Genome Sequence & Gene Expression

Chromatin & NuclearOrganization

Chromosome Inheritance& Genome Stability

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roles of DNA sequences, epigenetics, proteins, RNA/RNAi

impact on functions, eg cis regulation of expression

replication &chromatin assembly

packaging

chromatinmodifications

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organizationof 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

methylationacetylationphosphorylationSUMOylationubiquitination

mono, di, tri…

The ‘Histone Code’ Hypothesis

D. AllisT. 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 modificationsgene array (40% of genes), 2L tiling array

not genome wide, no heterochromatin, no intergenicanalyzed 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 Ac H3 Lys4 Me H3 Lys79 Meactive

inactive

+ + +

- - -

Blocks of H3 and CENP-A Nucleosomes are Interspersed in CEN Chromatin

extended chromatin fibers

CENP-A

H3

MERGE

0.3-1.5 Mb

human and fly

H1H4

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 repeatschromosome elements - CENs, TELs, replication origins/timingDNA repairnon-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 costlywant 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 changeP elements, defined deletions, and other mutationsgenome-wide RNAi screens, tilling

robust functional assays in the organism & cells

from sequence to chromatin to chromosomes to… gene expressionchromosome packagingnuclear organizationchromosome 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 sizecan 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 Mb many many Mbs

flies&

humans

Interspersed H3 different from flanking heterochromatin?

??? ???

pombe

Interspersed H3 like flanking heterochromatin?

H2A K119 monoubiquitin - PcG binding, X inactivation

H3 K27 methylation- PcG binding, domain-wide repression

H3 diMeK9(heterochromatin)

+ -H3 H3 CENP-A

Human and flyH3 diMeK4

(euchromatin)- +

Sullivan & Karpen, 2004

CENs are not Modified Like Heterochromatin