Histone Modifications

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Histone Modifications

• Seminar in Bioinformatics
• Saar Gershuni
• 2005

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Background DNA Packing

• The DNA is packed in various levels of
  condensation in the nucleous (10,000)
• Form of condensation biological role
• Chromosomes, Euchromatin, heterochromatin, DNA
  strand

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The Beads on a String

• The Histones form the 11nm strand.
• Are octamer build H3,H4,2HA,2HB monomers
• 146-147 bp are wrapped around every histone core
• The histone tail sequences account for 28 of the
  total amino acid content of the core histones
  chromatin fiber folding requirement for the
  Histone H4 n-terminal tail , Benedetta Dorigo,
  Thomas Schalch, Kerstin Bystricky, and
  timothy J. Richmond , journal of molecular
  biology 327, 1 , 14 march 2003
• Tail of histone h4 taken from cow

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The Histone Core Wrapped With DNA
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Chemical Review

• Acetyl
• Methyl
• Phosphoryl
• Ubiquitin

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Histone Modifications

• De/Acetylation
• Methylation
• Phosphorylation
• Ubiquitination
• ADP-Rybosilation
• Swi/Snf complex, which, in vitro, uses the energy
  of ATP hydrolysis to disrupt histone-DNA
  interactions

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Histone Modifications Map
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Histone Modifications - Role

• Transcription Acetylation/Methylation
• DNA repair H2A -Phosphorilation
• Mitosis chromosomal arrengement
• Chromatin assembly DNA replication

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Figure 1. Sites of post-translational
modificationson the histone tails
Yi Zhang et al. Genes Dev. 2001 15 2343-2360
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Examples of Biological Role of Histone
Modifications

• Acetylation Lisine
• Transcription loosening the strand
• Replication the positioning of histones
• Gcn5 h3k14

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Acetylation mechanism
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Examples of Biological Role of Histone
Modifications

• Phosphorylation serine, threonine (scienceweek)
• Chromosomal condensation H1,H3
• Rsk-2, an H3 kinase, Coffin Lowry syndrome
• Transcription regulation - drosophila sex
  chromosomes serine 10 H3 in concert with H4K16

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Coffin Lowry Syndrome

• Coffin-Lowry syndrome is a rare genetic disorder
  characterized by mental retardation
  abnormalities of the head and facial area large,
  soft hands with short, thin (tapered) fingers
  short stature and/or various skeletal
  abnormalities. Characteristic facial features may
  include an underdeveloped upper jawbone, an
  abnormally prominent brow, downslanting eyelid
  folds, widely spaced eyes, large ears, and/or
  unusually thick eyebrows. Skeletal abnormalities
  may include abnormal front-to-back and
  side-to-side curvature of the spine and unusual
  prominence of the breastbone.
• Coffin-Lowry syndrome is caused by mutations in
  the RSK2 gene and is inherited as an X-linked
  dominant genetic trait. Males are usually more
  severely affected than females.

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Examples of Biological Role of Histone
Modifications

• Methylation Arginine, Lisine
• Less studied, enzymology not known
• Can be mono-, bi-, tri- methylated
• Transcription regulation - CARM1,
  arginine-specific, histone h3-selective
  methyltransferase activity, coactivator, with
  p160 family

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Figure 2. Chemistry of arginine and lysine
methylation
Yi Zhang et al. Genes Dev. 2001 15 2343-2360
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Histone Code

• Code - a system of signals or symbols for
  communication (webster meriam online)
• Requirements from a code
• Consistent
• Combinatorial (Kurdistany Grunstein, 2003)

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Mapping Global Histone Acetylation Patternsto
Gene ExpressionSiavash K. Kurdistani, Saeed
Tavazoie,and Michael Grunstein
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Introduction

• The mechanism by which histone de/acetylation
  affect transcription involve two pathways
• By altering the folding properties of the
  chromatin fiber
• By providing binding surface for recruitment of
  other elements
• To date (6/2004) there is no evidence for
  consistent patterns of de/acetylation from gene
  to gene or for the combinatorial use of histone
  modification sites

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The Experiment Data Collection Methods

• Chromatin was extracted from YDS2 exponentially
  growing
• Using chip and DNA microarrays levels of
  modification was determined
• 11 sites of acetylation were examined
• H4k8,12,16
• H3k9,14,18,23,27
• H2ak7,h2bk11,16

