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RESEARCH PAPER
1 Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, Virginia 22908-0733, USA , 2 Laboratory of Chromatin Biology, The Rockefeller University, New York, New York 10021, USA , 3 Argonne National Laboratory, Biosciences Division/Structural Biology Center, Argonne, Illinois 60439, USA
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Abstract |
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 Top Abstract ResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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[Keywords: Polycomb; HP1; heterochromatin; histone H3; lysine methylation; epigenetic]
Received May 7, 2003; revised version accepted June 2, 2003.
). Histone modifications can be highly reversible, such as
histone acetylation, or more stable, such as histone (lysine) methylation
(Zhang and Reinberg 2001;
Lachner and Jenuwein 2002).
Thus, a wide range of chromatin-based regulatory options is available. These
include dynamic marks permitting rapid changes in gene expression in response
to physiological and environmental stimuli as well as more permanent indexing
systems required for the passage of heritable patterns of epigenetic
information from one cell generation to the next
(Fischle et al. 2003). The
identification of enzyme systems responsible for the steady-state balance of
posttranslational histone modifications, together with the discovery of
binding modules that "read" covalent marks on histones, have been
key for our present understanding of gene regulation in the context of the
chromatin polymer.
Bromodomains have been the first modules implicated in the read-out of
histone marks. They show affinity for acetylated lysines in histone and
nonhistone proteins (for review, see Zeng
and Zhou 2002), and local recruitment of bromodomain factors to
certain regions of chromatin might function in mediating
acetyl-histone-encoded antisilencing
(Ladurner et al. 2003). In
contrast, a second conserved module found in a variety of chromosomal
proteins, the chromodomain, has been implicated in binding to methylated
lysines on the histone tails (Bannister et
al. 2001; Jacobs et al.
2001; Lachner et al.
2001). Indeed, recently a biochemical pathway of gene repression
by heterochromatin assembly, involving methylation of Lys 9 of H3 by SET-type
histone methyltransferases (HMTs), and the read-out of this methylation mark
by the chromodomain of HP1 (heterochromatin protein
1), has been established
(Schotta et al. 2002;
Snowden et al. 2002;
Cheutin et al. 2003).
Furthermore, the three-dimensional structure of HP1 revealed that three
"caging" aromatic residues are necessary for methyl-Lys 9 binding
of this domain (Jacobs and Khorasanizadeh
2002; Nielsen et al.
2002). Many, but not all chromodomain proteins identified to date,
contain such aromatic residues at conserved positions
(Jacobs and Khorasanizadeh
2002). However, it is unclear if these additional chromodomains
indeed bind to methylated lysines and if they have preferences for specific
methyl marks on histones or other proteins.
HP1 is a conserved chromosomal protein that participates in chromatin
packaging and gene silencing (for review, see
Eissenberg and Elgin 2000).
Loss of HP1 leads to lethality in Drosophila and correlates with
metastasis in human breast cancer cells
(Kirschmann et al. 2000).
Factors of the Polycomb group (PcG) of proteins are part of a widely conserved
cell memory system that controls repressed transcriptional states of many loci
in the genome, including developmentally and cell-cycle-regulated genes. The
PcG proteins were first identified in the fruit fly Drosophila
melanogaster, but homologs have been identified in all higher organisms
(for review, see Jacobs and van Lohuizen
2002; Orlando
2003). These proteins are required for long-term transcriptional
silencing of the Drosophila homeotic genes, which are required for
proper embryonic development. In mammalian systems, PcG repressors are
implicated in hematopoesis, X inactivation, B-cell development and control of
cell proliferation. Mutations in PcG proteins have also been recently linked
to cancers of the immune system and prostate (for review, see
Simon 2003). However, the
mechanisms by which PcG proteins repress transcription are largely unknown. In
Drosophila, Polycomb response elements (PRE) have been identified and
implicated for PcG targeting. However, such DNA elements have been elusive in
mammalian systems. The PcG proteins are known to be present in multiprotein
complexes. The best-characterized complex is Polycomb-repressive complex (PRC)
1, which contains the Polycomb, Polycomb-like, Polyhomeotic, Posterior Sex
Combs, and Sex Combs on Midleg proteins, among others (for review, see
Simon and Tamkun 2002). The
gene-repressing activity of the PRC1 complex has been suggested to involve
activities that render target regions resistant to remodeling by
chromatin-remodeling complexes (Shao et
al. 1999).
Recent reports have provided breakthrough evidence that a second PcG
complex, the Esc-E(z) complex, contains HMT activity. This activity is
dependent on the E(z) (enhancer of zeste) SET domain protein, and the complex
has been reported to preferentially methylate Lys 27 and Lys 9 on the H3 tail.
