Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Dept. Wenner-Gren of Molecular Institute Biosciences, for TheExperimental Wenner-Gren Biology Institute Chromatin, the packaging of chromosomes, and the epigenome is a regulator of nuclear processes Ann-Kristin Östlund Farrants Stockholm University [email protected] Chromosomes The chromosomes take up most of the space in the eukaryotic nucleus. DNA is bound by proteins = chromatin www.cellnucleus.org Chromosomes The DNA is packaged with ptoteins to condense it about 1000 fold The chromosomes are even more highly packed at mitosis – 10 times more condensed hromosomes. The packaging of chromosomes DNA is wrapped around histone cores to form nucleosomes. 1.7 turns, 146 bp The nucleosomes, in turn, are further package by association by H1 to form the 30 mm fibre. Linker reagion 30-60 bp The 30 nm fibre is further package with non-histone protein. Alberts et al. Crystal structure 2.8 Å resolution Luger et al. The structure of the nucleosome Histone octamer – tetramer of H3-H4 2 dimers of H2A-H2B EM image of salt extracted chromosomes Beads-on-a-string Chromosomes have different regions with different functions. Heterochromatin – densly packed Telomeres, centromeres, silent regions Euchromatin – less densly packed Regions with actively transcribed genes What is specific for the different chromosome regions? Different histone modifications Different proteins associated with the different regions RNA At least 7 different chromatin structures Histone variants CEN-A – centromer, kinetochore H3 synthesised late in S-phase H3.3 – H3-form synthesised outside of Sactive transcribed regions phase, in H2Az/v – H2A formed during G1 and G2, in transcription. H2Ax – 2-25% of all H2A, phosphorylated damage upon DNA Other proteins Histone H1 – 30 nm fibre Non-histone proteins – HMG box proteins, structural proteins. Specific proteins for chromosome region – heterochromatin proteins, polycomb proteins (silenced), transcription factor (active) The different states are dynamic and are different in different cell type. - different in different cell type - changes depending on cell state Changes upon differentiation or on envronmental cues Differentiation – changes in gene expression Waddington’s epigenetic landscape Differentiation is following paths Differentiation is tipping the balance Saddled-node landscape: Commitment is the disappearing of valleys – Ferell Jr Processes in the nucleus Replication Transcription RNA processing (capping, splicing, polyadenylation of mRNA, rRNA modification) Recombination DNA-repair Export of RNPs, import of protein Histone modifications • 25% N-terminal part of the histones are not in the core region. • N-terminal tail – lysines, serines, argenines • N-terminal tails are modified by; acetylation, methylation, phosphorylation, ubiquitinylation, ribosylaton Histone code • Modifications located in heterochromatinmethylation of H3lysine9, H3K27, H4K20me3, H3R2 Allis and Jenuwein • Modifications in actively transcribed regions – Methylation of H3K4, Acetylation of H3K9, H3K27 Acetylation of H4 DNA-methylation also affects chromatin structure Methylation at the promoters inactivates transcription slideshare The dynamic chromatin structure Inactive Me me Enzymes: DNA methylases/demethylases Acetyl transfeases/deacetylatses TF Use energy to change the structure of the nucleosome A c Ac mRNA Active Epignetic alterations Enzyme puts on a modification, other protein binds, and yet other removes the modifications- modifier Binding protein Eraser Epignetic alterations Enzyme puts on a modification, other protein binds, and yet other removes the modifications- HAT Activator HDAC In addition -Move nucleosomes – nucleosome remodellers Histone modifications are differently distributed along active genes (J. Mellor) H3K4trimet K3K4dimeth H3K36 -metn H3K-Ac H4K-Ac MODIFIERS • Acetylation-Histon acetyltrasferases (HAT) • Deacetylation-histone deacetylases (HDAC) • Methylation- Methyl transferases • Demethylation- demethylases • Ubiquitinylation – Ligases • Deubiquitinylation deubiquitinylases • Phosphorylation –kinases • Dephosphorylation-phsosphtases The histone marks are decoded by proteins: Bromodomains – Ac Chromodomains – H3K9me3 PhD-domain – H3K4me3 Location of euchromatin and heterochromatin in the nucleus - EM S-phase early Replication is affected by the chromatin packaging, and the location of the different forms of chromatin middle late very late Transcription is also affected by location Between the chromosomes Chromosomes are organised in territories Chromosome loops – functional