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Eukaryotic cell nucleus
mircotubules
DNA
10 mm
heterochromatin
euchromatin
nucleolus
Chromatin organization of higher eukaryotes
DNA
Chromatin in the nucleus
• In 1884, Albrecht Kossel coined the term “histon” to describe the proteins he
found by extracting avian erythrocyte nuclei using diluted acids
• In 1973, Olins et al and Woodcock et al observed that chromatin shows a
“beads on a string” structure by EM
• treatment of chromatin with micrococcal nuclease preferentially cuts between the beads
Nucleosome structure
Roger Kornberg
• based on EM images, nuclease digestion patterns, X-ray diffraction data, and purification
of nucleoprotein complexes, proposed that the nucleosome is the repeating unit of
chromatin and that every ~200 bp of DNA forms a complex with four histone pairs (1974)
nucleosome
H1
core histone octamer=
2 copies each of
DNA
H2A
H2B
H3
H4
Core histones
• core histones (H2A, H2B, H3 and H4) are small (11 to 14 kD), highly basic proteins
• they are evolutionarily highly conserved (from yeast to humans)
• they all share similar structural motifs
N
C
N-terminal tail
helicies
C-term tail
histone fold
= “hand shake” motif
Assembly of a nucleosome
• histones can dimerize through their “hand shake motifs”
• H3 can only dimerize with H4 and H2A always dimerizes with H2B
• nucleosome assembly starts with two H3-H4 dimers forming a tetramer
• this is followed by addition of two H2A-H2B dimers to form the octamer
• DNA is wrapped around the histone octamer
H3
H4
Nucleosome crystal structure
Luger et al, Nature, 1997
H3
H2A
H4
H2B
Nucleosome crystal structure
Luger et al, Nature, 1997
Why is chromatin folding important in the cell?
DNA/chromatin has to condense and decondense during the cell cycle
Stable cell line expressing H3-GFP
How does chromatin folding affect nuclear functions?
• nucleosomes inherently function as barrier to nuclear factors that need to
access and bind to DNA elements
• e.g. chromatinized template inhibits transcription of underlying genes
• also affects other DNA-templated processes such as DNA replication,
repair etc.
• in order to activate gene expression, the cell has developed ways to “open”
up chromatin
a.
b.
c.
ATP-dependent chromatin remodeling factors
histone modifying enzymes
insert histone variants at strategic locations within genome
Post translational modifications on histones
ubiquitination
• different modifications occur on specific residues to perform specific
regulatory functions
Post translational modifications on histones
• Histone PTM has been a "hot” research topic in the last 15 yrs
Frequently asked questions:
• What biological processes are associated with/regulated by site-specific
histone modifications?
• What are the enzymes (acetylases, kinases, methyl-transferases) that
directly modify histones at specific sites?
• What are the upstream pathways that regulated these enzymes?
• What are the downstream effects of histone PTMs -- i.e. mechanism?
• What are the enzymes that remove specific histone PTMs?
• What pathways that regulate these de-acetylases, phosphatases, de-methylases etc?
Histone acetylation regulates transcription activation
• It has long been known that histones in vivo are acetylated, and as early as in
the 60’s, Vincent Allfrey has suggested that histone acetylation (and methylation)
regulate RNA synthesis
• e.g. by the 70’s, Allfrey et al showed that drugs that increase histone acetylation
in cells also increased DNase sensitivity of the cellular DNA
• by special labeling techniques, it was shown that more accessible chromatin
are enriched for acetylated histones
• However, the direct link between histone acetylation and transcription regulation wasn’t
discovered till 1996 when the first transcription-associated histone acetyltransferase
(HAT) was identified
Identification of the first histone acetyltransferase
The first transcription-associated histone acetyltransferase (HAT) was identified by
an “in gel” histone acetyltransferase assay
-
+
histone substrates
-
+
SDS
PAGE
cut out for
peptide
sequencing,
protein ID
denature and
renature proteins
in the gel
+ 3H Ac-CoA
Coomassie stain
Autorad
Brownell et al, Cell, 1996
Transcription is regulated by the balance of HATs and HDACs
• The first HAT identified was Gcn5, which was a well-studied transcription
co-activator identified by genetics studies in yeast
• Also in 1996, the first histone deacetylase (HDAC) was identified, and the enzyme
Rpd3 was also a long studied transcription repressor identified by yeast genetic
studies
• Many other transcription co-activators and repressors were found to be HATs and
HDACs respectively, and these enzymes are recruited to promoters during
transcription activation or repression
HATs
hypo Ac-histone
hyper Ac-histone
HDACs
transcription
repression
transcription
activation
Technical advances that helped the study of histone modifications
1. Development and refinement of in vitro assays
enzyme source
+
substrate
nuclear extracts
histones
IP’d protein
nucleosomes
recombinant protein
peptides
+
radioactive
co-factor
3H-Ac-CoA
(acetylation)
3H-SAM
(methylation)
32P-ATP
(phosphorylation)
32P-NAD
(ADP-ribosylation)
modified histones
Example: identification of a histone H3 methyltransferase
fractionate nuclear lysates by
chromatography techniques
collect fractions
add histone H3 substrate and 3H SAM
separate proteins by SDS PAGE
stain gel or do autoradiography
identify fractions that contain radio-actively labeled H3
repeat fractionation if necessary
identify histone modifying enzyme
Wang et al, Mol Cell, 2001
How to identify site of histone modification?