Furthermore, it has been suggested that the PcG chromodomain protein Polycomb
(Pc) could act as a binding module for methyl-Lys 9 and methyl-Lys 27 in the
H3 tail, thereby critically mediating the targeting of PcG complexes to
different sites of the epigenome (Cao et
al. 2002; Czermin et al.
2002; Kuzmichev et al.
2002; Muller et al.
2002). Interestingly, when the chromodomain of HP1 was substituted
by the chromodomain of Pc, the chimeric HP1Pc was recruited to
PcG-binding sites on polytene chromosomes
(Platero et al. 1995). This
implies that either the chromodomain is sufficient to recognize and be
recruited to different methyl marks on the H3 tail, or the Pc chromodomain
specifies critical interactions with other PcG components that mediate
recruitment to PcG sites. So far, the extent by which Pc binds preferentially
to either repressive methyl-Lys 9 or methyl-Lys 27 marks on the H3 tail is
unclear. Furthermore, the role of chromodomain binding to methyl marks for
recruitment to different target sites has not been established. Equally
unclear is whether any potential discrimination of Pc or HP1 for one lysine
over another is an intrinsic structural feature of their chromodomains.
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Results |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The chromodomains of Pc and HP1 are highly conserved (54% identity in
protein sequence; see Fig. 1A).
Moreover, the Pc chromodomain contains three "caging" aromatic
residues that were shown in HP1 to be necessary for methyl-Lys 9 binding
(Jacobs and Khorasanizadeh
2002). The amino acid sequences immediately surrounding Lys 9 and
Lys 27 in the H3 tail are very similar as shown in
Figure 1B and, in particular,
share a consensus sequence ARKS. However, the residues flanking this consensus
motif are unrelated. To determine the extent by which Pc and HP1 are able to
discriminate between the Lys 9 and Lys 27 methylation sites, we used a set of
synthetic methylated H3 peptides to measure the relative binding affinities of
their chromodomains to these histone H3 tail segments. Fluorescence
polarization measurements indicated a clear preference of the chromodomain of
Pc for the trimethylated Lys 27 site. Specifically, the dissociation constant
for the trimethyl-Lys 27 peptide was 5 µM, and that for the trimethyl-Lys 9
peptide was 125 µM (Fig.
1C). In contrast, the HP1 chromodomain bound to the trimethyl-Lys
9 peptide with an affinity of 4 µM and to the trimethyl-Lys 27 peptide with
an affinity of 64 µM (Fig.
1C; Jacobs and Khorasanizadeh
2002). These data indicate that both protein modules discriminate
methyl marks effectively, with Pc showing a 25-fold selectivity and HP1
showing a 16-fold selectivity for the cognate versus noncognate target
sequences.
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We also investigated to what extent the degree of methylation affected target selection. Binding of Pc to a peptide with mono- or dimethylated Lys 27 peptides was about five times weaker than binding to the trimethylated Lys 27 peptide, but still much stronger than binding to the trimethylated Lys 9 peptide (Fig. 1C). Furthermore, no significant interactions between the Pc chromodomain and mono- or dimethylated Lys 9 peptides were observed. HP1 binding to dimethyl- and monomethyl-Lys 9 was weakened 2-fold and 15-fold, respectively. These results indicate that the degree of methylation does affect the binding of both chromodomains to their target sites and that the trimethyl-lysine is the preferred level of modification for both proteins in vitro. In the context of trimethyl-lysine, each protein shows clear discrimination for its cognate site, likely because of differences in sequence context of the trimethyl-Lys in H3 as well as the binding groove of the chromodomain (see below).
