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Eukaryotic cell nucleus mircotubules DNA 10 mm heterochromatin euchromatin nucleolus Chromatin organization of higher eukaryotes DNA Chromatin in the nucleus • In 1884, Albrecht Kossel coined the term “histon” to describe the proteins he found by extracting avian erythrocyte nuclei using diluted acids • In 1973, Olins et al and Woodcock et al observed that chromatin shows a “beads on a string” structure by EM • treatment of chromatin with micrococcal nuclease preferentially cuts between the beads Nucleosome structure Roger Kornberg • based on EM images, nuclease digestion patterns, X-ray diffraction data, and purification of nucleoprotein complexes, proposed that the nucleosome is the repeating unit of chromatin and that every ~200 bp of DNA forms a complex with four histone pairs (1974) nucleosome H1 core histone octamer= 2 copies each of DNA H2A H2B H3 H4 Core histones • core histones (H2A, H2B, H3 and H4) are small (11 to 14 kD), highly basic proteins • they are evolutionarily highly conserved (from yeast to humans) • they all share similar structural motifs N C N-terminal tail helicies C-term tail histone fold = “hand shake” motif Assembly of a nucleosome • histones can dimerize through their “hand shake motifs” • H3 can only dimerize with H4 and H2A always dimerizes with H2B • nucleosome assembly starts with two H3-H4 dimers forming a tetramer • this is followed by addition of two H2A-H2B dimers to form the octamer • DNA is wrapped around the histone octamer H3 H4 Nucleosome crystal structure Luger et al, Nature, 1997 H3 H2A H4 H2B Nucleosome crystal structure Luger et al, Nature, 1997 Why is chromatin folding important in the cell? DNA/chromatin has to condense and decondense during the cell cycle Stable cell line expressing H3-GFP How does chromatin folding affect nuclear functions? • nucleosomes inherently function as barrier to nuclear factors that need to access and bind to DNA elements • e.g. chromatinized template inhibits transcription of underlying genes • also affects other DNA-templated processes such as DNA replication, repair etc. • in order to activate gene expression, the cell has developed ways to “open” up chromatin a. b. c. ATP-dependent chromatin remodeling factors histone modifying enzymes insert histone variants at strategic locations within genome Post translational modifications on histones ubiquitination • different modifications occur on specific residues to perform specific regulatory functions Post translational modifications on histones • Histone PTM has been a "hot” research topic in the last 15 yrs Frequently asked questions: • What biological processes are associated with/regulated by site-specific histone modifications? • What are the enzymes (acetylases, kinases, methyl-transferases) that directly modify histones at specific sites? • What are the upstream pathways that regulated these enzymes? • What are the downstream effects of histone PTMs -- i.e. mechanism? • What are the enzymes that remove specific histone PTMs? • What pathways that regulate these de-acetylases, phosphatases, de-methylases etc? Histone acetylation regulates transcription activation • It has long been known that histones in vivo are acetylated, and as early as in the 60’s, Vincent Allfrey has suggested that histone acetylation (and methylation) regulate RNA synthesis • e.g. by the 70’s, Allfrey et al showed that drugs that increase histone acetylation in cells also increased DNase sensitivity of the cellular DNA • by special labeling techniques, it was shown that more accessible chromatin are enriched for acetylated histones • However, the direct link between histone acetylation and transcription regulation wasn’t discovered till 1996 when the first transcription-associated histone acetyltransferase (HAT) was identified Identification of the first histone acetyltransferase The first transcription-associated histone acetyltransferase (HAT) was identified by an “in gel” histone acetyltransferase assay - + histone substrates - + SDS PAGE cut out for peptide sequencing, protein ID denature and renature proteins in the gel + 3H Ac-CoA Coomassie stain Autorad Brownell et al, Cell, 1996 Transcription is regulated by the balance of HATs and HDACs • The first HAT identified was Gcn5, which was a well-studied transcription co-activator identified by genetics studies in yeast • Also in 1996, the first histone deacetylase (HDAC) was identified, and the enzyme Rpd3 was also a long studied transcription repressor identified by yeast genetic studies • Many other transcription co-activators and repressors were found to be HATs and HDACs