alter the bindingdynamics of other stem cell factors on a global scale. There is emerging evidence that ESC factors such as Oct4 issubjected to sumoylation that enhances its stability and transactivation function (Wei et al., 2007). The Src familyof tyrosine kinases has also been reported to be important for ESC self-renewal (Anneren et al., 2004), emphasizingthe under-appreciated aspect of post-translation modifications in controlling transactivation activity and occupancyprofile. Thus, differential stability of TFs can determine chromatin occupancy and help establish the appropriategenetic networks.

4.5. Surrogates to somatic stem cells?

Stem cells and cancer cells share several traits. Distinct from normal somatic cells which contribute to tissuefunction and homeostasis, both cell types are capable of sustained self-renewal in vitro and in vivo. The scarcity ofcultured stem cells has, in part, led to the use of cancer cell lines such as Burkitt’s lymphoma cells (Li et al., 2003),colorectal cancer cells (Hatzis et al., 2008), gliomas (Bruce et al., 2004), and T-cell acute lymphoblastic leukemic cells(Palomero et al., 2006) as surrogates for genome-wide TF location analyses. For example, the immortalized humannon-neuronal T-lymphocyte cell line Jurkat is used to map binding sites for REST (Johnson et al., 2007b). Consistentwith its current knowledge as a repressor of neuronal gene expression, a significant proportion of its targets is related toneurons and their development, synaptic transmission, and nervous system development (Johnson et al., 2007b). Theoncogene c-Myc initially identified to be elevated in the lymph cells of patients suffering from Burkitt’s lymphomadue to chromosomal translocations (Blum et al., 2004; Boxer and Dang, 2001), has been later discovered as one ofthe key factors for inducing pluripotent stem cells from somatic cells (Takahashi and Yamanaka, 2006). The pervasiveimportance of c-Myc in underlying stem cell and cancer cell functions has led to its genome-wide mapping in Burkitt’slymphoma cells and ESCs (Kim et al., 2008; Li et al., 2003). In both self-renewing populations, c-Myc is recruitedto similar categories of genes that are involved in metabolism, cell growth and proliferation, and apoptosis. Theresemblance, in which a TF may be expressed in different cell types but operate in an apparently similar context, thusprovides compelling justifications for using cell lines as surrogates. However, it is imperative to note that surrogatesprovide at best a rough approximation of TF function. Even tissue stem cell lines arguably provide an estimation oftheir in vivo function owing to the effects of long-term culture adaptation and exposure to signals different from theniche context. On-going efforts in the generation of tandem affinity-tagged stem cell-specific TFs amendable to highquality immunoaffinity capture in transgenic animals constitutes the next step forward in unraveling transcriptionaltargets in the physiological context (Veraksa et al., 2005).

5. Reactivating pluripotency

Somatic cells are functionally specialized cells programmed to be in a stable state. In a landmark experiment,the somatic nucleus from Xenopus can be reverted to totipotency by an enucleated oocyte, and becomes capableof recapitulating complete animal development (Gurdon et al., 1958). Subsequently, the same phenomenon wasrecapitulated with the mammalian sheep somatic nucleus (Wilmut et al., 1997). The reversal of a differentiated cellto a developmentally more primitive state is termed ‘reprogramming’. Using an in vitro system, the generation of iPScells from somatic cells through careful manipulation of specific molecules has been demonstrated (Takahashi andYamanaka, 2006). By over-expressing a limited but variable combination of ESC-associated molecules comprised ofOct4, Sox2, c-Myc, n-Myc, Lin28, Nanog, Klf2 and Klf4, stem cells can be induced from embryonic and adult somaticcells such as fibroblasts, keratinocytes, liver and stomach epithelial cells of mouse and human (Aoi et al., 2008;Maherali et al., 2007; Meissner et al., 2007; Nakagawa et al., 2008; Park et al., 2008; Takahashi et al., 2007; Werniget al., 2008a; Wernig et al., 2007; Wernig et al., 2008b; Yu et al., 2007b). These karyotypically normal cells exhibitmorphology, growth properties and molecular signatures similar to ESCs. Notably, iPS cells can contribute to all germlayers of the chimeric mouse, and are capable of germ-line transmission. Here, an understanding of the transcriptiontargets of these ‘reprogramming factors’ has provided useful insights into the mechanism for pluripotency reactivation.

In ESCs, Oct4 and Sox2 bind and regulate downstream genes, which corroboratively contribute to reversethe differentiated cell phenotype, through controlling a host of genes responsible for maintaining pluripotency or

13

stembook.org


inhibiting differentiation described previously. The relevance of c-Myc and Klf4 targets appear to be more speculative.From recent studies, the role of c-Myc in sustaining self-renewal may be attributed to its many targets that enhanceproliferation, negatively regulate differentiation, cellular transformation, and regulation of chromosome accessibilityto other factors (Adhikary and Eilers, 2005; Niwa, 2007; Takahashi and Yamanaka, 2006). The permissiveness ofchromatin to TF binding and transcription activity is partly determined by histone modifications. The specific covalentmodifications on the histone moieties of nucleosomes can be correlated to the transcription activity of genes in eitherpromoting or repressing their expression (reviewed in (Strahl and Allis, 2000)). In ESCs, a large set of developmentallyimportant genes that are silent but activated upon differentiation carries the bivalent marks, consisting of the activatinghistone 3 lysine 4 trimethylation (H3K4me3) and the repressive H3K27me3 modifications (Bernstein et al., 2006;Mikkelsen et al., 2007). This suggests lineage-specifying genes are primed for activation upon ESC differentiation.

A global assessment of the bivalent histone marks of c-Myc target promoters has revealed that the majority(95%) bear the H3K4me3 mark (Kim et al., 2008), suggestive of a relationship between c-Myc occupancy and histonemodifications (Guccione et al., 2006). Indeed, these target genes are more frequently expressed in ESCs, comparedto other factors. The data indicates that c-Myc occupancy is associated with global epigenetic modification of thechromatin at its targets, distinct from the role of other core factors which may manifest bivalent domains comprisingof both H3K4me3 and H3K27me3 marks (Kim et al., 2008). This latter data is coherent with observations in humanand mouse ESCs where there is considerable overlap between H3K4me3 and H3K27me3 loci across the pluripotentgenomes (Azuara et al., 2006; Bernstein et al., 2006; Pan et al., 2007; Zhao et al., 2007). In addition to histonemethylation, c-Myc may also potentially modify the chromatin architecture to be more permissive for the binding ofOct4 and Sox2, through its regulation of histone acetyltransferase complexes that induce global histone acetylation(Takahashi and Yamanaka, 2006). The role of Klf4 in ESCs and in reprogramming is less clear, but circumstantialobservations hints that it could act as a tumor-suppressor gene in iPS cells. Klf4 inhibits cell growth in mouse (Shieldset al., 1996), and can reduce the tumorigenicity of colon cancer cells in humans (Dang et al., 2003). This may becorrelated to the observations that during the reprogramming process, bona fide iPS cells are sparsely scattered amidstlarge numbers of transformed colonies. Presumably, Klf4 could act as a selector for truly reprogrammed cells overtransformed or incompletely reprogrammed cells which are deregulated in proliferation control. Klf4 may also actvia repression of p53 which leads to the activation Nanog and other ESC-associated genes (Rowland et al., 2005;Takahashi and Yamanaka, 2006). The occupancy of Klf4 on Oct4, Sox2 and other common downstream targets thatinclude Nanog and c-Myc has revealed that it is an upstream regulator of positive feed-forward loops that sustain highlevels of the activated factors. Interestingly, Klf2 can replace Klf4 in reprogramming (Nakagawa et al., 2008), and it hasbeen shown that Klf2, Klf4 and Klf5 bind to similar targets in ESCs (Jiang et al., 2008). The amenability in which thecombinatorial use of reprogramming factors results in the generation of iPS cells has raised interesting speculationspertaining to the plasticity of reprogramming. On the one hand, certain TFs appear mutually replaceable to targetcommon genes possibly through motif recognition; on the other hand, the activation of distinct but functionally similartarget genes may, in concert, results in induced pluripotency.

Further attempts to dissect the mechanism of reprogramming through examination of partially reprogrammedcells have provided strong evidence of a direct connection between transcription regulation and epigenetic modificationduring the transition of cellular states. Whereas fully reprogrammed cells manifest gene expression and epigeneticprofiles that are similar to ESCs, partially reprogrammed cell lines do not completely repress lineage-specifying TFsand show DNA hypermethylation at the loci of pluripotency-related genes (Mikkelsen et al., 2008). Strikingly, silencingof specific lineage-specifying TFs and treatment with DNA methyltransferase inhibitors help improve the efficiency ofthe reprogramming process (Mikkelsen et al., 2008), supporting genome-wide observations that methylation of CpGsare dynamic epigenetic marks that are subject to extensive changes during differentiation (Meissner et al., 2008),and need to be appropriately reversed at specific genes during reprogramming. Presumably, partially reprogrammedentities are trapped in the intermediate state as a result of either the inappropriate silencing of pluripotency factors orthe mis-expression of differentiation-associated genes. A better understanding of the role of lineage-specifying TFs,and the requirements for epigenetic landscape that include DNA and histone modifications at specific gene loci, mayfacilitate in reprogramming differentiated cells to somatic stem cells which are clinically relevant.

6. Cell type-dependent regulation of distinct circuitries

The molecular mechanism for the specification of embryonic germ layers and extra-embryonic lineages duringpre-implantation development is an important area that is not well-understood. With genome-wide TF localizationanalyses, precise and detailed maps can provide a comprehensive view of the molecular circuitry governing embryonicdevelopment. Although much effort has focused on how different TFs act singly or collectively to regulate extensivenetworks (Boyer et al., 2005; Kim et al., 2008; Loh et al., 2006), there is scant knowledge on how the same TF may

14

stembook.org


behave in different stem or progenitor cells. This is largely due to the paucity of identified molecules that are commonin more than one stem cell types, but yet retain discrete functional significance in each lineage (Fortunel et al., 2003).