Colocalization of Pc and H3 Lys 27 methylation on polytene
chromosomes
Using immunofluorescence staining, we have previously shown that the HP1
protein is localized almost exclusively to the chromocentric heterochromatin
of Drosophila salivary gland polytene chromosomes, a chromosomal
domain highly enriched in H3 Lys 9 methylation
(Jacobs et al. 2001). To
investigate the specific localization pattern of the Pc protein and to
correlate its distribution with chromosomal regions of H3 Lys 9 and H3 Lys 27
methylation, we performed similar immunostaining experiments using newly
developed anti-H3-Me3K27-specific antibodies in combination with
antibodies specific for Pc. As shown in
Figure 2A, the
anti-H3-Me3K27-specific antibodies recognized many bands on the
arms of polytene chromosomes. A weaker immunofluorescence signal for this
modification was detected around the chromocentric regions. Whether the lower
staining in this area represents a low level of H3 Lys 27 trimethylation at
the chromocenter or might be caused by slight cross-reactivity of the
antibodies with the H3 Lys 9 methyl mark is unknown (data not shown; see
Materials and Methods). Importantly, the bands labeled by the
anti-H3-Me3K27-specific antibodies were highly correlated with
bands detected by anti-Pc-specific antibodies. Indeed, >90% of bands
labeled by these antibodies showed a clear overlap
(Fig. 2A, merged image). In
contrast, antibodies specific for H3-Me3K9 only labeled the
chromocenter, but did not stain regions overlapping with the anti-Pc-specific
antibodies (Fig. 2B). Similar
results were obtained using anti-H3-Me2K9-specific antibodies (data
not shown). The colocalization of H3 Lys 27 trimethylation with Pc complex
proteins was independently verified by double labeling experiments using an
anti-Psc (posterior sex comb)-specific
monoclonal antibody in combination with the anti-H3-Me3K27-specific
polyclonal antibodies (data not shown). The immunostaining experiments are
consistent with recruitment of Pc to regions of H3 Lys 27 trimethylation but
not to regions of H3 Lys 9 trimethylation. This interpretation is in excellent
agreement with the observed binding preferences of the chromodomains of HP1
and Pc for methylated H3 tails in vitro.
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Different subnuclear localizations of Pc and HP1 in S2 cells are
correlated with different histone H3 methyl marks
To further analyze and compare the localization of histone H3 methylation
on Lys 9 and Lys 27 with that of the HP1 and Pc proteins in vivo, we performed
indirect immunofluorescence studies on Drosophila Schneider S2 cells.
In these diploid male cells, antibodies specific for H3 dimethylated or
trimethylated on Lys 9 accumulated in defined, often more internal, subnuclear
regions (Fig. 3A). In contrast,
immunostaining with antibodies specific for H3 trimethylated on Lys 27 showed
a more diffuse, external nuclear staining pattern, which almost decorated the
whole cell nucleus (Fig. 3B).
Costaining with antibodies specific for HP1 showed a complete overlap with the
anti-H3-Me2K9 signal as indicated by the yellow spots in the merged
image (Fig. 3A). These findings
are in good agreement with previously published observations on other cellular
systems and reinforce the idea that HP1 is recruited to sites of Lys 9
methylation in vivo (Moazed
2001; Cheutin et al.
2003). However, little, if any, significant overlap was detected
between the anti-HP1 and anti-H3-Me3K27 immunostainings. HP1 is
rather excluded from regions enriched in Lys 27 methylation in this cell line
(Fig. 3B). Furthermore, Pc
showed a subnuclear distribution very similar to the anti-H3-Me3K27
signal. Again, almost no overlap in staining with HP1 was detected
(Fig. 3C). Thus, Pc-mediated
gene silencing and H3-Lys 27 methylation appear to be similarly distributed in
diploid S2 cells. Both are mainly localized to areas distinct from the
heterochromatic regions marked by enrichment in HP1 and H3-Lys 9
methylation.
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Importance of the different chromodomains of Pc and HP1 for distinct
subnuclear targeting
Based on the different binding preferences of the chromodomains of Pc and
HP1 for selective methyl marks on H3 tail peptides in vitro, and the distinct
cellular localization patterns of Pc and HP1, we next wanted to test if the
chromodomains of Pc and HP1 are sufficient for subnuclear targeting in vivo.
Therefore, we generated chimeric fusion protein constructs, replacing the
chromodomain of Pc with that of HP1 (PcHP1-CD-V5) and the
chromodomain of HP1 with that of Pc (HP1Pc-CD-V5). These chimeric
fusion protein constructs and their wild-type counterparts were transiently
transfected into S2 cells. The cellular distribution of the fusion proteins
was then analyzed by immunofluorescence using antibodies directed against the
common V5 tag. Equal expression levels of the fusion proteins were confirmed
by Western blotting (data not shown). As shown in
Figure 4A, the transiently
expressed HP1-V5 fusion protein showed a subnuclear staining pattern very
similar to that observed for the endogenous HP1 protein (see
Fig. 3A). In addition, good
overlap with heterochromatic regions stained by the
anti-H3-Me2K9-specific antibodies was detected. In contrast, the
HP1Pc-CD-V5 chimeric fusion protein did not show a focal
localization, but was rather dispersed throughout almost the whole cell
nucleus in many cells inspected. Very little colocalization with H3
dimethylated on Lys 9 was detected (Fig.
