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Chromatin>> CHIPs

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  1. 1. ChIP–SAGE 1st ChIP to purify chromatin 2nd crosslinks are reversed, a universal linker is ligated to DNA ends3rd NlaIII, which recognizes CATG, is used to digest DNA and a linkercontaining the recognition sequence of MmeI is ligated to the cleaved DNA ends 4th MmeI digestion produces 21–22 bp sequence tags, which are cloned into a sequencing vector and sequenced 5th The sequence tags then mapped tothe genome to identify modified regions
  2. 2. Histone VariantsDeposition of histone variants into nucleosomesconstitutes another type of chromatin modificationHtz1 ) H2A.Z ( marks the boundaries of heterochromatic regionsH2A.Z is localized to enhancers and promoters, with a positivecorrelation between occupancy and transcription activity at promotersH2A.Z might function to increase nucleosome mobility bydestabilizing nucleosome structure H3.3 marks boundaries of regulatory regions in D. melanogastergenomeDeposition of histone variants into a nucleosomefunctions to create hierarchical nucleosomal stability
  3. 3. Interpreting ChIP Experiments for Histone Modifications several points need to be regarded when interpreting ChIP results 1st The results absolutely rely on the specificity of the antibodies used in the experiments caution needed when working with a less well-characterized antibody2nd Different methods for chromatin preparation can lead to different results )sonication VS. micrococcal nuclease )MNase 3rd ChIP results that are obtained are just an average snap- shot of the modification status, which could contain contributions from very heterogeneous modification states of different cells
  4. 4. Nucleosome PositioningAdvantage of the preferential cleavage of linker DNA overnucleosomal DNA by MNaseMononucleosomes isolatedfrom MNase-digestion areanalyzed by either tilingmicroarrays or high-throughput sequencingChIP–Seq data for certain histone modifications can also beused to map nucleosomes in certain genomic regions
  5. 5. Genomic Profiles of Nucleosome PositionsPol II promoters had a 200 bp nucleosome free regionupstream of the transcription start siteIntergenic DNA was relatively depleted of nucleosomescompared to coding regions, and regions upstream ofactively transcribed genes are depleted of nucleosomesGenes with similar expression patterns have similar profilesof nucleosomes at their promotersChIP experiments recently combined with pyrosequencingto obtain a genome-wide profile of nucleosomes positions
  6. 6. Bioinformatic Studies of Nucleosome PositioningWith genome-wide maps of nucleosome positions availabilityInvestigating factors that influence positioning allowedRecent studies on extensive data sets have concluded thatalthough a fraction of nucleosomes positions determined by the underlying DNA sequence,most are notDNA structural features were strongly indicative ofnucleosome occupancy, while the AA/TT/TA dinucleotiderepeat pattern does not
  7. 7. Chromatin AccessibilityTarget-site accessibility is modulated by multiple factors,including DNA methylation and histone modificationsVarious reagents can be used to probe chromatin structureR.Es ’openness‘ or accessibility of a specific sequenceMNase define nucleosome boundariesDNase I define accessibility or hypersensitivity the presence of functionalcondensation/decondensation enhancer elements or status of a large chromatin region chromatin boundary elements
  8. 8. ChIP–Seq1st Purification of modified chromatin by immunoprecipitation using a specific antibody2nd ChIP DNA ends are repaired andligated to a pair of adaptors, followed by limited PCR amplification3rd Clusters of DNA are generated and sequencing by synthesis is performed4th The resulting sequences are mapped to a reference genome to obtain genomic coordinates that correspond to the immunoprecipitated fragments
  9. 9. Comparison Type ChIP–SAGE ChIP–Seq Qualities Depends on how Depends on the size of frequently restriction enzyme the chromatin fragments Resolution sites occur in the DNA of & ChIP The depth of sequencing No hybridization required No hybridization requiredQuantification Slightly less quantitative More quantitative Limited to recognition sites Does not require pre- for the restriction enzyme that Options is used to cleave the ChIP selection of genomic regions DNA
  10. 10. Characteristics of Epigenomes Regulation of gene expression, the cell memory ”decided“ by the interaction of ,DNA methylation histone modification, nucleosome positioning and other factors such Noncoding RNAs and three-dimensional chromatin architecture