From BIOTEC, Technische Universitaet Dresden, c/o Max
Planck Institute for Molecular Cell Biology and Genetics and the
¶ Max Planck Institute for Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, D-01307 Dresden, Germany and the
Department of Molecular Biology, University of Bergen,
Thromoehlenstrasse 55, N-5020 Bergen, Norway
Received for publication, September 18, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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Histone 3 lysine 4 (H3 Lys4)
methylation in Saccharomyces cerevisiae is
mediated by the Set1 complex (Set1C) and is dependent upon
ubiquitinylation of H2B by Rad6. Mutually exclusive methylation of H3
at Lys4 or Lys9 is central to chromatin
regulation; however, S. cerevisiae lacks Lys9 methylation. Furthermore, a different H3
Lys4 methylase, Set 7/9, has been identified in mammals,
thereby questioning the relevance of the S. cerevisiae
findings for eukaryotes in general. We report that the majority of
Lys4 methylation in Schizosaccharomyces
pombe, like in S. cerevisiae, is mediated by
Set1C and is Rad6-dependent. S. pombe Set1C
mediates H3 Lys4 methylation in vitro and
contains the same eight subunits found in S. cerevisiae,
including the homologue of the Drosophila trithorax Group
protein, Ash2. Three additional features of S. pombe Set1C each involve PHD fingers. Notably, the Spp1 subunit is dispensable for
H3 Lys4 methylation in budding yeast but required in
fission yeast, and Sp_Set1C has a novel proteomic hyperlink to a new
complex that includes the homologue of another trithorax Group protein,
Lid (little imaginal discs).
Thus, we infer that Set1C is highly conserved in eukaryotes but
observe that its links to the proteome are not.
The stable maintenance of gene expression patterns through mitotic
cell divisions, termed epigenetic regulation, is essential during
development of higher organisms. Searches for epigenetic mechanisms in
Drosophila development uncovered an opposition
between the trithorax (trxG)1
and Polycomb (PcG) groups. TrxG proteins appear to maintain patterns of
gene activation, whereas PcG proteins maintain patterns of gene
repression. TrxG encompasses several subclasses of gene regulatory factors (1). Although there are good models for action by some trxG
members (e.g. brahma, moira,
zeste, and GAGA), the activity of one subclass of the trxG, trxG3,
which includes Trx, Ash1, and Ash2 (2, 3), remains unclear. Several
trxG and PcG proteins contain a SET domain (Su(var) (3-9),
E(z), and Trithorax (4)), a well conserved
150-amino acid domain found in all eukaryotes, which mediates histone
methyltransferase activity (5, 6). Trx and Ash1 both contain a SET
domain. There is little evidence so far that Trx methylates histones;
however, binding to histones has been documented (7). The third trxG3
member, Ash2, does not contain a SET domain but has a PHD finger (8)
and a SPRY domain (9).
Although budding yeast does not have a Trx homologue, it has a protein,
Set1, with a very similar type of SET domain (10). For this reason, we
determined the composition of the Set1 complex, Set1C, and found that
it has H3 Lys4 methyltransferase activity in
vitro (3). Concomitantly, two other groups have identified most
members of Set1C (11, 12). The complete Set1C and the in
vitro specificity for H3 Lys4 methylation have since
been confirmed (13). The requirement for Set1 in H3 Lys4
methylation in vivo was identified (14) and extended to
Set1C members (12, 13). Set1C includes Bre2, the protein in
Saccharomyces cerevisiae that is most similar to the
Drosophila trxG protein, Ash2. Because we were exploring
trxG action, we speculated that the protein-protein linkage between
Set1 and Ash2/Bre2 in Set1C might consolidate trxG3 action to SET
domains and histone methylation. At that time, however, no Set1
homologue was apparent in the Drosophila genome. We mined
the Drosophila genome with deep bioinformatic tools to
identify the Set1 orthologue, buried in sequence misreads. This
discovery encouraged the trxG3-histone methylation proposition and the
possibility that the Set1-Ash2 association may be broadly conserved in
eukaryotes (3). The work reported here was begun with the motivation to
challenge our proposition by characterizing the protein complex
associated with Schizosaccharomyces pombe Set1.