respectively, and these enzymes are recruited to promoters during transcription activation or repression HATs hypo Ac-histone hyper Ac-histone HDACs transcription repression transcription activation Technical advances that helped the study of histone modifications 1. Development and refinement of in vitro assays enzyme source + substrate nuclear extracts histones IP’d protein nucleosomes recombinant protein peptides + radioactive co-factor 3H-Ac-CoA (acetylation) 3H-SAM (methylation) 32P-ATP (phosphorylation) 32P-NAD (ADP-ribosylation) modified histones Example: identification of a histone H3 methyltransferase fractionate nuclear lysates by chromatography techniques collect fractions add histone H3 substrate and 3H SAM separate proteins by SDS PAGE stain gel or do autoradiography identify fractions that contain radio-actively labeled H3 repeat fractionation if necessary identify histone modifying enzyme Wang et al, Mol Cell, 2001 How to identify site of histone modification? ubiquitination How to identify site of histone modification? histone methyltransferase + H3 peptide + 3H-SAM radioactively-labeled peptide protein sequencing (Edman degradation) detect radioactive amino acid Strahl et al, PNAS, 1999 Technical advances that helped the study of histone modifications 2. Development and usage of histone modification-specific antibodies • antibodies are very useful reagents for research • they can have exquisite specificities and sensitivities for detection of proteins • can generate and purify antibodies that specifically detect site-specifically modified histones Technical advances that helped the study of histone modifications Development and usage of histone modification-specific antibodies Recent article in BMC Bioinformatics on epigenetics and histone modifications Technical advances that helped the study of histone modifications 2. Development and usage of histone modification-specific antibodies • antibodies are very useful reagents for research • they can have exquisite specificities and sensitivities for detection of proteins • can generate and purify antibodies that specifically detect site-specifically modified histones • these antibodies can be used for Western blot analyses, immunofluorescence (IF) studies, and chromatin immunoprecipitation (ChIP) assays Uses of modification-specific histone antibodies 2a. Western blot analyses • modification-specific histone antibodies are useful for monitoring overall abundance and global changes of specific histone modifications Briggs et al, Genes Dev, 2001 Uses of modification-specific histone antibodies 2b. Immunofluorescence assays • modification-specific histone antibodies can be used to examine localization of the modified histones within the nucleus Chromosome enriched in Lys9-methylated H3 Me(Lys9) H3 Uses of modification-specific histone antibodies 2c. Chromatin immunoprecipitation assay • ChIP assay is useful for examining the enrichment of specific histonemodifications or binding of specific factors to the gene of interest in vivo Uses of modification-specific histone antibodies 2c. Chromatin immunoprecipitation (ChIP) assay • can be coupled to gene activation procedures to look at changes in histonemodifications or transcription factor binding to specific genes before and after transcription activation • can also be used in combination with microarray analyses (ChIP on chip) or deepDNA sequencing (ChIP-seq) to do genome-wide mapping of histone modifications and chromatin-binding proteins • while ChIP-chip or ChIP-seq provide correlational information, detailed ChIP analyses of specific genes can help eludicate step-wise mechanisms Transcription activation of the b-interferon gene • The b-interferon gene is highly activated upon viral infections and has served as a model system to study gene activations mRNA levels ChIP assays adapted from Agalioti et al, Cell, 2000 How does histone acetylation promote transcription? • Acetylation neutralizes the positively charged lysine residues on histones and thus reduces the interactions of the histones with the negatively charged DNA • Acetylated histones recruit and stabilize binding of transcription or chromatin remodeling factors via interactions of the acetylated lysines with the Bromodomains of these nuclear factors TAFII250 BD Ac H4 5/8 BD Ac 12/16 Jacobson et al; Science 2000 Histone acetylation precedes recruitment of transcription factors ChIP assays mRNA levels adapted from Agalioti et al, Cell, 2000 Different dynamics of histone modifications HATs Ac-histone histone HDACs highly dynamic kinases Phos-histone histone phosphatases HMT Me-histone histone de-methylase more stable