Chromatin Remodelling: Histone Modifications PDF

MSc Cancer UCL Cancer Institute Faculty of Medical Sciences Chromatin remodelling: histone modifications Professor Richard Jenner [email protected] 1 Part 1 Histone modification in normal cells 2 Learning outcomes After this topic you should be able to: 1. Understand that cells differentiat...

MSc Cancer UCL Cancer Institute Faculty of Medical Sciences Chromatin remodelling: histone modifications Professor Richard Jenner [email protected] 1 Part 1 Histone modification in normal cells 2 Learning outcomes After this topic you should be able to: 1. Understand that cells differentiate and maintain their identity through epigenetic mechanisms. 2. Describe the constituent parts of a nucleosome. 3. Understand that chromatin structure is dynamic and regulates access to genes. 4. Understand that histones are subjected to a large array of post-translational modifications. 5. Provide examples of histone modifications, their writers and readers, and their distribution around genes. 6. Discuss the functions of polycomb and trithorax proteins. 3 Cell differentiation • Progenitor cells differentiate along specific pathways into specialised cell types. • This occurs without changes to the DNA present within the cell. 4 Epigenetics Cell differentiation pathways can be thought of as a cell traversing an epigenetic landscape (Conrad Waddington, 1942). Progenitor cell Cell differentiation is a unidirectional process (like the ball rolling down the hill into one of the four valleys) Possible cell fates. Once reached, the cell cannot move between them “Epigenetics is the study of any potentially stable (ideally heritable) change in gene expression or cellular phenotype that occurs without changes in DNA sequence”. Goldberg, Allis and Bernstein, Cell 2008. 5 Imagining cancer cells on the epigenetic landscape Cancer cell gets stuck on the landscape Cancer cell rolls back up the landscape • Rather than following their prescribed course, cancer cells can be considered to get stuck traversing the epigenetic landscape or to move across or back up the landscape. 6 Epigenetic state is governed by chromatin • DNA is not free in the nucleus but instead is packaged with proteins to form chromatin. • Chromatin packages the 2 m of DNA present within every cell into the 10 m-wide nucleus. • Chromatin regulates cell differentiation and cell identity by controlling access to genes. • Some aspects of chromatin state are inherited by daughter cells. Chromatin was first visualised by Walther Flemming in 1878. 7 Heterochromatin and euchromatin • G-bands consist of tightly packaged heterochromatin, light bands of relatively open euchromatin (first seen by Geimsa staining by Heitz, 1928). • Heterochromatin is also visible in interphase cells by EM. • Euchromatin has a more open, accessible structure the heterochromatin absorbs more dye as a packed protein than the open euchromatin 8 The nucleosome • The nucleosome is the most basic unit of chromatin and is composed of DNA wrapped around a histone octamer. • The histone octamer contains two histone H3-H4 dimers and two histone H2A-H2B dimers with 1.7 turns of DNA wrapped around (147 bp). • Nucleosomes are spaced along the DNA at an average of 35 bp apart. But this varies Figure from Molecular Biology of the Cell (5th edition) 9 Chromatin regulates access to the genome • Chromatin can be open or closed to regulate access of the transcriptional machinery to genes. Open chromatin (euchromatin) Closed chromatin (heterochromatin) 10 Adapted from Yan et al., J App Physiol 2010 Chromatin state is thought to be the unit of epigenetic inheritance • Chromatin structure at individual genes is inherited by daughter cells, maintaining the same pattern of gene activity, and therefore maintaining cell identity. Signal received by the cell that initiates a change in chromatin structure Cell division Chromatin structure is maintained even though the original signal is no longer present. This allows cells to maintain their identity or state. 11 Modifying chromatin • Differences in gene expression between cells are thought to be due to differences in the way the genome is packaged into chromatin. DNA methylation Methyl marks added to C residues in CpG-dense regions repress transcription. Nucleosome remodelling Nucleosomes can be removed, slid up or down or different histones inserted. Histone modification Lysine residues in histone tails are methylated, creating binding sites for regulatory proteins can be moved or kicked off the DNA 12 Qui, Nature 2006 Nucleosomes must be remodelled to allow transcription to proceed Closed chromatin Allows transcription factor binding Open chromatin 13 Histone modifications • Histone tails are subject to more than 100 post-translational modifications, including acetylation, methylation, phosphorylation and ubiquitination. • The “histone marks” are binding sites for regulatory proteins. 14 Molecular Biology of the Cell (5th edition) Writing, erasing and reading histone modifications Example proteins Nat Immunol. 