domains: Silent regions, active regions Insulators CTCF is involved in mammalians cells Insulators – separates functional domains Prevent chromaitn domains from spreading Locus control region domains Monoallelic expressed locus -Imprinted genes Silencing Anchoring to structures Regulate Xchromosome activity Matharu 2015 TADs provide a 3-D structure to the nucleus Change in TAD usage – redistribution of TAD factors Setting the chromosome landscape such that specific genes are transcribed The TADs at enhancers, insulators, promoters also change histone modification Active Enhancer – H3K4me1 and H3K27ac Active Promoter – H3K4me3 and H3K27ac Actively transcribed gene body – H3K36me3 Inactive enhancers and promoters – H3K27me3 and H3K9me3 Some also have dual marks – very accessible Chromatin Immunoprecipitation The active sites are more open, accessible – They are regarded as nucleosome free Enzyme such as DNase I or MNase can cut The active sites exhibit a larger mutation rate than gene bodies. Changes transcription factor binding. RNA is involved in the setting of functional domains Many sites are transcribed – not just genes Required for heterochromatin fomation non-coding RNA is transcribed and recruits factors to specific sites – Centromers in S. pombe X-chromosome inactivation LncRNA can act as glue between sites in cis or in trans The expression of nc-genes determines the spatial organisation Enhancers and promoters are transcribed The ncRNAs binds proteins that targets the site The ncRNA hybridises to other RNA Transcription can also cause R-loops - RNA-DNA hybrids Result in replication forks and DNA breaks Cause recombination in repetitive regions Transcription of ribosomal genes and chromatin remodelling Repetitive genes with special chromatin Hot spot for recombination Transcription Actively transcribed chromosomes are located in the inside of the nucleus, whereas inactive chromosomes parts are located at the periphery. Does this always hold true? Inactive active Model: LCR associates with proteins – Ldb1 Leads to migration Recruitment of the transcription machinery Moving genes depending on their state of activity Inter- and intrachromosomal chromatin loops Schneider R, Grosschedl R Genes Dev. 2007;21:3027-3043 ©2007 by Cold Spring Harbor Laboratory Press Chromosomes translocate according to functional requirement RNA dependent mechanism? Could explain the movement of chromosomes during differentiation Different ncRNAs are expresssed ncRNA are seeding points for proteins Accumulate proteins in subnuclear bodies. 1.ChIP-seq (Chromatin immunoprecipitation sequencing), aimed against different histone modifications, can be used to identify chromatin states throughout the genome. Different modifications have been linked to various states of chromatin. 2.DNase-seq (DNase I hypersensitive sites Sequencing) uses the sensitivity of accessible regions in the genome to the DNase I enzyme to map open or accessible regions in the genome. 3.FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing) uses the chemical properties of protein-bound DNA in a two-phase separation method to extract nucleosome depleted regions from the genome.[16] 4.ATAC-seq (Assay for Transposable Accessible Chromatin sequencing) uses the Tn5 transposase to integrate (synthetic) transposons into accessible regions of the genome consequentially highlighting the localisation of nucleosomes and transcription factors across the genome. 5.DNA footprinting is a method aimed at identifying protein-bound DNA. It uses labeling and fragmentation coupled to gel electrophoresis to identify areas of the genome that have been bound by proteins.[17] 6.MNase-seq (Micrococcal Nuclease sequencing) uses the micrococcal nuclease enzyme to identify nucleosome positioning throughout the genome.[18][19] 7.Chromosome conformation capture determines the spatial organization of chromatin in the nucleus, by inferring genomic locations that physically interact. 8.MACC profiling (Micrococcal nuclease ACCessibility profiling) uses titration series of chromatin digests with micrococcal nuclease to identify chromatin accessibility as well as to map nucleosomes and non-histone DNA-binding proteins in both open and closed regions of the genome.[20] Where does transcription take place in the nucleus? • Specific locations – Transcription factories In specific spots In the nucleus 2000 in cell culture 200 in tissue Moving genes depending on their state of activity Inter- and intrachromosomal chromatin loops Schneider R, Grosschedl R Genes Dev. 2007;21:3027-3043 ©2007 by Cold Spring Harbor Laboratory Press