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First published online September 12, 2006
doi: 10.1242/10.1242/dev.02584
1 Department of Biology, Indiana University, Bloomington, IN 47405, USA.
2 Department of Biochemistry, Purdue Cancer Center, Purdue University, West
Lafayette, IN 47907, USA.
3 Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer
Center, University of North Carolina at Chapel Hill, NC 27599, USA.
4 Department of Genetics, Yale University School of Medicine, New Haven, CT
06520, USA.
Authors for correspondence (e-mail:
lbender{at}indiana.edu;
sstrome{at}indiana.edu)
Accepted 8 August 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: C. elegans, MES proteins, Histone methylation, Germ line, X-chromosome silencing
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
One level of chromatin modulation is via the covalent modification of
histones, including methylation, acetylation and ubiquitination (reviewed by
Fischle et al., 2003).
Modified histone residues can serve as docking sites for downstream effector
proteins: for instance, H3 methylated at Lys9 recruits HP1, and H3 methylated
at Lys27 recruits the Polycomb repressive complex PRC1 (reviewed by
Martin and Zhang, 2005).
Certain histone modifications are known to lead to either the activation or
repression of underlying genes (Fischle et
al., 2003; Sims et al.,
2003).
Within the germ line of Caenorhabditis elegans, both X chromosomes
in XX hermaphrodites and the single X in XO males are silenced by global
repression mechanisms involving modifications of histones
(Kelly et al., 2002). The
C. elegans proteins MES-2, MES-3, MES-4 and MES-6 have been
implicated in this repression. Mutations in the mes genes result in
maternal-effect sterility, due to defects in germ cell proliferation and
necrotic degeneration of germ cells
(Capowski et al., 1991;
Garvin et al., 1998). Several
findings suggest that this necrotic germline death is primarily a result of
the aberrant expression of X-linked genes when silencing fails. First,
mes mutant animals with two X chromosomes are more severely affected
than mes mutants with only one X; in fact, single X animals are
usually fertile (Garvin et al.,
1998). Second, in germ cells of wild-type hermaphrodites the X
chromosomes lack numerous marks of active chromatin
(Kelly et al., 2002), whereas
in germ cells of mes-2, mes-3 or mes-6 hermaphrodites the X
chromosomes display those marks (Fong et
al., 2002). Thus, the MES proteins are required for germ cell
viability and probably function, at least in part, to silence the X
chromosomes.
MES-2, MES-3 and MES-6 operate together in a complex
(Ketel et al., 2005;
Xu et al., 2001) and probably
participate directly in X-chromosome silencing. MES-2 possesses a SET domain,
a hallmark of histone methyltransferases (HMTs), and has been shown to have
HMT activity on Lys27 of histone H3 (H3K27)
(Bender et al., 2004), like its
fly and vertebrate orthologs, E(Z) and EZH2, respectively
(Cao et al., 2002;
Czermin et al., 2002;
Kuzmichev et al., 2002;
Muller et al., 2002). The HMT
activity of MES-2 requires the association of both MES-6, an ortholog of fly
ESC and vertebrate EED, and MES-3, a novel protein
(Ketel et al., 2005). Thus,
the MES-2/MES-3/MES-6 complex resembles the Polycomb Repressive Complex PRC2
in its HMT activity, substrate specificity and certain partner requirements.
In worms, the MES-2/MES-3/MES-6 complex is responsible for all detectable
H3K27 methylation in most regions of the germ line and in early embryos
(Bender et al., 2004). Notably,
the MES-2/MES-3/MES-6 complex concentrates trimethylated H3K27 (H3K27me3) on
the X chromosomes (Bender et al.,
2004). This repressive mark is likely to contribute to the
repressed state of the X chromosomes (Fong
et al., 2002).