ubiquitination
How to identify site of histone modification?
histone
methyltransferase
+
H3 peptide
+
3H-SAM
radioactively-labeled peptide
protein sequencing (Edman degradation)
detect radioactive amino acid
Strahl et al, PNAS, 1999
Technical advances that helped the study of histone modifications
2.
Development and usage of histone modification-specific antibodies
• antibodies are very useful reagents for research
• they can have exquisite specificities and sensitivities for detection of proteins
• can generate and purify antibodies that specifically detect site-specifically
modified histones
Technical advances that helped the study of histone modifications
Development and usage of histone modification-specific antibodies
Recent article in BMC Bioinformatics on epigenetics and histone modifications
Technical advances that helped the study of histone modifications
2.
Development and usage of histone modification-specific antibodies
• antibodies are very useful reagents for research
• they can have exquisite specificities and sensitivities for detection of proteins
• can generate and purify antibodies that specifically detect site-specifically
modified histones
• these antibodies can be used for Western blot analyses, immunofluorescence
(IF) studies, and chromatin immunoprecipitation (ChIP) assays
Uses of modification-specific histone antibodies
2a. Western blot analyses
• modification-specific histone antibodies are useful for monitoring
overall abundance and global changes of specific histone modifications
Briggs et al, Genes Dev, 2001
Uses of modification-specific histone antibodies
2b. Immunofluorescence assays
• modification-specific histone antibodies can be used to examine localization
of the modified histones within the nucleus
Chromosome enriched in
Lys9-methylated H3
Me(Lys9) H3
Uses of modification-specific histone antibodies
2c. Chromatin immunoprecipitation assay
• ChIP assay is useful for examining the enrichment of specific histonemodifications or binding of specific factors to the gene of interest in vivo
Uses of modification-specific histone antibodies
2c. Chromatin immunoprecipitation (ChIP) assay
• can be coupled to gene activation procedures to look at changes in histonemodifications or transcription factor binding to specific genes before and after
transcription activation
• can also be used in combination with microarray analyses (ChIP on chip) or deepDNA sequencing (ChIP-seq) to do genome-wide mapping of histone modifications
and chromatin-binding proteins
• while ChIP-chip or ChIP-seq provide correlational information, detailed ChIP
analyses of specific genes can help eludicate step-wise mechanisms
Transcription activation of the b-interferon gene
• The b-interferon gene is highly activated upon viral infections and has served as a
model system to study gene activations
mRNA levels
ChIP assays
adapted from Agalioti et al, Cell, 2000
How does histone acetylation promote
transcription?
• Acetylation neutralizes the positively charged lysine residues on histones and thus
reduces the interactions of the histones with the negatively charged DNA
• Acetylated histones recruit and stabilize binding of transcription or chromatin
remodeling factors via interactions of the acetylated lysines with the
Bromodomains of these nuclear factors
TAFII250
BD
Ac
H4
5/8
BD
Ac
12/16
Jacobson et al; Science 2000
Histone acetylation precedes recruitment of transcription factors
ChIP
assays
mRNA levels
adapted from Agalioti et al, Cell, 2000
Different dynamics of histone modifications
HATs
Ac-histone
histone
HDACs
highly
dynamic
kinases
Phos-histone
histone
phosphatases
HMT
Me-histone
histone
de-methylase
more
stable