BIO347 Epigenetics Lecture Notes PDF

10/7/24 BIO347 Epigenetics Katharina Braeutigam Office: Room DV 3035 Email: [email protected] Office hours:...

10/7/24 BIO347 Epigenetics Katharina Braeutigam Office: Room DV 3035 Email: [email protected] Office hours: bigstockDNA Tuesdays: 12-1pm or by appointment 1 BIO347 - Epigenetics - Lectures Chapters 4 and 5, Law2010 Chapters 6 and 7 Chapters 7, 12, 13 Focus on Fundamental Processes Law and Jacobsen 2010. Nat Rev Genet. 11:204 2 1  10/7/24 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT Induced Pluripotency 3 Histone modifications Covalent modifications: methylation (me) acetylation (ac) phosphorylation (p) ubiquitination (ub) sumoylation (su) [small ubiquitin-related modifier] ADP ribosylation (ar) deimination (cit) Alberts, 6th ed, Fig 4-34b 4 2  10/7/24 Methylation plants: differ Plants: H3K9me3 : mark of active genes H3K9me2: silencing of TEs Mammals: H3K9me3: heterochromatic mark 5 Histone methylation Alberts, 6th ed, Fig. 4-33 8 3  10/7/24 Histone methylation Effect on gene expression Many modifictions occur at lysine Alberts, 6th ed, Fig. 4-33 10 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT Induced Pluripotency 15 4  10/7/24 Epigenetic modulator enzymes Writer Eraser Reader http://being-bioreactive.com 16 Histone modifications and modifiers Protein Arginine Methyltransferase Peptidylarginine Deiminase Allis, Epigenetics, Fig.3-7 17 5  10/7/24 Epigenetic modulator enzymes Writer Eraser Reader http://being-bioreactive.com 19 Histone Modification Machinery Methyl group HMTs - histone methyltransferases lysine or arginine methylation (mostly tail) mono, di-, or tri methylation of lysine transcriptional activation or repression Other functions: - Transcriptional elongation - Pericentromeric heterochromatin - X chromosome inactivation 20 6  10/7/24 Histone methylation permissive or repressive chromatin – depending on exact position of lysine on histone tail – More “stable” than acetyl marks – also: non-histone proteins as substrates SAM SAH Methyl donor: S-adenosyl-L-methionine, same as in DNA methylation 21 Histone lysine methyltransferases HKMTs Conserved SET domain Catalytic unit in many known HKMTs evolutionary conserved, but relatively young ca. 130 amino acids Identified in Drosophila Su(var)3-9, E(z), Trx (homeobox gene regulator trithorax) Often SET domain proteins contain an additional domain (e.g. chromo domain in SUV39H1) Many HKMTs: part of multi protein complexes (e.g. EHZ2 in PRC) 22 7  10/7/24 Histone lysine methyltransferases HKMTs Examples SET 7/9 enzyme Xiao et al., Nature, 2003. H3K4me monomethylation activity mutation: Tyr3053 à Phe: dimethyltransferase EZH2 (PRC): H3K27me1/2/3 PRC2 spread H3K27me2/3 MLL-family proteins : H3K4 Mixed-Lineage Leukemia 1 (MLL1) master regulator of Hox genes homolog to Drosophila trithorax Is part of complex Non-SET-dependent HKMTs e.g. Dot1L: H3K79 (core) Requires adjacent ubiquitin 23 HKMT are highly specific 24 8  10/7/24 Histone arginine methyltransferases PRMTs Protein arginine methyltransferases SAM dependent Mono or di-methylation of arginine Can function as part of protein complexes SAM asymmetric dim ethylarginine arginine SAH m onom ethylarginine symmetric dim ethylarginine 25 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT Induced Pluripotency 26 9  10/7/24 Epigenetic modulator enzymes Writer Eraser Reader http://being-bioreactive.com 27 Histone lysine demethylases Lysine demethylation characterized in 2004 by oxidation: LSD1 (lysine-specific demethylase) FAD as electron acceptor H3K4me1/2 Member of amino oxidase superfamily by hydroxylation: Jumonji-containing demethylases iron(II)-dependent demethylate Kme3 Large family Wysocka et al., Cell, 2005. 28 10  10/7/24 Pathways of Lysine demethylation by Oxidation (LSD1) …shows the formation of H2O2 and formaldehyde as side products and the imine intermediate that requires protonated lysine and accounts for the inability of LSD1 to demethylate trimethyl residues Perillo et al. Experimental & Molecular Medicine (2020) 52:1936 29 HMT and histone demethylases can be highly specific Shi, Nature Reviews, 2007. 