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Epigenetics

Epigenetics. Epigenetics - Heritable changes in gene expression that operate outside of changes in DNA itself - stable changes in gene expression caused by changes in chromatin structure. Epigenetics. DNA methylation Histone tail post-translational modifications microRNA.

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Epigenetics

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  1. Epigenetics • Epigenetics - Heritable changes in gene expression that operate outside of changes in DNA itself - stable changes in gene expression caused by changes in chromatin structure

  2. Epigenetics • DNA methylation • Histone tail post-translational modifications • microRNA

  3. Mechanisms of Epigenetics: Nature 441, 143-145 May 2006.

  4. The five nucleotides that make up DNA

  5. Cytosine methylation occurs predominantly at CpG dinucleotides which are palindromic 5’ CpG 3’ 3’ GpC 5’

  6. Roles of DNA methylation • Transcriptional silencing • Protecting the genome from transposition • Genomic imprinting • X inactivation • Tissue specific gene expression

  7. HP1 (heterochromatin protein 1) binds to H3K9-Me3 HP1 oligomerization Spreads along chromatin Condenses chromatin into heterochromatin Model for Role of Methylation in Heterochromatin Formation

  8. DNA methylation and Transcription: • only known covalent modification of DNA • in mammals • occurs in context of CpGs • CpG islands: • regions of the genome rich in CpG dinucleotide • comprise 60% of promoters • hypomethylated in differentiated tissue but, variable methylation patterns found in tumors • Non-CpG island promoters: • 40% of promoters • often methylated • tissue-specific differentially methylated regions exist • primary or secondary event?

  9. CpG Islands: - Regions of the genome in which the CpG dinucleotide occurs at the EXPECTED frequency - Usually located in 5’ flanking sequence, around the proximal promoter, and/or within the first exon and intron - Refractory to methylation in somatic cells - Frequently methylated in tumor cells Exon 1 Exon 2

  10. How does one study DNA methylation ? • Bisulphite sequencing • Methylation-specific restriction endonucleases • e.g., HspII/MSPI • Methylation specific assays, e.g. MethylLight

  11. Epigenetics • DNA methylation • Histone tail post-translational modifications • microRNA

  12. Bednar et al., PNAS, 95, 1998 Wolffe and Hayes, NAR 27, 1999

  13. Post-translational Modification of Histone N-terminal Tails Wide Range of Histone Modifications to Regulate the “STATE” of Chromatin Proteins with bromodomains (acetate groups) or chromodomains (CH3)

  14. DNA methylation In establishing heterochromatin, which is the driving force – DNA methylation? - Histone modification?

  15. Variety of Histone modifications: Landmarks for Chromatin-binding Proteins • Chromodomain • CH3(Methyl)- recognition domain • HP1 has a chromodomain • Targets to Me-lys or H3K9me • Promote packed “closed” chromatin • Demethylation of Lys 9 in H3 tail • facilitates phosphorylation (P) of Ser 10 • Acetylation (Ac) of Lys 9 and 14 • leads to “OPEN” chromatin • Bromodomain • Binds to acetylated lysines “OPEN” • Wide range of histone modifications ->

  16. Repressors Regulate Gene Expression by Modulating Chromatin Structure to be Closed Fig. 7-38 -The DBD of repressors (like Ume6) bind a DNA element (URS1) and the Repression Domain (RD) recruits a protein complex containing a histone deacetylase like Rpd 3. -The subsequent deacetylation of histone N-terminal tails results in chromatin condensation which promotes gene repression.

  17. Activators Regulate Gene Expression by Modulating Chromatin Structure to be Open Fig. 7-38 -The DBD of Activators like Gcn4 bind their Upstream Activating Sequence (UAS). -Activation Domain (AD) attracts protein complexes containing histone acetylases (Gcn5) -Subsequent acetylation of histone tails serve to open up chromatin. -Thus HDACs and HATs are important global regulators of transcription

  18. Model for Heterochromatin Formation Condensation assisted by recruitment of HMT (histone methyltransferase), which methylates adjacent H3K9 Chromatin condensed until a boundary element is reached. Methylation of histone tails long lasting compared to acetylation Can be Inherited by daughter cells: Responsible for X-inactivation Epigenetics: chromatin structure controls gene expression rather than nt. sequence

  19. Histone Post-translational Modifications (PTMs) involved in transcription: • Histone modifying enzymes generate “the histone code” • PTMs dictate chromatin structure and serve as a scaffold for additional regulatory proteins • acetylation, methylation, ubiquitination sumolyation and phosphorylation • H3KAc, H4KAc, H3K4me, H3K36me H3K27me, H3K9me We examined histone PTMs across the fpgs to study their role in promoter choice and silencing. Workman et. al 2007, review

  20. Current thoughts on the role of chromatin in transcriptional initiation: Workman et. al 2007, review Does this model apply to most tissue-specific promoters?

  21. How does one detect histone PTMS ? • Chromatin Immunoprecipitation (ChIP) • -variations, i.e., ChIP walking, ChIP on chip • Or • Mass Spectrometry

  22. Identify proteins that interact with DNA Chromatin Immunoprecipitation Cross Link Sonicate Supe after IgG IP Primary Ab & Protein G Beads IP Ab Enriched DNA Quantitate by Real Time PCR INPUT

  23. Histone H3 lysine 4 tri-methylation across the fpgs gene: H3k4 Workman et. al 2007 Vakoc et. al 2006 Histone H3K4me3 decorates P2 in all tissues, but appears limited in liver and P1 K4me3 correlates with loss of DNA methylation and promoter activation.

  24. Histone H3 lysine 4 tri-methylation across the fpgs gene: H3k4 L1210 P1 P2 Liver P1 P2 Histone H3K4me3 decorates P2 in all tissues, but appears limited in liver and P1 K4me3 correlates with loss of DNA methylation and promoter activation. Brain P1 P2

  25. Histone H3 and H4 acetylation across the fpgs gene: Vakoc et. al, G and D, 2006 Histone H3 and H4 acetylation decorates P2 in all tissues, and P1 acetylation correlates with loss of DNA methylation and promoter activation. Racanelli et al, MCB, 28, 836-48, 2008

  26. 3Me 3Me Ac Ac Ac Ac Ac Ac Ac Ac Ac 2Me 2Me 3Me 1Me 1Me 1Me P P P P 1Me 2Me 1Me H3 2Me 1- 39 ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPH- 1Me P P P Ac 1Me ARTKQTARKSTGGKAPRKQL ATKAARKSAPATGGVKKPH 1Me 2Me 2Me ARTKQTARKSTGGKAPRKQL ATKAARKSAPATGGVKKPH Histone Post-Translational Modifications

  27. Summary: Layers of Gene Regulation Chromatin Remodeling Factors (ATPase) Histone Modifications DeAcetylation/Acetylation Phosphorylation, methylation, Ub Regulatory Factors-gene specific Also subject to post-translational modifications General Transcription Factors Pol II Once Transcription Complex Assembled, what starts transcription? FACT: CTD-becomes hyperphosphorylated repress activate HDATs Co-repressors HATs Co-activators

  28. The dynamic epigenome and its implications for modulating gene expression in the cell

  29. Chromatin modifications with impact on gene expression • K9 histone H3 and H4 acetylation • K9 H3 methylation • K4 H3 methylation • Ser 10 phosphorylation • Ubiquitination • Association of chromatin remodeling complexes

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