Adelphi University Epigenetics Essay

Epigenetics (21:120:454)
Article Summary – Rubric
Requirement
Introduction
Main points
Experimental approach
Strengths & weaknesses
Conclusion
Excellent Exceeds assignment objectives
Good Meets assignment objectives
20 points
Includes strong thesis statement that
clearly describes the purpose of the
paper.
40 points
Describes the main points of the paper
without including extraneous
information. Doesn’t just repeat what
is stated in the paper but rather
presents these points in an effective
and concise manner.
40 points
Describes in a concise manner but not
in details the main experiments (if
applicable) used. Also, describes the
results of these experiments. Analysis
is comprehensive.
15 points
Thesis statement is clear but not detailed
or descriptive.
10 point
Thesis statement is weak and does not
describe the purpose of the paper.
35 points
Describes the main points of the paper
but includes extraneous information that
are not pivotal.
30 points
Does not describe the main points of the
paper but includes extraneous
information that are not important.
35 points
Describes the main experiments and the
results but does not demonstrate an
understanding of the physiological
significance or the outcomes of these
experiments. Analysis is not
comprehensive or may overlook key
elements.
25 points
Presents some of the strengths and
weaknesses of the paper. Lacks
thoughtful and creative
recommendations for future research.
30 points
Experiments and results are not clearly
described. Lacks a comprehensive
analysis.
15 points
Summary states the concluding thoughts
of the paper.
10 point
Summary lacks key points and insights.
30 points
Presents effectively the main strengths
and weaknesses of the paper and
suggests alternatives to address the
weaknesses. Presents thoughtful and
creative recommendations for future
research.
20 points
Summarizes key points with insights.
1
Poor Assignment objectives minimally met
20 points
Does not identify correctly the main
strengths and weaknesses of the paper. .
Lacks thoughtful and creative
recommendations for future research.
Dr. Rola Bekdash
Rutgers University – Newark
Department of Biological Sciences
Epigenetics (21:120:454)
Article Assignments & Guidelines
I.
Article Assignments
We will discuss five articles this semester. These articles address current topics in the field of
Epigenetics and focus on four main themes such as 1) Epigenetics of stress, 2) epigenetics of
neurodegeneration, 3) epigenetics of mental health and 4) epigenetics of cancer. We will
spend time explaining the main physiological concepts and the experimental approaches used
in investigating the role of epigenetic mechanisms in human health and diseases.
Students are expected to read the assigned articles before coming to class and actively
participate in discussions. You will not receive credit for any missed discussion session. You are
also required to submit on Blackboard by the due date a copy of each paper summary. Your
summary should focus on main points of the assigned article and should be 800 – 1000 words.
You will then receive feedback from your instructor on how to improve the structure of your
summary writing. You are required to resend a revised final version of your summary by the
due date to receive credit. Refer to the “Summary Rubric” that I posted on Blackboard under
“Assignments” to understand how you will be assessed and graded.
II. Papers Summaries
You are required to submit on Blackboard in a docx. File a concise summary (first draft) of
selected articles by the due date. No late submission is allowed.
The main objectives of these assignments are to: 1) encourage you to read papers and grasp
information from outside the textbook, 2) improve your critical thinking and 3) incrementally
improve your written and verbal communication styles of what you learn.
Each summary should have the following five sections:
1. Introduction: State the essential take home message of the assigned paper and what it
is all about. Do not simply restate the abstract.
2. Key points: Describe the main points in the paper
3. Methods/Experiments: Discuss the experiments or methods used to prove these points
(if applicable)
4. Strengths/Weaknesses: Discuss the strengths and weaknesses of the paper. Indicate
additional points of investigation that you think should be pursued and were not
addressed in the paper
5. Conclusion
1
Dr. Rola Bekdash
Write 800 – 1000 words, 11 point Ariel Font, double spaced with one inch margin. After you
receive the instructor’s feedback for improvement, you are required to resubmit a revised
version of your summary on blackboard.
III.
Recitations & Paper Summaries
Useful website: https://learn.genetics.utah.edu/content/epigenetics/
Paper Reference
1
2
3
4
5
Felsenfeld, G. A brief history of epigenetics. Cold Spring Harbor perspectives in
Biology. 1, 6:1 (2014).
https://www.ncbi.nlm.nih.gov/pubmed/24384572
Environmental epigenomics and disease susceptibility. Jirtle & Skinner, 2007.
https://www.ncbi.nlm.nih.gov/pubmed/17363974
Pages 1-5, pages 7-8 and conclusion
Histone methylation versus histone acetylation: new insights into epigenetic
regulation. Rice & Allis. Current Opinion in Cell Biology 2001, 13:263–273.
https://www.ncbi.nlm.nih.gov/pubmed/?term=Histone+methylation+versus
+histone+acetylation%3A+new+insights+into+epigenetic+regulation.
Reversal of maternal programming of stress responses in adult offspring through
methyl supplementation: altering epigenetic marking later in life. Weaver et al.,
2005, J. Neuroscience, 25, 11045-11054.
https://www.ncbi.nlm.nih.gov/pubmed/?term=Reversal+of+maternal+programmin
g+of+stress+responses+in+adult+offspring+through+methyl+supplementation%3A
+altering+epigenetic+marking+later+in+life
Epigenetic mechanisms in neurological and neurodegenerative diseases.