The fission yeast S. pombe is widely held to be a more
representative model for higher eukaryotes than the budding yeast
S. cerevisiae. Indeed many S. pombe proteins
appear to be more similar to their mammalian homologues than to
S. cerevisiae counterparts (15). A number of cellular
aspects such as the nuclear cycle, structure of centromeres, and
aspects of histone methylation are similar between S. pombe
and higher eukaryotes but divergent in S. cerevisiae.
Although both yeasts have H3 Lys4 methylation, S. cerevisiae lacks the nearby H3 Lys9 methylation (14,
16). Emerging evidence in S. pombe and higher eukaryotes
indicates that methylations of H3 Lys4 and H3
Lys9 are mutually exclusive in chromatin domains (16, 17).
Because this mechanism cannot exist in S. cerevisiae,
extrapolations from the budding yeast to higher eukaryotes regarding H3
Lys4 methylation, Set1C, and Ash2 remain unsafe.
Hence, we examined H3 Lys4 methylation in S. pombe and compared it with S. cerevisiae.
Strains, TAP Purification, and Mass
Spectrometry--
Strains used in this study are isogenic to DB241
(h Protein Assays and Antibodies--
Assays were performed as
described previously (3). The antibodies used were peroxidase
anti-peroxidase (PAP; Sigma), rabbit polyclonal anti-dimethylated
Lys4-H3 (Abcam), and rabbit polyclonal anti-acetyl histone
H4 (Upstate Biotechnology Inc.). Superose 6 size exclusion
column (Amersham Biosciences) was loaded with 500 µl of cleared crude
cell extract from a TAP-tagged strain and run in Buffer E (20 mM Na-HEPES, pH 8.0, 350 mM NaCl, 10%
glycerol, 0.1% Tween 20). Fractions were resolved on 10%
SDS-PAGE and analyzed by immunoblotting against the TAP tag. Size
standards were run in parallel under the same conditions.
RNA and DNA Assays--
Total RNA from exponentially growing
yeast cultures was isolated (Qiagen) and subjected to RT-PCR. As a
control for DNA contamination, a reaction without reverse transcriptase
(RT) was run in parallel. Before reaching reaction plateau, (30 cycles)
aliquots were analyzed by agarose gel electrophoresis. Genomic DNA was
isolated from exponentially growing yeast cultures, digested with
EcoRI, and analyzed by standard methods using a 1.3%
agarose/TAE (Tris acetate-EDTA) gel. pAMP1 (21) was digested
with ApaI and EcoRI (New England Biolabs),
gel-purified (Qiagen), and labeled by random priming (Amersham Biosciences).
Composition of S. pombe Set1C--
We purified the complex
associated with S. pombe Set1 (Sp_Set1C) using tandem
affinity purification (TAP) and mass spectrometry (19, 20, 22).
Sp_Set1C is composed of the following eight proteins (Fig.
1) named in descending molecular weight
order as follows (with S. pombe genome database
reference in parentheses): Sp_Set1 (spcc306.04c), Sp_Ash2
(spbc13g1.08c), Sp_Spp1 (spcc594.05c), Sp_Swd3 (spbc354.03), Sp_Swd2
(spbc18h10.06c), Sp_Swd1 (spac23h3.05), Sp_Shg1 (spac17g8.09), and
Sp_Sdc1 (spcc18.11c). The overall composition differs only
slightly from S. cerevisiae Set1C (3, 13). The composition
of S. pombe Set1C (Sp_Set1C) was confirmed by tagging each
member except Sp_Shg1 (Fig. 1 and data not shown).
Purification of Sp_Set1C from the Sp_Set1-TAP strain indicated that
all cellular Set1 is incorporated in the complex because no
uncomplexed, free, cellular protein was apparent (Fig. 1a). As for Sc_Set1, Sp_Set1 contains an RNA recognition motif and a SET
domain flanked by an n-SET domain (3) and postSET peptide. In
all Set1C preparations, Sp_Set1 appeared in two forms, the shorter of
which lacked the N terminus (as determined by MALDI peptide mass maps;
data not shown). Whether this is a result of specific intracellular
processing or nonspecific degradation during the isolation of the
complex remains to be determined.