In several cell types, the mapping of lineage-determining TFs has placed them at the top of transcriptionhierarchies. These factors are largely auto-regulatory, but also orchestrate the expression of downstream effectors.These effectors may independently or collaborate with other factors to control the cellular response. The observationthat a TF controls its own expression is not surprising as auto-regulatory loop appears to be a general feature of ‘master’regulators (Odom et al., 2006). For example, HNF TFs are required for the normal function of liver and pancreaticislets. In human hepatocytes, HNF1α binds a substantial number of genes related to hepatocyte biochemistry such asgluconeogenesis, carbohydrate synthesis, lipid metabolism and detoxification. This network also includes HNF4α andHNF6 which co-occupy the promoters of one another, thus forming a core network (Odom et al., 2004).

6.1. Lineage-specific recruitment

One central theme has been the prevalent observation that a lineage-specific factor controls of a discretetranscription network. Recently, we have identified Sall4 as a key regulator common to two embryo-derived stemcell lines that are developmentally connected (accepted, Cell Stem Cell). Our deciphering of Sall4 targets is timelyas integrative studies from the mapping of multiple TFs has identified Sall4 to be a major regulatory node in ESCs(Kim et al., 2008). Many Sall4-regulated genes are known to play important roles in maintaining ESC pluripotency.Of significance, many homeodomain protein promoters occupied by multiple factors such as Nanog, Sox2, Oct4 andKlf4 (Kim et al., 2008), are also regulated by Sall4. These findings thus integrate Sall4 as a major component of thecore ESC circuitry.

In addition to defining its role in ESCs, we demonstrated the importance of Sall4 function in the primitiveendoderm stem cell derivative, XEN cells. Our studies reveal Sall4 as an upstream activator of key lineage-defininggenes in the primitive endoderm. Gata6 and Gata4 are two of the earliest determinants of the primitive endoderm.However, other factors appear necessary for the initial primitive endoderm specification as Gata6 and Gata4 mutants donot exhibit extra-embryonic endoderm defects until several days after blastocyst formation (Ralston and Rossant, 2005).The discovery that Sall4 lies upstream of Gata6 and Gata4, and drives the expression of other lineage-determininggenes, provides a plausible reasoning for why the loss of either factor does not lead to a complete disruption ofprimitive endoderm formation, but only affects visceral endoderm differentiation at later stages (Koutsourakis et al.,1999; Morrisey et al., 1998; Ralston and Rossant, 2005; Soudais et al., 1995). These observations thus support Sall4’srole as a central player in primitive endoderm and XEN cell maintenance.

Genome-wide mapping of the same TF in two distinct stem cell lines has revealed several novel findings (seeTable 1). Firstly, Sall4 regulates distinct genes in the different cell types. In ESCs, pluripotency-associated genespredominate; whereas in XEN cells, endoderm lineage-associated factors that include Gata4, Gata6, Sox7 and Sox17are targets (see Figure 2C). Secondly, while the recruitment of Sall4 and its interacting partners appear necessaryto coordinate the expression of a subset of genes in establishing a specific lineage program in one cell type, it isequally important that the promoters of the same subset of genes become inaccessible to Sall4 in another stem celltype to ensure the complete absence of Sall4 regulatory input. This study illustrates and provides a basis for futureinvestigations into how other similar factors could have unique roles in more than one progenitor cell type, as well asfurther our understanding on the genetic and epigenetic bases for self-renewal and lineage-potency in stem cells.

6.2. Cofactor-mediated differential recruitment

The interaction of a sequence-specific TF with co-factors plays a contributory role in its differential recruitment.Sall4 physically interacts with several factors that include Nanog, Dax1, Nac1, Zfp281 and Oct4 as protein complexesin ESCs (Wang et al., 2006; Wu et al., 2006). However, the identity of its partners in XEN cells is unknown. Whetherthe recruitment to specific promoters in different cell types is partially mediated by other sequence-specific co-factorsremains to be tested. Notably, the reliance on co-factors for conferring recruitment specificity appears to be a recurrenttheme that has been observed in non-stem cells. For example, FoxA1 (HNF3α), a member of the Forkhead familyof winged-helix TFs, is involved in the organogenesis of liver, kidney, prostate, and mammary gland (Friedman andKaestner, 2006). Interestingly, the high expression of FoxA1 in tumors arising from the prostate and breast has led tospeculation on the similarities or differences of its transcription dynamics between these cell types (Lin et al., 2002;Mirosevich et al., 2006). In breast cancer cells, FoxA1 behaves as a pioneer factor that mediates estrogen receptorα (ERα) recruitment to cis-regulatory elements (Carroll et al., 2005; Laganiere et al., 2005), whereas it interactswith the androgen receptor (AR) in prostate cancer cells (Gao et al., 2003). A comparison of the FoxA1 binding loci

15

stembook.org


between the breast and prostate cancer cells has shown that FoxA1 is recruited to largely different sites across thegenome (Lupien et al., 2008). Coherent with similar observations made in ESC and XEN cells, FoxA1 appears toregulate discrete transcription programs as a result of lineage-specific recruitment (see Table 1). While the transcriptionlandscape of ESC factors is primarily associated with promoter occupancy, the ERα or AR-dependent recruitment ofFoxA1 occurs at enhancers (Lupien et al., 2008). The Forkhead motif is enriched at the binding loci both cell types,but the recognition motifs for ERα and AR are specifically enriched in FoxA1 binding loci unique to either breast orprostate cancer cells, respectively. The extensive occupancy of TFs at enhancers on a global scale supports the notionof enhanceosomes which may be prevalent features in many cell types.

The transcription switch in the regulation of differential gene sets following cellular perturbation provides aninteresting framework for testing the dynamicity of factor localization to the chromatin (see Table 1), as well as therequirements of co-factors and/or epigenetic permissiveness. For example, NF-κB, a key mediator of inflammation,responds to lipopolysaccharide (LPS) stimulation in monocytic cells. On a global scale, NF-κB recruits E2F1 to fullyactivate the transcription of NF-κB target genes upon LPS induction, whereby this effect could be abolished throughthe depletion of E2F1 by RNAi (Lim et al., 2007). This fluid nature of TF recruitment raises interesting questionsof whether forced expression of co-factors not naturally present in the target cell could induce the binding to, andexpression of alternate genes, and elicit a change in phenotype. Through this manner, the forced ectopic expressionof ESC TFs in somatic cells has been shown to dramatically alter the transcription landscape and cellular identity,leading to the induction of pluripotent cells.

7. The integration of microRNAs in transcription circuits

The governance of stem cell identity is not limited to transcription networks. Post-transcriptional regulatorymechanisms modulate the expression of many genes, emphasized by the many points of control in eukaryotes(Hollams et al., 2002). It is now apparent that a class of non-coding RNAs, microRNAs, modulates mRNA decay andtranslation rates (Seitz et al., 2004). MicroRNAs are necessary for many aspects of animal development and stem cellmaintenance. For instance, the miR-17∼92 cluster which is highly abundant in mouse ESCs has extensive roles in thedevelopment of the heart, lungs, and immune system, as well as tumorigenesis (Koralov et al., 2008; Ventura et al.,2008; Xiao et al., 2008; Yu et al., 2007a). The skin microRNA, miR-203, represses p63 and promotes differentiationof stratified epithelial stem cells (Yi et al., 2008b). In mouse ESCs, miR-134 and miR-1 can induce differentiation intothe neuroectoderm and cardiac muscle lineage, respectively (Ivey et al., 2008; Li and Gregory, 2008; Tay et al., 2008).The importance of microRNAs in stem cells is further highlighted by the observation that ESCs lacking the microRNAprocessing machinery comprised of either Dicer or Drosha cannot differentiate into most lineages (Kanellopoulouet al., 2005; Murchison et al., 2005; Wang et al., 2007).

Despite our increasing knowledge of microRNA functions and their regulation of target gene translation, thereis a dearth of information regarding the control of their expression. Unlike most protein-coding transcription unitswhich contain obvious transcription start sites and other initiation landmarks, microRNA promoters are not well-characterized. The occupancy of a TF on a microRNA gene is at best determined by its proximity to infer someregulatory function. In human ESCs, at least 14 microRNA loci are bound closely by Oct4, Sox2 or Nanog, wheremiR-137 and miR-301, are co-occupied by all three TFs (Boyer et al., 2005). In the mouse counterpart, five microRNAloci are bound by Oct4 or Nanog, where miR-296 and miR-302 are co-occupied by both (Loh et al., 2006). These twomicroRNAs are highly expressed in ESCs (Houbaviy et al., 2003), and thought to contribute towards ESC function.Interestingly, the mouse miR-302a-e cluster is the homologue of the human miR-372/3 family which is highly abundantin human ESCs, and acts as oncogenes in testicular germ cell tumors. When introduced into primary human cells,miR-372/3 can induce cellular transformation (Voorhoeve et al., 2006), suggesting a common role for this microRNAfamily in cell proliferation. Although many of these ESC-specific microRNAs appear to be under the control of keyTFs, there is surprisingly little demonstration of their direct regulation.