4B). Conversely, the Pc-V5 fusion protein localized to regions
outside of the heterochromatic domains stained by the
anti-H3-Me2K9-specific antibodies, similar to the endogenous Pc
protein (Fig. 4C). Swapping the
chromodomain of Pc with that of HP1 resulted in a focal subnuclear
distribution reminiscent of that of HP1 (PcHP1-CD-V5;
Fig. 4D). Furthermore, this
chimeric fusion protein was often closely associated with the heterochromatic
regions stained by the anti-H3-Me2K9-specific antibodies
(Fig. 4D). The importance of
the chromodomain for Pc localization was further emphasized by the loss of
specific subnuclear targeting of an Ile 69-Asp 70 deletion mutant, which had
previously been identified in an unbiased genetic screen
(Messmer et al. 1992; data not
shown). In our in vitro binding assays, this mutant Pc chromodomain was not
able to interact with the trimethyl-Lys 27 H3 peptide presumably because of
loss of necessary structure for peptide binding (see below). Our chromodomain
swapping experiments implicate an important role for the different
chromodomains of Pc and HP1 in both target site binding and discrimination.
They further emphasize the critical targeting role of the chromodomains for
the biological function of these factors.
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Overall structure of the Pc chromodomain-methyl-Lys 27 H3 peptide
complex
To visualize how the Pc protein binds the methyl-Lys 27-containing histone H3 tail, we crystallized its chromodomain in complex with a synthetic peptide corresponding to residues 15-32 of histone H3 with a trimethyl-lysine at residue 27. The crystals diffracted to 1.8 Å resolution, and the structure was solved using molecular replacement. Table 1 summarizes the quality of the X-ray diffraction data and the structure refinement parameters. The electron density map was interpretable throughout the entire chromodomain region (residues 23-73). Analysis of the |2Fo - Fc| and |Fo - Fc| difference maps clearly indicated electron density for the bound position of the H3 peptide in the complex as shown in Figure 5A. The bound H3 peptide density observed corresponds to residues 20-28, including clear density for trimethyl-Lys 27.
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The histone peptide forms a 
-strand structure that lies between two
-strands from one face of the chromodomain, completing a three-stranded
sheet and the overall -sandwich architecture of the protein. We
previously observed this binding mode in the structure of the HP1 chromodomain
in complex with H3-peptide-bearing trimethyl-Lys 9. The methyl-Lys 27
interacts with the Pc protein via a cation-
interaction reminiscent of the
mode seen in the HP1 chromodomain interaction with methyl-Lys 9
(Jacobs and Khorasanizadeh
2002). The aromatic cages of the two complexes superimpose with an
RMSD of 0.7 Å, supporting our previous prediction that aromatic cages in
diverse chromodomains act as a recognition substructure for methyl-lysines in
target sequences (Jacobs and
Khorasanizadeh 2002). Within the aromatic cage, one of the
residues of the Pc chromodomain, Trp 50, is different from its counterpart in
HP1, Tyr 48 (Fig. 5B). Mutation
of Trp 50 to Tyr does not change the affinity of Pc for its target peptide
significantly (KD = 7 µM for the binding of the W50Y variant to
trimethyl-Lys 27 H3 peptide). Other architectural features of the HP1 and Pc
chromodomains are highly related. Comparison of the HP1 and Pc complex
structures reveals that the chromodomains and H3 tail peptides superimpose
with an RMSD of 0.74 Å and 1.0 Å over all of their overlapping
C
atoms, respectively
(Fig. 5C).
Molecular basis for discrimination of methyl-Lys 9 and methyl-Lys 27
by Pc and HP1
Despite the fact that the chromodomains of HP1 and Pc are similarly
structured, their peptide-binding grooves show distinct features
(Fig. 6A). The most striking
difference is the extent of peptide-protein interactions in these two
complexes. A total of six residues of the H3 tail were observed and ordered in
the HP1 complex (Jacobs and Khorasanizadeh
2002). These correspond to the sequence stretch Gln 5 to Ser 10.
In the structure of Pc complex with trimethyl-Lys 27 H3, a total of nine
residues corresponding to the sequence stretch Leu 20 to Ser 28 are observed.
As a result of a more extended peptide-binding groove in the Pc protein, its
interactions with the Lys 27 site bury 1482 Å2, whereas the
interaction of HP1 with the Lys 9 site buries 1063 Å2. The
remaining peptide residues in each case were present in the crystals as
confirmed by mass spectrometry, but are unobserved because of disorder,
presumably caused by their lack of binding interaction with the chromodomain
surface.