The function of MES-4 has until now been a mystery. MES-4 shows the unique
property of associating with the five autosomes but not with the X chromosome
(Fong et al., 2002). Here, we
show that MES-4 is a histone H3 HMT that it is responsible for all detectable
H3K36 dimethylation in most regions of the germ line and in early embryos, and
that it concentrates H3K36me2 marks on the autosomes. In contrast to
Set2-related H3K36 HMTs, which associate with elongating RNA polymerase II and
methylate H3K36 within the coding regions of genes (e.g.
Kizer et al., 2005;
Morris et al., 2005), the
binding of MES-4 to chromatin and its H3K36 HMT activity do not appear to
depend on RNA polymerase II. This suggests that methylation of H3K36 can serve
different roles in regulating chromatin function. Microarray analysis,
performed on gonads dissected from wild type and mes-4 mutants,
revealed that loss of MES-4(+) function results primarily in the upregulation
of genes on the X chromosome. Our results suggest that in germline tissue
MES-4 cooperates with MES-2/MES-3/MES-6 to achieve proper silencing of
X-linked genes.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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LGII: rol-1(e91), mes-2(bn11), mnC1.
LGIV: mes-6(bn38), DnT1(IV;V), DnT1[qIs51](IV;V).
LGV: dpy-11(e224), mes-4(bn23, bn50, bn58, bn67, bn73, bn85, bn87), pgl-3(bn104), mnT12(IV;X).
RNAi analysis
RNAi was performed to deplete RNA Pol II/AMA-1, CDK-9 and TLK-1 as
described by Kamath et al. (Kamath et al.,
2003). Either wild-type or mes-4(bn73) L3 hermaphrodites
were placed on plates containing dsRNA-expressing bacteria, at room
temperature, and stained 36-40 hours later.
Immunofluorescence staining
Samples were fixed using methanol/paraformaldehyde
(Han et al., 2003) or
methanol/acetone (Strome and Wood,
1983). Rabbit antibodies to MES-4 were raised against the
C-terminal 19 amino acids+Cys, or against amino acids 530-898, then affinity
purified and used at 1:100 to 1:500 dilution. Other primary antibodies used
were affinity-purified rabbit anti-H3K36me2
(Tsukada et al., 2006) at
1:200, mouse monoclonal antibody H5 to RNA Pol II CTD pSer2 (Covance) at
1:200, rabbit anti-H4K20me2 [Upstate and a gift from Yi Zhang
(Fang et al., 2002)] at 1:100,
chicken anti-H3K27me2 (Upstate) at 1:25, rabbit anti-H3K27me3 (Upstate) at
1:200, rabbit anti-acetylated histone H4 (Upstate) at 1:5000, mouse monoclonal
antibodies PA3 at 1:1000 and PL4-2 at 1:2000 [a gift from M. Monestier
(Monestier et al., 1994)],
mouse monoclonal antibody OIC1D4 (Hird et
al., 1996) at 1:5, and rat anti-PGL-3
(Kawasaki et al., 2004) at
1:10,000. Secondary antibodies from Jackson Immunologicals (TRITC-conjugated
anti-rabbit IgG and anti-mouse IgM) and Molecular Probes (Alexa 488-conjugated
anti-rat and anti-rabbit IgG) were used at 1:200. Images were acquired with an
UltraVIEW LCI spinning-disk confocal laser and Nikon Eclipse TE200 microscope
with UltraVIEW software (Perkin-Elmer), assembled with Adobe Photoshop, and
displayed as projections of images taken at 0.5 µm intervals through the
sample.
Bacterial expression of MES-4 and HMT assays
Full-length MES-4 cDNA was subcloned into a pET28b bacterial
expression vector (Novagen). Transformed BL21-Gold (DE3) E. coli were
grown to an OD600 of 0.6 and induced with 0.1 mM
isopropyl-D-thiogalactoside for 24 hours at 20°C. His-tagged MES-4 was
detected in western blots using mouse monoclonal SC-8036 anti-His antibodies
(Santa Cruz Biotechnology). HMT assays were performed by incubating 4 µl of
bacterial lysate with 16 µg of chicken oligonucleosomes and 1.0 µCi of
S-adenosyl-L-[methyl-3H]methionine (Amersham Pharmacia Biotech) in
methyltransferase buffer (25 mM Tris-HCl pH 8.0, 5% glycerol) for 30 minutes
at 20°C in a total volume of 20 µl. Half of the reaction was analyzed
by SDS-PAGE, followed by Coomassie staining and fluorography.