The repressor REST is observed to bind several microRNA promoters which contain REST regulatory sequencesin ESCs (Singh et al., 2008). One of these microRNAs, miR-21, has been previously characterized as an ‘oncomir’that promotes transformation in cells by targeting tumor suppressors (Asangani et al., 2008; Lu et al., 2008; Zhu et al.,2007; Zhu et al., 2008). Its levels also appear elevated in certain primary cancer cells (Meng et al., 2007; Slaby et al.,2007). While REST regulates ESC pluripotency possibly through its silencing of developmental genes associatedwith the neural lineage (Ballas et al., 2005; Bruce et al., 2004; Kim et al., 2008), it also blocks the expression oftarget microRNAs which may promote differentiation (Singh et al., 2008). Elevating the level of miR-21 reducedthe self-renewal capacity of ESCs (Singh et al., 2008); and it is proposed that miR-21 may potentially alter theproteins levels of its computationally predicted targets, Sox2 and Nanog, although this has yet been demonstrated.

16

stembook.org


miR-1

miR-133

LRH1

Tcf3

REST

Oct4

Nanog

Sox2

miR-296

miR-290~295

miR-17~92

miR-9

miR-302

miR-22

miR-124a

miR-134

Sall4

let-7Lin28

miR-21

miR-106a

?

?

?

?ES

C-asso

ciated

Neuroectoderm

Mesoderm

?

?

ActivationRepression

Undetermined regulation

Experimentally confirmed interaction

Indirect evidence or speculative

? Unidentified factor

Figure 5. MicroRNAs are integral to transcription networks in ESCs. The circuit map that integrates transcription regulation and microRNA functionsis constructed based on existing data. A core set of TFs regulates themselves, as well as the expression of microRNAs. MicroRNAs, whose expressions arepositively regulated, are frequently associated with self-renewal, whereas those negatively regulated in ESCs are associated with differentiation. In addition,microRNAs feedback into the transcription circuitry through post-transcriptional modulation of their TF targets, controlling their levels and activity. Severalother microRNAs appear to have roles in either self-renewal or differentiation, but their upstream regulators have yet been identified.

Parallel work on miR-134 has shown that it suppresses the translation of ESC factors Nanog and LRH1, therebyleading to cellular differentiation (Tay et al., 2008). It would indeed be fascinating if miR-21 feeds back into thetranscription circuitry by suppressing the activity of its target TFs. The transcriptional control of microRNAs and theirre-integration into the circuit presents a novel facet of gene regulatory network that is currently not well-elucidated.There is emerging evidence that microRNAs are integral components of transcription circuitries, both regulating, aswell as being regulated by, TFs (see Figure 5). Computation models predict that microRNA-mediated feedback andfeed-forward loops are prevalent (Tsang et al., 2007). The TF input can either positively or negatively co-regulate amicroRNA and its targets simultaneously, thereby enhancing the robustness of gene regulation and stability. This maybe particularly important for reinforcing the gene expression program of differentiated cellular states (Tsang et al.,2007; Yoo and Greenwald, 2005).

The recent genome-wide analyses of multiple TF occupancy could help reveal microRNAs bound by multiplefactors (Chen et al., 2008; Kim et al., 2008). It is interesting to test whether the distinctive classes of factors comprisingof Oct4/Sox2/Nanog and n-Myc/c-Myc/Rex1 could regulate delineating classes of microRNAs. If this is true, it wouldthen suggest that different classes of microRNAs perform specific roles that complement their upstream regulators.From a clinical perspective, the set of microRNAs bound by reprogramming factors is potentially important to examine.It remains to be tested whether any of these microRNA targets can replace or enhance TF-mediated reprogrammingof somatic cells into iPS cells. The chemical simplicity and ease of synthesis of microRNAs are key advantages in asmall molecule approach to generate medically useful stem cells.

In epithelial stem cells where p63 maintains the undifferentiated state (McKeon, 2004; Senoo et al., 2007),the interaction of miR-203 and p63 in repressing ‘stemness’ and inducing differentiation into stratified epitheliumhas been shown (Yi et al., 2008b). However, the manner in which p63 could trigger other microRNAs which help

17

stembook.org


maintain undifferentiated stem cells is not known. Like transcription repressors, microRNAs are integral nodes ofmany gene regulatory networks. While microRNAs appear to predominantly operate through a repression mechanism,their functions in the context of networks need not be repressive (Tsang et al., 2007). Presumably, microRNAs couldsuppress certain TFs, leading to the activation of a second order hierarchy that initiates the next transcription program.The identification of microRNA signatures specific to many stem cell types such as HSCs, mammary and lung epithelialprogenitor cells, and mesenchymal stem cells (Hatfield and Ruohola-Baker, 2008; Ibarra et al., 2007; Lakshmipathyand Hart, 2008; Liao et al., 2008; Lu et al., 2007), has provided the opportunity to intersect the regulatory networks oftranscription regulators with microRNA and their target genes.

8. Perspectives

Investigations into transcription regulation have progressed from the elucidation of very limited gene interactionnetworks to the mapping of complex extensive interactions on a genome-wide scale. This is, in part, facilitated bythe employment of high-throughput technologies in detecting and analyzing TF binding sites. Knowledge of howdominant factors control gene expression in stem cells and the progression of tissue lineage commitment during devel-opment is particularly important. As the stage-wise transition of pluripotency to multipotency and finally terminallydifferentiated cells requires the timely activation of a cascade of unique transcription programs, the identification ofkey regulators in the hierarchy and their targets provides valuable clues into the manipulation of stem cells. Twoaspects of this manipulation predominates the field of stem cell biology. Firstly, uncovering the transcription programof a differentiated cell type can provide information on directing the stem cell program to proceed towards that ofthe desired cell type. Understanding how to mature ESCs into stable functional somatic cells is the key to generatinguseful cell types for transplantation. Secondly, the derivation of patient-specific transplantable cells is a major chal-lenge. Obtaining patient-specific ESCs has proved surmountable and has raised ethical concern. Hence, unravelingthe somatic stem cell program can aid the reprogramming of terminal differentiated cells into all the various somaticstem cell types. This can potentially bypass the need for extensive differentiation regimes that would be required forthe effective use of ESCs.

The observation that certain key transcription regulators previously thought to define a particular lineage is alsoabundantly expressed in other cell lineages is not new. As intrinsic determinants of the cellular identity, TFs provide anentry point for uncovering how stem or progenitor cells attain their phenotype, and how lineage-specific differentiationis programmed. However, insights into the mechanism for the differential function of these TFs are critically lacking.The simple explanation that a single factor controls a small handful of divergent lineage-restricted genes necessary forestablishing distinct cellular identities does not satisfactorily resolve a complex developmental problem. The genome-wide identification of TF binding targets has brought us one step closer in our attempt to deconstruct the regulatorymechanism of stem cells. With the elucidation of complex gene regulatory networks, the field is only starting toreveal its secrets. Much of the on-going challenges will lie in our ability to dissect the mechanistic interaction ofTFs with the chromatin. In particular, how the epigenetic state determines the specificity of factor recruitment, therequirement and function of co-regulators, the dissection of a combinatorial code for transcription output, as well asthe activity and biochemical properties of TFs, will be key areas to study. Arguably, pioneering work in understandingthe comprehensive stem cell regulatory network will form the basis for probing the greater depths of developmentalbiology, and our ontogeny.

9. Acknowledgements

This work is supported by the Agency for Science, Technology and Research (Singapore) and the SingaporeStem Cell Consortium grant (SSCC-06-03). The work is also partially supported by National Institutes of Health (NIH)grants to B.L. (DK47636 and AI54973). We are grateful to Leah Vardy and Yuin-Han Loh in the Genome Institute ofSingapore for critical comments.

10. References

Acosta-Alvear, D., Zhou, Y., Blais, A., Tsikitis, M., Lents, N.H., Arias, C., Lennon, C.J., Kluger, Y., and Dynlacht,B.D. (2007). XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Mol Cell 27,53–66.

Adams, I.R., and McLaren, A. (2004). Identification and characterisation of mRif1: a mouse telomere-associatedprotein highly expressed in germ cells and embryo-derived pluripotent stem cells. Dev Dyn 229, 733–744.

18

stembook.org


Adhikary, S., and Eilers, M. (2005). Transcriptional regulation and transformation by Myc proteins. Nat Rev Mol CellBiol 6, 635–645.

Anneren, C., Cowan, C.A., and Melton, D.A. (2004). The Src family of tyrosine kinases is important for embryonicstem cell self-renewal. J Biol Chem 279, 31590–31598.

Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T., and Yamanaka, S. (2008). Generationof pluripotent stem cells from adult mouse liver and stomach cells. Science.

Asakura, A., Fujisawa-Sehara, A., Komiya, T., and Nabeshima, Y. (1993). MyoD and myogenin act on the chickenmyosin light-chain 1 gene as distinct transcriptional factors. Mol Cell Biol 13, 7153–7162.

Asangani, I.A., Rasheed, S.A., Nikolova, D.A., Leupold, J.H., Colburn, N.H., Post, S., and Allgayer, H. (2008).MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, in-travasation and metastasis in colorectal cancer. Oncogene 27, 2128–2136.

Azuara, V., Perry, P., Sauer, S., Spivakov, M., Jorgensen, H.F., John, R.M., Gouti, M., Casanova, M., Warnes, G.,Merkenschlager, M., and Fisher, A.G. (2006). Chromatin signatures of pluripotent cell lines. Nat Cell Biol 8, 532–538.

Ballas, N., Grunseich, C., Lu, D.D., Speh, J.C., and Mandel, G. (2005). REST and its corepressors mediate plasticityof neuronal gene chromatin throughout neurogenesis. Cell 121, 645–657.

Ballas, N., and Mandel, G. (2005). The many faces of REST oversee epigenetic programming of neuronal genes. CurrOpin Neurobiol 15, 500–506.

Beato, M., Herrlich, P., and Schutz, G. (1995). Steroid hormone receptors: many actors in search of a plot. Cell 83,851–857.