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The two complexes show differences in the recognition of the n - 4 position (Gln 5 vs. Lys 23), where n corresponds to the methyl-lysine (see Fig. 1B for the H3 sequence). This appears to have important consequences for the Pc peptide interactions. Within the peptide, Lys 23 donates a hydrogen bond to the backbone carbonyl of Ala 21, thus stabilizing the extended conformation bound in the Pc groove (Fig. 6B). Gln 5 does not form a similar stabilizing interaction when bound to the HP1 chromodomain. Another important difference is the n - 5 position of the peptides (Thr 22 vs. Lys 4). In the Pc complex, Thr 22 of the peptide is appropriately positioned to donate a hydrogen bond to the side chain of a conserved chromodomain residue, Lys 48. A lysine in this position of the peptide would not be able to make this interaction, and in fact would cause charge repulsion with the lysine of the chromodomain. In addition, Thr 22 together with Leu 20 of the peptide form backbone hydrogen bonds with the Arg 67 side chain of the Pc protein. This interaction would not be possible with the HP1 protein, as Arg 67 is not conserved and is instead substituted by Asp 62 in HP1. These observations indicate that the n - 4 through n - 7 positions interact with the Pc protein through several backbone contacts. As such, these do not provide specific recognition per se, but stabilize the overall strand conformation of the H3 tail. As an important consequence, the side chain of n - 5 is oriented to hydrogen-bond with the Pc protein, and the side chains of n - 4 and n - 6 are allowed to form complementary van der Waals contacts on the Pc surface. Together these interactions provide the necessary specificity for discrimination of residues n - 4 through n - 7 by the Pc protein.
Whereas the Pc chromodomain recognizes an extended surface encompassing n - 4 through n - 7 residues, HP1 appears to be more discriminating for residues in the immediate vicinity of the methyl-Lys corresponding to the n - 1 through n - 3 residues (TARK9 vs. AARK27). This is shown through mutagenesis studies in which the n - 3 residue alone is changed from one target site to the other. Mutation of Thr 5 to Ala (corresponding to residue 24) reduced the peptide-binding affinity of HP1 by sixfold (KD = 27 ± 4 µM for the binding of HP1 to the T5A variant of a trimethyl-Lys 9 H3 peptide). Conversely, when Ala 24 is changed to Thr, the peptide-binding affinity of Pc did not change significantly (KD = 8 ± 1 µM for the binding of Pc to the A24T variant of a trimethyl-Lys 27 H3 peptide). Together, these results indicate that the HP1 protein is much more discriminating than Pc for the residue in the n - 3 position.
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Discussion |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Structural aspects of chromodomain methyl-lysine binding
In both the HP1 and Pc complexes, histone tail interactions are stabilized
by the formation of hydrogen bonds and a complementary surface, whereas the
recognition of the methyl-lysine mark is mediated by an aromatic cage
consisting of three residues. An intriguing feature of our structural analyses
of the HP1 and Pc methyl-lysine H3 tail complexes is that the recognition of
specific target sites is highly restricted to the N-terminal
"faces" of the Lys 9- and Lys 27-binding sites. No residues
C-terminal to n + 1 (Ser 10 or Ser 28) are seen in the
three-dimensional structures. Therefore, the specificity of interaction has to
be derived from hydrophobic and van der Waals interactions between the
chromodomain and residues not only N-terminal to the methyl-lysine mark but
also N-terminal to the n - 2 residue because both Lys 9 and Lys 27
are embedded into the same sequence motif ARKS. Whereas the Pc chromodomain
forms an extended groove that discriminates its target via the n - 4
to n - 7 residues of the H3 tail, the HP1 chromodomain does not form
stable interactions with the corresponding residues in its target, but is more
discriminating for the n - 3 position. This interpretation of the
complex structures is consistent with our mutagenesis analysis of the H3 tail
motif surrounding the Lys 9 and Lys 27 methyl marks, where mutation of the
n - 3 position had a significant effect on the binding of HP1 to its
cognate target, but did not impair Pc affinity for methyl-Lys 27. Therefore,
we conclude that more than a single key residue is important for target
selection by the chromodomains of HP1 and Pc. This asymmetric and selective
binding to a cognate mark and its surrounding residues is reminiscent of SH2
domains and other cellular signaling docking modules
(Pawson et al. 2001). Future
structural studies of other chromodomains that recognize a methyl-lysine in
distantly related sequence context should provide additional insights about
the target selectivity. For example, Lys 4 in histone H3 can also be
methylated, and the sequence context of this methyl mark is very different
from Lys 9 and Lys 27 (Fig.
1B).