Landgrave-Gomez et al., 2015. Frontiers in Cellular Neuroscience, 9, 1-11.
https://www.ncbi.nlm.nih.gov/pubmed/25774124
Subject to changes with prior notice from the Instructor.
2
263
Histone methylation versus histone acetylation: new insights
into epigenetic regulation
Judd C Rice and C David Allis
Post-translational addition of methyl groups to the aminoterminal tails of histone proteins was discovered more than
three decades ago. Only now, however, is the biological
significance of lysine and arginine methylation of histone tails
being elucidated. Recent findings indicate that methylation of
certain core histones is catalyzed by a family of conserved
proteins known as the histone methyltransferases (HMTs).
New evidence suggests that site-specific methylation,
catalyzed by HMTs, is associated with various biological
processes ranging from transcriptional regulation to epigenetic
silencing via heterochromatin assembly. Taken together, these
new findings suggest that histone methylation may provide a
stable genomic imprint that may serve to regulate gene
expression as well as other epigenetic phenomena.
Addresses
Department of Biochemistry and Molecular Genetics, University of
Virginia, Health Sciences Center, Box 800733 Jordan Hall,
Room 6222, Charlottesville, Virginia 22908-0733, USA
Correspondence: C David Allis; e-mail: [email protected]
Current Opinion in Cell Biology 2001, 13:263–273
0955-0674/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
CAF-1 chromatin assembly factor 1
CD
chromodomain
ChIP
chromatin immunoprecipitation
chromo chromatin organization modifier
CSD
chromo shadow domain
HATs
histone acetyltransferases
HDACs histone deacetylases
HDM
histone demethylase
HMTs
histone methyltransferases
PEV
position effect variegation
PMEFs primary mouse embryonic fibroblasts
SET
Su(var), E(z) trithorax
Su(var) suppressor of variegation
TXR
transcriptional regulator proteins
Introduction
Nucleosomes, the fundamental structural units of chromatin, are comprised of the core histone octamer (H2A,
H2B, H3 and H4) and the associated DNA that wraps
around them. Although the crystal structure of a nucleosome core particle has provided considerable insight into
the protein–protein and protein–DNA interactions that
govern nucleosome structure [1], little is known about how
distinct functional domains of chromatin are established
and maintained [2]. The precise organization of chromatin
is critical for many cellular processes, including transcription, replication, repair, recombination and chromosome
segregation. Dynamic changes in chromatin structure are
directly influenced by post-translational modifications of
the amino-terminal tails of the histones [3,4]. These highly
basic histone tails are predicted to be less structured than
the histone fold regions and are believed to interact with
the negatively charged DNA backbone or with other
chromatin-associated proteins [2,3,5–9]. Specific amino
acids within these histone tails are targets for a number of
post-translational modifications, including acetylation,
phosphorylation, poly(ADP-ribosylation), ubiquitination
and methylation [10–12]. These covalent modifications
may alter the interaction of the histone tail with DNA or
with chromatin-associated proteins that may be required for
different downstream cellular processes [13•,14•,15].
Histone methylation was first discovered more than thirtyfive years ago [16]. Until quite recently, however, little was
known about the biological significance of this covalent modification due, in part, to a lack of convenient electrophoretic
assays or immunological tools to detect its presence.
Therefore, little information was available as to the enzyme
systems responsible for the establishment and maintenance
of histone methylation. Unlike the dynamic ‘on–off’ nature
of histone acetylation, early studies found that mammalian
histones H3 and H4 were highly methylated with little
turnover of the methyl groups [17–20]. The apparent stability
of this modification in bulk histone preparations led to the
belief that histone methylation was a generic and static modification. In contrast, other findings hinted that histone
methylation, like histone acetylation, is a dynamic process
involved in a number of diverse biological processes
including transcriptional regulation, chromatin condensation,
mitosis and heterochromatin assembly [21–26,27••,28•,29••].
In this review we compare and contrast the histone
methyllysine modification with the well studied acetyllysine modification to provide contemporary insights into
the functional and biological consequences of these
histone modifications. We propose that methylation of
specific lysine residues in histone tails functions as a stable
epigenetic mark that directs particular biological functions,
ranging from transcriptional regulation to heterochromatin
assembly. Furthermore, we speculate that, in certain
circumstances, context-dependent histone methylation can
provide a critical ‘mark’ on the histone tails, leading to the
recruitment and binding of chromatin-associated proteins,
which ultimately results in a distinct biological response.
It should be noted that arginine residues in histone tails
are also targets for in vitro methylation catalyzed by a new
class of coactivators functionally linked to a variety of
nuclear receptors (i.e. CARM1 and PRMT1) [27••,30,31].
This review focuses predominantly on lysine methylation,
but a current review on arginine methylation of histones is
highly recommended [32•].
264
Nucleus and gene expression
Figure 1
(a)
H3C
H
O
N
+
H3N
C
H
HDAC
COO–
ε–N-acetyllysine
H
+
H3N
H
H3C
N
HAT
(CH2)4
H
H
C
N
HMT
(CH2)4
H
HDM?
C
H COO–
+
H3N
N
HMT
(CH2)4
C
H
HDM?
H3C
+
H3N
COO–
ε–N-monomethyllysine
Lysine
H
H3C
H3C
C
H
N
HMT
(CH2)4
HDM?
