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Gene Expression

Protein DNA RNA

Metabolites, stress, environment

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EPIGENETICS

The study of alterations in gene function that cannot be explained by changes in DNA sequence. Epigenetic gene regulatory mechanisms include: chromatin (heterochromatin, remodeling, histone modifications), DNA methylation, RNA interference.

• Epigenetic modifications are heritable, but reversible, changes associated with chromatin that affect gene expression without alteration of the DNA sequence.

• Epigenetics – gene activation or silencing, ‘extends the information potential of DNA’.

• Epigenomics – characterization of the set of epigenetic modifications associated with an entire genome.

Important concepts behind the study of epigenetics/epigenomics

• “How does a fixed DNA blueprint allow flexibility in managing changes to environmental signals? Environmental inputs such as nutrition can modulate cell metabolism…” Sassone-Corsi Science 2013

• “External (infections, chemical agents & drugs) and internal (cytokines, hormones) environmental stimuli can modify the epigenetic profile of a gene, directly influencing its expression and, ultimately, the cell type and immune response.”

• “The epigenome connects the genome with the cellular environment and determines cellular identity and functionality.” Suarez-Alvarez et al Epigenetics 2013

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• Developmental regulation: X-inactivation, imprinting • Cell cycle • Viral latency • Transgene expression • Somatic gene therapy • Cancer gene expression • DNA damage repair • Apoptosis • Immune gene expression

Epigenetics is involved in: (partial list)

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Epigenetic regulation of immune genes

• VDJ recombination • Ig expression - µ and κ chain enhancers, isotype switching • CD4+ T cell differentiation • B cell maturation • Cytokine gene expression IFN-γ, IL-12, IL-2, IL-4 • Allelic silencing • MHC class I, II, CIITA • Costimulatory genes CD40, CD80, CD86

“The ability to package large genomes is associated with a fundamental shift in the logic of gene regulation between prokaryotes and eukaryotes. In prokaryotes, the ground state is non-restrictive; in eukaryotes, transcriptional activity is generally impeded by nucleosomal packaging. Activators and repressors influence gene expression by recruiting chromatin modifying activities to promoters.” Richards and Elgin 2002.

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Lewin, B. Genes VII. Oxford University Press Inc., New York: 578, 2000

Chromatin

Eukaryotic chromatin is a complex environment with regions of differential compaction.

Nuclear processes must occur in the context of chromatin:

Transcription

Replication

DNA damage repair

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Jenuwein, T., David Allis, CD. Science 293: 1074, 2001

Chromatin and transcription Eukaryotic transcription involves:

• large multiprotein polymerase complexes

• sequence specific transcription factors.

Chromatin context plays a large role in regulating gene transcription through accessibility.

Various protein complexes regulate chromatin compaction, nucleosome position, DNA accessibility and protein interaction sites.

Chromosomal environment influences gene expression

1. X-inactivation and imprinting - identical sequence of genes but functionally different

2. Chromosomal rearrangements that juxtapose euchromatin next to heterochromatin

3. Transgenes expressed at different levels depending on integration site

4. LCRs - can overcome the effects of chromosomal environment. Overall control of several genes at a distance

5. Cytologically uncondensed, ‘open’ chromatin [euchromatin] is transcriptionally active. Generally located near the center of the interphase nucleus. The histones are often hyperacetylated and CpG dinucleotides unmethylated. Open chromatin sites are 2-10 fold more sensitive than bulk chromatin to DNase and restriction enzymes.

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Komberg, RD. Cell 98: 285-294,1999

Nucleosome positioning in relation to promoter activation

• Preset promoters DNase I HS in proximal promoter. Open to transcription factor binding. Remodeling of specifically positioned nucleosomes required for activity e.g., Hsp70

• Promoters requiring remodeling Promoters repressed by chromatin and remodeling of nucleosomes required for transcriptional factor binding e.g., nuclear receptor

ATP-dependent chromatin remodeling complexes

Protein complexes that use ATP hydrolysis to alter the physical structure of chromatin by loosening nucleosomal DNA, sliding/repositioning nucleosomes, ejecting or inserting variant histones. Different classes of complexes function in forming nucleosomes during replication or local remodeling of chromatin for transcription initiation and elongation.

