L14 Epigenetics: Histone Modification and Gene Expression PDF

L14 Epigenetics: Histone modification and gene expression Dr Stuart Knight 5BBG0205 Molecular Basis of Gene Expression Histone modification and gene expression Department Biochemistry...

L14 Epigenetics: Histone modification and gene expression Dr Stuart Knight 5BBG0205 Molecular Basis of Gene Expression Histone modification and gene expression Department Biochemistry Learning Outcomes Describe chromatin structure Differentiate the function of open and closed chromatin Describe the process of chromatin remodelling Outline the different types of histone modification Understand the link between chromatin modifications and changes to gene expression Outline how drugs can be used to change gene expression by altering chromatin modifications Describe the process of X-chromosome inactivation  Epigenetics Inheritance of differences in gene function that are not caused by changes in the primary sequence of DNA Pattern of gene expression is inherited by cells after cell division Changes don’t affect the nucleotides they affect the pattern of expression. These can be inherited by daughter cells. Chromatin describes structures where DNA binds to proteins. Unit = nucleosome. Packaging of DNA Human genome is highly condensed i.e. Chr22 DNA stretched end-to-end is 1.5cm long As a mitotic chromosome, Chr22 is 2μm Therefore compaction ratio ~10,000 fold Role of chromatin Package DNA into a small volume to fit into a cell Regulate access to the DNA sequence during transcription and replication Lock-in patterns of gene expression Packaging with chromatin allows for a 10,000-fold compaction. Level of activity of a gene can be varied by chromatin. 2  Higher Order DNA Packaging DNA molecules are highly condensed in chromatin Histones and non-histone chromosomal proteins organise higher level structure Histones make up the first order of structure, the nucleosome Nucleosomes coil into 30 nm Chromatin fibre Chromosome scaffold proteins provide higher order organisation Fibres loop out from central core in chromosome. Nucleosome Structure Five histone subunits H1, H2A, H2B, H3 and H4 Nucleosome core assembled sequentially from 2x H2A-H2B dimers plus H3-H4 tetramer – an ocatmeric structure 1.8 turns of DNA wrap around nucleosome core Histone H1 binds to the linker region Octameric central core of histones. Two copies of each protein. 1.8 turns of DNA wrapped around nucleosome core. Histones (H1) link nucleosomes together. 3  Matrix-attachment regions (MAR) / Scaffold- attachment regions (SAR) Scaffold/matrix attachment regionScaffold/matrix attachment region Fig 12.14 Genetics: Analysis and Principles : Robert Brooker : McGraw Hill Loops important for arrangement of gene expression – often contain genes that are expressed in a similar way or contain locus control regions e.g. globin genes are grouped together in the chromosome and are expressed sequentially, suggesting coordinated regulation. Pattern of Gene Expression Tissue-specific variation in gene expression Variation in which parts of the genome are transcriptionally active Chromatin plays a role in regulating this Tissue diversity defined by gene expression. Genes turn on and off in response to external stimuli, e.g. cAMP pathways Chromatin regulates access of transcription factors to the DNA. 4  Heterochromatin and Euchromatin Chromatin in interphase nuclei found in two forms highly condensed heterochromatin : INACTIVE less condensed euchromatin : ACTIVE Heterochromatin is condensed so transcription factors cannot get access to it. What Does Active Chromatin Mean? Active Genes are active Genes are being expressed Transcription is taking place Open Transcription factors and RNA polymerase has access to the DNA 5  Chromatin Remodelling Nucleosome structure or position can be altered Chromatin remodelling complexes e.g. SWI/SNF Hydrolysis of ATP provides energy Change in the location of nucleosome Change in how tight the DNA is bound Promotes the exchange of histones in nucleosomes Chromatin is not static, it is dynamic in order to facilitate gene expression. Illustration of Chromatin remodelling Histones move to change which part of the DNA is exposed for the transcriptional machinery to get access. 6  Histone variants H3.1 / H3.2 / H3.3 very similar to H3 – involved in transcriptional changes in development H3t is specific to the nucleosomes in testis CENP-A is replaces H3 in nucleosomes of the highly condensed centromeric region – only 46% identical to H3 H2A.X found in nucleosomes in inactive X-chromosome CENP-A ensures heterochromatin stays condensed. Histones don’t have introns, so they are very highly conserved between species. Histone function in humans and yeast is identical. Modification of Histones N-terminal tails of Histones H2A, H2B, H3 and H4 can be modified Acetylation of lysines Histone acetyltransferase HAT Histone deacetylases HDAC Methylation of lysines and arginines Mono-, di- and tri-methyl derivatives are possible Specific histone methytransferases (HMT) and demethylases Phosphorylation of serines and threonines Ubiquitination of lysines Sumoylation of lysines Small Ubiquitin-like Modifier (or SUMO) Lysine = K Arginine = R Serine = S Threonine = T 7  N terminal tails are sticking out from core region of histones, Modification of lysine by acetylation and methylation Kme1 Kme2 Kme3 ‘Histone Code’ Each nucleosome contains histones that are potentially modified in a large number of different ways Pattern of modification is duplicated during DNA replication R: arginine, K: lysine, S: serine, T: threonine Considered to be second genetic code as changes to histones have important consequences on whether a gene is expressed or not expressed. Set of residues that are modified will very and these changes will be inherited following mitosis. Changes can be inherited in the germ line 8  Acetylation and Chromatin Histone Acetyl Transferase (HAT) Donor is acetyl-CoA p300 / CBP (CREB-binding protein) complex p300 / CBP is transcriptional activator - response of cAMP activation GNAT family and MYST family Hyperacetylation of histones leads to active /open chromatin e.g. H3K14ac and H4K16ac High levels of acetylation leads to an OPEN conformation, i.e. genes are ACTIVE. Histone deacetylases HDAC 4 families > 11 enzymes Specificity e.g. H4K5ac is preferentially deacetylated by HDAC3 Non histone substrates e.g. HDAC1 Some cytoplasmic and nuclear location e.g. HDAC4 Leads to a REDUCTION in expression. 