Berkes, C.A., Bergstrom, D.A., Penn, B.H., Seaver, K.J., Knoepfler, P.S., and Tapscott, S.J. (2004). Pbx marks genesfor activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential. Mol Cell 14,465–477.

Bernstein, B.E., and Kellis, M. (2005). Large-scale discovery and validation of functional elements in the humangenome. Genome Biol 6, 312.

Bernstein, B.E., Mikkelsen, T.S., Xie, X., Kamal, M., Huebert, D.J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath,K., et al. (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125,315–326.

Bertrand, N., Castro, D.S., and Guillemot, F. (2002). Proneural genes and the specification of neural cell types. NatRev Neurosci 3, 517–530.

Blum, K.A., Lozanski, G., and Byrd, J.C. (2004). Adult Burkitt leukemia and lymphoma. Blood 104, 3009–3020.

Bohm, J., Kaiser, F.J., Borozdin, W., Depping, R., and Kohlhase, J. (2007). Synergistic cooperation of Sall4 and CyclinD1 in transcriptional repression. Biochem Biophys Res Commun 356, 773–779.

Botquin, V., Hess, H., Fuhrmann, G., Anastassiadis, C., Gross, M.K., Vriend, G., and Scholer, H.R. (1998). New POUdimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2.Genes Dev 12, 2073–2090.

Boxer, L.M., and Dang, C.V. (2001). Translocations involving c-myc and c-myc function. Oncogene 20, 5595–5610.

Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray,H.L., Jenner, R.G., et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122,947–956.

19

stembook.org


Boyer, L.A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L.A., Lee, T.I., Levine, S.S., Wernig, M., Tajonar,A., Ray, M.K., et al. (2006). Polycomb complexes repress developmental regulators in murine embryonic stem cells.Nature 441, 349–353.

Bruce, A.W., Donaldson, I.J., Wood, I.C., Yerbury, S.A., Sadowski, M.I., Chapman, M., Gottgens, B., and Buckley,N.J. (2004). Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencingfactor (REST/NRSF) target genes. Proc Natl Acad Sci USA 101, 10458–10463.

Buckingham, M., Bajard, L., Chang, T., Daubas, P., Hadchouel, J., Meilhac, S., Montarras, D., Rocancourt, D., andRelaix, F. (2003). The formation of skeletal muscle: from somite to limb. J Anat 202, 59–68.

Cao, Y., Kumar, R.M., Penn, B.H., Berkes, C.A., Kooperberg, C., Boyer, L.A., Young, R.A., and Tapscott, S.J. (2006).Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters. Embo J 25,502–511.

Carroll, J.S., Liu, X.S., Brodsky, A.S., Li, W., Meyer, C.A., Szary, A.J., Eeckhoute, J., Shao, W., Hestermann, E.V.,Geistlinger, T.R., et al. (2005). Chromosome-wide mapping of estrogen receptor binding reveals long-range regulationrequiring the forkhead protein FoxA1. Cell 122, 33–43.

Cartwright, P., McLean, C., Sheppard, A., Rivett, D., Jones, K., and Dalton, S. (2005). LIF/STAT3 controls ES cellself-renewal and pluripotency by a Myc-dependent mechanism. Development 132, 885–896.

Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith, A. (2003). Functional expressioncloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655.

Chambers, I., Silva, J., Colby, D., Nichols, J., Nijmeijer, B., Robertson, M., Vrana, J., Jones, K., Grotewold, L., andSmith, A. (2007). Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234.

Chang, K., Elledge, S.J., and Hannon, G.J. (2006). Lessons from Nature: microRNA-based shRNA libraries. NatMethods 3, 707–714.

Chen, G., and Courey, A.J. (2000). Groucho/TLE family proteins and transcriptional repression. Gene 249, 1–16.

Chen, X., Xu, H., Yuan, P., Fang, F., Huss, M., Vega, V.B., Wong, E., Orlov, Y.L., Zhang, W., Jiang, J., et al. (2008).Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133,1106–1117.

Chew, J.-L., Loh, Y.-H., Zhang, W., Chen, X., Tam, W.-L., Yeap, L.-S., Li, P., Ang, Y.-S., Lim, B., Robson, P., andNg, H.-H. (2005a). Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonicstem cells. Mol Cell Biol 25, 6031–6046.

Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang, Y.S., Lim, B., Robson, P., and Ng,H.H. (2005b). Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonicstem cells. Mol Cell Biol 25, 6031–6046.

Chickarmane, V., Troein, C., Nuber, U.A., Sauro, H.M., and Peterson, C. (2006). Transcriptional dynamics of theembryonic stem cell switch. PLoS Comput Biol 2, e123.

Cole, M.F., Johnstone, S.E., Newman, J.J., Kagey, M.H., and Young, R.A. (2008). Tcf3 is an integral component ofthe core regulatory circuitry of embryonic stem cells. Genes Dev 22, 746–755.

Coulson, J.M. (2005). Transcriptional regulation: cancer, neurons and the REST. Curr Biol 15, R665–668.

Cowan, C.A., Atienza, J., Melton, D.A., and Eggan, K. (2005). Nuclear reprogramming of somatic cells after fusionwith human embryonic stem cells. Science 309, 1369–1373.

Dang, D.T., Chen, X., Feng, J., Torbenson, M., Dang, L.H., and Yang, V.W. (2003). Overexpression of Kruppel-likefactor 4 in the human colon cancer cell line RKO leads to reduced tumorigenecity. Oncogene 22, 3424–3430.

20

stembook.org


Diehl, F., Rossig, L., Zeiher, A.M., Dimmeler, S., and Urbich, C. (2007). The histone methyltransferase MLL is anupstream regulator of endothelial-cell sprout formation. Blood 109, 1472–1478.

Dodge, J.E., Kang, Y.K., Beppu, H., Lei, H., and Li, E. (2004). Histone H3-K9 methyltransferase ESET is essentialfor early development. Mol Cell Biol 24, 2478–2486.

Eastman, Q., and Grosschedl, R. (1999). Regulation of LEF-1/TCF transcription factors by Wnt and other signals.Curr Opin Cell Biol 11, 233–240.

Edmondson, D.G., and Olson, E.N. (1989). A gene with homology to the myc similarity region of MyoD1 is expressedduring myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev 3, 628–640.

Elling, U., Klasen, C., Eisenberger, T., Anlag, K., and Treier, M. (2006). Murine inner cell mass-derived lineagesdepend on Sall4 function. Proc Natl Acad Sci USA 103, 16319–16324.

Evans, M.J., and Kaufman, M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature292, 154–156.

Fode, C., Gradwohl, G., Morin, X., Dierich, A., LeMeur, M., Goridis, C., and Guillemot, F. (1998). The bHLH proteinNEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons. Neuron 20, 483–494.

Fode, C., Ma, Q., Casarosa, S., Ang, S.L., Anderson, D.J., and Guillemot, F. (2000). A role for neural determinationgenes in specifying the dorsoventral identity of telencephalic neurons. Genes Dev 14, 67–80.

Fortunel, N.O., Otu, H.H., Ng, H.H., Chen, J., Mu, X., Chevassut, T., Li, X., Joseph, M., Bailey, C., Hatzfeld, J.A.,et al. (2003). Comment on “ ‘Stemness’: transcriptional profiling of embryonic and adult stem cells” and “a stem cellmolecular signature”. Science 302, 393; author reply 393.

Friedman, J.R., and Kaestner, K.H. (2006). The Foxa family of transcription factors in development and metabolism.Cell Mol Life Sci 63, 2317–2328.

Gao, N., Zhang, J., Rao, M.A., Case, T.C., Mirosevich, J., Wang, Y., Jin, R., Gupta, A., Rennie, P.S., and Matusik,R.J. (2003). The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptionalregulation of prostatic genes. Mol Endocrinol 17, 1484–1507.

Gowan, K., Helms, A.W., Hunsaker, T.L., Collisson, T., Ebert, P.J., Odom, R., and Johnson, J.E. (2001). Crossinhibitoryactivities of Ngn1 and Math1 allow specification of distinct dorsal interneurons. Neuron 31, 219–232.

Graham, V., Khudyakov, J., Ellis, P., and Pevny, L. (2003). SOX2 functions to maintain neural progenitor identity.Neuron 39, 749–765.

Grass, J.A., Jing, H., Kim, S.I., Martowicz, M.L., Pal, S., Blobel, G.A., and Bresnick, E.H. (2006). Distinct functionsof dispersed GATA factor complexes at an endogenous gene locus. Mol Cell Biol 26, 7056–7067.

Green, M.R. (2000). TBP-associated factors (TAFIIs): multiple, selective transcriptional mediators in common com-plexes. Trends Biochem Sci 25, 59–63.

Gu, P., LeMenuet, D., Chung, A.C., Mancini, M., Wheeler, D.A., and Cooney, A.J. (2005). Orphan nuclear recep-tor GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem celldifferentiation. Mol Cell Biol 25, 8507–8519.

Guccione, E., Martinato, F., Finocchiaro, G., Luzi, L., Tizzoni, L., Dall’ Olio, V., Zardo, G., Nervi, C., Bernard, L.,and Amati, B. (2006). Myc-binding-site recognition in the human genome is determined by chromatin context. NatCell Biol 8, 764–770.

Guenther, M.G., Levine, S.S., Boyer, L.A., Jaenisch, R., and Young, R.A. (2007). A chromatin landmark and tran-scription initiation at most promoters in human cells. Cell 130, 77–88.

21

stembook.org


Guhaniyogi, J., and Brewer, G. (2001). Regulation of mRNA stability in mammalian cells. Gene 265, 11–23.

Guillemot, F. (1999). Vertebrate bHLH genes and the determination of neuronal fates. Exp Cell Res 253, 357–364.

