roles of DNA sequences, epigenetics, proteins, RNA/RNAi impact on functions, eg cis regulation of expression replication & chromatin assembly packaging chromatin modifications organization of chromosomes & proteins in nucleus “….not all types of elements, such as centromeres, telomeres, and other yet-to-be defined elements, will be surveyed in the pilot project.”
‘active’ or ‘open’ chromatin‘silent’ chromatin methylation acetylation phosphorylation SUMOylation ubiquitination mono, di, tri… The ‘Histone Code’ Hypothesis D. Allis T. Jenuwein
Histone Variants CENP-A - centromere-specific H3-like protein H2AX - DNA repair H3.3 - gene expression replication independent deposition
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
ChIP with antibodies recognizing modifications gene array (40% of genes), 2L tiling array not genome wide, no heterochromatin, no intergenic analyzed limited number of modifications more complex patterns emerge when more regions, modifications, and functions are assayed expression levels correlated with degree of modification H3 & H4 AcH3 Lys4 MeH3 Lys79 Me active inactive +++ ---
Blocks of H3 and CENP-A Nucleosomes are Interspersed in CEN Chromatin extended chromatin fibers CENP-A H3 MERGEMERGE 0.3-1.5 Mb human and fly H1 H4
CEN, EUCH, and HET Contain Distinct Modifications distinct from ‘classical’ Euchromatin & Heterochromatin
What are the distributions of histone modifications and variants ? epigenomic ‘landscape’ including repeats, intergenic regions Genome-wide analysis of chromatin structure & function 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 How do these ‘landscapes’ correlate with gene expression & chromosome functions ? How do ‘landscapes’ differ among tissues/stages/cells ? NOT like genome sequence - there is no real baseline Are there other ‘islands’ of distinct modification patterns ? Are patterns evolutionarily conserved, even if sequences are not ?
ChIP with modification/variant specific antibodies isolate nuclei/chromatin (cross-link ?) (reverse cross-links ?) purify IP’d DNA (random amplification ?) label DNA, probe arrays data analysis and correlation with functions sonicate or micrococcal (to mononucleosomes)
Requirements Genome tiling arrays - including heterochromatin melanogaster - Affymetrix, NimbleGen ?, others ? other fly species - ????? 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 ?
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 Why Flies ??
Are Centromeres Heterochromatic? ‘Histone code’ defines different CEN-region functional domains?? 0.3-1.5 Mbmany many Mbs flies & humans Interspersed H3 different from flanking heterochromatin? ??? pombe Interspersed H3 like flanking heterochromatin?