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

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. Model for Role of Methylation in Heterochromatin Formation HP1 (heterochromatin protein 1) binds to H3K9-Me 3 HP1 oligomerization Spreads along chromatin Condenses chromatin into heterochromatin

8. 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? DNA methylation and Transcription:

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 1Exon 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. Wolffe and Hayes, NAR 27, 1999 Bednar et al., PNAS, 95, 1998

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 (CH 3 )

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 CH 3 (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 -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. Fig. 7-38

17. Activators Regulate Gene Expression by Modulating Chromatin Structure to be Open -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 Fig. 7-38

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: Does this model apply to most tissue-specific promoters? Workman et. al 2007, review

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

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

23. H3k4 Histone H3 lysine 4 tri-methylation across the fpgs gene: Vakoc et. al 2006 Workman et. al 2007 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. H3k4 Histone H3 lysine 4 tri-methylation across the fpgs gene: L1210 Liver Brain P1P2 P1 P2P1 Histone H3K4me3 decorates P2 in all tissues, but appears limited in liver and P1 K4me3 correlates with loss of DNA methylation and promoter activation.

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

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

30. 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 repressactivate HDATs Co-repressors HATs Co-activators

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

33. 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