Sp_Ash2 is orthologous to Drosophila melanogaster Ash2
protein and is more closely related to it than to its counterpart in Sc-Set1C, Sc_Bre2, which has no PHD finger. Homologies flanking the
SPRY domains of Sp_Ash2, Bre2, Dm_Ash2, Mm_Ash2l, Hs_Ash2L, and
Hs_Ash2l2 extend beyond the defined limits of the SPRY domain (3, 9).
The Sp_Ash2 PHD finger is closely related to the PHD finger of Dm_Ash2
and to the unconventional PHD fingers of Mm_Ash2l, Hs_Ash2L, and
Hs_Ash2l2 but not to the PHD finger of Spp1 (data not shown).
Purification of Sp_Set1C from the Sp_Spp1-TAP strain indicated that all
cellular Spp1 is incorporated in the complex, because no uncomplexed
free cellular protein was apparent. Both yeast Spp1s contain nearly
identical PHD fingers, which are closely related to the PHD finger of
CGBP, a protein that binds preferentially to unmethylated CpGs
(23). Thus, in contrast to Sc_Set1C, the complex from S. pombe contains two PHD fingers (in Sp_Ash2 and Sp_Spp1).
Sp_Swd1, Sp_Swd2, and Sp_Swd3 belong to the WD40
Sp_Shg1, like its homologue in the Set1C of S. cerevisiae,
appeared in Sp_Set1C as a minor component. Only two other proteins with
similarity to Sp_ and Sc_Shg1s are evident in the data bases, one in
Candida albicans and the other in Caenorhabditis
elegans, with highest similarity at the N termini (data
not shown).
In Sc_Set1C, Sdc1 appeared as a minor component. We did not identify
its S. pombe homologue in the Sp_Set1 TAP preparations, probably because of its small size and poor mass spectrometry signature. However, when the Sp_Sdc1 homologue was tagged, Sp_Set1C was
retrieved, thus proving its presence in the complex. Notably, a
significant excess of Sp_Sdc1 was retrieved over Set1C members and
other proteins, indicating that Sp_Sdc1 may also exist as free protein
in the cell (Fig. 1b; Table
I). Sp_Sdc1 and Sc_Sdc1 show similarity
to the C. elegans dosage compensation protein, Dpy-30. The
similarity includes a short motif related to the dimerization motif in
the regulatory subunit of protein kinase A (3).
Identification of a New Protein Complex Containing
Lid--
Interestingly, when either Sp_Ash2 or Sp_Sdc1 was TAP-tagged
and purified, Sp_Set1C was retrieved along with three new high molecular weight proteins, now called Sp_Lid2 (Spbp19A11.06), Sp_Ecm5 (Spbc83.07), and Sp_Snt2 (Spac3h1.12c). Sp_Lid2 is the S. pombe homologue of the Drosophila trxG
protein, Lid (little imaginal discs
(25)). The Lid family of proteins, which includes Sc_Lid2 and mammalian
Xe169 and Rbp2, contains three PHD fingers, a BRIGHT domain, and a JmjC
domain. The BRIGHT domain is a helix-turn-helix DNA binding domain with
preference for AT-rich regions (26). The JmjC domain is found in a wide
variety of organisms from bacteria to humans in at least seven families
of proteins. The domain has no known function but may be involved in
regulation of chromatin remodeling (27, 28). Sp_Snt2 contains three PHD
fingers, a SANT domain (29), and a BAH domain (30). In budding
yeast, the homologues of Ecm5 and Snt2 interact physically; however, they do not appear to interact with Sc_Sdc1 or Sc_Lid2 (data not shown). Sp_Ecm5 contains a JmjC domain together with the N-terminal domain JmjN (27), often associated with JmjC.
To confirm the composition of the Sp_Lid2 complex (Sp_Lid2C), Sp_Lid2
was TAP-tagged and purified. Although Sp_Sdc1 was not identified, again
presumably because of its small size and consequent difficulties with a
clear mass spectrometry signature, Sp_Ash2, Sp_Ecm5, and Sp_Snt2 were
retrieved (Fig. 1b). By dissection of protein-protein
interactions within Sc_Set1C, we showed previously that Bre2 and Sdc1
directly interact with each other (3). Here we show that Sp_Ash2 and
Sp_Sdc1 both associate with Sp_Set1C and Sp_Lid2C. Thus, we conclude
that Sp_Ash2 and Sp_Sdc1 serve as proteomic hyperlinks between two
complexes and probably form a module through interaction with each other.
