Chromatin and Epigenetics PDF

Chromatin and Epigenetics Epigenetics The study of how environmental and behavioural factors can a6ect how genes function For example, the way the chromatin is structured Nucleosomes located at a gene promoter can limit the transcription e6iciency Unless hist...

Chromatin and Epigenetics Epigenetics The study of how environmental and behavioural factors can a6ect how genes function For example, the way the chromatin is structured Nucleosomes located at a gene promoter can limit the transcription e6iciency Unless histones are acetylated or methylated a certain way for example Chromatin Tightly packed eukaryotic DNA and proteins Allows DNA to fit within nucleus Controls gene expression Allows segregation of chromosomes during mitosis Protects DNA from lesions Heterochromatin Highly condensed form of chromatin Typically found near centromeres and telomeres Also found elsewhere Makes up ~10% of DNA in mammalian cells Contains few genes Any euchromatin that is changed to heterochromatin has its genes switched o6 "The position e6ect" Sometimes the cause of di6erential gene expression in di6erent cell types in the early embryo Euchromatin Nucleosomes Fundamental subunits of chromatin 147bp of DNA wrapped around an octamer of paired histones H3, H4, H2A, H2B Linked by DNA of 38-53bp Simplest form of repeating nucleosome units; beads on a string Image Protection against nuclease digestion Method used to study nucleosomes Micrococcal nuclease (Mnase) Endo- and exo-nuclease Preferentially digests naked DNA between nucleosomes Mnase-seq  Sequence undigested DNA to determine protected sequences Design of nucleosomes Image Roughly 11nm in diameter and 5.7nm thick Histone dimerisation Helix bundle Histones Highly conserved, small, basic proteins Core histones Make up nucleosome 2 of each, H3, H4, H2A, H2B Linker histone H1 H1 is largest - H4 is smallest Linker DNA 38-53bp Amino acid content High levels of lysine and arginine Important for binding DNA Histone Design H3 and H4 combine to make H3-H4 dimer H2A and H2B combine to make H2A-H2B dimer Two dimers combine to form octamer Dimensions 11nm diameter 5.7nm thickness 147 bp of DNA wrapps around in 1.65 turns of flat left-handed superhelix Binds predominantly through hydrogen bonding Between phosphodiester backbone and lysine or arginine residues Histone Fold  Structural motif contained in globular domain Consists of 3 helicases connected by 2 loops Loops make crescent-like shape Also found in other non-histone proteins TAF's (TATA box protein-associated factor Histone Dimerization Histone fold motifs form basis of dimerization interface Allows histones to interact with each other in specific pairings Interaction known as "handshake" Helix bundle interactions H3-H4 dimers interact via 4-helix bundle to form (H3-H4)2 tetramer H2A-H2B dimers are bound to the H3-H4 tetramer via another 4-helix bundle interaction Forms histone octamer Histone Tails Each histone also has an N-terminal flexible tail H2A and H2B also have flexible tails at C-terminal Tails contain many basic residues Lysine, arginine Tails are important for modification Methylation, acetylation, etc. Histone Modification  Examples Acetylation of lysine residues Removes positive charge Loosens nucleosome Mono, di, or trimethylation of lysine or arginine residues Serine phosphorylation Most modifications occur at N-terminal tail Also more than 20 side chain modifications in globular core Enzymes Acetylation Driven by histone acetyl transferases (HATs) Removed by histone deacetyl complexes (HDACs) Methylation Driven by histone methyl transferases (HMTs) Removed by histone demethylases (HDMs) Recruitment of enzymes often requires transcription factors Histone modifications can attract other proteins to the modified chromatin Trimethylation recruits HP1 Contributes to spread of heterochromatin Modifications occur in sets More than 15 have been discovered Di6erent positions of modifications on DNA have di6erent functions Led to histone code hypothesis Is still accepted, but is far more intricate and complex than initially hypothesised How are they read? Di6erent domains on proteins can recognise methyl modifications very specifically PHD domains Present in BPTF (largest subunit of ISWI) Chromodomains HP1 TUDOR SGA HAT module MBT Bromodomains recognise acetylated histone tails Di6erent versions can recognise modifications at certain residue positions and can discriminated between mono-, di-, and trimethylation Histone Modifying enzymes  Writers add modifications Erasers remove modifications Readers read modifications Histone modifying enzymes (writers) SAGA complex Functions Acetylation through Gcn5 HAT subunit Helps activate transcription Deubiquitination through deubiquitinase (DUB) module Primarily targets H2B Removes ubiquitin from histones Writer reader model Writers methylate nucleosomes Readers form heterochromatin by pulling nucleosomes together Creates positive feedback loop Keeps regions repressed Modification example: H3K4me3 Trimethylation on lysine 4 of histone H3 tail Always in region of promoters Can be used to determine activity of genes by simply determining amount of H3K4me3 Histone Code suggests that the combination of specific chemical modifications on histone proteins (like methylation, acetylation, phosphorylation, and ubiquitination) forms a code that determines how tightly or loosely DNA is wrapped around histones, ultimately influencing gene expression Examples One type of marking demonstrates that a stretch of DNA has been newly replicated Another signals that DNA is damaged and needs to be repaired Others signal when and how gene expression should take place Histone variants Each histone (other than H4) has a set of less common variants Only di6er at a couple amino acids Examples H2A variants H2AX DNA repair and recombination H2AZ Gene expression Chromosome segregation macroH2A Transcriptional repression H3 variants H3.3 Transcriptional activation CENP-A Centromere function Kinetochore assembly  Each are present at certain points of chromosomes DNA methylation At cytosine Functions Gene expression repression Development