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The Experiment - Methods

• Two Microarrays 6700 IGR, 6200lt ORF
• 2-4 repetitions
• Normalization by the ration of total intensities.
• Coefficient of variation lt 0.5 between replicate
  experiment were counted
• End up with 2206 IGR, 2403 ORF
• Data were again normalized over the 11 sites per
  histone

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ChIP
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Results Raw Data
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Results Correlation With Gene Expression
These results though significant might be
artificially low due to technicalities.
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Results - Clustering
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Results coexpression of Clusters
Problem what is the expression level of randomly
selected cluster of genes? The study also checked
expression levels at different stress conditions
(255) correlation was found in 6 IGRs and 13
ORFs
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Results Clusters Biological Relevance

• Annotations for all the genes in a cluster were
  taken from MIPS, GO, MDS
• 12/53 IGRs, 13/68 ORFs with significant
  results
• Motif search using AlignACE algorithm found 102
  of 29/53 IGRs, 110 of 34/68 ORFs

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Does the Modifications Constitute a Code?

• The authors believe that the answer is no
  because
• The total number of modifications does not
  contain more information than the sum of
  individual modification.
• Problem it has been shown to be combinatorial
  bdf1 in vitro preference for tetra acetylated H4.

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Problems

• Cutting the chromatin fiber was done using
  sonicator bath thus creating various size of
  fiber with various number of nucleosome
  problem with measuring acetylation levels.
• Microarrays are 1kb in length can contain up to 5
  nucleosomes.

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Problems

• Normalization step 1 average number of 1
  through all the genome.Step 2 normalizing
  groups of 11 lysines in each and every locus
  (?0, var1)
• All the problems relates with k means algorithm,
  AlignACE, and gene expression data

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Genomic Maps and Comparative Analysis of Histone
Modifications in Human and Mouse

• Bradley E. Bernstein, Michael Kamal, Kerstin
  Lindblad-Toh, Stefan Bekiranov, Dione K. Bailey,
  Dana J. Huebert, Scott McMahon, Elinor K.
  Karlsson, Edward J. Kulbokas III, Thomas R.
  Gingeras, Stuart L. Schreiber, and Eric S. Lander

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The Experiment

• Large scale study of histone modifications
  (methylation, acetylation) patterns in human and
  mouse cells
• Methods ChIP, RTPCR for validating the data,
  tiling oligonucleotide arrays 35 bp intervals
• Focus chromosomes 21,22, (H3K4 di/trimethylation
  and H3K9,14 acetylation) and cytokine cluster,
  IL4 Receptor, and Hox clusters (H3K4
  dimethylation)

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Results Raw Data

• 90lt correlation between methylated H3K4, and
  acetylated H3K9,14
• Di/Trimethyl gene start

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Conservation of Modification pettern Between
Human and Mouse

• For this purpose the IL4R methylation analysis
  were preformed
• 55 conservation in human, 68 conservation in
  mouse, (7 than random) with no correlation with
  sequence conservation

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Hox Clusters

• A group of linked regulatory homeobox genes that
  are involved in patterning the animal body axis
  during development. Homeobox genes are defined as
  those that contain an 180-base-pair sequence that
  encodes a DNA-binding helixlturnhelix motif (a
  homeodomain).(Nature)
• The remaining orthologous regions between human
  and mouse

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Methylation Patterns in Hox Clusters

• Completely unique.
• Contain huge methylated regions encompass
  multiple genes
• Evolutionary conserved (human-mouse)
• Methylation correlates with expression both in
  ORFs and IGRs unlike IL4R

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Problems

• The method of creating the lysate is still
  sonication.
• No relation with genes functionality, cell cycle
  phase can change nucleosome concentration

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What Is It Good for?

• Genome wide understanding of chromatin role
  (structure, functionality( in biology
• The use of all the methods we learned
• Improve our understanding regarding various
  mechanisms and processes within the nucleous
• Develop new bioinformatic and biological methods
  for research (advanced ChIP technics, combination
  with tiling microarrays, and data analyzing tools
  normalizations, intergrated tools)

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Where Do We Go Next?

• Characterization of lysine 56 of histone H3 as an
  acetylation site in Saccharomyces
  cerevisiae.Ozdemir A, Spicuglia S, Lasonder E,
  Vermeulen M, Campsteijn C, Stunnenberg HG, Logie
  C.
• Epigenomic mapping in Arabidopsis using tiling
  microarrays.Martienssen RA, Doerge RW, Colot V.
• The epigenetic breakdown of cancer cells from
  DNA methylation to histone modifications.Ballesta
  r E, Esteller M.

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Questions?