Hence Sp_Set1C and Sc_Set1C show almost the same
polypeptide and domain compositions, differing only by the absence of a
PHD finger in Bre2 and unexpected proteomic hyperlinks through Sp_Ash2 and Sp_Sdc1 to Sp_Lid2C. Except for Shg1, homologues and orthologues are present in diverse eukaryotic databases, indicating that Set1C is
highly conserved.
The Apparent Sizes of the Two Yeast Set1Cs and Their in Vitro
Methylation Specificities Are Identical--
We estimated the
size of the Sp_Set1C by means of Superose 6 size-exclusion column
chromatography (Fig. 2a). Two
bands were observed, corresponding to the full-length and
shorter versions of Set1 with the longer Set1 migrating in a
complex(es) at ~1 MDa and the shorter migrating around 800 kDa
(assuming an overall globular configuration). We do not know whether
the smaller complex(es) represents an authentic second complex(es) or a
degradation product. The estimated size of 1 MDa is the same as
Sc_Set1C (11)2 but differs
from the smaller apparent estimation of ~500 kDa made after cell
extraction in higher salt (12).
The relative stoichiometry of Sp_Set1C subunits was estimated by
densitometric measurement of images from the Coomassie-stained gels and then adjusted for molecular weight (Table I). Given a 1-MDa
complex, we propose the complex composition presented in Fig.
2b. This model is concordant with our data; however, it should be regarded as an initial proposition. Mass estimations from
Coomassie staining intensities can be only approximations for several
reasons, including understaining because of the highly acidic content.
This may not be a problem for Sp_Set1C because all subunits have
isoelectric points between 5 and 6.5 except for Set1, which is 9.0. We
also point out that the question as to whether Set1C is present in
cells as a single complex or in several slightly different forms cannot
be answered yet.
The histone methyltransferase activity of Sp_Set1C was tested using the
complex isolated from the Sp_Set1-TAP strain and incubation with core
histones in the presence of [3H]SAM. It showed
methyltransferase activity directed toward histone H3 (Fig.
3a), which was specific for
Lys4 (Fig. 3b). In this case, unlike Sc_Set1C
(3), fusion of the TAP tag to the C terminus next to the SET domain did
not inhibit the enzymatic activity in vitro.
In Vivo Methylation Requirements for Set1C Subunits--
The
contribution of individual complex members to the enzymatic activity
in vivo was examined. Disruption strains were readily obtained for all complex members except for Sp_Sdc1, which was elusive
until recently. Hence, no complex member is essential for S. pombe. Individual disruption of all members totally ablated H3
Lys4 methylation except for Sp_Ash2, Sp_Sdc1, and Sp_Shg1,
which showed about one-tenth, one-half, or wild type levels of H3
Lys4 methylation, respectively (Fig.
4a). These results are
identical to those obtained from the same experiments in S. cerevisiae2 (Refs. 12 and 13) except for Spp1.
Deletion of Spp1 in S. cerevisiae has no effect on H3
Lys4 methylation, whereas in fission yeast it leads to
complete loss of H3 Lys4 methylation. All of the WD40
repeat proteins are required for H3 Lys4 methylation,
demonstrating that they are not redundant to each other. WD40 repeat
proteins have been shown to serve as platforms for protein complex
assembly; however, some of these proteins bind to histones
(e.g. TBL1, Groucho, TUP1, and RCC1; e.g. Ref. 31). As seen in Fig. 4a, no dimethylated H3 Lys4