11, 565-568 (2010) Examples of histone modifications and their readers BPTF, CHD1 and ING2 (nucleosome remodelling factors) can only bind to the K at position 4 if it is methylated creating a specific binding site with a specific effect • The combination of histone modifications is like a “code” that tells us the structure of chromatin at each position in the genome. Outcome Transcription Gene repression initiation (heterochromatin) Gene repression (euchromatin) more dynamic Constitutive Transcription through gene Readers Histone ”marks” Writers 4 MLL SET1 9 SUV39H1 SETDB1 KAP1 14 Aurora-B MSK2 Bottom are enzymes that add these modifications 27 36 CBP PRC2 p300 PCAF GCN5 H3 H4 SETD2 Myst 16 Histone acetylation • Catalysed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs). • HATs and HDACs are recruited to chromatin by DNA-binding transcription factors. Acetylation ALWAYS promotes euchromatin • Acetylation alters histone charge, reducing interaction between histone tails and DNA and increasing nucleosome mobility. • Acetylation also creates a binding site for proteins with bromodomains: H3K9/K14ac recruits BRD4, which in turn recruits P-TEFb, that activates transcription elongation. 17 Histone methylation • Lysines in histone tails can be mono, di or tri-methylated. doesn’t change the charge of N but changes the binding site can either be activatory or repressive • Different methyltransferase enzymes methylate different lysines and these create binding sites for specific proteins that contain chromodomains or WD-40 domains. • Depending on the lysine, methylation can be activating or repressive. For example:  H3K4 methylation is associated with transcriptional initiation.  H3K27me3 is associated with transcriptional repression. 18 Polycomb versus Trithorax proteins • The balance between opposing activating and repressive histone modifying complexes regulates gene expression and cell differentiation Polycomb H3K27me3 Repressive Trithorax H3K4me3 Activatory Piunti and Shilatifard, Science 2016 Polycomb group proteins are required for cell differentiation Antennapedia • Drosophila mutant for Su(z)12 Small wings Sex combs present on rear legs Birve et al., 2001 • Polycomb group proteins form three main complexes, canonical polycomb repressive complex 1 (cPRC1), vPRC1 and PRC2, which together compact chromatin. H2AK119Ub Loubiere, Martinez and Cavalli 2019 Polycomb maintains genes specific for other cell types in a repressed state How‘blankets’ are different cell identities maintained? PRC2 genes encoding Upon cell differentiation, PRC2 and Celltranscription types developmental regulator PRC1 are selectively lost from genes factors specificproteins for othermodify cell types. • Polycomb chromatin to repress for the new specialised cell important developmental regulator genes specificstate. for other cell types Change in targeting during differentiation ESC = Embryonic stem cell ES cells NPC = neural precursor cell ESC PRC2 NPC Olig2 CpG island Lee et al., Cell 2006 Lee, Jenner et al., Cell 2006 Mikkelsen et al., 2007 Trithorax group proteins • Trithorax (Trx) was identified due to its mutant phenotype in Drosophila (Ingham 1985). • The orthologous human gene MLL1 was discovered in 1992 due to its mutation in myeloid and mixed-lineage leukemia (hence name). • Trx and MLL1 catalyse trimethylation of H3K4 (H3K4me3). • There are 5 other MLL orthologues in mammals and additional Trithorax group proteins that function in other steps of gene activation. Trx wild-type Trx mutant MLL Trx/MLL promotes gene activation In this case - methylation is activating RNA Pol II is blocked by nucleosomes positioned in the gene PAF1 and P-TEFb bind to RNA Poll II PAF1 recruits MLL, which methylates histone H3 at K4 NURF H3K4me3 is recognised by NURF chromatin remodeling complex which moves histones to allow Pol II to pass Summary 1. Epigenetic state is dependent on chromatin structure – the way in which DNA is packaged with proteins. 2. DNA is wrapped around histone proteins to form the nucleosome. 3. Chromatin can be tightly packed (heterochromatin) or more accessible (euchromatin) and this is regulated dynamically. 4. Histones are subjected to a large array of post-translational modifications and “writers”, “erasers” and “readers” are highly specific for particular modified amino acids. 5. Different histone modifications have different effects on chromatin structure and gene expression. 6. Histone acetylation opens chromatin, whereas histone methylation can have various effects. 24 Part 2 Dysregulation of histone modification in cancer 25 Learning outcomes After this topic you should be able to: 1. Understand the different ways in which chromatin can become dysregulated in cancer. 2. Describe the effect of gene fusions on the function of MLL and how this leads to leukaemia. 3. Explain why chromosomal translocations can cause dysregulation of C-Myc gene expression and lymphoma. 4. Recount the effect of EZH2 gain-of-function mutations on gene expression in B cell lymphoma. 5. Describe the effect of H3K27M mutations on histone modification in glioma. 6. Understand how EZH2 and BET domain inhibitors block 26 the growth of certain tumour types. Dysregulation of histone modification in cancer Alterations to histone mark writers, readers and erasers, and even histones themselves, have all been found to cause cancer. These proteins can be dysregulated by: • Gene deletion, amplification or over-expression. • Chromosomal translocations that fuse the gene encoding a chromatin regulator with another gene. • Chromosomal translocations that disrupt the expression pattern of a chromatin regulator gene. • Point mutations that alter chromatin regulator function. 