and cell di6erentiation Genome stability Repression of repetitive elements (transposons etc.) and heterochromatic regions Prevents movement of transposons Epigenetic inheritance Methylation pattern is passed from one cell generation to the next Example: when stem cell di6erentiates, methylation keeps unneeded genes silenced Cancer and disease Loss of methylation can lead to activation of oncogenes Can lead to tumorigenesis Increased methylation can silence tumour suppressor genes Cannot stop continuous proliferation More methylation is lost with age May cause increase in cancer risk in elderly Nucleosome assembly Histones don’t naturally form nucleosomes Instead, bind non-specifically to naked DNA Histone chaperones are required for nucleosome assembly Positioning is mostly influenced by other DNA-bound proteins Some favour a nucleosome adjacent Some provide obstacles and force nucleosome to move ATP- Dependent Remodelling Complexes Allow for DNA access ATPase subunit binds to protein core and wound DNA Uses energy from ATP hydrolysis to move DNA relative to the core Nucleosome sliding Remodelling complexes can also interact with chaperones Can partially (H2A-H2B dimer) or fully remove nucleosomes from DNA Estimated that nucleosomes are replaced every 1-2 hours Cells contain dozens of remodelling complexes Most are large and contain 10+ subunits Some bind specifically to histone modifications Activity is carefully controlled Epigenetics and Gene Expression Transcription Factor binding Most transcription factors Cooperativity allows nucleosome/chromatin binding Simultaneous binding with other factors Pioneer transcription factors Independent nucleosome/chromosome binding Precedes other factors binding Chromatin dynamics Inaccessibility leads to a dependence on chromatin dynamics Achieved by ATP dependent chromatin remodelling Nucleosome Positioning Nucleosome positioning is extremely well defined on active gene Nucleosomes present at peaks of graph NDR Nucleosome depleted region Present at transcription start site ATP- Dependent Remodelling Complexes Allow for DNA access ATPase subunit binds to protein core and wound DNA Uses energy from ATP hydrolysis to move DNA relative to the core Nucleosome sliding  Act like two hands that push and pull DNA molecule Pushes away nucleosomes Remodelling complexes can also interact with chaperones Can partially (H2A-H2B dimer) or fully remove nucleosomes from DNA Estimated that nucleosomes are replaced every 1-2 hours Cells contain dozens of remodelling complexes Most are large and contain 10+ subunits Some bind specifically to histone modifications Activity is carefully controlled Types of remodellers SWI/SNF complex Makes DNA accessible for transcription Slides nucleosomes Ejects nucleosomes Alters nucleosome structure Second most mutated gene across all cancers INO80 complex Nucleosome sliding Histone exchange and replacement Especially H2A.Z (good for transcription DNA damage repair Important for making damaged DNA accessible to repair machinery Replication stress response Stabilises replication forks and prevents replication stress Any condition that slows down or disrupts replication ISWI (imitation switch) complexes Involved in nucleosome spacing and assembly Also used to reassemble nucleosomes after replication forks have passed Examples NURF CHRAC CHD (chromatin helicase DNA-binding) complexes Play roles in nucleosome sliding and histone variant exchange Some CHDs recognise methylated histones and modify chromatin in response to specific marks Crucial for regulating chromatin structure in response to histone modifications FACT (facilitates chromatin transcription) complex Helps to disassemble and reassemble nucleosomes during transcription Temporarily removes one of the H2A-H2B dimers from nucleosome  Allows RNA to transcribe through nucleosome High Order Chromatin organisation Chromosome conformation capture (3C) assays Techniques used to study 3D organisation of genome Allows scientists to map physical interactions between di6erent regions of DNA Principle 1. Crosslinking DNA and associated proteins are chemically crosslinked to freeze their interactions in place 2. Digestion Crosslinked chromatin is then digested with restriction enzyme DNA is cut at specific sites Interacting regions are kept due to crosslinking 3. Ligation Fragments are re-ligated Fragments that were physically close in the 3D space are more likely to ligate together 4. Detection Hybrid molecules are purified and analysed Identifies which regions of the genome are interacting Uses Gene regulation Understand how distant enhancers regulate genes by physically looping to their promoters Chromatin compartmentalisation Identifying regions of eu- and heterochromatin Structural domains Mapping topologically associating domains (TADs) ChiA-PET Specialised type of 3C Used to investigate DNA-protein interactions Can be useful in determining 3D structure of transcriptional regulation Steps 1. Crosslinking Same as 3C 2. Chromatin immunoprecipitation (ChIP) Antibodies specific to a protein of interest are used to pull down chromatin regions bound by that protein Enriches for DNA interactions mediated specifically by targeted protein 3. Digestion and ligation Pulled down DNA fragments are digested and nearby regions are religated 4. Paired-end tag sequencing Ligated fragments are sequenced using paired-end sequencing Provides info about interacting regions Identifies loci bound by protein and physical proximity in 3D space Hi-C  High-throughput version of 3C Used to provide genome-wide map of chromatin interactions Can capture interactions across all chromosomes simultaneously How to read Hi-C map Shown is the small green region of chromosome 14 Blue region for example To determine if it has interacted with anything, you must read in the direction of the arrows Comes across dot on graph Regions were able to be linked as they were in close prox Means blue region has tendency to interact with orange region Means there are parts of chromatin that tend to form loops Topologically associated domains (TADS) Genes within TADs tend to be activated at the same time