COO–
ε–N-dimethyllysine
CH3
H3C
+
H3N
(CH2)4
C
H
COO–
ε–N-trimethyllysine
Hydrophobicity and basicity
(b)
Me
Ac
Ac
Me
Human H3
Ac
Me
Me
N–ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHR…
K
K
K
K
K
K
KK
4
9
14
18
23
27
3637
Ac
Human H4
Ac
Ac
Ac
Ac
Me
Ac–N-SGRGKGGKGLGKGGAKRHRKVLRDN…
K K
K
K
K
5
8
12
16
20
Me
= methyl
Ac
= acetyl
Current Opinion in Cell Biology
Histone methylation versus histone acetylation. (a) Molecular
structure of lysine, acetyllysine and methyllysine. Conserved lysine
residues in the amino-terminal histone tails can be posttranslationally modified by acetylation or methylation. Acetylation is
catalyzed by HATs and is removed by opposing HDACs. Acetyl
substitution of the ε-amino group of lysine occurs once and results in
a more acidic, hydrophilic residue. Methyl substitution is catalyzed by
the HMTs and may be removed by yet unidentified histone
demethylases (HDMs?). It remains unclear how increasing degrees
of methyl addition (mono, di and tri) is brought about and whether
the same HMT can catalyze all of these methyl additions.
Nevertheless, increasing methyl addition increases the basicity and
hydrophobicity of the lysine without altering its overall charge.
(b) Known acetyl and methyl modifications of lysine residues in the
amino-terminal tails of human histone H3 and H4 are shown.
Conserved lysines are in bold and their amino-acid position in the
histone tail is indicated by the numbers. The in vivo sites for lysine
acetylation and methylation are shown. Current findings suggest that
each lysine is either acetylated or methylated with the exception of
lysine 9 in the H3 tail (see text for details).
Lysine methylation and acetylation of histones
decreases their overall positive charge (Figure 1a). As these
tails are highly basic, it has long been postulated that acetylation decreases their affinity for the negatively charged
DNA and facilitates the binding of proteins that regulate
transcription to chromatin templates [37–40]. In addition,
recent evidence suggests that histone acetylation may alter
the structure of histone tails by increasing their alpha-helical
content [41]. In contrast, methylation does not alter the
overall charge of the histone tails, however, increasing methyl
addition (mono, di or tri) does increase its basicity and
hydrophobicity (Figure 1a). Furthermore, increased methyl
addition on histone tails increases their affinity for anionic
molecules (i.e. DNA) and results in increased resistance to
trypsin digestion [42,43]. Taken together, these observations
suggest a tight association between the methylated histone
tail and DNA and/or chromatin. Therefore, similar to acetylation, methylation of histones may alter the interaction of
the histone tails with the DNA and/or chromatin-associated
proteins and, hence, nucleosomal structure and function.
The packaging of eukaryotic DNA into nucleosomal arrays
presents a major obstacle to transcription that must be
dealt with to allow the transcriptional machinery to access
the DNA template. The discovery of enzyme complexes
dedicated to chromatin remodeling, whether by directly
modifying histone proteins or by ATP-dependent nucleosomal remodeling complexes, has led to new insights into
the mechanisms of transcription [2,12,33,34]. Compelling
evidence obtained over the past six years indicates that
acetylation of core histones, which is catalyzed by the histone acetyltransferases (HATs) and removed by the
histone deacetylases (HDACs) (Figure 1a), is causally
linked to transcriptional activation. Interestingly, a surprisingly large number of previously identified components of
the transcriptional apparatus are HATs [35,36].
Enzymatic acetylation of the ε-amino group of conserved
lysine residues in the amino-terminal tails of histones
Histone methylation versus histone acetylation Rice and Allis
Methylation of histones is catalyzed by histone methyltransferases (HMTs), which use S-adenosylmethionine
(SAM) as a cofactor in much the same way that HATs
utilize acetyl-coenzyme A as a cofactor (Figure 1a). In contrast to acetylation, the majority of data available suggests
that the methyl modification is relatively irreversible
[19,43,44]. Because there is little evidence for large scale
decreases in methylated histones from bulk chromatin, the
existence of a global histone demethylase (HDM) seems
unlikely. However, like acetylation, it is plausible that
regulated or targeted demethylation of histones occurs on
specific residues at specific loci or promoters, although
these activities have yet to be discovered (Figure 1a).
265
during DNA replication and reassembled by G2/M,
concomitant with the observed peak activity of histone
methylation [50]. Once established (following replication),
telomeric silent chromatin is relatively stable. These early
observations suggested that histone methylation, in certain
circumstances, may serve as a stable epigenetic mark that
aides in the establishment of discrete chromosomal regions
involved in specific chromatin-mediated events. Moreover,
histone methylation may have the capacity to impart different biological functions depending upon the specific
methylated histone, lysine residue and chromatin-associated
protein involved (see below).
Histone methylation as an epigenetic mark
The major targets for post-translational acetylation and
methylation are conserved lysine residues located in the
amino-terminal tails of histones H3 and H4, although these
modifications also occur on the H2A and H2B tails [45].
Acetylation of specific lysine residues by specific HATs is
well documented [35,36]. Similarly, histone methylation is
also a non-random event in vivo, as specific lysines are
selectively methylated (Figure 1b; [45]). Therefore, similar
to HATs, each HMT is likely to have its own unique set of
kinetic parameters, as well as histone and lysine preferences. Furthermore, various methyllysine species (mono, di
or tri) have been observed for each methylated lysine
residue in vivo, however, the biological significance of these
differences remains undetermined (Figure 1a).