27 Chromatin dysregulation due to gene fusion: MLL • MLL is translocated in myeloid and mixed-lineage leukemias (hence name) • Over 50 fusion partners. Most frequently fused to the super elongation complex components AF4 or ENL (also called MLLT1). • The super elongation complex recruits the H3K79 methyltransferase DOT1L and RNA Pol II kinase P-TEFb (CyclinT1-CDK9). • P-TEFb and DOT1L activate transcriptional elongation. 28 MLL-AF4 activates genes ectopically MLL-AF4 fusion protein occupies genes not normally bound by MLL or AF4 and activates their expression. Guenther et al. Genes Dev 2008 29 MLL-AF4 interacts with BRD4, which activates transcriptional elongation BRD4 interacts with components of the super elongation complex. BRD4 also binds H3K9/K14ac. Small molecule inhibitors have been designed to fit into this histone binding pocket. Popovic and Licht, 2012 Purification of BRD4 co-purifies components on the SEC and vice versa (identified by mass spectrometry). 30 BRD4 inhibitors block MLL-mediated oncogenesis • The BRD4 inhibitor I-BET-151 repress growth of MLL-AF4-driven leukemic cells in vitro and in mice (Dawson et al. Nature 2011). Cell count and organ weight in SCID-mice injected with human leukemia cells. 31 Dysregulation due to point mutations: PRC2 subunit EZH2 • Follicular lymphoma (FL) and germinal centre B cell diffuse large B cell lymphoma (GCB-DLBCL) arise in B cells in lymph nodes. • The cancer cells resemble germinal centre B cells that are blocked in their differentiation to mature, antibody-secreting plasma cells. 32 Dysregulation due to point mutations: PRC2 subunit EZH2 • Mutations (usually Y641F) in the EZH2 catalytic domain are found in 712% of FL and 22% of GCB-DLBCL cases. • These mutations increase EZH2 catalytic activity (ability to convert H3K27me2 to H3K27me3). Y641 H2AK119Ub 33 Dysregulation due to point mutations: PRC2 subunit EZH2 • EZH2-Y641F promotes germinal centre hyperplasia and accelerates lymphomagenesis in mice, demonstrating it to be a driver mutation. Souroullas et al., Nat Med 2016 34 EZH2-Y641F ectopically silences genes that inhibit B cell proliferation EZH2 represses Blimp1 and Irf4 expression, blocking plasma cell differentiation. Caganova et al., J Clin Invest 2013 EZH2 represses the pro-apoptotic gene Cdkn1a (p21WAF1), maintaining cell survival. Beguelin et al., Cancer Cell 2013 35 EZH2 inhibitors block DLBCL and FL cell proliferation • EZH2 histone methyltransferase inhibitors compete with the methyl donor S-adenosylmethionine (SAM) for the EZH2 active site. • EZH2 inhibition decreases proliferation of DLBCL cells and inhibits the growth of EZH2 mutant DLBCL xenografts in mice (McCabe et al., 2012; Qi et al., 2012; Knutson et al., 2012). • Tazemetostat (EPZ-6438) is now licensed for treatment of relapsed refractory FL. McCabe et al., 2012 36 Dysregulation due to histone mutation: H3K27M in paediatric glioblastoma • 80% of cases of diffuse intrinsic pontine glioma (DIPG) exhibit mutation of two of the genes encoding histone H3 (H3F3A and HIST3H1B), changing K27 to M27 (Lewis et al., Science 2013). • These mutations cause reduction in the level of H3K27me3 and an increase in H3K27ac. • But some genes exhibit increased H3K27me3, including CDKN2A (p16INK4A) (Mohammad et al., Nat Med 2017). Lewis et al., 2013 37 BRD4 and EZH2 inhibitors block DIPG tumour growth • DIPG tumour growth can be blocked by BET inhibitors, which antagonise BRD4 binding to H3K27ac (Piunti et al., 2017). • EZH2 inhibitors also inhibit DIPG tumour growth… ...due to reactivation of CDKN2A (p16INK4A), repressing cell proliferation. Dox-inducible p16 construct. Mohammad et al., 2017 38 more common that the histone regulator is mutated but there are examples of the histone having mutations too eg DIPG Summary 1. Chromatin can become dysregulated due to chromosomal translocations or mutation of genes encoding histone modifiers, or histones themselves. 2. MLL is a proto-oncogene whose dysregulation by chromosome translocations can cause leukaemia or lymphoma, respectively. 3. Gain-of-function EZH2 mutations cause FL and GCB-DLBCL by keeping pro-differentiation and apoptosis genes repressed. 4. H3K27M mutations cause DIPG. 5. BET domain protein inhibitors counteract the oncogenic effects of C-Myc and H3K27M. 6. EZH2 inhibitors counteract the oncogenic effects of gain-offunction mutant EZH2 and H3K27M. 39 Further reading Baylin and Jones. (2011). A decade of exploring the cancer epigenome — biological and translational implications. Nature Reviews Cancer 11: 726-734. Shi and Vakoc. (2014). The mechanisms behind the therapeutic activity of BET bromodomain inhibition. Mol Cell 54(5):728-36. Schuettengruber et al., (2017). Genome Regulation by Polycomb and Trithorax: 70 Years and Counting. Cell 21;171(1):34-57. Comet et al., (2016). Maintaining cell identity: PRC2-mediated regulation of transcription and cancer. Nat Rev Cancer 16(12):803-810. Ribich et al., (2017). Drug Discovery and Chemical Biology of Cancer Epigenetics. Cell Chem Biol. 24(9):1120-1147. 40