Guo, Y., Costa, R., Ramsey, H., Starnes, T., Vance, G., Robertson, K., Kelley, M., Reinbold, R., Scholer, H., andHromas, R. (2002). The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression. Proc Natl Acad Sci USA 99, 3663–3667.

Gurdon, J.B., Elsdale, T.R., and Fischberg, M. (1958). Sexually mature individuals of Xenopus laevis from thetransplantation of single somatic nuclei. Nature 182, 64–65.

Hanna, L.A., Foreman, R.K., Tarasenko, I.A., Kessler, D.S., and Labosky, P.A. (2002). Requirement for Foxd3 inmaintaining pluripotent cells of the early mouse embryo. Genes Dev 16, 2650–2661.

Hansen, J., Floss, T., Van Sloun, P., Fuchtbauer, E.M., Vauti, F., Arnold, H.H., Schnutgen, F., Wurst, W., von Melchner,H., and Ruiz, P. (2003). A large-scale, gene-driven mutagenesis approach for the functional analysis of the mousegenome. Proc Natl Acad Sci USA 100, 9918–9922.

Hasty, P., Bradley, A., Morris, J.H., Edmondson, D.G., Venuti, J.M., Olson, E.N., and Klein, W.H. (1993). Muscledeficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364, 501–506.

Hatfield, S., and Ruohola-Baker, H. (2008). MicroRNA and stem cell function. Cell Tissue Res 331, 57–66.

Hatzis, P., van der Flier, L.G., van Driel, M.A., Guryev, V., Nielsen, F., Denissov, S., Nijman, I.J., Koster, J., Santo,E.E., Welboren, W., et al. (2008). Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancercells. Mol Cell Biol 28, 2732–2744.

Hill, C.S., and Treisman, R. (1995). Transcriptional regulation by extracellular signals: mechanisms and specificity.Cell 80, 199–211.

Hirabayashi, Y., Itoh, Y., Tabata, H., Nakajima, K., Akiyama, T., Masuyama, N., and Gotoh, Y. (2004). The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131, 2791–2801.

Hochedlinger, K., Yamada, Y., Beard, C., and Jaenisch, R. (2005). Ectopic expression of Oct-4 blocks progenitor-celldifferentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477.

Hollams, E.M., Giles, K.M., Thomson, A.M., and Leedman, P.J. (2002). MRNA stability and the control of geneexpression: implications for human disease. Neurochem Res 27, 957–980.

Hombria, J.C., and Lovegrove, B. (2003). Beyond homeosis–HOX function in morphogenesis and organogenesis.Differentiation 71, 461–476.

Houbaviy, H.B., Murray, M.F., and Sharp, P.A. (2003). Embryonic stem cell-specific MicroRNAs. Dev Cell 5, 351–358.

Huang, G., Zhang, P., Hirai, H., Elf, S., Yan, X., Chen, Z., Koschmieder, S., Okuno, Y., Dayaram, T., Growney, J.D.,et al. (2008). PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis. Nat Genet 40,51–60.

Huang, H.P., Liu, M., El-Hodiri, H.M., Chu, K., Jamrich, M., and Tsai, M.J. (2000). Regulation of the pancreaticislet-specific gene BETA2 (neuroD) by neurogenin 3. Mol Cell Biol 20, 3292–3307.

Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G., and Birchmeier, W. (2001). beta-Catenin controls hair folliclemorphogenesis and stem cell differentiation in the skin. Cell 105, 533–545.

Ibarra, I., Erlich, Y., Muthuswamy, S.K., Sachidanandam, R., and Hannon, G.J. (2007). A role for microRNAs inmaintenance of mouse mammary epithelial progenitor cells. Genes Dev 21, 3238–3243.

22

stembook.org


Ivanova, N., Dobrin, R., Lu, R., Kotenko, I., Levorse, J., DeCoste, C., Schafer, X., Lun, Y., and Lemischka, I.R. (2006).Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538.

Ivey, K.N., Muth, A., Arnold, J., King, F.W., Yeh, R.F., Fish, J.E., Hsiao, E.C., Schwartz, R.J., Conklin, B.R., Bernstein,H.S., and Srivastava, D. (2008). MicroRNA regulation of cell lineages in mouse and human embryonic stem cells.Cell Stem Cell 2, 219–229.

Iyer, V.R., Horak, C.E., Scafe, C.S., Botstein, D., Snyder, M., and Brown, P.O. (2001). Genomic binding sites of theyeast cell-cycle transcription factors SBF and MBF. Nature 409, 533–538.

Jiang, J., Chan, Y.S., Loh, Y.H., Cai, J., Tong, G.Q., Lim, C.A., Robson, P., Zhong, S., and Ng, H.H. (2008). A coreKlf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 10, 353–360.

Johnson, K.D., Boyer, M.E., Kang, J.A., Wickrema, A., Cantor, A.B., and Bresnick, E.H. (2007a). Friend of GATA-1-independent transcriptional repression: a novel mode of GATA-1 function. Blood 109, 5230–5233.

Johnson, K.M., Mitsouras, K., and Carey, M. (2001). Eukaryotic transcription: the core of eukaryotic gene activation.Curr Biol 11, R510–513.

Johnson, T.J., Kariyawasam, S., Wannemuehler, Y., Mangiamele, P., Johnson, S.J., Doetkott, C., Skyberg, J.A., Lynne,A.M., Johnson, J.R., and Nolan, L.K. (2007b). The genome sequence of avian pathogenic Escherichia coli strainO1:K1:H7 shares strong similarities with human extraintestinal pathogenic E. coli genomes. J Bacteriol 189, 3228–3236.

Kanellopoulou, C., Muljo, S.A., Kung, A.L., Ganesan, S., Drapkin, R., Jenuwein, T., Livingston, D.M., and Rajewsky,K. (2005). Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.Genes Dev 19, 489–501.

Kassar-Duchossoy, L., Gayraud-Morel, B., Gomes, D., Rocancourt, D., Buckingham, M., Shinin, V., and Tajbakhsh,S. (2004). Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice. Nature 431, 466–471.

Kiefer, J.C. (2007). Back to basics: Sox genes. Dev Dyn.

Kim, I., Saunders, T.L., and Morrison, S.J. (2007). Sox17 dependence distinguishes the transcriptional regulation offetal from adult hematopoietic stem cells. Cell 130, 470–483.

Kim, J., Chu, J., Shen, X., Wang, J., and Orkin, S.H. (2008). An extended transcriptional network for pluripotency ofembryonic stem cells. Cell 132, 1049–1061.

Kim, W.Y., Fritzsch, B., Serls, A., Bakel, L.A., Huang, E.J., Reichardt, L.F., Barth, D.S., and Lee, J.E. (2001).NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development128, 417–426.

Koralov, S.B., Muljo, S.A., Galler, G.R., Krek, A., Chakraborty, T., Kanellopoulou, C., Jensen, K., Cobb, B.S.,Merkenschlager, M., Rajewsky, N., and Rajewsky, K. (2008). Dicer ablation affects antibody diversity and cellsurvival in the B lymphocyte lineage. Cell 132, 860–874.

Koutsourakis, M., Langeveld, A., Patient, R., Beddington, R., and Grosveld, F. (1999). The transcription factor GATA6is essential for early extraembryonic development. Development 126, 723–732.

Laganiere, J., Deblois, G., Lefebvre, C., Bataille, A.R., Robert, F., and Giguere, V. (2005). From the Cover: Locationanalysis of estrogen receptor alpha target promoters reveals that FOXA1 defines a domain of the estrogen response.Proc Natl Acad Sci USA 102, 11651–11656.

Lagutin, O.V., Zhu, C.C., Kobayashi, D., Topczewski, J., Shimamura, K., Puelles, L., Russell, H.R., McKinnon, P.J.,Solnica-Krezel, L., and Oliver, G. (2003). Six3 repression of Wnt signaling in the anterior neuroectoderm is essentialfor vertebrate forebrain development. Genes Dev 17, 368–379.

23

stembook.org


Lakshmipathy, U., and Hart, R.P. (2008). Concise review: MicroRNA expression in multipotent mesenchymal stromalcells. Stem Cells 26, 356–363.

Lee, T.I., Jenner, R.G., Boyer, L.A., Guenther, M.G., Levine, S.S., Kumar, R.M., Chevalier, B., Johnstone, S.E., Cole,M.F., Isono, K., et al. (2006). Control of developmental regulators by Polycomb in human embryonic stem cells. Cell125, 301–313.

Lee, T.I., Rinaldi, N.J., Robert, F., Odom, D.T., Bar-Joseph, Z., Gerber, G.K., Hannett, N.M., Harbison, C.T., Thompson,C.M., Simon, I., et al. (2002). Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804.

Letting, D.L., Chen, Y.Y., Rakowski, C., Reedy, S., and Blobel, G.A. (2004). Context-dependent regulation of GATA-1by friend of GATA-1. Proc Natl Acad Sci USA 101, 476–481.

Li, Q., and Gregory, R.I. (2008). MicroRNA regulation of stem cell fate. Cell Stem Cell 2, 195–196.

Li, Z., Van Calcar, S., Qu, C., Cavenee, W.K., Zhang, M.Q., and Ren, B. (2003). A global transcriptional regulatoryrole for c-Myc in Burkitt’s lymphoma cells. Proc Natl Acad Sci USA 100, 8164–8169.

Liao, R., Sun, J., Zhang, L., Lou, G., Chen, M., Zhou, D., Chen, Z., and Zhang, S. (2008). MicroRNAs play a role inthe development of human hematopoietic stem cells. J Cell Biochem.

Lim, C.A., Yao, F., Wong, J.J., George, J., Xu, H., Chiu, K.P., Sung, W.K., Lipovich, L., Vega, V.B., Chen, J., et al.(2007). Genome-wide mapping of RELA(p65) binding identifies E2F1 as a transcriptional activator recruited byNF-kappaB upon TLR4 activation. Mol Cell 27, 622–635.

