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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

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

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

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.

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

11 Becker & Workman CSH Perspectives in Biology 2013

Four ATPase subfamilies

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

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

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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

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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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

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

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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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

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

Epigenetic inactivation of tumor-suppressor genes

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

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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