13_10 DNA Methylation and Gene Regulation (2).pdf

UCL Cancer Institute Faculty of Medical Sciences DNA methylation and gene regulation Dr Sarah Koushyar [email protected] MSc Cancer 2 Part 1 Epigenetics and DNA methylation Learning Outcomes Following this topic, students should be able to: • Demonstrate an understanding of epigenetics. •...

UCL Cancer Institute Faculty of Medical Sciences DNA methylation and gene regulation Dr Sarah Koushyar [email protected] MSc Cancer 2 Part 1 Epigenetics and DNA methylation Learning Outcomes Following this topic, students should be able to: • Demonstrate an understanding of epigenetics. • Understand and explain the types of epigenetic mechanisms. 3 4 What is epigenetics? Heritable change not due to changes in the DNA sequence. Information is ’added’ to DNA. “Branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being” Waddington, 1942 Lifestyle and epigenetics 5 “You are what your grandparents ate” Lamarck´s theory of evolution states that an organism can pass physical characteristics acquired during its lifetime on to its offspring. 6 Epigenetic mechanisms Primarily two mechanisms identified. 1. DNA methylation. Methyl groups added to bases to inhibit gene transcription. 2. Histone modification. Different molecules attach to the tails of the histones, altering chromatin structure. 7 DNA methylation Does not change DNA sequence. Stable. Inherited through cell division due to maintenance methylases. They copy the methylation pattern from the parent strand during DNA replication in some species, meiosis can pass down epigenetic patterns. Epigenetic changes may explain why some individuals are more at risk for common diseases (more research is needed). 8 9 DNA methyation DNMT1 DNMT3A DNMT3B in bacteria, adenine can be methylated too • Cytosine C5 methylation-covalent modification of DNA (stable). • Occurs throughout the genome. • Particularly on CpG dinucleotides repeats (in mammals 60-90% CpG islands are methylated). CpG islands (have around 200 of the dinucleotides but are un-methylated) • Methylated adds information to the DNA sequence but doesn’t disrupt the DNA base pairing. Histone modifications Core histone tails are free from the nucleosome and are accessible Often are modified • Methylated (no charge) • Acetylated (-ve charge) • Phosphorylated (-ve charge) Covalent modifications 10 11 Other epigenetic modifications In addition to DNA methylation and histone modifications, RNA can also play a role in epigenetics. Aristizabal et al., PNAS 2020. 12 Part 2 DNA methylation and transcription Learning Outcomes Following this topic, students should be able to: • Explain what DNA methylation is. • Discuss how the biological functions of DNA methylation depend on context and location. • Explain the mechanisms through which DNA methylation affects transcription. 13 14 DNA methylation Water mediated de-amination (through metabolism of certain foods ?) can cause passive demethylation 15 DNA methylation can silence gene expression 1. Promoter DNA methylation-> TFs cannot bind to the promoter. 2. Recruits other proteins that recognises methylated DNA (MeCP2) that recruits histone modifiers and nucleosome remodelling proteins (HDACs). 3. Methylation can also occur at enhancer regions. e.g. MeCP2 e.g. at germline specific gene locus Typical DNA methylation landscape they are mobile DNA sequences capable of replicating themselves within genes independently of the host cell DNA All methylated • Stable modification: Patterns can be transmitted for more than 80 cell divisions. un-methylated but CpG shores can be • Predominantly found at CpG dinucleotides. usually methylated • Human genome contains 28 millions CpG. • At least 70% of all CpGs are methylated. 16 Spontaneous mutations CpG is under-represented in the mammalian genome (due to propensity for mC to deaminate into T) 17 CpG islands • First identified as small fragments post digestion with methylation-sensitive restriction enzymes with higher than expected CG. Digested nature indicates UNMETHYLATED. Function of the CpG islands: usually in the promoter regions, but not all promoter regions have CpG islands • - Definitions vary: Bioinformatic definition is: > 200bp GC >50% O/E (observed/expected) ratio of CpG >60%. • There are around 25,000 CpG islands in the human genome. • Around 50% of human genes have a CpG island in their promoter region. 