9  Acetylation link to open chromatin Hyper-acetylation of histones reduces the close packaging of neighbouring nucleosome Acetylated histones creates nucleosome that bind the DNA less tightly Removal of acetyl group allows the same lysine to be methylated instead leading to condensation and inactivation of chromatin H3K9ac – activation H3K9me - repression Lysine is positively charged and basic Acetylation reduces positive charge so the negatively charged DNA is not bound as tightly. If the same residue is methylated, then transcription turns off. Therapeutic HDAC inhibitor SAHA (suberanilohydroxamic acid) Binds to the active site of HDAC and chelates zinc ions involved in the enzyme reaction Inhibits the removal of acetyl groups and chromatin remains in open state – genes being active Licenced for treatment of Cutaneous T cell Lymphoma (CTCL) Changing gene expression is a significant drug target. HDAC removes acetyl group, so blocking HDAC keeps the gene active. Drug is nonspecific, but can have a therapeutic advantage by activating genes. 10  Histone Methylation and Chromatin Methylation of histones does not affect the charge of to the amino group Methylation of some specific lysines associated with active chromatin Trithorax group (trxG) proteins methylates H3 lysine 4 (H3K4) Methylation of some specific lysines associated with inactive chromatin Polycomb group (PcG) proteins methylates H3 lysine 9 (H3K9) Protein HP1 binds to H3K9me3 and condenses chromatin Other proteins bind to nucleosome that has methylated nucleosomes – effect depends on which protein binds. DNA methylation Methylation of DNA also affects chromatin structure Methylation of cytosine (5’-methyl cytosine), ~5% bases most in CpG dinucleotides 5’ Cytosine-Phosphate-Guanidine-3’ DNA methyltransferase (DNMT1, DNMT2, DNMT3) Transfer of methyl group from S-adenosylmethionine (SAM) DNA DNA https://www.mdpi.com/cells/cells-08-00953/article_deploy/html/images/cells-08-00953-g001.png 11  CpG locations CpG sites often clustered into islands Hyper-methylation of CpG islands located at promoters linked with inactive chromatin Hyper-methylation of CpG at other locations is not linked to changes in transcription CpG island = lots of CpGs Methylated cytosine associated with region of inactive chromatin by promotors. – Therefore, gene expressed more. Role of DNA methylation Tissues-specific variation in methylation Pattern of methylation is maintained during DNA replication Methylated DNA binds to MeCP2 (methyl CpG binding Protein 2) MeCP2 recruits HDAC which leads to inactive chromatin Mutation in MeCP2 gene causes neurodevelopmental disorder Rett syndrome MeCP2 recruits HDAC histone deacetylase so gene expression is less likely. 12  Therapeutic DNA methylase inhibitors Some tumours become resistant to chemotherapy due to epigenetic changes in gene expression of tumour suppressor genes 5-azacytidine is a cytidine analogue – gets incorporated into DNA but cannot be methylated https://media.springernature.com/lw685/springer- static/image/art%3A10.1186%2Fs13148-016-0237- y/MediaObjects/13148_2016_237_Fig1_HTML.gif Prevents DNA methylation X-chromosome inactivation At fertilization female cells both Xs are active In humans random inactivation occurs at gastrulation 50% cells active maternal and 50% cells active paternal X - somatic mosaic Inactive X is bound with Xist (X-inactive specific transcript) Xist is 17kb transcript expressed from inactive X gene Binding of Xist recruits polycomb group methylases Inactive X is heterochromatin present in nucleus as Barr body 13  Genetic difference between sexes Female XX homogametic sex Male XY heterogametic sex Functionally female cells do not have two Xs X-inactivation is the mechanism of dosage compensation. Female cells only have a single active X chromosome (same as male cells) In birds male cells are homogametic ZZ and female cells are heterogametic sex ZW Chromatin changes in inactive X Increase in DNA methylation Reduced histone H3 lysine-4 methylation High levels of histone H3 lysine-9 methylation (H3K9me2 and H3K9me3) H3K9me2 and H3K9me3 bind to Heterochromatin protein 1 (HP1) High levels of H3 lysine-27 methylation Increased ubiquitination of H2A Incorporation of H2A.X into nucleosomes 14  Genomic Imprinting Diploid cells have two alleles for genes on autosomes (non- sex chromosomes) Both alleles are active Some genes are imprinted Parental origin of gene determines which gene is active Insulin-like growth factor 2 (IGF2) only expressed from paternal allele Epigenetic regulation is fixed due to parental origin Mutations can affect the epigenetic modification of imprinted genes Angelman syndrome / Prader-Willi syndrome Important because it only comes from paternal and if there is a mutation on the paternal line, it can cause disease. Summary Chromatin structure (review) Basis of epigenetics Nucleosome remodelling Histone modification X-chromosome inactivation Genomic imprinting 15