As depicted in Figure 1b, specific lysine residues in both
the H3 and H4 tails appear to be targeted for either acetylation or methylation. However, one lysine in the H3 tail
(Lys9) can be targeted for both modifications, presumably
in different biological contexts (see below). It is possible
that other acetylated lysines could also be targets for
methylation, as early studies using bulk histone fractions
would not have been able to detect minor changes in
methylation. With the advent of more sensitive methods
and techniques, such as mass spectrometry and immunological reagents, we look forward to a critical re-examination
of this issue. Taken together, these observations suggest
that certain combinations of acetyl and methyl modifications of lysines in histone tails may have antagonistic or
cooperative biological effects. For example, hyperacetylated H4 from transcriptionally active chromatin preparations
is a preferential target of histone H3 methylation, suggesting that these modifications may act synergistically to
promote transcription in a way that remains unclear [26].
Histone acetylation occurs throughout the cell cycle, whereas histone methylation peaks in G2 phase, subsequent to
DNA replication and histone synthesis, and during heterochromatin assembly [17,18,46–48]. In addition, developing
rat neurons have robust HMT activity, whereas non-replicating adult neurons exhibit marked decreases in HMT activity
[49]. Furthermore, using telomere position effect variegation
(TPE) as a model for epigenetic silencing, data in yeast suggest that this repressive chromatin state is disassembled
Eukaryotic genomes are often conveniently described as
transcriptionally active (euchromatin) or transcriptionally
silent (heterochromatin). Heterochromatin was originally
defined as the fraction of the genome that remained visibly
condensed during interphase. More recently, heterochromatin has been defined as genomic regions that are gene
poor, contain large blocks of repetitive DNA, are inaccessible to DNA-modifying reagents and replicate late in the
cell cycle [51,52]. Interestingly, when a transcriptionally
active gene is displaced from its normal euchromatic position to the vicinity of heterochromatin, the gene becomes
and remains inactivated [53–55]. Remarkably, epigenetic
inheritance of this inactivated state is propagated during
mitosis and through the germ-line during meiosis [56,57].
This epigenetic phenomenon, known as position effect
variegation (PEV), provides an attractive model to understand the heritable molecular imprint that specifies the
transcriptional state of a gene, as well as the factors that
influence its stability.
Genetic screens for suppressors or enhancers of PEV in
Drosophila melanogaster [53,58] and Schizosaccharomyces
pombe [59,60] have identified numerous genes whose products probably stabilize and/or propagate higher-order
chromatin structure. For example, about 30–40 loci, collectively referred to as the Su(var) group (suppressor of
variegation) [53], include catalytic components such as
HDACs, protein phosphatases and S-adenosylmethionine
(SAM) synthetases [61,62], as well as heterochromatinassociated proteins, such as heterochromatin-associated
protein 1 (HP1) (Su(var)2-5) [63], which is thought to play
an architectural role (see below).
Consistent with the paradigm established between histone
acetylation and transcription, the recent discovery that a
Su(var) protein is a lysine histone methyltransferase has provided a critical link between histone methylation and
heterochromatin assembly [29••]. The Drosophila Su(var)3-9
protein is localized in condensed chromatin and is a key regulator in the organization of repressive chromatin; however,
its precise function was not known [64]. Rea et al. [29••]
demonstrated that the human and S. pombe homologues of
Su(var)3–9, SUV39H1 and Clr4, respectively, are HMTs that
specifically methylate H3 Lys9 in vitro. The methylation of
266
Nucleus and gene expression
Figure 2
(a)
PCAF HAT
TAFII250 HAT
HAT
Bromo
Double bromo
Ac
Ac
Ac
Ac
Lys
Lys
Lys
Lys
Transcription
(b)
SUV39H1, Clr4 HMTs
Chromo
Cys
SET
HP1, Swi6
Cys
Chromo
Shadow
?
?
Me
Me
Me
Lys
Lys
Lys
Heterochromatin
Current Opinion in Cell Biology
H3 Lys9 was catalyzed by the conserved SET domain
(Su(var)3-9, E(z), trithorax) and two flanking cysteine-rich
regions in SUV39H1 and Clr4 (Figure 2b). All three of these
regions are required for HMT activity in vitro since other
SET domain-containing proteins that lack one or more of
these regions, including human EZH2 and HRX, fail to
methylate histones. Interestingly, most Suv39h-null mice
were non viable. The small fraction that remained viable
were growth retarded compared to control mice, suggesting a
role for histone methylation in normal development.
Furthermore, primary mouse embryonic fibroblasts
(PMEFs) derived from the Suv39h-null mice exhibited
increased division defects during mitosis and had weakly
defined heterochromatic regions compared to normal
PMEFs. Consistent with this, overexpression of SUV39H1
induced ectopic heterochromatin formation. Taken together,
these novel findings implicate H3 Lys9 methylation in the
proper assembly of heterochromatin in a conserved pathway
leading to epigenetic silencing.
Histone H3 Lys9 methylation, HP1 recruitment
and heterochromatin assembly
Several nuclear HATs (A-type) contain an evolutionarily
conserved motif known as the bromodomain (Figure 2a).