31 11  10/7/24 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT Induced Pluripotency 32 Epigenetic modulator enzymes Writer Eraser Reader http://being-bioreactive.com 33 12  10/7/24 Histone modifications and modifiers & more! Protein Arginine Methyltransferase Peptidylarginine Deiminase Allis, Epigenetics, Fig.3-7 34 Histone methylation is recognized by a variety of domains ③.1 ① ④ ③.2 ③.3 ② web.stanford.edu/group/gozani/ 35 13  10/7/24 ① PhD finger – a versatile epigenome reader Plant homeodomain common among chromatin remodelers > 100 human proteins often adjacent to other reader modules Examples: BPTF (nucleosome-remodeling factor subunit): H3K4methylation also present in HATs (p300) Pcl, Trx, HMTs (ASH1/2L), NuRD bromodomain PHD finger transcription factor Allis, “Epigenetics, 2nd ed., Fig 6-2 37 ② Bromo adjacent (BAH) domain recognizes methylated histones BAH domain: shown to be reader in 2012 NuRD subunit, DNMT1, CMT3 Flanked by bromodomains or PhD domains Law & Jacobsen 2010, Nature Allis, “Epigenetics, 2nd ed., Fig 6-3 38 14  10/7/24 ③Royal family of reader modules Allis, “Epigenetics, 2nd ed., Fig 6-4 HP1, polycomb Male-specific lethal A polycomb-like protein Brom odom ain and plant hom eodom ain finger 1 CMT3: structure also recognize with highly conserved function relationship methylarginine Pro-Trp-Trp-Pro motif between DNA and histone methylation Tandem royal family modules 39 Increased binding through linked modules Allis, “Epigenetics, 2nd ed., bromodomain PHD finger transcription factor PhD finger: H3K4me3 Bromo: H4K16ac (& others) 41 15  10/7/24 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT Induced Pluripotency 44 Histone modifictions Covalent modifications: methylation acetylation phosphorylation ubiquitination suomylation ADP ribosylation deamination Alberts, 6th ed, Fig 4-34b 45 16  10/7/24 Histone phosphorylation Serine/threonine kinases: H3S10 ribosomal S6 kinases (RSKs) mitogen- and stress-activated kinases 1 & 2 (MSK1/2) aurora kinases (centrosome-localized) Serine/threonine kinases: H2BS14 MST1 (sterile 20 protein family, apoptosis) Phosphatases Prigent & Dimitrov 2003. Journal of Cell Science 116 (18):3677 46 Roles of Ser/Thr Phosphorylation Cheung et al., Cell, 2000. 47 17  10/7/24 48 Cross talk among histone modifications Mutually exclusive: a position can be modified either with an activating or repressive mark – H3K9ac vs. H3K9me One modification recruits a modifying enzyme that places/removes another modification on the same/different histone tail – H3S10P [promotes H3K14ac by GCN5] The binding of a protein may be disrupted by modification of an adjacent position – HP1 binding to H3K9me disrupted by H3S10P Cooperative recruitment PHF8 binds H3K4me3 most strongly if H3K9ac and H3K14ac Methyl/Pospho switch Hirota et al. Nature 2005 49 18  10/7/24 Recognition of histone modifications www.activemotif.com 50 Histone modifications & their modulators www.activemotif.com 51 19  10/7/24 52 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT Induced Pluripotency 53 20  10/7/24 What is epigenetics? One genome – Different cell types Conrad Hal Waddington cc.scu.edu.cn/G2S/; Taiz, Zeiger 6th ed; jessicalucasphd.com 54 Waddington‘s epigenetic landscape Waddington, CH. The Strategy of the Genes. A Discussion of Some Aspects of Theoretical Biology, Alen & Unwin, 1957 Waddington 1942: Canalization of development and the inheritance of acquired characters, Nature 3811, 563 55 21  10/7/24 Differentiation Waddington, CH. The Strategy of the Genes. A Discussion of Some Aspects of Theoretical Biology, Alen & Unwin, 1957 Waddington 1942: Canalization of development and the inheritance of acquired characters, Nature 3811, 563 57 Differentiation ba sis of a ll e mb ryo ge ne sis Waddington, CH. The Strategy of the Genes. A Discussion of Some Aspects of Theoretical Biology, Alen & Unwin, 1957 Waddington 1942: Canalization of development and the inheritance of acquired characters, Nature 3811, 563 58 22  10/7/24 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC – Embryonic stem cells SCNT Induced Pluripotency 59 Differentiation Oocyte Morula Blastocyst blastomeres Inner cell mass (ICM) totipotent pluripotent Embryonic Stem Cells Allis 2015 60 23  10/7/24 Maitenance of pluripotency in ES cells prolonged self-renewal can differentiate into cells of the 3 germ layers have potential to activate all of the genome’s gene expression programs (embryo to adult cells) tight and dynamic epigenetic control 61 mouse Embryonic Stem Cells (mESC) - An important tool for research Cell and developmental biology: - cell cycle regulation, - cellular interactions