18 Becker & Workman CSH Perspectives in Biology 2013

ATP-dependent nucleosome remodeling

ATP-dependent multiprotein chromatin remodeling complexes

About 12 multiprotein complexes [5-20 subunits] falling into 4 groups of complexes depending on ATPase subunit:

1) SWI/SNF – SNF, BAF, BRG/BRM

2) ISWI - ISW1/2, RSF, NURF, CHRAC, ACF

3) NURD/Mi-2/CHD

4) INO80


Four ATPase subfamilies

Nomenclature

SWI Switching of mating type

SNF Sucrose non-fermenting

NURF Nucleosome remodeling factor

NURD Nucleosome remodeling & deacetylase

RSC Remodeling the structure of chromatin

CAP Chromatin assembly protein

SIR Silent information regulator

CREB Cyclic AMP response element binding protein

BRG 1 Brahma related gene 1

SAGA Spt Ada GCN5-acetyltransferase


ATPase subunits shared among several remodeling factors

SWI/SNF nucleosomal remodeling

• Remodeling accompanied by altered DNase I digestion pattern and increased transcription factor binding

• Remodeling complexes move along DNA without separating the DNA strands

• Complex movement and ATPase activity cause translational and rotational displacement of DNA


Mechanism of nucleosome remodeling


An example of the complexity of remodeling factors


Diversity and complexity of remodeling factors


Action of a Swi/Snf-type remodeler and regulation by acetylation

Regulation by remodeling complexes

• Different transcriptional activators may recruit different complexes

• Subsets of genes may be affected by specific modifying complexes

• Alternative histone incorporation (e.g. H2A.Z, H2A.X, H3.3, etc)

• A spectrum of amino acids may be modified to recruit specific readers and determine outcome.

• Chromatin remodelers are required during transcription for initiation, elongation and repression.

Maze et al (2014) 15: 259-71

Regulation by remodeling complexes

• Different transcriptional activators may recruit different complexes

• Subsets of genes may be affected by specific modifying complexes

• Alternative histone incorporation (e.g. H2A.Z, H2A.X, H3.3, etc)

• A spectrum of amino acids may be modified to recruit specific readers and determine outcome.

• Chromatin remodelers are required during transcription for initiation, elongation and repression.

The Chromatin Group, Department of Anatomy, The Medical School, Birmingham, B15 2TT, U.K.

Epigenetic repression of gene expression

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Lewin, Benjamin Genes VII. Oxford University Press Inc., New York: 582, 2000

The ‘histone code’

“Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins which in turn dictate dynamic transitions between transcriptionally active or silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a ‘histone code’ that considerably extends the information potential of the genetic code.”

Jenuwein and Allis. Science 2001, 293:1074-80

We commonly refer to ‘readers’, ‘writers’ and ‘erasers’ of epigenetic marks.

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Cheung, P. et. al., Cell, Vol. 103, p. 263–271, October 13, 2000

9 14 18 23 27

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Epigenetic marks on histones

Methylation (lysine, arginine)

Nucleosome Histones

Ubiquitination (lysine)

Phosphorylation (serine, threonine, tyrosine, histidine)

Acetylation (lysine)

HDACs

HATs

HMTs (MLL, Set, Dot, PRMT)

HDMs (LSD, JMJ)

ubiquitination deubiquitination

Kinases Phosphatases

36 Rothbart & Strahl BBA 2014

Reading and interpreting histone modifications

Histone acetylation • Induces new DNase I hypersensitivity sites within the nucleosome by opening the chromatin structure • Enhances binding of transcription factors • Activates transcription of certain genes • Nucleosome incorporation of unacetylated histones is repressive • Reduces capacity of histone HI to compact chromatin • Transcriptionally active chromatin [euchromatin] manifests enhanced acetylation compared to inactive chromatin [heterochromatin] • Activates integrated retroviral sequences – HIV • Can compete or synergize with other modifications