Recent discoveries indicate that the bromodomain and
double bromodomain of PCAF and TAFII250, respectively, bind preferentially and specifically to acetylated lysines
on histone tails in vitro (Figure 2a; [65••,66••]). Therefore,
as predicted by the ‘histone code’ hypothesis [13•,14•],
Function of conserved motifs within certain
chromatin-modifying proteins. (a) Schematic
drawing of conserved motifs within the
transcriptional regulators PCAF, a GCN5
homologue, and TAFII250, a subunit of the
TFIID complex. PCAF and TAFII250 contain a
HAT catalytic domain that acetylates specific
lysine residues on the histone tails (left; not
shown for TAFII250). In addition, each protein
contains an evolutionarily conserved
bromodomain (bromo) and double
bromodomain, respectively, that binds to the
appropriately acetylated lysines on the histone
tails to promote transcription (right).
(b) Schematic representation of the
Su(var)3–9 family of HMTs and
heterochromatin-associated proteins. Human
SUV39H1 and fission yeast Clr4 proteins
contain a conserved catalytic SET domain
flanked by two cysteine-rich regions (Cys),
which are required for methyltransferase
activity (left). The evolutionarily conserved
chromodomain (chromo) of human HP1 and
fission yeast Swi6 proteins bind to the
appropriately methylated histone tail (i.e. H3
Lys9) to induce the assembly of
heterochromatin (right). The exact functions of
the HMT chromodomain and HP1 chromo
shadow domain (shadow) are not known.
Note: these drawings are not to scale.
one functional consequence of histone acetylation may be
that the acetyl modification serves as a ‘mark’ on the histone tail that leads to the recruitment and binding of
bromodomain-containing HATs and other coactivators to
chromatin for transcription [67]. These findings strongly
support the theory that alterations in nucleosomal structure, induced or perturbed by covalent histone
modifications, may be important in the recruitment of
chromatin-associated proteins that ultimately influence
distinct cellular processes [41,68•].
Recent findings indicate that a similar conserved pathway
may exist for histone methylation and heterochromatin
assembly, with the methyl modification serving as the ‘mark’
on the histone tails to recruit heterochromatin-associated
proteins. A potential methyl-histone binding candidate is
HP1 (Figures 2b and 3a). Interestingly, HP1 and its S. pombe
homologue, Swi6, are required for heterochromatin formation and colocalize with SUV39H1 and Clr4, respectively
[63,69,70•]. Many heterochromatin-associated proteins, like
HP1 and Su(var)3-9, share a common evolutionarily conserved domain known as the chromodomain (chromatin
organization modifier) (Figure 2b; [63,71]). In addition,
numerous heterochromatin-associated proteins contain a single or repeated chromodomain (CD) followed by a chromo
shadow domain (CSD) [72]. These domains were thought to
mediate protein–protein interactions responsible for targeting these proteins to their proper chromosomal positions by
mechanisms that remained unclear until recently [57,73].
Histone methylation versus histone acetylation Rice and Allis
267
Figure 3
Temporal model of heterochromatin assembly.
(a) Key players involved in heterochromatin
assembly in D. melanogaster and S. pombe.
The transcriptionally silent pericentric
heterochromatin in flies and the silent matingtype locus (mat) and centromeric repeats
(cen) in fission yeast are depicted as red
bricked structures. Several well-studied,
conserved chromatin-associated proteins are
shown for both organisms, these are HDACs,
HMTs or heterochromatin-associated proteins
(e.g. HP1). *Rpd3 was previously shown to be
an enhancer of PEV [61]. (b) One proposed
temporal pathway leading to the
establishment of transcriptionally silent
heterochromatic regions with regard to the
covalent modifications in the histone H3 tail.
The acetyl group on H3 Lys9, a modification
often associated with transcriptionally active
regions, is removed by an HDAC prior to
methylation by an HMT. The CD of an HP (i.e.
HP1) selectively recognizes and binds to the
H3 Lys9-methyl modification resulting in the
self-assembly and propagation of
heterochromatin and transcriptional silencing.
Other trans-acting factors and covalent
histone modifications are likely to influence
the temporal sequence, kinetic steps and final
outcome of this proposed pathway (see text
for details).
(a)
D. melanogaster
S. pombe
∗Rpd3
HDAC
clr3,clr6
Su(var)3-9
HMT
clr4
Su(var)2-5
HP1
swi6
mat and cen loci
Pericentric heterochromatin
(b)
Active
Ac
Lys9
Histone H3
HDAC
Ac
Lys9
HMT
HP1
Me
CD
HP1
Propagation
CD
Propagation
Me
Lys9
Silent
Current Opinion in Cell Biology
Two independent research groups have recently shown that
the chromodomain, but not the chromo shadow domain, of
HP1 and Swi6 preferentially bind to methylated H3 Lys9
in vitro [74••,75••]. The chromodomain of HP1 was selective
for the H3 Lys9-methyl modification, as demonstrated by
the lack of binding to an H3 unmodified or H3 Lys4-methyl
peptide [75••]. In addition, the H3 Lys9-methyl modification
appears to be highly specific for the HP1 chromodomain, as
other chromodomain-containing proteins, including
SUV39H1, polycomb (M33) and Mi-2, failed to bind to an
H3 Lys9-methyl peptide in these assay conditions.
Interestingly, HP1 chromodomain binding affinity was
significantly less than that of the full-length HP1, suggesting
that native HP1 (or an HP1 dimer) is required for high-affinity binding in vivo [74••,75••]. In Suv39h1-null PMEFs, HP1
is dispersed from heterochromatic regions and ‘rescue’ of
SUV39H1 by retroviral expression in these cells induced
localization of HP1 to heterochromatin foci [74••]. Similarly,
in S. pombe, functional Clr4 protein is required for Swi6 localization and transcriptional silencing at centromeric regions
[75••]. Therefore, analogous to the bromodomain-binding
acetylated histone peptides, chromodomain-containing
proteins, such as HP1 and Swi6, are predicted to bind to
the H3 Lys9-methyl modification catalyzed by the
Su(var)3-9 family of HMTs in order to establish silent regions
of heterochromatin.
