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Genome Sequence & Gene Expression

Chromatin & Nuclear Organization. Genome Sequence & Gene Expression . Chromosome Inheritance & Genome Stability . organization of chromosomes & proteins in nucleus . packaging . replication & chromatin assembly. chromatin modifications.

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Genome Sequence & Gene Expression

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  1. Chromatin & Nuclear Organization Genome Sequence & Gene Expression Chromosome Inheritance & Genome Stability

  2. organization of chromosomes & proteins in nucleus packaging replication & chromatin assembly chromatin modifications roles of DNA sequences, epigenetics, proteins, RNA/RNAi impact on functions, eg cis regulation of expression “….not all types of elements, such as centromeres, telomeres, and other yet-to-be defined elements, will be surveyed in the pilot project.”

  3. ‘active’ or ‘open’ chromatin ‘silent’ chromatin The ‘Histone Code’ Hypothesis methylation acetylation phosphorylation SUMOylation ubiquitination mono, di, tri… D. Allis T. Jenuwein

  4. Histone Variants CENP-A - centromere-specific H3-like protein H2AX - DNA repair H3.3 - gene expression replication independent deposition

  5. What is Known about the Distribution of Histone Modifications and Variants in Flies ? The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote Dirk Schübeler, David M. MacAlpine, David Scalzo, Christiane Wirbelauer, Charles Kooperberg, Fred van Leeuwen, Daniel E. Gottschling, Laura P. O’Neill, Bryan M. Turner,Jeffrey Delrow, Stephen P. Bell, and Mark Groudine GENES & DEVELOPMENT 18:1263–1271 2004

  6. H3 & H4 Ac H3 Lys4 Me H3 Lys79 Me active + + + inactive - - - ChIP with antibodies recognizing modifications gene array (40% of genes), 2L tiling array expression levels correlated with degree of modification not genome wide, no heterochromatin, no intergenic analyzed limited number of modifications more complex patterns emerge when more regions, modifications, and functions are assayed

  7. H1 H4 CENP-A H3 MERGE 0.3-1.5 Mb Blocks of H3 and CENP-A Nucleosomes are Interspersed in CEN Chromatin extended chromatin fibers human and fly

  8. CEN, EUCH, and HET Contain Distinct Modifications distinct from ‘classical’Euchromatin&Heterochromatin

  9. How do these ‘landscapes’ correlate with gene expression & chromosome functions ? transcription factor binding sites and CRMs, hypersens. sites expression domain regulators (PREs) heterochromatin - genes and repeats chromosome elements - CENs, TELs, replication origins/timing DNA repair non-protein-coding / micro RNAs Genome-wide analysis of chromatin structure & function What are the distributions of histone modifications and variants ? epigenomic ‘landscape’ including repeats, intergenic regions Are there other ‘islands’ of distinct modification patterns ? How do ‘landscapes’ differ among tissues/stages/cells ? NOT like genome sequence - there is no real baseline Are patterns evolutionarily conserved, even if sequences are not ?

  10. isolate nuclei/chromatin (cross-link ?) sonicate or micrococcal (to mononucleosomes) ChIP with modification/variant specific antibodies (reverse cross-links ?) purify IP’d DNA (random amplification ?) label DNA, probe arrays data analysis and correlation with functions

  11. ChIP-grade antibodies to histone modifications and variants most commercially available, but costly want widest spectrum to reveal combinatorial patterns Data generation and array analysis use existing tools Database integration for correlating chromatin with functions how to standardize results from different platforms ? different tissues/stages ? Requirements Genome tiling arrays - including heterochromatin melanogaster - Affymetrix, NimbleGen ?, others ? other fly species - ?????

  12. Why Flies ?? knowledge of biology & tools extensive collection of gene disruptions-assay how patterns change P elements, defined deletions, and other mutations genome-wide RNAi screens, tilling robust functional assays in the organism & cells from sequence to chromatin to chromosomes to… gene expression chromosome packaging nuclear organization chromosome replication & segregation ready access to different dev. stages/tissues, cell culture ready access to multiple layers of unique data sets, for integration cis-reg. data, heterochromatin sequence / annotation, etc. small genome size can get ‘whole’ genome on a single array availablility of sequence from close and distant species

  13. pombe flies & humans 0.3-1.5 Mb many many Mbs ??? ??? Interspersed H3 like flanking heterochromatin? Interspersed H3 different from flanking heterochromatin? Are Centromeres Heterochromatic? ‘Histone code’ defines different CEN-region functional domains??

  14. H3 K27 methylation- PcG binding, domain-wide repression H2A K119 monoubiquitin - PcG binding, X inactivation

  15. H3 H3 CENP-A + - H3 diMeK9(heterochromatin) - + H3 diMeK4(euchromatin) CENs are not Modified Like Heterochromatin Human and fly Sullivan & Karpen, 2004

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