Microarray analysis of RNA from dissected gonads
Dissection of gonad arms from hermaphrodites, isolation of RNA, and linear
amplification were performed as described by Chi and Reinke
(Chi and Reinke, 2006). Gonad
arms were dissected from wild-type and mes-4(bn85)
M+Z- young adult hermaphrodites containing one to two
fertilized embryos. During each of four dissection sessions, 100 gonad arms
were collected from each genotype. Fluorescently labeled cDNA samples were
prepared and hybridized to microarrays as previously described
(Reinke et al., 2000). DNA
microarrays were prepared as described elsewhere
(Jiang et al., 2001;
Reinke et al., 2004). Two
hybridization experiments were performed with Cy3-labeled wild-type cDNA and
Cy5-labeled mes-4 cDNA, and two with the dyes swapped. For every gene
in each microarray hybridization experiment, the ratio of wild type
(wt)/mes-4 was transformed into a log2 value and the mean
log2 ratio calculated. Confidence levels were determined using a
two-tailed paired t-test. Genes were considered to be significantly
altered in the level of mRNA accumulation in wild type versus mes-4
if they displayed a mean fold-difference ratio of 1.8 or higher, and a
confidence level of greater than 95% (P<0.05)
(Whetstine et al., 2005). The
GEO accession number for microarray data is GSE5454.
Real-time PCR analysis of RNA from dissected gonads
Fifty gonad arms were dissected from wild-type and mes-4(bn85)
M+Z- young adult hermaphrodites and total RNA isolated
as described by Chi and Reinke (Chi and
Reinke, 2006). Poly-adenylated cDNA was prepared using the
SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time
PCR was performed in triplicate using iQ SYBR Green Supermix (Bio-Rad) and the
iCycler iQ Multi-Color Real-Time PCR Detection System (Bio-Rad). The Autoprime
program
(www.autoprime.de)
was used to design primers to span an exon-exon junction. All data were
normalized to him-3 and F14B4.2, and the Pfaffl method
(Pfaffl, 2001) was used to
calculate relative fold changes.
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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) predicted
that it has histone methyltransferase (HMT) activity. The mouse SET-domain
protein bearing the closest similarity to MES-4
(Fong et al., 2002), NSD1, has
been reported to methylate histones H3 and H4 in vitro
(Rayasam et al., 2003). As
shown in Fig. 1A, full-length
6his::MES-4 expressed in bacteria and incubated with chicken oligonucleosomes
methylates H3. No other histones are detectably methylated.
We used antibodies specific for H3 and H4 peptides containing methylated
lysines to investigate the residue specificity of MES-4. Mouse NSD1 has been
reported to dimethylate H3K36 and H4K20
(Rayasam et al., 2003). Based
on the staining of dissected C. elegans germ lines and early embryos
with an antibody specific for H3 dimethylated at K36 (H3K36me2)
(Tsukada et al., 2006), this
mark is abundant on chromatin in wild-type worms but is absent from
mes-4 mutants (Fig.
1B-I). Thus, MES-4 is required for H3K36 dimethylation in vivo, at
least in germline tissue (see below). Given its H3 HMT activity in vitro, we
propose that MES-4 functions as an H3K36 HMT in vivo. Based on the staining of
wild type and mes-4 mutants, MES-4 is not required in vivo for any of
the other H3 methyl marks we tested (H3K4me2 or me3, H3K9me2, H3K27me2 or me3,
H3K79me2) or for H4K20me2 (Fong et al.,
2002) (see Fig. S2E,F in the supplementary material; data not
shown). The latter result agrees with the lack of H4 HMT activity of MES-4 in
vitro. Furthermore, we were unable to detect H4 methylation activity for
recombinant human NSD1 (amino acids 1556-1950) in vitro under conditions where
strong H3 methylation was seen (data not shown). Therefore, we propose that
both MES-4 and human NSD1 are specific for histone H3K36.