In S. pombe and D. melanogaster, genes known to encode
heterochromatin-associated proteins have been identified
and include HDACs, HMTs and HP1-like proteins
(Figure 3a). Importantly, homologues of these proteins
exist in yeast, flies and mammals underscoring what is
likely to be a highly conserved pathway of heterochromatin assembly. As these proteins are found together in
repressive complexes, it is thought that they act cooperatively to induce a silent epigenetic state that propagates
along distinct chromosomal regions. However, the precise
function(s) of these components and the kinetic steps
leading to the initiation and maintenance of this epigenetic
pathway have remained elusive until quite recently.
268
Nucleus and gene expression
Proposed pathway of heterochromatin
assembly leading to epigenetic silencing
As depicted schematically in Figure 3b, an understanding
of what may be one major pathway of heterochromatin
assembly and heterochromatin-induced gene silencing is
beginning to emerge. In this model, the status of covalent
modifications on the H3 tail plays a critical role in the final
outcome of heterochromatin assembly, leading to an epigenetically silent state. For example, H3 Lys9 is acetylated
prior to histone deposition in some species [76], suggesting
that one role of HDACs in these complexes may be to
deacetylate Lys9 to ‘clear’ the ε-amino group of Lys9 so
that it can be methylated by members of the Su(var)3-9
family of HMTs. Conversely, acetylation of Lys9 may serve
to maintain transcriptionally competent regions of the
genome by blocking methylation of Lys9 and preventing
the downstream assembly of heterochromatin. We envision that once the acetyl modification is enzymatically
removed by HDACs, the responsible HMT is free to
methylate Lys9, resulting in the recruitment of HP1.
Binding of HP1 ultimately leads to the formation of heterochromatin via a self-propagating pathway that may
involve dimerization of HP1 molecules through chromoshadow–chromo-shadow domain interactions.
The proposed link between HP1-mediated heterochromatin
assembly and H3 Lys9 methylation depicted in Figure 3
leaves many important questions unresolved. This model
relies on the recognition of chromodomains in proteins such
as HP1 with appropriately methylated histone tails. Whether
active demethylases exist to reverse this state, or whether the
long suspected methyl ‘marks’ are only removed by histone
replacement or repeated rounds of DNA replication, is not
clear. It also remains unclear whether H3 Lys9 is the physiological target of the SET-domain-containing HMTs in vivo
[74••,75••]. In addition, Figure 3 predicts that the H3 Lys9methyl modification and HP1 modification may be localized
exclusively to heterochromatic regions. If this model is accurate, it is currently unresolved where the boundaries
between H3 Lys9 methylation and euchromatin begin and
end. Furthermore, if HP1 localization is functionally
dependent upon H3 Lys9 methylation, it is unclear how
overexpression of HP1 can ‘spread’ heterochromatin [77].
This could be explained by an HP-1–HMT interaction,
which would serve to recruit additional HMTs, methylate
additional histones and propogate the spreading phenomenon (see also Update).
Other chromatin-associated proteins or other covalent
histone modifications may be required for proper
heterochromatin assembly. During chromatin assembly,
H4 acetylated at Lys5 and Lys12 is deposited onto newly
synthesized DNA and, once deposited, these residues are
usually deacetylated [14•,76]. The association of chromatin
assembly factor 1 (CAF-1), deposition-related HAT
complexes and certain repressive HDAC complexes
containing RbAp48/p46 could play a fundamental role in
heterochromatin assembly [78]. CAF-1 is a polypeptide
complex that mediates histone deposition on newly
replicated DNA. The large subunit of CAF-1, p150, binds
to the chromo shadow domain of HP1 and is concentrated
at regions of heterochromatin [79]. Deletion of the HP1
binding region of p150 results in derepression of a transfected reporter gene in mammalian cells [80]. These data
suggest that CAF-1 has a heterochromatin-associated
function that may be linked to the well known late
replication property of heterochromatin.
RbAp48/p46 proteins appear to facilitate the nucleosome
remodeling that is necessary for the efficient
acetylation/deacetylation of lysines in H4 that are covered
by the natural wrapping of DNA around the histone
octamer [81]. It is interesting to note that H4 Lys20, which
is very close to the first alpha helix of H4, is methylated
late in the cell cycle, near to the G2/M boundary [41].
Whether methylation of H4 Lys20 and/or acetylation at
Lys5 and Lys12 are covalent ‘markings’ that facilitate the
binding of HP1 or other chromodomain-containing, heterochromatin-associated proteins is not known but remains
an intriguing possibility. In addition, other covalent histone modifications, such as H3 Ser10 phosphorylation,
antagonize H3 Lys9 methylation [15,29••]. Thus, it
remains a formal, and likely, possibility that other transacting factors besides those outlined in Figure 3a, and
other histone modifications besides those illustrated in
Figure 3b, play critical, yet undetermined, roles in the
overall heterochromatin assembly pathway. As immunological reagents are developed against these proteins and
as physiologically relevant covalent histone modifications
are identified, we look forward to in vivo tests of these and
related models using chromatin immunoprecipitation
(ChIP) assays (see also Update).
