Transcript Slide 1

Deciphering the Histone Code
Dandan Li, Karen Lohnes, Kathleen Sanders, Nuttinee Teerakulkittipong
How the Idea of the Code Came About
•The history of the study of histone modifications began over 40 years ago with Murray’s identification
of lysine methylation in the N-terminal domains of calf histones.
• Identification of these proteins and peptides can be determined from fragmentation patterns obtained
by mass spectrometric analysis (MS).
• Lysine acetylation and serine phosphorylation were discovered soon after in histone tails from such
broadly displayed sources as human lymphocytes, rat liver, peas, and calf thymus1.
• There are two general approaches to mass spectrometry of protein complexes: “bottom-up” and “topdown”7.Current methodology utilizes a combination of these two approaches to get the most accurate
determination of PTMs8.
• In 1975, ubiquitylation and ADP-ribosylation were added to the spectrum, with novel histone
modifications such as SUMO (the small ubiquitin-related modifier) being discovered as recently as
20031,2,3.
• These covalent posttranslational modifications (PTMs) allow for regulating contacts with the
underlying DNA2,4 (Fig 1A).
• In 2000, awareness that enzymes created both remarkable diversity and biological specificity through
these distinct histone modification patterns led Strahl and Allis to propose that distinct histone tail PTMs
may act sequentially or in combination to form a “histone code” that could be read by other proteins or
protein modules4,5 (Fig 1B,C).
• The most extensively studied histone tail modification is acetylation by histone acetyltransferases
(HATs) and deacetylation by histone deacetylase (HDACs) of the ε-amino groups of conserved lysine
residues associated with transcriptional activity2,5.
• It is thought that the neutralization of the histone tails’ basic charge through acetylation reduces their
affinity for DNA and alters histone-histone interactions between adjacent nucleosomes as well as
histone interactions with other regulatory proteins2,5 (Fig 2).
• Convincing molecular evidence directly linking acetylation and transcription was finally obtained when
acetyltansferase (HAT) activity was discovered in the conserved transcriptional regulator Gcn5. Since
then, numerous other coactivator proteins existing as families of acetyltransferases (comprised of more
than 20 enzymes!) have been found to possess HAT activity2,4.
• It is likely that short preferred consensus
motifs exist for individual HATs and HDACs
that assist in establishing the histone code5.
Discovering the Modifications
A
B
The actions of histone deacetylase (HDAC) inhibitors are to induce histone hyperacetylation, reactivate
suppressed genes, and cause pleiotropic cellular effects that inhibit tumor-cell growth and survival. Almost
all HDAC inhibitor mechanisms cause induction of cell cycle arrest which leads to mediated cell
differentiation or apoptosis in vitro20,21. Many also have potent antitumor activities in vivo by activation of the
host immune response22 and inhibition of angiogenesis14,23. The molecular and biological functions of these
agents are being used in preclinical cancer models and clinical trials that represent a new wave of
anticancer drugs and are exciting prospects for a more rational approach to chemotherapy.
• The bottom-up approach allows for comprehensive and quantitative measurement of distinct histone
modifications while the top-down approach aides one in determining the interdependencies between
clustered modifications. Mass spectrometry not only allowed a much faster detection of PTMs, but also
revealed a much higher abundance of PTMs than previously expected3 (Fig 1).
Fig 5. A comparison of treatment
of normal and certain tumor cell
lines with HDAC inhibitors23.
• In 2002, the research monopoly of the histone code tail ended with the application of mass spectrometry,
opening the door to the study of the modifications of the core histone domain1.
• These core PTMs are postulated to have different mechanisms of action than their tail domain counterparts1.
Their importance has been illustrated with point mutations for single modifiable residues which demonstrated
sometimes dramatic effects on transcriptional silencing, formation and/or maintenance of chromatin structure
and DNA damage repair9.
• In fact the role these modifications play may be more critical than tail domain PTMs given that deletion of tail
domains in H3, H2A or H2B proteins was found to have no effect on the ability of these proteins to organize
into nucleosomes10.
• Clustering of these modifications in particular locations led Marsfelder and Parthun to organize them into
three classes;
(1) the solute accessible face: believed to play a role in the regulation of chromatin (i.e. assembly of silent
chromatin structure), the ability of non-histone proteins to bind to the nucleosome and influencing
nucleosome-nucleosome interactions1, One modification on the solute accessible face, methylation of histone
H3 lysine 79, has also been shown to play a role in the functionality of DNA damage checkpoints11.
(2) the histone lateral face: that regulates histone-DNA interactions by altering the mobility of the
nucleosomes, which results in a change in higher order chromatin structure or accessibility of specific
sequences of DNA such as those needed to be available for DNA damage repair1. Many of these
modifications act in concert with the cell cycle to enhance the general dynamic nature of eukaryotic
chromosomes12,13.
(3) the histone-histone interface: essential in the regulation of nucleosome stability. Weakening of these
interactions can allow access of DNA damage repair machinery and orderly assembly of nucleosomes onto
DNA following replication, transcription and DNA repair1.
• The study of the N-terminal tail domains
Coffin Lowry Syndrome: A Disease Associated with Histone
Phosphorylation
Coffin-Lowry syndrome (CLS) is a genetic disorder characterized by craniofacial dysmorphisms,
progressive skeletal abnormalities, severe mental retardation and short stature24,25. It is caused by a
mutated gene, RSK-2, which is located on the X chromosome (Xp22.2-p22.1), and is the sole gene
known to be associated with CLS26. RSK-2 is a growth factor-regulated serine/threonine kinase that can
change the activity of many transcription factors by phosphorylation27.