was present upon disruption of the complex, indicating that Sp_Set1C is
the major, possibly only, H3 Lys4 methyltransferase
activity in S. pombe and extending the recently published
result of Noma and Grewal (32) who show that deletion of set1 from
S. pombe abolishes H3 Lys4 dimethylation.
Disruption of Set1 and other Sc_Set1C components causes complex
phenotypes in S. cerevisiae including defects in cell wall formation, growth, and DNA repair, as well as alterations in silencing and telomere length (10, 14, 33-36). We examined two of these phenotypic markers, growth and telomere length, for Sp_Set1C members (Fig. 4, b and c). Concordance between loss of H3
Lys4 methylation and phenotypic impact was observed in both
assays. Loss of Shg1 and Sdc1 had the least impact on H3
Lys4 methylation and phenotype. The next mildest
impact in all three assays was loss of Ash2. The only notable
deviation from qualitative equivalence in all three assays is the
effect of loss of Spp1 on telomere length. This suggests selectivity
for Spp1 in the regulation of telomere length. Notably, in S. cerevisiae, loss of Set1C members results in shorter telomeres (3,
10, 34), whereas we observed lengthening in S. pombe. Either the telomere length changes due to Set1C
disruption are not directly due to disturbance of chromatin at
the telomeres, or the difference between S. cerevisiae and S. pombe may reflect the
possibility that loss of H3 Lys4 methylation in S. pombe alters the boundaries of H3 Lys9
methylation domains.
H3 Lys4 Methylation in S. pombe Requires the Rad6
Homologue--
In S. cerevisiae, H3 Lys4
methylation is dependent upon both Set1C and ubiquitinylation of H2B by
Rad6 (37, 38). When the S. pombe Rad6 orthologue Rhp6
(Spac18b11.07c) was disrupted, there was a complete loss of H3
Lys4 methylation (Fig.
5a). The possibility that this
effect was indirect via abolished expression of a Set1C member was
excluded by semiquantitative RT-PCR (Fig. 5b). Hence, the
regulation and enzymology of H3 Lys4 methylation is highly
conserved between budding and fission yeasts.
Set1C in S. cerevisiae includes Bre2, the homologue of
the Drosophila trxG member, Ash2. To enquire whether the
association between Set1 and Ash2 may also be found in other
eukaryotes, we characterized Set1C from S. pombe. We found
that both yeast Set1Cs are highly conserved, hence suggesting that
Set1C will be highly conserved throughout eukaryotes, including the
retention of the Set1-Ash2 linkage in flies. Our observations also
strengthen the proposition that H3 Lys4 methylation is a
common mechanistic feature of the trxG3 subgroup. Very recently,
further evidence for this proposition has emerged with the biochemical
identification of H3 Lys4 methyltransferase activity in
both of the other trxG3 members, Ash1 (42) and Mll (43, 44). We also
found that Ash2 associates with Lid2 in S. pombe. Because
Lid is a trxG protein in Drosophila (25), the
identification of Ash2 as a trxG protein may relate to its interaction
with Lid rather than with Set1 or possibly to its involvement in both complexes.
In both yeasts, mutations of both Ash2 (Bre2) and Sdc1 decreased but
did not abolish H3 Lys4 methylation. Whether these
reductions represent a global reduction of methylation or loss of
methylation at specific sites and not others remains to be determined.
In any event, the protein-protein linkage of Ash2 and Sdc1 suggests
that these reductions are based on a similar mechanistic loss. If Set1C
selectively targets specific nucleosomes, then it is likely that the
nonessential Ash2, Sdc1 or Shg1 subunits play roles in selectivity.
Conversely, if Set1C methylates nucleosomes by a general mechanism, for
example to methylate every nucleosome in a chromatin domain, then it is
likely that the three WD40 proteins, Swd1, Swd2, and Swd3, play roles in substrate binding.
Our results reveal the striking conservation surrounding H3
Lys4 methylation. However, prior evidence from the two
yeasts highlight differences rather than similarities. Budding yeast
lacks H3 Lys9 methylation, but it appears that H3
Lys4 and Lys9 methylations are mutually
exclusive in other eukaryotes (16, 17). Furthermore, the first evidence
from budding yeast indicates that Set1 and H3 Lys4
methylation is associated with chromatin repression at telomeres and
rDNA (13, 14, 33, 36), whereas it correlates with active chromatin in
fission yeast (16, 32). Resolution of this discrepancy awaits further
work; however, recent evidence from budding yeast also associates H3
Lys4 methylation with active chromatin, in particular with
actively transcribed coding regions (39, 40).
