1. Eukaryotic cell nucleus
10 mm
mircotubules
DNA
heterochromatin
euchromatin
nucleolus

2. Chromatin organization of higher eukaryotes
DNA
Chromatin organization of higher eukaryotes

3. 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

4. Nucleosome structure nucleosome H1 core histone octamer=
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)
core histone octamer=
2 copies each of
H2A
H2B H3 H4
DNA H1
nucleosome

5. 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
helicies
N-terminal tail
C-term tail
histone fold
= “hand shake” motif

6. 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

7. Nucleosome crystal structureH3 H4
Nucleosome crystal structure
Luger et al, Nature, 1997

8. Nucleosome crystal structureH3 H4
H2A
H2B
Nucleosome crystal structure
Luger et al, Nature, 1997

9. 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

10. 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
ATP-dependent chromatin remodeling factors
histone modifying enzymes
insert histone variants at strategic locations within genome

11. Post translational modifications on histones
ubiquitination
different modifications occur on specific residues to perform specific
regulatory functions

12. 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?

13. 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

14. 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

15. 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
hyper Ac-histone
hypo Ac-histone
HATs
HDACs
transcription
activation
repression

16. Technical advances that helped the study of histone modifications
1. Development and refinement of in vitro assays
radioactive
co-factor
enzyme source +
substrate +
modified histones
3H-Ac-CoA (acetylation)
nuclear extracts
histones
IP’d protein
nucleosomes
3H-SAM (methylation)
32P-ATP (phosphorylation)
recombinant protein
peptides
32P-NAD (ADP-ribosylation)

17. 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

18. How to identify site of histone modification?
ubiquitination

19. How to identify site of histone modification?
methyl-
transferase +
H3 peptide +
3H-SAM
radioactively-labeled peptide
protein sequencing (Edman degradation)
detect radioactive amino acid
Strahl et al, PNAS, 1999

20. Technical advances that helped the study of histone modifications
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

21. Technical advances that helped the study of histone modifications
Development and usage of histone modification-specific antibodies

22. Recent article in BMC Bioinformatics on epigenetics and histone modifications

23. Technical advances that helped the study of histone modifications
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

24. 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

25. 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

26. Uses of modification-specific histone antibodies
2c. Chromatin immunoprecipitation assay
ChIP assay is useful for examining the enrichment of specific histone-
modifications or binding of specific factors to the gene of interest in vivo

27. Uses of modification-specific histone antibodies
2c. Chromatin immunoprecipitation (ChIP) assay
can be coupled to gene activation procedures to look at changes in histone-
modifications 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 deep-
DNA 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

28. 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

29. 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
Jacobson et al; Science 2000

30. Histone acetylation precedes recruitment of transcription factors
adapted from Agalioti et al, Cell, 2000
mRNA levels
ChIP
assays

31. Different dynamics of histone modifications
Ac-histone
HATs
HDACs
highly
dynamic
histone
Phos-histone
kinases
phosphatases
HMT
more
stable
histone
Me-histone
de-methylase