18 19 CpG islands DNA methylation at/close to promoters can change according to expression state but not within CpG islands. exons introns CpG Island promoter PBK Sperm ES Cell Fibroblast Brain upstream and downstream of the CpG islands are variable levels of methylation (activation/deactivation) DNA methylation is thought to play an important role in the silencing of tissuespecific genes to prevent them from being expressed in the wrong tissue. Transposable elements TE: ‘jumping genes’. DNA sequences that move from one region of the genome to another. • Can disrupt the genome architecture by mediating large structural genomic variation such as deletions, inversions, duplications and translocations. • TEs litter the genome and therefore are major determinants of genome size and composition in eukaryotes. 20 Transposable elements TE: ‘jumping genes’. DNA sequences that move from one region of the genome to another. Human version Mouse version Bestor, 2003. 21 Types of transposable elements • Around 45% of mammalian genome is transposons. • Retrotransposons (class 1):  Long Terminal Repeat (LTR)  Non-LTR: 1. Long INterspersed Elements (LINE). 2. Short INterspersed Elements (SINE). • DNA Transposons (class II). 22 23 Genomic distribution of CpG and mCpG CpG Distribution Predominately found in SINE and LINE repeats mCpG Distribution -Most CpGs are found in repeats -Most methylation is found in repeats Rollins et al., Genome Res. 2006 cancer healthy Retrotransposons are hypomethylated in cancer 24 Tubio, Science 2014 DNA methylation affects gene expression Mechanisms: Direct 1) DNA methylation within target genes (promoter, gene body) controls correct gene expression. 2) DNMTs control correct gene expression. Indirect 1) DNA methylation at enhancers controls correct gene expression. 2) DNA methylation at repeats controls correct gene expression. 3) DNMT protein activity controls expression of transcriptional regulators that directly affect perturbed genes. 4) DNA methylation is necessary for genome stability. 25 Regions that can acquire DNA methylation Transcription Factor Promoter Repeats % mCG Enhancer Active Gene Enhancer Inactive Gene CG-poor Promoter CGI Promoter - Intermediate/Poor CpG Islands (e.g. during in vitro cellular differentiation). - CpG Island Shores-regions 2kb from classical CpG islands. - Gene body (correlates with transcription). Note: 70% of CpG islands associated with promoters normally unmethylated irrespective of gene transcription state. Adapted from Schubeler, 2015 26 How DNA methylation can affect transcription 1. DNA methylation prevents TF binding. 2. DNA methylation recruits Methyl Binding Proteins (MBP), which recruit co-repressor complexes. 3. DNMTs are physically linked to HDAC and HMT activities, which couples methylation to transcription repression and chromatin modification. 27 Moore et al, 2013 Maintaining mono-allelic silencing 28 DNA methylation plays a key role in Xchromosome inactivation, a process that achieves dosage compensation for Xencoded gene products. Developmental stages • X inactivation is stable • Initiated by Xist • Xist is non-coding RNA • Maintained by DNA methylation • Highly compact inactive chromatin structure DNA methylation regulates imprinted loci Some sec ons of the genome are imprinted on either the maternal or paternal chromosome Diploid cells (soma c cells) have two alleles (copies) of a gene that is both expressed On chr 15 if the same large sec on is imprinted On the maternal chr = Angelman syndrome On the paternal chr = Prader-Willi syndrome Is it DNA methyla on that determines which gene is switched off Whilst imprin ng is a normal part of physiology, if it occurs in certain gene loci, disorders can arise 29 DNA methylation regulates imprinted loci 30 ICR – imprinting control region ICR is methylated on the paternal Chr. CTCF cannot bind to the insulator and the enhancer can loop back and switch on Igf2 Deregulated methylation of Igf2 (chr 11): Beckwith-Wiedemann Syndrome (BWS) DNA methylation is important for genome stability 31 Hypomethylation of satellite DNA in chromosomes 1, 9 and 16: Immunodeficiency, Centromeric region Instability, Facial Anomalies (ICF) Syndrome. Chromosomal breaks Xu et al., Nature 1999 Summary DNA methylation is an example of an epigenetic modification. Predominantly occurs at CpG dinucleotides. Most CpGs are found in repeats-these repeat sequences can cause transcriptional interference. Outside of repeats, methylation states of CpG islands depend on context. Promoter associated CpGs typically unmethylated, irrespective of gene transcription states. Biological function of methylation depends on context! 