Genome Sequence

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Genome Sequence Gene Expression
Chromosome Inheritance Genome Stability
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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.
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The Histone Code Hypothesis
methylation acetylation phosphorylation SUMOylatio
n ubiquitination mono, di, tri
D. Allis T. Jenuwein
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Histone Variants
CENP-A - centromere-specific H3-like protein
H2AX - DNA repair
H3.3 - gene expression
replication independent deposition
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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. ONeill, Bryan M.
Turner,Jeffrey Delrow, Stephen P. Bell, and Mark
Groudine
GENES DEVELOPMENT 1812631271 2004
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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
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Blocks of H3 and CENP-A Nucleosomes are
Interspersed in CEN Chromatin
extended chromatin fibers
human and fly
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CEN, EUCH, and HET Contain Distinct Modifications
distinct from classical Euchromatin
Heterochromatin
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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 ?
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isolate nuclei/chromatin
(cross-link ?)
sonicate or micrococcal (to mononucleosomes)
ChIP with modification/variant specific
antibodies
(reverse cross-links ?)
purify IPd DNA
(random amplification ?)
label DNA, probe arrays
data analysis and correlation with functions
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Requirements
Genome tiling arrays - including
heterochromatin melanogaster - Affymetrix,
NimbleGen ?, others ? other fly species - ?????
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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
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Are Centromeres Heterochromatic?
Histone code defines different CEN-region
functional domains??
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H3 K27 methylation- PcG binding, domain-wide
repression
H2A K119 monoubiquitin - PcG binding, X
inactivation
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CENs are not Modified Like Heterochromatin
Human and fly
Sullivan Karpen, 2004