Interestingly, the interacting peptide regions of the H3 tail that are in
contact with the chromodomains in both, the Pc and HP1 complexes, contain
additional residues known to be posttranslationally modified. These include
phospho marks on Ser 10 and Ser 28 and an acetyl mark on Lys 23. At present it
is not clear to what extent combinations of these marks with the methyl-lysine
marks of Lys 9 and Lys 27 exist. However, additional modifications could
influence the binding affinity and selectivity of chromodomain modules to
methyl-lysine marks additively, synergistically, or antagonistically.
Therefore, the analysis of such effects will be important for future
biochemical and structural analyses and should help to delineate the
regulation of modules binding to histone marks
(Strahl and Allis 2000;
Jenuwein and Allis 2001).
Polycomb, HP1, and targeting of chromatin modifiers
Our studies show a clear preference of the Pc chromodomain for the H3 Lys
27 methyl mark. How does this activity of Pc contribute to PcG function? In
the case of the formation of heterochromatin and the initially genetically
defined pathway of suppression of variegation, it has been suggested that
methylation of H3 on Lys 9 by Suv3-9 generates a docking site for the HP1
(also known as Suv2-5) chromodomain. Further recruitment of Suv3-9 by the
chromo shadow domain of HP1 has been postulated to lead to a perpetuation and
spreading of a heterochromatic domain until blocked by yet unknown mechanisms
(Bannister et al. 2001;
Dillon and Festenstein 2002;
Grewal and Elgin 2002;
Snowden et al. 2002).
Similarly, Esc-E(z)-dependent methylation of Lys 27 (and possibly Lys 9) and
consecutive recruitment of Pc and Pc-containing complexes might contribute to
the stability of the PcG complex, particularly in the early stages of assembly
at a PRE by permitting complex formation to spread to neighboring sequences
(Poux et al.
2001a,b). This
interpretation of the specific binding of Pc to methyl-Lys 27 is in agreement
with studies demonstrating loss of chromosome binding for several components
of PRC1 upon inactivation of E(z)
(Rastelli et al. 1993) and is
consistent with several other in vivo results that imply synergy between these
complexes (Simon and Tamkun
2002). However, it is unclear at present if dynamic perpetuated
spreading of a Lys 27 mark indeed exists and is dependent on Pc recruitment
and Esc-E(z) enzymatic activity. Other possible functions for the binding of
Pc to the Lys 27 methyl mark include a more static maintenance effect that
could contribute to epigenetic memory. In this model, the recruitment of
Esc-E(z) would be independent of and precede any involvement of Pc binding.
Alternatively, although complex recruitment could be constitutive, the
decision to repress or not could depend on an epigenetic switch mediated by
Lys 27 methylation and its interaction with local Pc/PRC1
(Breiling et al. 2001;
Czermin et al. 2002).
However, alternative routes and mechanisms of Pc and PcG recruitment to
local sites of chromatin besides recognition of Lys 27 methylation might also
exist. For example, studies involving the localization of a chimeric HP1
protein containing the chromodomain of Pc on polytene chromosomes implied
critical interactions of the Pc chromodomain with other PcG components that
are recruited to PcG sites (Platero et al.
1995,
1996). Therefore, the
particular localization patterns observed for the wild-type and chimeric
proteins in our swapping experiments might be the result of additive effects,
including other targeting mechanisms besides methyl-lysine binding.
Nevertheless, it is intriguing to note that the subnuclear localization
patterns for wild-type and chimeric Pc and HP1 proteins are coincident with
the localization of specifically recognized methyl-lysine marks on the histone
H3 tail. It is unclear to what extent additional regions of the proteins
C-terminal to the chromodomains could contribute to subnuclear localization
and function. For example, different studies have implicated the C-terminal
chromo shadow domain and hinge regions of HP1 in addition to the chromodomain
in the specific subnuclear targeting of this factor
(Smothers and Henikoff 2001;
Muchardt et al. 2002;
Cheutin et al. 2003). However,
a C-terminal truncation of Pc did not affect its specific chromosomal
localization (Messmer et al.
1992). Additional studies will have to address the exact
contribution of the chromodomains and their recognition and binding of
particular methyl-lysine marks to the specific functions of the Pc and HP1
proteins.
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Materials and methods |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Polyclonal antibodies against H3-K9 (Me)2 and H3-K9
(Me)3 were from Upstate Biotech, Inc.; the monoclonal antibody
against HP1 was obtained from the Hybridoma Bank at the University of Iowa.
Antibodies against H3-Me3K27
(Silva et al. 2003), Pc
(Messmer et al. 1992), and Psc
(Martin and Adler 1993) were
kind gifts from Thomas Jenuwein (Research Institute of Molecular Pathology,
The Vienna Biocenter, Vienna, Austria), Renato Paro (ZMBH, University of
Heidelberg, Heidelberg, Germany), and Paul Adler (Biology Department and
Cancer Center, University of Virginia, Charlottesville, VA), respectively. The
monoclonal anti-V5 antibody was purchased from Invitrogen.