MES-4 is required for H3K36 dimethylation in most regions of the germ line and in early embryos
We used the antibody specific for H3K36me2 to investigate the tissue
distribution of H3K36 dimethylation. H3K36me2 is abundant on the chromatin of
most or all nuclei in wild-type worms (Fig.
1B-I; see also Fig. S1A in the supplementary material). To learn
which tissues and stages require MES-4 function, we compared the H3K36me2
patterns in wild type to those in mes-4 adult hermaphrodites,
specifically in the fertile F1 progeny of mes-4/+ heterozygotes.
These F1 progeny have a maternal load of mes-4 products but no
expression from the zygotic genome (we refer to them as
M+Z-). The embryos produced by
M+Z- hermaphrodites are M-Z- and
develop into sterile adults. In mes-4 M+Z- germ
lines, H3K36me2 is undetectable in nuclei from the mitotically dividing distal
region of the gonad (Fig. 1C)
through the pachytene region (Fig.
1E). Some H3K36me2 signal is visible in late-pachytene/diplotene
nuclei and in oocytes (Fig.
1G). In addition, some H3K36me2 staining is detectable in the
polar body products of oocyte meiosis, although H3K36me2 staining is
undetectable in the blastomere nuclei of mes-4 embryos through the
35-cell stage (Fig. 1I,
see Fig. 4B). Thus, MES-4 is
responsible for all detectable H3K36 dimethylation in most regions of the
adult germ line and in early stages of embryogenesis. A different H3K36 HMT(s)
apparently is active in adult somatic cells (arrowheads in
Fig. 1C,E), in the
diplotene/diakinesis region of the oogenic germ line, and in >40-cell-stage
embryos (Fig. 4D,F,H). These
findings are consistent with the accumulation of MES-4 in the germ line and in
early embryos (Fong et al.,
2002), and with mes-4 mutants displaying defects
primarily in the germ line (Capowski et
al., 1991).
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), we
predicted that the chromosomes lacking H3K36me2 are the X chromosomes.
H3K36me2 staining of X:autosome translocations in embryos confirmed that
prediction: H3K36me2 is absent from the presumed X-chromosome portion of each
translocation chromosome (Fig.
2B). Interestingly, in embryos containing an X:autosome
translocation with a free left end of the X chromosome, we noticed a pinpoint
spot of MES-4 (or of H3K36me2) staining on the exposed tip of the X portion
(Fig. 2A,B). A `dot' of MES-4
was detected on 16 out of 31 X-chromosome left ends examined. A dot of MES-4
was never found on the right end (18 X-chromosome right ends examined; data
not shown). Clues as to the significance of the dot of X-chromosome staining
are considered in the Discussion.
Targeting of MES-4 to chromosomes requires the first PHD finger
MES-4 contains three CysHis fingers previously classified as PHD (plant
homeodomain) motifs, plus an AWS-like domain and a post-SET domain flanking
the SET domain (Fong et al.,
2002) (Fig. 3A). We
sequenced seven EMS- or gamma radiation-induced mes-4 mutations
(Capowski et al., 1991;
Fong et al., 2002)
(Fig. 3A; see also Table S1 in
the supplementary material). Some of the mes-4 lesions shed light on
features of MES-4 required for its association with chromatin, in particular
the first PHD domain.