Lin, L., Miller, C.T., Contreras, J.I., Prescott, M.S., Dagenais, S.L., Wu, R., Yee, J., Orringer, M.B., Misek, D.E.,Hanash, S.M., et al. (2002). The hepatocyte nuclear factor 3 alpha gene, HNF3alpha (FOXA1), on chromosome band14q13 is amplified and overexpressed in esophageal and lung adenocarcinomas. Cancer Res 62, 5273–5279.

Lin, T., Chao, C., Saito, S., Mazur, S.J., Murphy, M.E., Appella, E., and Xu, Y. (2005). p53 induces differentiation ofmouse embryonic stem cells by suppressing Nanog expression. Nat Cell Biol 7, 165–171.

Loh, Y.H., Wu, Q., Chew, J.L., Vega, V.B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., et al.(2006). The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet38, 431–440.

Lu, Y., Thomson, J.M., Wong, H.Y., Hammond, S.M., and Hogan, B.L. (2007). Transgenic over-expression of themicroRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells.Dev Biol 310, 442–453.

Lu, Z., Liu, M., Stribinskis, V., Klinge, C.M., Ramos, K.S., Colburn, N.H., and Li, Y. (2008). MicroRNA-21 promotescell transformation by targeting the programmed cell death 4 gene. Oncogene.

Luo, J., Sladek, R., Bader, J.A., Matthyssen, A., Rossant, J., and Giguere, V. (1997). Placental abnormalities in mouseembryos lacking the orphan nuclear receptor ERR-beta. Nature 388, 778–782.

Lupien, M., Eeckhoute, J., Meyer, C.A., Wang, Q., Zhang, Y., Li, W., Carroll, J.S., Liu, X.S., and Brown, M. (2008).FoxA1 translates epigenetic signatures into enhancer-driven lineage-specific transcription. Cell 132, 958–970.

Lurie, L.J., Boyer, M.E., Grass, J.A., and Bresnick, E.H. (2008). Differential GATA factor stabilities: implications forchromatin occupancy by structurally similar transcription factors. Biochemistry 47, 859–869.

Ma, Q., Chen, Z., del Barco Barrantes, I., de la Pompa, J.L., and Anderson, D.J. (1998). neurogenin1 is essential forthe determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20, 469–482.

Ma, Q., Fode, C., Guillemot, F., and Anderson, D.J. (1999). Neurogenin1 and neurogenin2 control two distinct wavesof neurogenesis in developing dorsal root ganglia. Genes Dev 13, 1717–1728.

24

stembook.org


Ma, Q., Kintner, C., and Anderson, D.J. (1996). Identification of neurogenin, a vertebrate neuronal determinationgene. Cell 87, 43–52.

Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tchieu, J.,Jaenisch, R., et al. (2007). Directly reprogrammed fibroblasts show global epigenetic remodeling and widespreadtissue contribution. Cell Stem Cell 1, 55–70.

Mangan, S., and Alon, U. (2003). Structure and function of the feed-forward loop network motif. Proc Natl Acad SciUSA 100, 11980–11985.

Mangan, S., Zaslaver, A., and Alon, U. (2003). The coherent feedforward loop serves as a sign-sensitive delay elementin transcription networks. J Mol Biol 334, 197–204.

Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark,M., Chambon, P., and Evans, R.M. (1995). The nuclear receptor superfamily: the second decade. Cell 83, 835–839.

Marson, A., Kretschmer, K., Frampton, G.M., Jacobsen, E.S., Polansky, J.K., MacIsaac, K.D., Levine, S.S., Fraenkel,E., von Boehmer, H., and Young, R.A. (2007). Foxp3 occupancy and regulation of key target genes during T-cellstimulation. Nature 445, 931–935.

Massie, C.E., and Mills, I.G. (2008). ChIPping away at gene regulation. EMBO Rep 9, 337–343.

McAdams, H.H., and Arkin, A. (1997). Stochastic mechanisms in gene expression. Proc Natl Acad Sci USA 94,814–819.

McKeon, F. (2004). p63 and the epithelial stem cell: more than status quo?. Genes Dev 18, 465–469.

Meissner, A., Mikkelsen, T.S., Gu, H., Wernig, M., Hanna, J., Sivachenko, A., Zhang, X., Bernstein, B.E., Nusbaum,C., Jaffe, D B., et al. (2008). Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature.

Meissner, A., Wernig, M., and Jaenisch, R. (2007). Direct reprogramming of genetically unmodified fibroblasts intopluripotent stem cells. Nat Biotechnol 25, 1177–1181.

Meng, F., Henson, R., Wehbe-Janek, H., Ghoshal, K., Jacob, S.T., and Patel, T. (2007). MicroRNA-21 regulatesexpression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 133, 647–658.

Menssen, A., and Hermeking, H. (2002). Characterization of the c-MYC-regulated transcriptome by SAGE: identifi-cation and analysis of c-MYC target genes. Proc Natl Acad Sci USA 99, 6274–6279.

Merrill, B.J., Pasolli, H.A., Polak, L., Rendl, M., Garcia-Garcia, M.J., Anderson, K.V., and Fuchs, E. (2004). Tcf3: atranscriptional regulator of axis induction in the early embryo. Development 131, 263–274.

Mikkelsen, T.S., Hanna, J., Zhang, X., Ku, M., Wernig, M., Schorderet, P., Bernstein, B.E., Jaenisch, R., Lander, E.S.,and Meissner, A. (2008). Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55.

Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.K.,Koche, R.P., et al. (2007). Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature448, 553–560.

Mirosevich, J., Gao, N., Gupta, A., Shappell, S.B., Jove, R., and Matusik, R.J. (2006). Expression and role of Foxaproteins in prostate cancer. Prostate 66, 1013–1028.

Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M., andYamanaka, S. (2003). The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and EScells. Cell 113, 631–642.

25

stembook.org


Mitsunaga, K., Araki, K., Mizusaki, H., Morohashi, K., Haruna, K., Nakagata, N., Giguere, V., Yamamura, K., andAbe, K. (2004). Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germcell number in mouse embryos. Mech Dev 121, 237–246.

Miyata, T., Maeda, T., and Lee, J.E. (1999). NeuroD is required for differentiation of the granule cells in the cerebellumand hippocampus. Genes Dev 13, 1647–1652.

Morrisey, E.E., Tang, Z., Sigrist, K., Lu, M.M., Jiang, F., Ip, H.S., and Parmacek, M.S. (1998). GATA6 regulatesHNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev 12, 3579–3590.

Murchison, E.P., Partridge, J.F., Tam, O.H., Cheloufi, S., and Hannon, G.J. (2005). Characterization of Dicer-deficientmurine embryonic stem cells. Proc Natl Acad Sci USA 102, 12135–12140.

Myer, V.E., and Young, R.A. (1998). RNA polymerase II holoenzymes and subcomplexes. J Biol Chem 273, 27757–27760.

Nabeshima, Y., Hanaoka, K., Hayasaka, M., Esumi, E., Li, S., and Nonaka, I. (1993). Myogenin gene disruption resultsin perinatal lethality because of severe muscle defect. Nature 364, 532–535.

Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa,N., and Yamanaka, S. (2008). Generation of induced pluripotent stem cells without Myc from mouse and humanfibroblasts. Nat Biotechnol 26, 101–106.

Nguyen, H., Rendl, M., and Fuchs, E. (2006). Tcf3 governs stem cell features and represses cell fate determination inskin. Cell 127, 171–183.

Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H., and Smith, A.(1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.Cell 95, 379–391.

Nishimoto, M., Fukushima, A., Okuda, A., and Muramatsu, M. (1999). The gene for the embryonic stem cell coactivatorUTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. MolCell Biol 19, 5453–5465.

Niwa, H. (2007). How is pluripotency determined and maintained?. Development 134, 635–646.

Niwa, H., Miyazaki, J., and Smith, A.G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dediffer-entiation or self-renewal of ES cells. Nat Genet 24, 372–376.

Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R., and Rossant, J. (2005). Interaction betweenOct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123, 917–929.

Nord, A.S., Chang, P.J., Conklin, B.R., Cox, A.V., Harper, C.A., Hicks, G.G., Huang, C.C., Johns, S.J., Kawamoto,M., Liu, S., et al. (2006). The International Gene Trap Consortium Website: a portal to all publicly available gene trapcell lines in mouse. Nucleic Acids Res 34, D642–648.

Odom, D.T., Dowell, R.D., Jacobsen, E.S., Nekludova, L., Rolfe, P.A., Danford, T.W., Gifford, D.K., Fraenkel, E.,Bell, G.I., and Young, R.A. (2006). Core transcriptional regulatory circuitry in human hepatocytes. Mol Syst Biol 2,2006 0017.

Odom, D.T., Zizlsperger, N., Gordon, D.B., Bell, G.W., Rinaldi, N.J., Murray, H.L., Volkert, T.L., Schreiber, J., Rolfe,P.A., Gifford, D.K., et al. (2004). Control of pancreas and liver gene expression by HNF transcription factors. Science303, 1378–1381.

Okumura-Nakanishi, S., Saito, M., Niwa, H., and Ishikawa, F. (2005). Oct-3/4 and sox2 regulate oct-3/4 gene inembryonic stem cells. J Biol Chem 280, 5307–5317.

Orkin, S.H. (2000). Diversification of haematopoietic stem cells to specific lineages. Nat Rev Genet 1, 57–64.

26

stembook.org


Orkin, S.H., and Zon, L.I. (2008). Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132, 631–644.