Peptide preparation
Synthetic peptides of the H3 tail were prepared at the Core Facility of
Baylor College of Medicine. Peptides corresponding to the Lys 9 and Lys 27
regions include residues 1-15 and 15-32, respectively (see
Fig. 1A). Unmodified as well as
modified lysines (mono-, di-, and trimethylation) were incorporated at the Lys
9 and Lys 27 positions. A nonnative Tyr residue at the C terminus of each
peptide was used for concentration determination by UV absorption
measurements. Peptides were labeled with fluorescein as previously described
(Jacobs et al. 2001).
Molecular biology
For binding studies, the chromodomain of Pc (residues 1-90) was amplified
by PCR and cloned into the BamHI/NdeI sites of the pET16b
vector (Novagen). For crystallization, Pc residues 15-77 were fused to a
His6-tag by PCR and cloned into the BamHI/NdeI
sites of the pET11a vector. The construct for the expression of the
chromodomain of HP1 (residues 17-76) has been described previously
(Jacobs and Khorasanizadeh
2002). Full-length Drosophila HP1 (residues 1-206) and Pc
(1-390) were PCR-amplified and cloned into the EcoRI/XhoI
sites of the pMT/V5-His A vector (Invitrogen). The chromodomain of HP1
(residues 23-77) and Pc (residues 21-74) were swapped using staggered PCR by
incorporation of overlapping oligonucleotides. Chimeric cDNAs were then cloned
into the pMT/V5-His A vector. Site-directed mutagenesis was performed
according to the QuikChange protocol (Stratagene).
Binding assays
Fusion proteins with N-terminal His-tag were expressed in Escherichia
coli strain BL21 (DE3) (Novagen) and purified by Ni2+-affinity
chromatography (QIAGEN). Protein concentrations were determined by absorbance
spectroscopy using predicted extinction coefficients (for Pc chromodomain,
280 = 22,190 M-1 cm-1; for HP1
chromodomain, 280 = 17,780 M-1 cm-1).
Peptide concentrations were determined using absorbance spectroscopy
(extinction coefficient for tyrosine, 280 = 1280
M-1 cm-1; extinction coefficient for fluorescinated
peptides, 492 = 68,000 M-1 cm-1).
Fluorescence polarization binding assays were performed under conditions of 20
mM imidazole (pH 7.0), 25 mM NaCl, and in the presence of 100 nM
fluorescein-labeled peptide following a previously described protocol
(Jacobs et al. 2001). Data
were obtained using a Teacan Polarion 96-well plate reader by setting it on
automatic gain and 100 flashes. Sample plates were kept on ice until
fluorescence reading at room temperature.
S2 cell transfection and immunofluorescence
S2 cells were grown at room temperature in Schneider's Drosophila
medium (Invitrogen) supplemented with 10% FBS. Cells were transfected using
the calcium phosphate method as instructed by the manufacturer (Invitrogen).
Expression of fusion proteins was induced by adding 250 µM CuSO4
for 12 h. For immunofluorescence staining, 
5 x 106 cells
were spun onto glass coverslips in 6-well tissue culture dishes (2000 rpm, 4
min). Cells were fixed in solution I (1x PBS, 3.7% formaldehyde, 1%
Triton X-100, 2% NP-40) for 10 min, washed in 1x PBST (PBS with 1%
Triton X-100) three times for 10 min. Slides were blocked for 1 h and
incubated with the indicated primary antibodies overnight in a humidified
atmosphere. Dilutions for primary antibodies were anti-H3-Me2K9
(1:500), anti-H3-Me3K27 (1:500, preabsorbed with
H3-Me3K9 peptide at 5 µg/mL), anti-HP1 (1:500), anti-Pc (1:400),
anti-Psc (1:100), and anti-V5 (1:500). Slides were washed in 1x PBST
three times for 10 min and incubated with the appropriate secondary antibodies
for 2 h in a humidified atmosphere. After washing in 1x PBST, DNA was
stained with DAPI (1 µg/mL) for 10 sec. Pictures were taken on a Zeiss
Axiopod II equipped with a 60x lens. Cells with intermediate levels of
fusion protein expression were selected.
Polytene chromosome immunofluorescence
Staining of polytene chromosomes was performed essentially as previously
described (Jin et al. 2000).
In brief, salivary glands from third instar larvae were dissected in 1x
PBS, fixed in solution I for 60 sec, followed by incubation in solution II
(50% glacial acetic acid, 3.7% formaldehyde in H2O) for 2 min.
