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Transcript
Histone
Modifications
Seminar in Bioinformatics
Saar Gershuni
2005
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
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
The Histone Core Wrapped With
DNA
Chemical Review
• Acetyl
• Methyl
• Phosphoryl
• Ubiquitin
Histone Modifications
•
•
•
•
•
•
De/Acetylation
Methylation
Phosphorylation
Ubiquitination
ADP-Rybosilation
Swi/Snf complex, which, in vitro, uses the
energy of ATP hydrolysis to disrupt histoneDNA interactions
Histone Modifications Map
Histone Modifications - Role
•
•
•
•
Transcription – Acetylation/Methylation
DNA repair – H2A -Phosphorilation
Mitosis – chromosomal arrengement
Chromatin assembly – DNA replication
Figure 1. Sites of post-translational modificationson the histone tails
Yi Zhang et al. Genes Dev. 2001; 15: 2343-2360
Examples of Biological Role of
Histone Modifications
Acetylation –Lisine
• Transcription – loosening the strand
• Replication – the positioning of histones
• Gcn5 – h3k14
Acetylation mechanism
Examples of Biological Role of
Histone Modifications
Phosphorylation – serine, threonine
• Chromosomal condensation – H1,H3
• Rsk-2, an H3 kinase, Coffin Lowry
syndrome
• Transcription regulation - drosophila sex
chromosomes serine 10 H3 in concert with
H4K16
(scienceweek)
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.
Examples of Biological Role of
Histone Modifications
Methylation – Arginine, Lisine
– Less studied, enzymology – not known
– Can be mono-, bi-, tri- methylated
– Transcription regulation - CARM1, argininespecific, histone h3-selective methyltransferase
activity, coactivator, with p160 family
Figure 2. Chemistry of arginine and lysine methylation
Yi Zhang et al. Genes Dev. 2001; 15: 2343-2360
Histone Code
• Code - a system of signals or symbols for
communication (webster meriam online)
• Requirements from a code:
– Consistent
– Combinatorial (Kurdistany & Grunstein, 2003)
Mapping Global Histone
Acetylation Patterns
to Gene Expression
Siavash K. Kurdistani, Saeed
Tavazoie,
and Michael Grunstein
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
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
The Experiment - Methods
•
•
•
•
Two Microarrays: 6700 IGR, 6200< ORF
2-4 repetitions
Normalization by the ration of total intensities.
Coefficient of variation < 0.5 between replicate
experiment were counted
• End up with 2206 IGR, 2403 ORF
• Data were again normalized over the 11 sites per
histone
ChIP
Results – Raw Data
Results – Correlation With Gene
Expression
These results – though significant might be artificially low due to
technicalities.
Results - Clustering
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
IGR’s and 13 ORF’s
Results – Clusters Biological
Relevance
• Annotations for all the genes in a cluster
were taken from MIPS, GO, MDS
• 12/53 IGR’s, 13/68 ORF’s – with significant
results
• Motif search using AlignACE algorithm
found 102 of 29/53 IGR’s, 110 of 34/68
ORF’s
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.
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.
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, var=1)
• All the problems relates with k means
algorithm, AlignACE, and gene expression
data
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
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)
Results – Raw Data
• 90%< correlation between methylated
H3K4, and acetylated H3K9,14
• Di/Trimethyl – gene start
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
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 helix–lturn–helix
motif (a homeodomain).
(Nature)
• The remaining orthologous regions between
human and mouse
Methylation Patterns in Hox
Clusters
• Completely unique.
• Contain huge methylated regions
encompass multiple genes
• Evolutionary conserved (human-mouse)
• Methylation correlates with expression both
in ORF’s and IGR’s unlike IL4R
Problems
• The method of creating the lysate is still
sonication.
• No relation with genes functionality, cell
cycle phase – can change nucleosome
concentration
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)
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.
Ballestar E, Esteller M.
Questions?