1. Biol 456/656 Molecular Epigenetics Lecture #5 Wed. Sept 2, 2015

2. http://www.nature.com/nrm/journal/v2/n6/fig_tab/nrm0601_422a_F1.htmlMarmostein, Nature Reviews Molecular Cell Biology, 2001

3. -Chromatin consists of nucleosomes -145-147 bp of DNA around octomeric core -core: 2 molecules each of H2A, H2B, H3, H4 -NH 2 -terminal is basic charged histone tail region NH 2

4. Turner, NSMB, 2005 Note (mistake from last class): N terminal is end of protruding tail. Number starts at N-terminus

5. Charting histone modifications Chromatin organization differs between cell types enables differential access to regulatory cis- elements Chromatin structure influenced by transcription factors and transcription itself

6. Chromatin Immunoprecipitation (ChIP) http://joe.endocrinology- journals.org/content/201/1/1/F1.large.jpg

7. http://www.bnl.gov/bnlweb/pubaf/pr/photos/2011/11/chip_seq_illustration_final-hr.jpg ChIP-seq

8. ChIP Chromatin Immunoprecipitation Steps 1- Cross-link proteins to DNA with formaldehyde 2- Sonicate DNA to shear to ~500 bp fragments 3- Immunoprecipitate with antibody targeting epitope of interest (histone modification, transcription factor, etc). 4- wash the bead-antibody-protein-DNA complex to remove non-specific chromatin 5-reverse cross-links 6- remove proteins with proteinase K 7-isolate DNA

9. With isolated DNA, can proceed with – 1. PCR/qPCR (ChIP-PCR) – 2. microarray (ChIP-chip) – 3. deep sequencing (ChIP-seq) The focus has shifted from single genes to a genome wide of where proteins bind DNA Generalities (universal rules) can be extrapolated from experiments on individual genes

10. What do histone modifications mark? Genome-wide studies show correlation of marks with – Promoters: high GC content vs low GC content (high vs low CpG) – Enhancers: positively influence transcription at distal promoters – Insulators: block enhancer activity – Transcribed regions – Repressed regions

11. Zhou et al., Nature Reviews Genetics, 2011 Transcribed gene Not-transcribed Insulator binding protein H3K36me3 H3K79me2 H3K9me3 H3K27me3 H3K4me3 H3K79me2, H3K36me3 associated with transcribed genes H3K9me3, H3K27me3 associated with non- transcribed regions H3K4me3 marks promoters of transcribed genes

12. Dashboard of histone modifications @ Promoters: Histone modifications contribute To fine tuning of gene expression @ Gene bodies: Active vs Inactive conformations

13. Mammalian Promoter Regions Can be high or low GC content – High CpG content promoter- HCP – Low CpG content promoter-LCP (the p is for phosphate!)

14. -open/active by default -Applies to housekeeping genes -Applies to developmental regulator gene promoters in Embryonic Stem (ES) cells -Inaccessible to RNA Pol II. Not Transcribed

15. Active Inactive -selective activation by transcription factors No histone methylation DNA methylation -Inactive by default Low CpG promoters tend to be DNA methylated. High CpG promoters tend to not.

16. Heterochromatin Tightly packed DNA (vs euchromatin) Important for genome organization/stability, gene regulation Lots of repetitive sequence H3K9me3 enriched Can use H3K9me3 to identify heterochromatin domains Despite the repressive environment, some expressed genes reside in heterochromatic regions of the genome

17. H3K9me3 marks heterochromatin Ho et al., Supp Fig 33 size Gray bars are unmappable regions

18. Ho et al., Supp Fig 33

19. What do chromatin marks look like on individual genes?

20. Ho et al., supp fig 37 H3K9me3 marks heterochromatin

21. Ho et al., supp fig 37 H3K36me3 marks transcribed genes H3K27me3 marks suppressed genes

22. H3K4me3 marks TSS (Isn’t so clear for this example, right??) -remember, genome-wide trends won’t be observable for each gene example!

25. Ho et al., Supp Fig 36

27. From Human to fly to worm, Histone methylation mark (H3K9me3) “tracks” the same

28. Ho et al., Supp Fig 36

29. LAD: Lamina associated domain Differentiated cells have more H3K9me3 regions than embryonic cells

30. c Homework: Explain the important findings from this figure in words. You can paraphrase what is in the text or give your own interpretation. Send by email to pmiura@unr.edu before Friday.pmiura@unr.edu See Ho et. al paper. Or go to link: http://www.nature.com/nature/journal/v512/n7515/full/nature13415.html#extended-data

31. Summary Histone modifications indicate: – Functional genomic elements (promoters, enhancers) – Expressed vs silenced genes

32. “epigenetics” does not appear in the text!!! Supplemental data section has 42 figures

33. Chromatin composition and organization – Worm, fly, human Techniques: – ChIP-seq: Chromatin IP, deep sequencing – ChIP-chip: Chromatin IP, microarray Profiles: – core histones, histone variants, histone modifications, chromatin associated proteins

34. Histone modifications – H3K27me3, H3K4me3 Non-histones – CHD3 (part of NuRD complex which deacetylates histones) – EZH2 (Histone methyltransferase) – KDM4A (Lysine demethylase) – RNA Pol II DNAse I hypersensitive sites (DHS) Nascent transcript sequencing (GRO-seq) Paper Summary: Marks profiled

35. DHS: DNAse I hypersensitivity site -susceptible to cleavage by DNAse I enzyme https://en.wikipedia.org/wiki/DNase_I_hypersensitive_site DHS

36. https://en.wikipedia.org/wiki/Nuclear_run-on#/media/File:GRO-Seq.png http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2833333/ GRO-seq

38. Transcription start site

40. Genome Architectureat promoters fly/worm vs human Very similar to human cells – Nucleosome density – Methylation marks at TSS E.g. H3K4me3, H3K27ac Very different from human cells – GC content – Antisense nascent transcription

41. Ext Data Fig 1B- Fly

45. Heterochromatin Tightly packed DNA (vs euchromatin) Important for genome organization/stability, gene regulation Lots of repetitive sequence H3K9me3 enriched Can use H3K9me3 to identify heterochromatin domains Despite the repressive environment, some expressed genes reside in heterochromatic regions of the genome

46. c Homework: Explain the important findings from this figure in words. You can paraphrase what is in the text or give your own interpretation. Send by email to pmiura@unr.edu before Monday.pmiura@unr.edu See Ho et. al paper. Or go to link: http://www.nature.com/nature/journal/v512/n7515/full/nature13415.html#extended-data