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10  m mircotubules DNA Eukaryotic cell nucleus heterochromatineuchromatinnucleolus.

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Presentation on theme: "10  m mircotubules DNA Eukaryotic cell nucleus heterochromatineuchromatinnucleolus."— Presentation transcript:

1 10  m mircotubules DNA Eukaryotic cell nucleus heterochromatineuchromatinnucleolus

2 DNA Chromatin organization of higher eukaryotes

3 Chromatin in the nucleus 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 In 1884, Albrecht Kossel coined the term “histon” to describe the proteins he found by extracting avian erythrocyte nuclei using diluted acids

4 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) core histone octamer= 2 copies each of H2A H2B H3 H4 DNA H1 nucleosome

5 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 NC N-terminal tailC-term tail helicies histone fold = “hand shake” motif

6 histones can dimerize through their “hand shake motifs” Assembly of a nucleosome 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

7 H3 H4 Luger et al, Nature, 1997 Nucleosome crystal structure

8 H3 H4 H2A H2B Luger et al, Nature, 1997 Nucleosome crystal structure

9 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

10 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. ATP-dependent chromatin remodeling factors b. histone modifying enzymes c. insert histone variants at strategic locations within genome

11 ubiquitination Post translational modifications on histones different modifications occur on specific residues to perform specific regulatory functions

12 Histone PTM has been a "hot” research topic in the last 15 yrs Post translational modifications on histones 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?

13 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

14 Identification of the first histone acetyltransferase The first transcription-associated histone acetyltransferase (HAT) was identified by an “in gel” histone acetyltransferase assay histone substrates + 3 H Ac-CoA Coomassie stainAutorad cut out for peptide sequencing, protein ID SDS PAGE denature and renature proteins in the gel Brownell et al, Cell, 1996

15 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 hyper Ac-histone hypo Ac-histone HATs HDACs transcription activation transcription repression

16 Technical advances that helped the study of histone modifications 1. Development and refinement of in vitro assays enzyme source substrate radioactive co-factor modified histones + + nuclear extracts IP’d protein recombinant protein histones nucleosomes peptides 3 H-Ac-CoA (acetylation) 3 H-SAM (methylation) 32 P-ATP (phosphorylation) 32 P-NAD (ADP-ribosylation)

17 Wang et al, Mol Cell, 2001 Example: identification of a histone H3 methyltransferase add histone H3 substrate and 3 H 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 fractionate nuclear lysates by chromatography techniques collect fractions

18 ubiquitination How to identify site of histone modification?

19 histone methyl- transferase H3 peptide 3 H-SAMradioactively-labeled peptide + + protein sequencing (Edman degradation) Strahl et al, PNAS, 1999 detect radioactive amino acid

20 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

21 Development and usage of histone modification-specific antibodies Technical advances that helped the study of histone modifications

22 Recent article in BMC Bioinformatics on epigenetics and histone modifications

23 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 Technical advances that helped the study of histone modifications

24 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

25 2b.Immunofluorescence assays modification-specific histone antibodies can be used to examine localization of the modified histones within the nucleus Me(Lys9) H3 Chromosome enriched in Lys9-methylated H3 Uses of modification-specific histone antibodies

26 2c.Chromatin immunoprecipitation assay Uses of modification-specific histone antibodies ChIP assay is useful for examining the enrichment of specific histone- modifications or binding of specific factors to the gene of interest in vivo

27 2c.Chromatin immunoprecipitation (ChIP) assay can be coupled to gene activation procedures to look at changes in histone- modifications 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 deep- DNA 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 Uses of modification-specific histone antibodies

28 Transcription activation of the  -interferon gene adapted from Agalioti et al, Cell, 2000 mRNA levels ChIP assays The  -interferon gene is highly activated upon viral infections and has served as a model system to study gene activations

29 How does histone acetylation promote transcription? Jacobson et al; Science 2000 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 Acetylation neutralizes the positively charged lysine residues on histones and thus reduces the interactions of the histones with the negatively charged DNA

30 Histone acetylation precedes recruitment of transcription factors adapted from Agalioti et al, Cell, 2000 mRNA levels ChIP assays

31 Different dynamics of histone modifications histone Ac-histone HATs HDACs histone Phos-histone kinases phosphatases histone Me-histone HMT de-methylase highly dynamic more stable


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