Although strikingly conserved, the two yeast Set1Cs differ in three
ways, each of which relates to PHD fingers. First, Ash2 in Sp_Set1C
includes a PHD finger, whereas Bre2 in Sc_Set1C does not. Second, the
PHD finger protein Spp1 is required for H3 Lys4 methylation
in S. pombe but not in S. cerevisiae. Third,
Sp_Ash2 and Sp_Sdc1 form a proteomic hyperlink between Sp_Set1C and
Lid2C, which includes several PHD fingers. Although PHD fingers have been found exclusively in chromatin proteins so far, their mode of
action remains elusive. Possibly the missing PHD finger relationships of Sc_Set1C relate to the absence of H3 Lys9 methylation in
budding yeast. This correlation suggests that the PHD fingers of Ash2
and Spp1 may help in the recognition, perhaps directly, of the
post-translational states of the nucleosomes upon which Set1C acts. If
so, then some PHD fingers would interpret the histone code like certain
chromo and bromo domains (8, 40). Furthermore the intriguing proteomic
association of Set1C to another trxG protein, Lid, is either a highly
unusual coincidence involving two trxG proteins or a hint that Lid
proteins and complexes may also involve reading of the histone code.
The close linkage of Sdc1 to Set1C and Lid2C also has implications for
dosage compensation in C. elegans through the Sdc1
homologue Dpy30 (45).
As for Set1C, ubiquitinylation of H2B (46) and Rad6 homologues are
apparent in all eukaryotes examined. Because the protein machinery for
H3 Lys4 methylation and its relationship to Rad6 is highly
conserved between budding and fission yeasts, it is likely that the
Set1C/Rad6 axis in H3 Lys4 methylation is highly conserved
in eukaryotes. If so, it will be interesting to understand why mammals
appear to have two orthologues of Set1 (3). Because Set1C appears to be
built on a dimeric platform, both mammalian Set1s may be incorporated
into a single Set1C, or there could be two or more Set1Cs with possible
differences in function. It will also be interesting to understand how
the putative mammalian Set1Cs relate to the other known H3
Lys4 activities mediated by Set7/9 (47, 48) and Ash1
(42).
Our work also presents new proteomic insights. The remarkable
conservation of the nonessential Set1C in the two yeasts bodes well for
future extrapolations from lower to higher eukaryotes regarding other
protein complexes. However the proteomic environment surrounding the
two Set1Cs appears to differ. The only identified proteomic hyperlink
in Sc_Set1C was Swd2, which is also a subunit of cleavage
polyadenylation factor (3). This proteomic hyperlink is not conserved
in S. pombe.2 Furthermore, we have identified a
new hyperlink between Sp_Set1C and Sp_Lid2. The S. cerevisiae orthologues of Ecm5 and Snt2 also interact with
each other but do not appear to interact with Sc_Bre2, Sc_Sdc1, or
Sc_Lid2.2
This work, in conjunction with our previous paper (3), presents, to our
knowledge, the first case in which fine mapping of one complex and the
surrounding proteomic environment from two organisms has been compared.
Although we find that the Set1Cs from S. cerevisiae and
S. pombe are highly conserved, their proteomic environments
appear to differ, thus pointing to an inherent limitation of the
"orthologous proteome" concept (49) and the need for comparative
proteomic analyses (50). Whether our observations define a new
proteomic principle regarding strong conservation within complexes and
weaker conservation of linkages between complexes remains to be
determined, but this explanation certainly makes intuitive sense.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
; ura 4-D18; ade 6-M210; leu
1-32). Gene disruptions and protein tagging was done as described
(18). TAP purification and identification of proteins by mass
spectrometry were performed essentially as described for budding yeast
(19, 20). All the detectable bands in all gel lanes were analyzed, and
only those specific to the complex are designated in Fig. 1. Persistent
background proteins were different from those of budding yeast (20) and
will be reported separately.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The compositions of Sp_Set1C and
Sp_Lid2C. a, affinity-purified Sp_Set1C using
Sp_Set1-TAP (left), Sp_Swd1-TAP (middle), or
Sp_Spp1-TAP (right) resolved on 7-25% SDS-PAGE and
visualized with Coomassie Blue staining. All bands present in the gels
were identified by MALDI mass spectrometry, and only those specific to
Sp_Set1C are indicated. Each protein is depicted with
identifiable domains and motifs, as indicated in the key at the
bottom of the figure. b, same as in a,
except proteins co-purifying with Sp_Sdc1-TAP (left) and
Sp_Ash2 (right) included the entire Sp_Set1C and three new
proteins (Lid2, Spbp19A11.06 (S. pombe genome data
base reference); Ecm5, Spac3h1.12c; and Snt2, Spbc83.07) that form
Sp_Lid2C. The composition of Lid2C was examined using Lid2-TAP
(middle), which did not co-purify Set1C.
propeller protein superfamily (24). Based on the Hidden Markov model and profile-based self-dot-plot analyses, each of these three proteins contains seven statistically significant WD40 repeats (Fig.