32 33 Part 3 Regulation of DNA methylation and cancer Learning Outcomes Following this topic, students should be able to: • Explain the mechanisms regulating DNA methylation. • Detail the types of DNA methylation changes that occur in cancer. 34 Enzymes mediating DNA methylation 35 DNMT1 • Main function in maintenance of global methylation patterns. 36 Expression of IAP in red • Deficiency in embryos/ES cells results in methylation at 5% of normal levels. • Essential for development. • Biallelic expression of imprinted loci. • Reactivation of silenced retrotransposons (e.g. IAP RNA). IAP is an example of a retrotransposon • Increased genome instability. Walsh et al., Nature 1998 DNMT3A and DNMT3B • Predicted to exist based on fact that Dnmt1deficient ES cells can establish methylation at newly integrated Mo-MLV (retrovirus). 37 DNMT3B -/- doesn’t make it past the embryo stage • Deficiency causes different phenotypes, but partial functional redundancy exists. • Both are important in DNA methylation establishment. • DNMT3A more important in de novo methylation in gametogenesis; DNMT3B required for minor satellite methylation. Knockout of A or B results in stunted growth - but isn’t lethal Okano et al., Cell 1998 DNA methylation abnormalities in cancer 38 Both hypo- and hyper- methylation can occur in cancers. Are seen in virtually all types of cancer. Have patterns that often differ among different types of cancer and different specimens. Can occur very early in tumorigenesis. Epigenetic gene inactivation can be as common as mutational events in the development of cancer. Often increases with tumor progression. Hypomethylation is global whereas hypermethylation is focal. 39 Hypomethylation in cancer The first epigenetic event seen in change was hypomethylation microsatellites = repeats • can be extended Global hypomethylation in early stages of colon cancer, breast cancer and chronic lymphocytic leukemia (CLL). Hypomethylation seen in prostate metastatic tumours vs primary. Hypomethylation of Sat2 repeats in Wilms tumour, ovarian and breast cancer. Wilson et al., BBA 2007 Hypomethylation in cancer Hypomethylation seen in prostate metastatic tumours vs primary. doi: 10.1158/0008-5472.CAN-07-6088 40 41 deregulated methylation in cancer Genetic model for colorectal tumorigenesis. CRC occurs through accumulation of genetic and epigenetic events. Fearon & Vogelstein, Cell 1990 DOI:10.1038/nrgastro.2011.173 Hypermethylation in cancer CpG Island Methylator Phenotype (CIMP) • hypermethylation of numerous CpG islands surrounding the promoter regions of several genes. • First described in colorectal tumors; since identified in multiple types of cancers (bladder, gastric, glioblastoma). CIMP associated with MI. • MI+ CRC tumors have faster rate of adenoma to carcinoma progression. 42 43 Hypermethylation in cancer Example of a gene that is hypermethylated in CRC is MLH1. Post replicative DNA mutations and Microsatellite instability (MSI). These tumours account for 15% of all CRCs And have a distinct prognosis (MSI-CIMP-H) Mutations in DNMT3A and cancer Brunetti et al,. Cold Spring Harb Perspect Med 2017 • 20% AML patients have heterozygous mutation in DNMT3A. • ~60% of mutations found at R882. In the catalytic site of the gene • Mutation functions as dominant negative, inhibiting methyltransferase activity. 44 DNMTs are involved in tumourigenesis 45 Mutations in TET 46 Rasmussen & Helin, Genes Dev. 2016 Summary • DNA methylation patterns are established & maintained through the combined actions of DNMT1, DNMT3 & TET family enzymes. • Genomic patterns of DNA methylation are perturbed in cancer. Two changes commonly observed: -Global hypomethylation (principally at repeats) -Focal hypermethylation (promoters and other regions) • Mutations in DNMTs have been identified using exon-sequencing approaches; best example to date is DNMT3A R882H mutation in AML. • Genetic data in animal models indicate that DNMTs are involved in cancer but exact roles & mechanisms (protein, DNA methylation) remain unclear. 47