Orlando, V., and Paro, R. (1993). Mapping Polycomb-repressed domains in the bithorax complex using in vivoformaldehyde cross-linked chromatin. Cell 75, 1187–1198.

Paddison, P.J., Silva, J.M., Conklin, D.S., Schlabach, M., Li, M., Aruleba, S., Balija, V., O’Shaughnessy, A., Gnoj,L., Scobie, K., et al. (2004). A resource for large-scale RNA-interference-based screens in mammals. Nature 428,427–431.

Pal, S., Cantor, A.B., Johnson, K.D., Moran, T.B., Boyer, M.E., Orkin, S.H., and Bresnick, E.H. (2004). Coregulator-dependent facilitation of chromatin occupancy by GATA-1. Proc Natl Acad Sci USA 101, 980–985.

Palmieri, S.L., Peter, W., Hess, H., and Scholer, H.R. (1994). Oct-4 transcription factor is differentially expressed inthe mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. DevBiol 166, 259–267.

Palomero, T., Odom, D.T., O’Neil, J., Ferrando, A.A., Margolin, A., Neuberg, D.S., Winter, S.S., Larson, R.S., Li, W.,Liu, X.S., et al. (2006). Transcriptional regulatory networks downstream of TAL1/SCL in T-cell acute lymphoblasticleukemia. Blood 108, 986–992.

Pan, G., Tian, S., Nie, J., Yang, C., Ruotti, V., Wei, H., Jonsdottir, G.A., Stewart, R., and Thomson, J.A. (2007).Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell StemCell 1, 299–312.

Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., and Daley, G.Q. (2008).Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146.

Park, I.K., Qian, D., Kiel, M., Becker, M.W., Pihalja, M., Weissman, I.L., Morrison, S.J., and Clarke, M.F. (2003).Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423, 302–305.

Pennesi, M.E., Cho, J.H., Yang, Z., Wu, S.H., Zhang, J., Wu, S.M., and Tsai, M.J. (2003). BETA2/NeuroD1 null mice:a new model for transcription factor-dependent photoreceptor degeneration. J Neurosci 23, 453–461.

Pereira, L., Yi, F., and Merrill, B.J. (2006). Repression of Nanog gene transcription by Tcf3 limits embryonic stemcell self-renewal. Mol Cell Biol 26, 7479–7491.

Pesce, M., and Scholer, H.R. (2001). Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19,271–278.

Pesce, M., Wang, X., Wolgemuth, D.J., and Scholer, H. (1998). Differential expression of the Oct-4 transcription factorduring mouse germ cell differentiation. Mech Dev 71, 89–98.

Plageman, T.F., Jr., and Yutzey, K.E. (2005). T-box genes and heart development: putting the “T” in heart. Dev Dyn232, 11–20.

Ralston, A., and Rossant, J. (2005). Genetic regulation of stem cell origins in the mouse embryo. Clin Genet 68,106–112.

Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C., and Melton, D.A. (2002). “Stemness”: transcriptionalprofiling of embryonic and adult stem cells. Science 298, 597–600.

Rao, C.V., Wolf, D.M., and Arkin, A.P. (2002). Control, exploitation and tolerance of intracellular noise. Nature 420,231–237.

Ren, B., Robert, F., Wyrick, J.J., Aparicio, O., Jennings, E.G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N.,Kanin, E., et al. (2000). Genome-wide location and function of DNA binding proteins. Science 290, 2306–2309.

27

stembook.org


Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. (2000). Embryonic stem cell lines from humanblastocysts: somatic differentiation in vitro. Nat Biotechnol 18, 399–404.

Robertson, G., Hirst, M., Bainbridge, M., Bilenky, M., Zhao, Y., Zeng, T., Euskirchen, G., Bernier, B., Varhol, R.,Delaney, A., et al. (2007). Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitationand massively parallel sequencing. Nat Methods 4, 651–657.

Rodda, D.J., Chew, J.L., Lim, L.H., Loh, Y.H., Wang, B., Ng, H.H., and Robson, P. (2005a). Transcriptional regulationof Nanog by Oct4 and Sox2. J Biol Chem 280, 24731–24737.

Rodda, D.J., Chew, J.L., Lim, L.H., Loh, Y.H., Wang, B., Ng, H.H., and Robson, P. (2005b). Transcriptional regulationof nanog by OCT4 and SOX2. J Biol Chem 280, 24731–24737.

Roeder, R.G. (1996). The role of general initiation factors in transcription by RNA polymerase II. Trends BiochemSci 21, 327–335.

Roose, J., and Clevers, H. (1999). TCF transcription factors: molecular switches in carcinogenesis. Biochim BiophysActa 1424, M23–37.

Rosner, M.H., Vigano, M.A., Ozato, K., Timmons, P.M., Poirier, F., Rigby, P.W., and Staudt, L.M. (1990). A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345, 686–692.

Rowland, B.D., Bernards, R., and Peeper, D.S. (2005). The KLF4 tumour suppressor is a transcriptional repressor ofp53 that acts as a context-dependent oncogene. Nat Cell Biol 7, 1074–1082.

Rudnicki, M.A., Schnegelsberg, P.N., Stead, R.H., Braun, T., Arnold, H.H., and Jaenisch, R. (1993). MyoD or Myf-5is required for the formation of skeletal muscle. Cell 75, 1351–1359.

Sadikovic, B., Andrews, J., Carter, D., Robinson, J., and Rodenhiser, D.I. (2008). Genome-wide H3K9 histoneacetylation profiles are altered in benzopyrene-treated MCF7 breast cancer cells. J Biol Chem 283, 4051–4060.

Sakaki-Yumoto, M., Kobayashi, C., Sato, A., Fujimura, S., Matsumoto, Y., Takasato, M., Kodama, T., Aburatani,H., Asashima, M., Yoshida, N., and Nishinakamura, R. (2006). The murine homolog of SALL4, a causative gene inOkihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart,brain and kidney development. Development 133, 3005–3013.

Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A.H. (2004). Maintenance of pluripotency in humanand mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat Med 10, 55–63.

Scholer, H.R., Ciesiolka, T., and Gruss, P. (1991). A nexus between Oct-4 and E1A: implications for gene regulationin embryonic stem cells. Cell 66, 291–304.

Scholer, H.R., Dressler, G.R., Balling, R., Rohdewohld, H., and Gruss, P. (1990a). Oct-4: a germline-specific tran-scription factor mapping to the mouse t-complex. Embo J 9, 2185–2195.

Scholer, H.R., Ruppert, S., Suzuki, N., Chowdhury, K., and Gruss, P. (1990b). New type of POU domain in germline-specific protein Oct-4. Nature 344, 435–439.

Seitz, H., Royo, H., Bortolin, M.L., Lin, S.P., Ferguson-Smith, A.C., and Cavaille, J. (2004). A large imprintedmicroRNA gene cluster at the mouse Dlk1-Gtl2 domain. Genome Res 14, 1741–1748.

Senoo, M., Pinto, F., Crum, C.P., and McKeon, F. (2007). p63 Is essential for the proliferative potential of stem cellsin stratified epithelia. Cell 129, 523–536.

Seo, S., Lim, J.W., Yellajoshyula, D., Chang, L.W., and Kroll, K.L. (2007). Neurogenin and NeuroD direct transcrip-tional targets and their regulatory enhancers. Embo J 26, 5093–5108.

28

stembook.org


Shields, J.M., Christy, R.J., and Yang, V.W. (1996). Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem 271, 20009–20017.

Silva, J.M., Li, M.Z., Chang, K., Ge, W., Golding, M.C., Rickles, R.J., Siolas, D., Hu, G., Paddison, P.J., Schlabach,M.R., et al. (2005). Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37, 1281–1288.

Silva, J.M., Mizuno, H., Brady, A., Lucito, R., and Hannon, G.J. (2004). RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells. Proc Natl Acad Sci USA 101, 6548–6552.

Singh, S.K., Kagalwala, M.N., Parker-Thornburg, J., Adams, H., and Majumder, S. (2008). REST maintains self-renewal and pluripotency of embryonic stem cells. Nature.

Slaby, O., Svoboda, M., Fabian, P., Smerdova, T., Knoflickova, D., Bednarikova, M., Nenutil, R., and Vyzula, R.(2007). Altered expression of miR-21, miR-31, miR-143 and miR-145 is related to clinicopathologic features ofcolorectal cancer. Oncology 72, 397–402.

Solomon, M.J., Larsen, P.L., and Varshavsky, A. (1988). Mapping protein-DNA interactions in vivo with formaldehyde:evidence that histone H4 is retained on a highly transcribed gene. Cell 53, 937–947.

Soudais, C., Bielinska, M., Heikinheimo, M., MacArthur, C.A., Narita, N., Saffitz, J.E., Simon, M.C., Leiden, J.M.,and Wilson, D.B. (1995). Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stemcells disrupts visceral endoderm differentiation in vitro. Development 121, 3877–3888.

Stein, S., and Liang, P. (2002). Differential display analysis of gene expression in mammals: a p53 story. Cell MolLife Sci 59, 1274–1279.

Strahl, B.D., and Allis, C.D. (2000). The language of covalent histone modifications. Nature 403, 41–45.

Struhl, K. (1995). Yeast transcriptional regulatory mechanisms. Annu Rev Genet 29, 651–674.

Stryke, D., Kawamoto, M., Huang, C.C., Johns, S.J., King, L.A., Harper, C.A., Meng, E.C., Lee, R.E., Yee, A.,L’Italien, L., et al. (2003). BayGenomics: a resource of insertional mutations in mouse embryonic stem cells. NucleicAcids Res 31, 278–281.

Su, X., Kameoka, S., Lentz, S., and Majumder, S. (2004). Activation of REST/NRSF target genes in neural stem cellsis sufficient to cause neuronal differentiation. Mol Cell Biol 24, 8018–8025.