Rsk-2 is involved in a Ras-dependent mitogen-activated protein kinase (MAPK) cascade that results in
the transcriptional activation of immediate-early responsive genes. During the immediate-early response
of mammalian cells to mitogens, rapid and transient phosphorylation of histone H3 occurs. Various
stimuli induce a MAPK cascade and RSK-2 is required for the epidermal growth factor (EGF)-stimulated
phosphorylation28 of H3. Fibroblasts derived from a CLS patient cannot exhibit EGF-stimulated
phosphorylation of H3, and transcriptional activation in response to stimuli is altered in CLS cells28. H3
appears to be a target of Rsk-2, suggesting a direct role for H3 phosphorylation in regulating gene
transcription possibly through chromatin remodeling and resulting in decondensation29.
dominated research in the field for over 4
decades.
• One classical method for analyzing PTMs used
specialized gel systems and/or the incorporation
of radioactive precursor molecules followed by
protein hydrolysis and analysis of the resulting
amino acid.
• Edman degredation was also used as a
primary method for discovering histone
modifications however it favored the analysis of
the first 20-30 amino acids of the tail.
• Both these methods required multiple arduous
purification steps making them non-applicable for
the analysis of histones from small cell numbers
or for mapping PTMs at defined genomic loci
which prompted researchers to search for novel
more sensitive reagents3.
Fig 6. Specific
phosphorylation site of
histone H3 by Rsk-2 in
vitro29.
C
Fig 3. The A. aeolicus HDLP has 35.2% sequence
homology to human HDAC119.
Fig 1. Models for “on-off” transcription states
reflected by differential histone tail modifications4,5.
• In the late 1980’s specific antibodies were
developed that could be used in western blots to
identify PTMs and these immunofluorescence
studies finally allowed a localization of specifically
modified histones within particular regions of the
genome3.
Beyond Histone Tails
Alteration of Histone Modification Patterns in Cancer Cells
Today there is more and more evidence that cancer cells present an altered pattern of histone modifications
within constitutive heterochromatin15. Aberrant transcription of the genes that result in improper targeting of
HATs or HDACs to certain loci, functional inactivation of HATs, overexpression of HDACs or epigenetic
changes due to DNA hyper- and hypomethylation, can induce abnormal expression of genes that regulate
cellular differentiation, the cell cycle and apoptosis, thereby enhancing the potential to mediate neoplastic
transformation, tumor onset and progression14.
• Unfortunately, similarities between different
modification sites caused cross reactivity in
antibodies which weakened specificity. Also, in
the presence of combined PTMs, many antisera
showed a strong interference in epitope binding,
necessitating the development of specific
antibodies for each individual combination. This
proved to be virtually impossible due to the large
number of modifications within a short stretch of
amino acids.
• Finally, modern mass spectrometry provided the
key technique for PTM analysis because of its
high resolution and the development of “soft”
ionization techniques.
Fig 4. TSA binds inside the pocket making contacts to
residues at the rim, walls and bottom of the pockets19.
Fig 7. Deficiency of histone H3 phosphorylation by EGF-stimulated pathway and decrease of Rsk-2-associated
H3 kinase activity in CLS cells29.
DAPI
normal
fibroblasts
+ EGF
CLS
fibroblasts
+ EGF
New Approach for the Treatment of Cancer by Chromatin Remodeling
Expression of the aberrant gene in cancer cells always involves epigenetic gene silencing mechanisms that
occur mainly through two pathways:
(1) Dense hypermethylation of the CpG islands located in the promoter region of tumor suppression genes.
This has been targeted in cancer therapeutics using DNA demethylating agents that restore the functionality
of silent genes, but a significant percentage of these drugs have toxic effects15,16.
(2) Different transcriptional repressors aberrantly target the HDACs of the gene promoter and cause histone
hypoacetylation. The archetypical gene silenced in this manner in human cancer is the cyclin-dependent
kinase inhibitor (p21WAF1)17. This causes loss of the P21WAF1 gene expression in a broad spectrum of
tumor types, and its experimental overexpression in deficient cancer cells can cause growth arrest18.
Fig 2. The hypothesis of the “histone code”. Diverse modifications occur
at selected histone amino acid residues. Some patterns are linked to
biological events (such as acetylation and transcription). Distinct H3 (red)
and H4 (black) tail modifications are believed to act in sequential and
combinatorial fashion in their regulation of unique biological functions.
• Mass spectrometry is an instrumental technique that determines mass to charge ratios (m/z) of ionized
molecules and/or fragments. These m/z are used to calculate weights of proteins, protein fragments and
peptides6.
• A translated genomic database can be used to determine the predicted molecular weight of a given
protein and its peptides and deviations in the predicted weight from observed masses, termed ‘mass shifts’
are often indicative of PTMs.
CLS
fibroblasts
+ EGF
Phos. H3
Fig 8. Rsk-2 deficiency effects on
mitogen-stimulated but not mitotic
phosphorylation of histone H3. Indirect
immuno-fluorescence microscopy of
transformed normal (the first pair of
panels) and CLS (the second pair of
panels) fibroblasts incubated with EGF
and costained with DAPI and pS10
antiserum. The last pair of panels
shows another fields of CLS fibroblasts
in which a cell in mitosis is visible. The
location of phosphorylated H3 in cells
is indicated by arrowheads29.
Conclusion
The discovery of the histone code and subsequent attempts to decipher it has opened many
avenues of research into the essential role that the histone modifications play in gene expression.
Unraveling the mechanisms and consequences of this epigenetic control has both extended our
understanding of chromatin regulation and offered far reaching implications for human biology and
disease.
References
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