during development, - and the control of gene expression during development - to creating transgenic or knock-out mice (HR) for in vivo disease models - identifying factors that maintain pluripotency - genetic disease research - identifying novel differentiation factors - cancer biology 62 24  10/7/24 Maitenance of pluripotency in ES cells What are the molecular characteristics of ESC? Apply logic and your current knowledge! Generic Characteristics of ESCs What happens during cell Differentiation Compile in class 63 Differentation of ESC & bivalent chromatin domains: an interesting case 65 25  10/7/24 Bivalent chromatin domains “Dashboard” of histone modifications Zhou et al., 2011. Nature Rev Genet. 12:7 66 Genome regulation occurs at multiple levels Large heterochromatin domains – Large Organized Chromatin K9 modifications: LOCKs Wen et al., 2009. Nat Genet 41(2): 246; Jorgensen & fisher 2009. Cell Stem Cell 4, 192 67 26  10/7/24 Stability and reversibility of epigenetic patterns Maintain Respond to cell type identity environmental triggers www.cam.ac.uk/ Epigenetic patterns established during cell differentiation: stable 68 Establishment of cell identity ba sis of a ll e mb ryo ge ne sis Waddington, CH. The Strategy of the Genes. A Discussion of Some Aspects of Theoretical Biology, Alen & Unwin, 1957 Waddington 1942: Canalization of development and the inheritance of acquired characters, Nature 3811, 563 69 27  10/7/24 Epigenomic reprogramming? Po ssi ble ? ?? Waddington, CH. The Strategy of the Genes., Alen & Unwin, 1957 Waddington 1942: Canalization of development and the inheritance of acquired characters, Nature 3811, 563 70 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT – Somatic cell nuclear transfer Induced Pluripotency 71 28  10/7/24 Epigenomic reprogramming: Somatic cell nuclear transfer Genome can be reset to totipotent state. Gene expression leading to all different cell types is result of reversible epigenetic change. But: cloning of mammals: challenging 72 Meet CC ! Born in 2001 mun.ca 73 29  10/7/24 Epigenomic reprogramming: Somatic Cell Nuclear Transfer (SCNT) Finn Dorset Scottish Blackface 74 Olsson et al., 2022. Sci Rep 12: 11209 76 30  10/7/24 SCNT - what happens to the genome? Pretty crude Brute force Low efficiency Underlying mechanisms poorly understood Source: http://gph.to/2zl1RXf 77 How can we get pluripotency? - Sources of pluripotent stem cells - Allis, 2015, Fig. 28-2 - Cell extracts can also be used to reprogram cells e.g. epithelial cells 78 31  10/7/24 Today‘s Roadmap Part I - MOLECULAR BACKGROUND Histones & histone modifications Histone methyltransferases Histone demethylases Readers of methylated histones Others PART II – EPIGENETIC PHENOMENA Differentiation in Mammals ESC SCNT Induced Pluripotency 80 Induced Pluripotency Ground breaking work by Takahashi and Yamanaka 2006. Takahashi & Yamanaka 2006. Cell 126:663 81 32  10/7/24 Induced Pluripotency Overexpression of a small set of transcription factors (embryonic factors in somatic cells) -> cells resemble ESC without going through development Minimally required core set of 4 transcription factors: Oct4, Sox2, Klf4, c-Myc (low efficiency, incomplete reprogramming) 82 Establishment of cell identity ba sis of a ll e mb ryo ge ne sis Waddington, CH. The Strategy of the Genes. A Discussion of Some Aspects of Theoretical Biology, Alen & Unwin, 1957 Waddington 1942: Canalization of development and the inheritance of acquired characters, Nature 3811, 563 83 33  10/7/24 iPS can be generated from a variety of different cell types various types Pancreatic cells Liver cells of fibroblasts Stomach cells 84 iPS can be generated from a variety of different cell types Fig. 28-6, Allis 2015, Epigenetics 85 34  10/7/24 Reprogramming during iPS induction Fig. 28-7, Allis 2015, Epigenetics 86 Induced Pluripotency Quo vadis? Slow process Stochastic elements Kick-start pluripotency-associated processes in minority of cells New: secondary systems: inducible promoters DNA methyltransferase inhibitor: boost reprogramming TSA trichostatin HDAC inhibitor did not. DNA methylation involved 87 35  10/7/24 Induced Pluripotency Current: - screens for chemicals and siRNAs - Changed view of development - Patient-specific iPS cells Future: - Advances in understanding of toti – and pluripotency - Temporal and cell-specific resolution 88 89 36  10/7/24 100 37