Suzuki, A., Raya, A., Kawakami, Y., Morita, M., Matsui, T., Nakashima, K., Gage, F.H., Rodriguez-Esteban, C., andIzpisua Belmonte, J.C. (2006). Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiationof embryonic stem cells. Proc Natl Acad Sci USA 103, 10294–10299.

Swillens, S., and Pirson, I. (1994). Highly sensitive control of transcriptional activity by factor heterodimerization.Biochem J 301(Pt 1),, 9–12.

Tada, M., Takahama, Y., Abe, K., Nakatsuji, N., and Tada, T. (2001). Nuclear reprogramming of somatic cells by invitro hybridization with ES cells. Curr Biol 11, 1553–1558.

Takahashi, K., Okita, K., Nakagawa, M., and Yamanaka, S. (2007). Induction of pluripotent stem cells from fibroblastcultures. Nat Protoc 2, 3081–3089.

Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblastcultures by defined factors. Cell 126, 663–676.

Tam, W.L., Lim, C.Y., Han, J., Zhang, J., Ang, Y.S., Ng, H.H., Yang, H., and Lim, B. (2008). Tcf3 regulates embryonicstem cell pluripotency and self-renewal by the transcriptional control of multiple lineage pathways. Stem Cells.

29

stembook.org


Tapscott, S.J., Davis, R.L., Thayer, M.J., Cheng, P.F., Weintraub, H., and Lassar, A.B. (1988). MyoD1: a nuclearphosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. Science 242, 405–411.

Tay, Y.M., Tam, W.L., Ang, Y.S., Gaughwin, P.M., Yang, H., Wang, W., Liu, R., George, J., Ng, H.H., Perera,R.J., et al. (2008). MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causespost-transcriptional attenuation of Nanog and LRH1. Stem Cells 26, 17–29.

Thanos, D., and Maniatis, T. (1995). Virus induction of human IFN beta gene expression requires the assembly of anenhanceosome. Cell 83, 1091–1100.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998).Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147.

To, C., Epp, T., Reid, T., Lan, Q., Yu, M., Li, C.Y., Ohishi, M., Hant, P., Tsao, N., Casallo, G., et al. (2004). The centrefor modeling human disease gene Trap resource. Nucleic Acids Res 32, D557–559.

Tokuzawa, Y., Kaiho, E., Maruyama, M., Takahashi, K., Mitsui, K., Maeda, M., Niwa, H., and Yamanaka, S. (2003).Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.Mol Cell Biol 23, 2699–2708.

Tomioka, M., Nishimoto, M., Miyagi, S., Katayanagi, T., Fukui, N., Niwa, H., Muramatsu, M., and Okuda, A. (2002).Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res 30,3202–3213.

Tsai, M.J., and O’Malley, B.W. (1994). Molecular mechanisms of action of steroid/thyroid receptor superfamilymembers. Annu Rev Biochem 63, 451–486.

Tsang, J., Zhu, J., and van Oudenaarden, A. (2007). MicroRNA-mediated feedback and feedforward loops are recurrentnetwork motifs in mammals. Mol Cell 26, 753–767.

Ventura, A., Young, A.G., Winslow, M.M., Lintault, L., Meissner, A., Erkeland, S.J., Newman, J., Bronson, R.T.,Crowley, D., Stone, J.R., et al. (2008). Targeted deletion reveals essential and overlapping functions of the miR-17through 92 family of miRNA clusters. Cell 132, 875–886.

Veraksa, A., Bauer, A., and Artavanis-Tsakonas, S. (2005). Analyzing protein complexes in Drosophila with tandemaffinity purification-mass spectrometry. Dev Dyn 232, 827–834.

Voorhoeve, P.M., le Sage, C., Schrier, M., Gillis, A.J., Stoop, H., Nagel, R., Liu, Y.P., van Duijse, J., Drost, J.,Griekspoor, A., et al. (2006). A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testiculargerm cell tumors. Cell 124, 1169–1181.

Wang, J., and Orkin, S.H. (2008). A Protein Roadmap to Pluripotency and Faithful Reprogramming. Cells TissuesOrgans.

Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D.N., Theunissen, T.W., and Orkin, S.H. (2006). A protein interactionnetwork for pluripotency of embryonic stem cells. Nature 444, 364–368.

Wang, S.H., Tsai, M.S., Chiang, M.F., and Li, H. (2003). A novel NK-type homeobox gene, ENK (early embryospecific NK), preferentially expressed in embryonic stem cells. Gene Expr Patterns 3, 99–103.

Wang, Y., Medvid, R., Melton, C., Jaenisch, R., and Blelloch, R. (2007). DGCR8 is essential for microRNA biogenesisand silencing of embryonic stem cell self-renewal. Nat Genet 39, 380–385.

Wei, C.L., Wu, Q., Vega, V.B., Chiu, K.P., Ng, P., Zhang, T., Shahab, A., Yong, H.C., Fu, Y., Weng, Z., et al. (2006).A global map of p53 transcription-factor binding sites in the human genome. Cell 124, 207–219.

Wei, F., Scholer, H.R., and Atchison, M.L. (2007). Sumoylation of Oct4 enhances its stability, DNA binding, andtransactivation. J Biol Chem 282, 21551–21560.

30

stembook.org


Werner, T. (2001a). Cluster analysis and promoter modelling as bioinformatics tools for the identification of targetgenes from expression array data. Pharmacogenomics 2, 25–36.

Werner, T. (2001b). Target gene identification from expression array data by promoter analysis. Biomol Eng 17, 87–94.

Wernig, M., Meissner, A., Cassady, J.P., and Jaenisch, R. (2008a). c-Myc is dispensable for direct reprogramming ofmouse fibroblasts. Cell Stem Cell 2, 10–12.

Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R.(2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324.

Wernig, M., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Constantine-Paton, M., Isacson, O.,and Jaenisch, R. (2008b). Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brainand improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA 105, 5856–5861.

Wijchers, P.J., Burbach, J.P., and Smidt, M.P. (2006). In control of biology: of mice, men and Foxes. Biochem J 397,233–246.

Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., and Campbell, K.H. (1997). Viable offspring derived from fetaland adult mammalian cells. Nature 385, 810–813.

Wu, Q., Chen, X., Zhang, J., Loh, Y.H., Low, T.Y., Zhang, W., Sze, S.K., Lim, B., and Ng, H.H. (2006). Sall4 interactswith Nanog and co-occupies Nanog genomic sites in embryonic stem cells. J Biol Chem 281, 24090–24094.

Xiao, C., Srinivasan, L., Calado, D.P., Patterson, H.C., Zhang, B., Wang, J., Henderson, J.M., Kutok, J.L., andRajewsky, K. (2008). Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression inlymphocytes. Nat Immunol 9, 405–414.

Yeom, Y.I., Fuhrmann, G., Ovitt, C.E., Brehm, A., Ohbo, K., Gross, M., Hubner, K., and Scholer, H.R. (1996).Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894.

Yi, F., Pereira, L., and Merrill, B.J. (2008a). Tcf3 Functions as a steady state limiter of transcriptional programs ofmouse embryonic stem cell self renewal. Stem Cells.

Yi, R., Poy, M.N., Stoffel, M., and Fuchs, E. (2008b). A skin microRNA promotes differentiation by repressing‘stemness’. Nature 452, 225–229.

Yoo, A.S., and Greenwald, I. (2005). LIN-12/Notch activation leads to microRNA-mediated down-regulation of Vavin C. elegans. Science 310, 1330–1333.

Yu, F., Yao, H., Zhu, P., Zhang, X., Pan, Q., Gong, C., Huang, Y., Hu, X., Su, F., Lieberman, J., and Song, E. (2007a).let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131, 1109–1123.

Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti,V., Stewart, R., et al. (2007b). Induced pluripotent stem cell lines derived from human somatic cells. Science 318,1917–1920.

Yuan, H., Corbi, N., Basilico, C., and Dailey, L. (1995). Developmental-specific activity of the FGF-4 enhancerrequires the synergistic action of Sox2 and Oct-3. Genes Dev 9, 2635–2645.

Yutzey, K.E., Rhodes, S.J., and Konieczny, S.F. (1990). Differential trans activation associated with the muscleregulatory factors MyoD1, myogenin, and MRF4. Mol Cell Biol 10, 3934–3944.

Zhang, J., Tam, W.L., Tong, G.Q., Wu, Q., Chan, H.Y., Soh, B.S., Lou, Y., Yang, J., Ma, Y., Chai, L., et al. (2006).Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulationof Pou5f1. Nat Cell Biol 8, 1114–1123.

31

stembook.org


Zhao, X.D., Han, X., Chew, J.L., Liu, J., Chiu, K.P., Choo, A., Orlov, Y.L., Sung, W.K., Shahab, A., Kuznetsov,V.A., et al. (2007). Whole-genome mapping of histone H3 Lys4 and 27 trimethylations reveals distinct genomiccompartments in human embryonic stem cells. Cell Stem Cell 1, 286–298.

Zhou, Q., Chipperfield, H., Melton, D.A., and Wong, W.H. (2007). A gene regulatory network in mouse embryonicstem cells. Proc Natl Acad Sci USA 104, 16438–16443.

Zhu, S., Si, M.L., Wu, H., and Mo, Y.Y. (2007). MicroRNA-21 targets the tumor suppressor gene tropomyosin 1(TPM1). J Biol Chem 282, 14328–14336.

Zhu, S., Wu, H., Wu, F., Nie, D., Sheng, S., and Mo, Y.Y. (2008). MicroRNA-21 targets tumor suppressor genes ininvasion and metastasis. Cell Res 18, 350–359.

32

stembook.org

genome-wide transcription factor localization and function in … · 2020. 1. 6. · genome-wide...

Documents