1a and data not shown). They also share significant sequence similarity with their homologues from S. cerevisiae and
several species including flies, Arabidopsis, and man,
indicating that they are individually very conserved (data not shown).
Relative molar subunit composition of Set1C
, denotes an
inability to determine a relative estimation due to the co-migration of
another protein.
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Fig. 2.
Size estimation and proteomic environment of
Sp_Set1C. a, size estimation of Sp_Set1C using a crude
cell extract from the Sp_Set1-TAP strain and Superose 6 sizing column.
Fractions were analyzed by Western blotting against protein A of the
TAP tag. b, the proteomic environment of Set1C is depicted,
which includes a working model of Set1C based on densitometric
quantification of Coomassie staining intensities as presented in Table
I. The proteomic hyperlink between Set1C and Lid2C is shown by a
double-headed arrow. The protein-protein
interactions included in the diagram are based on information from
Sc_Set1C (3).
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Fig. 3.
Sp_Set1C methylates histone 3 lysine 4 in vitro. a, Sp_Set1C methyltransferase activity was
assayed with Sp_Set1C-TAP-purified extracts using core histones as
substrates in the presence of [3H]SAM. Following
incubation, the reactions were resolved by SDS-PAGE followed by
Coomassie Blue staining and fluorography. Clr4, the S. pombe H3 Lys9 methyltransferase was used as a positive
control. b, Sp_Set1C has a preference for H3
Lys4. The complex isolated from the Sp_Set1-TAP strain was
incubated with H3 N-terminal (1-16) peptides mutated at lysine 4 (K4L)
or lysine 9 (K9L) in the presence of [3H]SAM, and
incorporated 3H was determined by scintillation
counter.
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Fig. 4.
H3 Lys4 methylation in S. pombe requires Sp_Set1C members and correlates with
phenotypic markers. a, Western blot using an antibody
specific to methylated H3 Lys4 showing the effect of
disruption of all the members of Sp_Set1C on the levels of H3
Lys4 methylation in vivo. An antibody specific
to acetylated H4 was used as a loading control. b, doubling
times of strains lacking Sp_Set1C members. Results are from three
experiments with error bars showing standard
deviations. c, the length of the telomere repeats in strains
lacking Sp_Set1C members determined from Southern blots and scans from
phosphorimaging data.
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Fig. 5.
H3 Lys4 methylation in S. pombe requires the Rad6 homologue, Rhp6. a,
Western blot using an antibody specific to methylated H3
Lys4 showing the effect disruption of Rhp6 or Set1 on H3
Lys4 methylation in vivo. b, RT-PCR
analysis for transcripts of different members of Sp_Set1C in Rhp6
(top), wild type, and
Set1C member strains, as indicated,
showing that all members of Set1C are expressed unless specifically
deleted. Each RT-PCR reaction was run in duplicate (with and without
reverse transcriptase) as a control for DNA contamination.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Damian Brunner for the yeast strains and the crash course on fission yeast, Thomas Jenuwein for reagents and discussion, David Drechsel for help with the sizing columns, Vincent Geli for communication of unpublished data, Junko Kanno for pAMP1, Henrik Thomas for assistance with mass spectrometric sample preparation, and to all members of the Stewart laboratory and Sabine Goldmann for useful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
** Supported by Grant 146652/431 from the Norwegian Research Council.
To whom correspondence should be addressed. E-mail:
stewart@mpi-cbg.de.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M209562200
2 A. Roguev, D. Schaft, A. Shevchenko, R. Aasland, A. Shevchenko, and A. Francis Stewart, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TrxG, trithorax Group; PcG, Polycomb Group; SET, Su(var), E(z), and Trithorax; TAP, tandem affinity purification; H, histone; RT, reverse transcriptase; MALDI, matrix-assisted laser desorption ionization; SAM, S-adenosyl methionine.
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