Slides were transferred to solution III (50% acetic acid, 16.7% lactic acid in
H2O) for 2 min. The fixed salivary glands were squashed, frozen in
liquid N2, dehydrated in 95% ethanol, and washed two times in
1x PBST, 30 min each. Immunostaining was essentially performed as
described for S2 cells. For sequential double labeling using two polyclonal
antibodies from rabbits, the first primary antibodies (anti-Pc, 1:400) were
incubated with the polytene tissue at room temperature for 2 h. After washing
(1x PBST, three times for 10 min), FITC-conjugated goat anti-rabbit
secondary antibodies (1:200, Fab fragment) were applied at room
temperature for 1 h. After washing (1x PBST, three times for 10 min),
slides were blocked with the corresponding unlabeled goat anti-rabbit
antibodies (Fab fragment at 70 µg/mL, at room temperature for 2
h). After washing, the second primary antibodies (either
anti-H3-Me3K9 or anti-H3-Me3K27) were applied (at room
temperature for 2 h), followed by incubation with Cy3-conjugated goat
anti-rabbit secondary antibodies.
Crystallization, data collection, and refinement
Purified Pc chromodomain (residues 15-77) was dialyzed into 20 mM Tris-HCl
(pH 8.0), 50 mM NaCl, and concentrated to 5 mg/mL before addition of
H3-Me3K27 peptide to reach a final protein-to-peptide molar ratio
of 1:5. Single crystals in space group
I212121 (a = 32.12, b =
75.81, c = 80.46) were grown by the hanging drop vapor diffusion
method at 10°C or 4°C by mixing 1.5 µL of the protein-peptide
solution with 1.5 µL of a reservoir solution containing 0.1 M Tris-HCl (pH
8.5), 0.2 M Li2SO4, and 30% polyethylene glycol 4000.
Crystals were cryoprotected in the same solution supplemented with 25%
ethylene glycol and flash-frozen in liquid nitrogen for data collection at the
Advanced Photon Source Beamline SBC 19-ID. Data were processed and scaled with
HKL2000 (Otwinowski and Minor
1997). Phases were solved by molecular replacement with MOLREP
(Vagin and Teplyakov 1997)
using the HP1 chromodomain crystal structure (PDB code 1KNA
[PDB]
) as the model.
MOLREP produced a clear solution with a correlation coefficient of 0.31 and
Rcrystal of 0.52. Rigid-body refinement of this solution using
REFMAC5 (Murshudov et al.
1997) reduced the Rfactor to 0.42 and Rfree
to 0.46. This model was then submitted to ARP/wARP
(Perrakis et al. 1999) to be
used only as a source of phases for automatic main-chain tracing and
side-chain docking as well as refinement. ARP/wARP successfully traced
residues 25-73 of the Pc chromodomain and 21-28 of the H3 tail, reducing the
Rcrystal and Rfree to 0.27 and 0.36, respectively.
Residues 23 and 24 of Pc and 20 of H3 were manually built as there was clear
density present in a simulated annealing composite omit map calculated using
CNS (Brunger et al. 1998).
Subsequent rounds of manual rebuilding and refinement using O
(Jones and Kjeldgaard 1994)
and CNS and addition of water molecules led to the converged
Rcrystal and Rfree values reported in
Table 1.
Coordinates
Atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 1PDQ).
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Footnotes |
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Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1110503.
4 These authors contributed equally to this work. 
5 E-MAIL
alliscd{at}rockefeller.edu;
FAX (212) 327-7849.
6 E-MAIL
khorasan{at}virginia.edu;
FAX (434) 924-5069.
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in which the H3 tail peptide was omitted for map
calculation. Side chains that make critical contacts are depicted; the
chromodomain backbone is in brown, and the electron density of the H3 peptide
is in magenta. Residues 15-19 and 29-32 of the peptide appear to be
unstructured, suggesting that only Leu 20-Ser 28 of the histone H3 tail
participate in binding to the chromodomain. Broken lines indicate
intermolecular hydrogen bonds. (B) The chromodomains of both Pc
(blue) and HP1 (green) contain three aromatic residues that form
superimposable cages around the methyl-ammonium groups of Lys 27 (red) and Lys
9 (yellow), respectively. The van der Waals radii of the Pc aromatic rings and
the methyl-Lys 27 methyl-ammonium atoms are shown. (C) Superposition
of the backbone structures of the Pc (brown) and HP1 (blue) chromodomains
bound to Me3K27 and Me3K9 H3 tail peptides,
respectively.