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). Studies of ISWI
and p300 implicate their PHD domains in nucleosome binding
(Eberharter et al., 2004;
Ragvin et al., 2004), and more
recently the PHD domains of ING2 and of BPTF have been shown to bind H3K4me3
(Li et al., 2006;
Pena et al., 2006). Although
the first PHD domain of MES-4 is fairly atypical
(Bienz, 2006), it appears to be
crucial for MES-4 to associate with chromatin. The in-frame deletion allele bn85, which disrupts the SET domain, results in partial dissociation of MES-4 from chromosomes (Fig. 3B, row 3; see Table S1 in the supplementary material). The small amount of bn85 mutant protein bound to chromosomes does not lead to detectable H3K36me2 signal (data not shown). The partial dissociation of bn85 protein from chromosomes may implicate the SET and/or post-SET domains in having a minor role in the localization of MES-4 to chromatin.
The point mutation bn58, within the region between the third PHD
domain and the AWS-like domain, also results in partial dissociation of MES-4
from chromosomes (Fig. 3B, row
2). bn58 is the only mes-4 allele that displays detectable
H3K36me2 signal (Table S1 in the supplementary material), suggesting that the
fraction of mutant protein that is associated with chromatin has at least
partial HMT activity. This residual activity may confer upon bn58
animals the ability to produce more germ nuclei than other mes-4
mutants (Capowski et al.,
1991).
Exclusion of MES-4 and H3K36me2 from the X requires MES-2, MES-3 and MES-6, and also depends on gamete history
Previously we showed that the SET-domain protein MES-2, in a complex with
MES-3 and MES-6, is required for H3K27 di- and trimethylation in the C.
elegans germ line (Bender et al.,
2004), and that MES-4 patterns are altered in mes-2,
mes-3 and mes-6 mutants. Specifically, in mes-2, mes-3
and mes-6 M+Z- germ lines, MES-4 appears
ectopically on X chromosomes in late oogenesis
(Fong et al., 2002). Here, we
have examined both MES-4 and H3K36me2 patterns in mes-2, mes-3 and
mes-6 M-Z- early embryos
(Fig. 2C,D). Consistent with
our previous study, in early embryos MES-4, and also H3K36me2, spread to the
oocyte-derived X chromosome. Thus, the activity of the MES-2/MES-3/MES-6
complex participates in repelling MES-4 and H3K36me2 from the X chromosomes at
late stages of oocyte differentiation and in early embryos.
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). This imprint is likely to explain the failure of MES-4 and
H3K36me2 to decorate the sperm X in mes-2, mes-3 and mes-6
mutants. The MES-2/MES-3/MES-6 complex may repel MES-4 and its HMT activity not only from the oocyte-derived X chromosome but also from particular regions of the autosomes. The pattern of H3K36me2 on autosomes is patchy or banded in appearance in wild type (Fig. 1D, see also Fig. S2 in the supplementary material), and appears more uniform and intense in mes-2, mes-3 and mes-6 nuclei (Fig. 2, compare rows B and D; Fig. S2, compare rows A and B, and rows C and D). An attractive mechanistic model to explain this finding is that MES-2-catalyzed methylation of H3K27 prevents MES-4 from binding to and methylating a nearby residue of the H3 tail, K36.
Another H3K36 HMT becomes active by the 40-cell stage of embryogenesis
Prior to the 35-cell stage of embryogenesis in mes-4
M-Z- embryos, no H3K36me2 is visible on chromosomes
(Fig. 4B). As mes-4
embryos reach the 40-cell stage, several nuclei show a faint H3K36me2
signal (Fig. 4D). The intensity
of staining increases as the embryos develop
(Fig. 4F). These results
demonstrate that at least one other H3K36 HMT, in addition to MES-4, becomes
active in embryos. Notably, this non-MES-4 H3K36 HMT does not appear to be
active in the primordial germ cell P4 or its newly formed daughters Z2 and Z3
(Fig. 4D,F), where MES-4 is
active (Fig. 4C,E). Later,
during the comma stage of embryogenesis, H3K36me2 appears in Z2 and Z3 of
mes-4 embryos (Fig.
4H). H3K36me2 levels are somewhat higher in all nuclei of older
wild-type embryos and L1 larvae when compared with mes-4 mutants
(Fig. 4G,H), probably because
of the contribution of MES-4 activity to overall levels of H3K36me2.