Histone methylation and transcriptional
regulation
Although transcriptional activation is typically attributed to
histone acetylation, the specific acetylation of H4 Lys12 is
associated with silent chromosomal regions in various organisms [82,83]. In contrast to H3 Lys9, methylation of H3 Lys4
is specifically associated with the transcriptionally active
macronuclei, but not the inactive micronuclei, in
Tetrahymena [28•]. Moreover, H3 Lys4-methyl modification
is conserved throughout evolution. Interestingly, acetylated
isoforms of H3 and H4 are the preferential targets of histone
methylation, suggesting that HMTs and HATs act synergistically to promote transcription by mechanisms that remain
unclear [24–26,84]. These findings suggest a correlation
between H3 Lys4 methylation, histone acetylation and transcriptional competency. Further studies are required to
clarify this since methylation of arginine residues in H3 and
H4 have also been implicated in transcriptional regulation
[32•]. In addition, histone acetyltransferase CBP/p300
(CREB-binding protein) coimmunoprecipitates with an
HMT [85] and, although unidentified, the HMT is likely to
be the SET domain-containing, CBP-associated ASH1 protein [86]. Interestingly, the preferred site for methylation of
Histone methylation versus histone acetylation Rice and Allis
269
Figure 4
Histone acetylation or methylation: a matter of
choice. (a) Lysine residues in the histone tails
can be acetylated by a HAT that can be
rapidly removed by an antagonistic HDAC
(bold arrows). Following a balanced increase
in histone acetylation, the acetyl modification
may recruit and bind to a conserved domain
within a transcriptional regulator protein
(TXR), such as the bromodomain of HATs
(Figure 2A). The specific acetylated lysine(s)
and the biological function of the TXR
determine the final outcome leading to a
transcriptionally active or silenced state.
(b) Prior to lysine methylation of a histone tail
by an HMT, the acetyl modification must first
be ‘cleared’ by an HDAC. Once methylated, a
conserved domain within a TXR may bind to
the methyl modification resulting in
transcriptional activation or silencing
depending upon the specific methylated lysine
and biological function of the TXR. It is
currently not known to what extent this
pathway is reversible; a pathway that would
require the activity of an, as yet, unknown
histone demethylase (HDM?). Depending on
the (ir)reversibility of pathway (b), histone
methylation may lead to a more stable
epigenetic state compared to histone
acetylation.
Histone tail
(a)
(b)
Lys
Ac
HAT
HMT
Ac
Me
Me
TXR
TXR
HDAC
Ac
Active
this HMT in vitro was H3 Lys9, suggesting that H3 Lys9
methylation may play a role in both transcriptional activation and silencing. Could differences in methylated species
of H3 Lys9 (i.e. mono, di or tri) explain these seemingly
contradictory data (Figure 1a)?
Different covalent modifications, alone or in combination,
on the same or different histone tails are associated with
distinct cellular process by the recruitment of chromatinassociated proteins, as predicted by the ‘histone code’
[13•,14•]. The acetylation status of certain lysine residues
in histone tails appears to result from the opposing activities of HATs and HDACs (Figure 4a). The acetyl
modification can be rapidly turned over in some chromatin
environments [2,87], suggesting that the ‘strength’ or ‘transient’ nature of gene expression may be coupled to the
degree of acetylation. This acetyl modification may serve
as a labile ‘mark’ permitting the binding of conserved
motifs within specialized transcriptional regulator proteins
(TXR), such as the bromodomain(s) within PCAF or
TAFII250 (Figure 2a). The biological consequences of this
interaction would be dependent upon the specific acetylated lysine residue(s) and the inherent properties of the
TXR, resulting in either transcriptional activation or
silencing. For example, the double bromodomain of the
TAFII250 HAT binds to diacetylated Lys5/Lys12 or
Lys8/Lys16 in histone H4 resulting in transcription. In
contrast, H4 Lys12 acetylation is coincident with silencing,
however, the chromatin-associated protein and its
conserved binding motif are currently unknown.
HDM?
Me
Lys
Lys
TXR
TXR
Ac
Me
Lys
Lys
Silent
Active
Silent
Current Opinion in Cell Biology
For histone methylation to take place, we envision that the
target lysine residue must first be cleared of any other preexisting modifications, for example, acetylation (Figure 4b).
Because no HDMs have yet been identified, methylation of
histones may serve as a more stable epigenetic mark, in contrast to the rapidly turned-over populations of acetylated
chromatin. Once methylated by an HMT, the methyl
‘mark’, like the acetyl ‘mark’, may bind to conserved motifs
within specialized TXRs, such as the chromodomain of
HP1. Again, the effects of this interaction on transcription
would be dependent upon the specific methylated lysine
residue(s) and the inherent properties of the TXR
(Figure 4b). For example, the chromodomain of HP1 binds
to the H3 Lys9-methyl ‘mark’, resulting in heterochromatin
assembly and epigenetic silencing. In contrast, H3 Lys4
methylation appears to be associated with transcription,
however, the chromatin-associated protein and its conserved
binding motif are currently unknown.
Although not all chromodomain-containing proteins bind
to H3 Lys4 or Lys9 methylated peptides in vitro
[74••,75••], we note that there are chromodomain-containing HATs that seem to facilitate transcription, such as
MOF and Esa1 [35,36]. Could the chromodomains of these
proteins bind to methylated histones in vivo to promote
transcription and, if so, which methylated lysines do they
bind? In addition, there are other chromodomain-containing proteins associated with repressive chromatin
remodeling complexes, such as Mi-2 of NuRD [88–90].