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Mizzen, C.A. et al., Cell Mol Life Sci 54:6, 1998

Different HATs display specificity for histones and residues:

• GCN5 - H3 [K14]

• CBP/p300 - H3 [K14 and K18] and H4 [K5 and K8]

• PCAF - H3 [K14] and H4 [K8]

Histone deacetylation • There are families of HDACs with different specificities

I – HDAC1,2,3,8 IIA – HDAC4,5,7,9 IIB – HDAC6,10 III – SIRT1-7 IV – HDAC11

• These deacetylases are recruited to various repressive complexes

e.g. NCoR/SMRT/HDAC3, Sin3a/b/HDAC1/2.

• Differential substrate usage, subcellular localization and expression contribute to the selective function of deacetylases.

• HDAC inhibitors are in clinical use.

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Histone deacetylases

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DNA methylation

• Mostly at CpG dinucleotides • Roles in X chromosome inactivation, genomic

imprinting, retrotransposon silencing • Disruptions in methylation or readers associated

with pathology • MBD readers (MBD and MeCP2 proteins), Zn

finger readers (SRA -- SET and RING assoc domain -- proteins)

• ‘erasers’ – TET oxidation, UDG BER

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Rothbart & Strahl BBA 2014

Reading and interpreting DNA modifications

UHRF1 UHRF1 UHRF2 UHRF2

Mechanisms of DNA methylation

Persistence of epigenetic states

Maintenance of repressed state by methylation

• Asymmetrically methylated CpG segregates one CH3 group to each daughter chromatid resulting in hemi-methylated DNA

• Dnmt-1 [maintenance DNA methyltransferase] binds to the replication fork and completes methylation

• CpG binding proteins [MeCP2] bind methylated CpGs and recruit HDAC to reestablish repression in each daughter cell

Fig. 3 Transmission of epigenetic states.

R Bonasio et al. Science 2010;330:612-616


DNA methylation during organismal development

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Coordinate genetic and epigenetic mechanisms regulate transcription factor binding

Transcriptional suppression by methylation of CpG islands

• Direct block to transcription factor/complex binding at promoter sites

• Some sequence specific TFs are methylation dependent (KLF)

• Methylated cytosine binding proteins recruit HDACs

• Compaction of higher order chromatin

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Writing, erasing and reading the histone and DNA modification landscape

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DNA methylation in normal and cancer cells

Baylin,S. Nature Clin Practice Oncology 2:S4-S11, 2005

DNA is generally found to be hypomethylated in tumors BUT CpG islands in gene promoters are frequently hypermethylated.

Epigenetic alterations in tumor progression

Esteller M. N Engl J Med 2008;358:1148-1159

Epigenetics in cancer management


Epigenetic inactivation of tumor-suppressor genes


Epigenetic Aberrations among Different Tumor Types


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Silencing of immune genes. MHC I, II, CD40. Silencing phenotype H3K4me1,2, H3K9me3, H3K27me3, accompanied by ↓ acetylation of H3, H4, H3K56ac

HDACi (TSA, SAHA, MS-275) enhance

histone acetylation and trigger DNA demethylation

Epigenetics in cancer

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Gene reactivation by Azacytidine

Baylin,S. Nature Clin Practice Oncology 2:S4-S11, 2005

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Epigenetic regulation of immune responses

Suarez-Alvarez et al Epigenetics 2013

DNA methylation and demethylation are represented by black and white lollipops, respectively; histone modifications are shown as circles: green, H3K4me3; red, H3K27me3; purple, H3K9me3; blue, acetylation of H3 or H4

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Epigenetic regulation CD4+ T cell differentiation

Suarez-Alvarez et al Epigenetics 2013

Fig. 1 Cis and trans epigenetic signals.

R Bonasio et al. Science 2010;330:612-616

Published by AAAS

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