MES-4 HMT activity does not appear to be linked to transcription elongation
Because MES-4 is concentrated on autosomes, and because autosomes, but not
X chromosomes, are actively transcribed in the germ line, we investigated
whether MES-4 H3K36 HMT activity is directly associated with the progression
of transcription. Studies in Saccharomyces cerevisiae showed that
Set2, an H3K36 HMT, associates with the elongating form of RNA Pol II
(Krogan et al., 2003;
Li et al., 2002;
Li et al., 2003;
Schaft et al., 2003;
Xiao et al., 2003). The
emerging view is that Pol II phosphorylated on Ser2 of its C-terminal domain
(CTD) recruits Set2, which in turns methylates nearby nucleosomes.
Transcription elongation-coupled methylation of H3K36 appears to be widely
conserved across eukaryotic species
(Adhvaryu et al., 2005;
Kizer et al., 2005;
Morris et al., 2005;
Sun et al., 2005).
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To investigate whether the non-MES-4 H3K36 HMT activity that becomes active
in >40-cell C. elegans embryos is linked to transcription
elongation, we examined whether the residual H3K36me2 mark in >40-cell
mes-4 embryos is affected by ama-1(RNAi). H3K36me2 was
undetectable in mes-4; ama-1(RNAi) 100-cell embryos
(Fig. 6A-D). Depletion of CTD
Ser2 kinases (CDK-9 or TLK-1) (Han et al.,
2003; Shim et al.,
2002) in a mes-4 background also eliminated H3K36me2
signal (see Fig. S3 in the supplementary material), revealing that the
residual H3K36me2 signal in mes-4 embryos requires the phosphorylated
form of CTD Ser2 in embryos. These results suggest that: (1)
non-MES-4-mediated dimethylation of H3K36 is directly linked with
transcription; and/or (2) the non-MES-4 H3K36 HMT must be transcribed from the
embryonic genome.
Loss of MES-4 results in desilencing of X-linked genes
To investigate directly the impact of MES-4 chromatin regulation on gene
expression, microarray analysis was used to compare the profile of mRNA
accumulation in wild-type and mes-4 mutant germ lines. Ideally, we
would compare the nascent germ lines in young wild-type and
M-Z- mutant larvae, prior to the onset of germline
degeneration in mutants. Because those germ lines contain very few cells, we
instead analyzed isolated gonads dissected from wild-type hermaphrodites and
from fertile mes-4 M+Z-hermaphrodites. Several
observations justified analyzing the M+Z- generation.
First, MES-4 levels are below detection in M+Z- adults
(see Fig. S1C in the supplementary material). Second, as described above,
MES-4-mediated H3K36me2 is also undetectable in M+Z-
germ lines. Third, mes-4 M+Z- germ lines are
compromised: transgenes are desilenced, brood size is reduced, and RNAi
depletion of other chromatin regulators renders mes-4
M+Z- worms, but not wild-type worms, sterile
(Capowski et al., 1991;
Kelly and Fire, 1998;
Xu and Strome, 2001). We
reasoned that altered patterns of gene expression are likely to underlie these
M+Z- germline phenotypes, and that elucidating the
alterations would provide insights into MES-4 function.
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16,400 of the 20,000 predicted C.
elegans genes (Reinke et al.,
2004). Seventy-one genes displayed significantly different
accumulation in mes-4 relative to wild type (>1.8 average fold
difference, P<0.05, two-tailed paired t-test, see
Materials and methods; Fig.