Interestingly, the Mi-2 ATPase subunit of NuRD contains
270
Nucleus and gene expression
a double chromodomain and is thought to be associated
with transcriptional silencing by DNA methylation (see
below). Analogous to the double bromodomain of
TAFII250 (Figure 2a), we wonder to what extent the double chromodomain of Mi-2 might selectively bind to
appropriated methylated histone tails. We also wonder if
this double chromodomain may exhibit a preference for
the appropriately dimethylated lysines within the same or
distinct histone tails.
Histone methylation and potential links to
DNA methylation and cancer
The methylation of cytosines in CpG island promoters is
associated with the transcriptional silencing of human
tumor suppressor genes by the recruitment of methyl-binding proteins (MBPs) and their associated repressive
complexes [91]. Some of these repressive complexes, such
as Sin3A and NuRD, contain HDACs that deacetylate H3
and H4 [90,92]. Although a few transcriptionally silenced
tumor suppressor genes in cancer cell lines can be partially
reactivated in the presence of a DNA methylase inhibitor
and histone deacetylase inhibitor, complete reactivation is
rarely achieved [93]. On the basis of these observations, we
speculate that these MBP-associated repressive complexes
may contain HMTs that serve to ‘lock’ the promoter and
chromatin into a transcriptionally inactive state that is maintained through replication regardless of histone acetylation
and DNA methylation status.
The disruption of many chromatin-modifying proteins are
coincident with various human diseases, including cancer
[94]. Although it has long been known that changes in cellular heterochromatin content are associated with more
aggressive cancers [77,95], a recent report shows a direct correlation between HP1 relocalization and invasive/metastatic
breast cancer [96]. On the basis of recent findings that HP1
binds the H3 Lys9-methyl modification, it is possible that
the altered HP1 localization may be directly linked to the
methylation status of Lys9. Besides altering genomic stability, the increase or decrease of H3 Lys9 methylation and
HP1 binding could have a number of effects on the progression of a metastatic phenotype, including the aberrant
silencing of tumor suppressor genes and/or activation of
oncogenes, respectively.
Conclusions and future directions
In this review, we have used histone acetylation as the paradigm to discuss the functional and biological importance
of histone methylation in different cellular processes. Past
reports and recent findings suggest that the methylation of
lysine residues in histone tails functions as a stable epigenetic mark to localize chromatin-associated proteins in
order to direct specific chromatin-mediated events.
Furthermore, methylation of different lysine residues on
the same histone tail in vivo can be associated with opposing biological processes such as transcriptional upregulation
and heterochromatin assembly leading to epigenetic silencing. We suspect that histone methylation will have
widespread and far reaching implications in numerous
epigenetic phenomena including, but not limited to,
imprinting, X-inactivation, differentiation, transposition
and programmed DNA rearrangements.
In vivo sites of histone methylation remain to be determined, especially for H2A and H2B, and as presented
earlier, arginine methylation has yet to be documented in
cellular histones. Further studies are required to determine the biological significance of each modified residue.
To this end it will be useful to generate an arsenal of antibodies specific for each known methylated residue in the
histone tails, arginine or lysine. The use of these antibodies in ChIP experiments, in combination with high density
microarrays of human gene promoters, would yield considerable insights into the global effects and mechanisms of
histone methylation and transcriptional regulation in normal and diseased cells. In addition, it is likely that many
more HMTs have yet to be discovered, each with their
own substrate and site specificity. The discovery of new
HMTs will probably result in the identification of novel
conserved families of proteins that contain unique catalytic domains distinct from the well defined SET domain.
In keeping with the flurry of research in the histone
acetylation field during the past six years, ignited by the
discovery of the transcription-associated HATs and
HDACs [97,98], we anticipate that the discovery of the
first HMTs [27••,29••] will lead to many more exciting
discoveries centered around histone methylation. In fact,
we predict that the methyl modification of histones may
prove to be as significant in the regulation of chromatin
structure and function as histone acetylation. Only time
will tell.
Update
A recent article by Nakayama et al. [99•] demonstrates the
enzymatic methylation of H3 Lys9 is required for proper
heterochromatin assembly in vivo. Using H3 Lys9-methyl
specific antibodies in ChIP assays and fission yeast as the
model system, it was shown that functional Clr4 was
required for H3 Lys9 methylation, appropriate localization
of Swi6 to the mat and cen loci, heterochromatin formation
and epigenetic silencing. Furthermore, it was shown that
other histone modifications, such as acetylation of H3 Lys9
and Lys14, negatively regulate this process. These findings provide novel insights into a conserved pathway of
heterochromatin assembly, whereby sequential histone
modifications lead to epigenetic silencing in organisms
ranging from yeast to humans (Figure 3).
Acknowledgements
In some cases, readers are directed to excellent reviews wherein many of
the key primary literature are referenced. We apologize for not being able
to cite all of the primary literature due to space limitations. We wish to
thank Patrick Grant and current laboratory members for their input,
especially Jim Bone, Peter Cheung and Craig Mizzen. In addition, we
would also like to thank Upstate Biotechnology for their continued
support. Research support to CDA is provided by a MERIT Award from
the National Institutes of Health.
Histone methylation versus histone acetylation Rice and Allis
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