7A,C; see also Table S2 in the supplementary material). The
microarray results were validated by real-time PCR for 15 out of 15 genes
(P<0.05) selected at random from the upregulated, downregulated,
and non-regulated classes (see Table S3 in the supplementary material). Two aspects of the altered gene expression profile in mes-4 are particularly striking and informative. First, 67 of the 71 affected genes displayed higher expression (upregulated) in mes-4 mutants relative to wild type, and four genes displayed lower expression (downregulated). Second, 61 of the 67 genes upregulated in mes-4 mutants are located on the X chromosome. Given the relative sizes of the six chromosomes and gene representation on our microarrays, random chance alone would predict that 10 of the 67 upregulated genes would map to the X. Thus, the primary effect of loss of MES-4 function on gene accumulation patterns in M+Z- germ lines is upregulation of genes on the X chromosome.
Given the concentration of MES-4 on autosomes, we considered the
possibility that an apparent upregulation of genes on the X chromosome in fact
reflects widespread downregulation of autosomal genes in mes-4
mutants. In this scenario, MES-4 would serve as an activator of autosomal
genes; loss of MES-4 would cause a reduced accumulation of autosomal
transcripts and therefore of total mRNA, and our use of equivalent input mRNA
to prepare microarray probes would result in artificially elevated levels of
X-linked mRNAs. This scenario is highly unlikely for several reasons. (1)
Absolute hybridization intensities for all 16,400 genes represented on
the microarrays showed similar profiles in wild type and mes-4
mutants (Fig. 7B). (2) Given
the banded appearance of MES-4 and H3K36me2 on the autosomes, downregulation
of autosomal genes in mes-4 mutants would be expected to show
gene-to-gene variation. Only 10 autosomal genes of the 14,000 represented
on the microarrays showed a >1.8-fold difference in accumulation in
mes-4 gonads relative to in wild-type gonads, and only four of those
were downregulated in mes-4 mutants
(Fig. 7A; see also Table S2 in
the supplementary material). (3) If the downregulation of autosomal genes led
to an apparent upregulation of X-linked genes in mes-4 gonads, then
those genes on the X chromosome would be expected to be fairly uniformly
affected. Instead, only 61 of the 2400 X-linked genes represented on the
microarrays showed a >1.8-fold difference in accumulation in mes-4
gonads relative to wild type. (4) Real-time PCR analysis using non-amplified
RNA from 50 dissected mes-4 and wild-type gonads verified the up- or
downregulation of 10 genes, and the approximately equivalent accumulation of 5
genes (see Table S3 in the supplementary material).
Taken together, our results lead to the surprising conclusion that MES-4, a chromatin regulator concentrated on the five autosomes, functions to repress genes on the X chromosome. How X-chromosome silencing may be achieved by MES-4, in collaboration with the MES-2/MES-3/MES-6 complex, is discussed below.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). We do
not see evidence that MES-4 participates in H4K20 methylation. Mouse NSD1 was
further reported to methylate oligonucleosomes, core histones, and recombinant
H3 and H4. In our assays, MES-4 and human NSD1 methylated oligonucleosomes,
but not core histones or recombinant H3 or H4 (not shown). Our results suggest
that both MES-4 and human NSD1 prefer nucleosomal substrates and are specific
for H3K36. Mouse NSD1 may have a broader substrate range.
MES-4 is apparently the sole active H3K36me2 HMT in the regions of the germ
line extending from the distal mitotic stem cells through the meiotic
pachytene region. In those regions, MES-4 and H3K36me2 are excluded from all
of the X chromosome except the leftmost tip
(Fong et al., 2002) (this
report), and the X chromosomes are silenced
(Kelly et al., 2002). During
oogenesis, the X chromosomes become at least partially activated late in
pachytene (Kelly et al.,
2002). This turn-on of the X chromosomes is not accompanied by a
detectable appearance of MES-4 on them, which argues, as do our microarray
results, against a model in which MES-4 is required to activate gene
expression in the germ line. However, concomitant with X activation, an
H3K36me2 HMT distinct from MES-4 becomes active on the X chromosomes as well
as the autosomes, leading to methylation of H3K36 on all chromosomes. This
non-MES-4 HMT may serve a Set2-like role during transcription elongation (e.g.
Kizer et al., 2005).
In embryos, MES-4 remains autosomally concentrated until at least the
100-cell stage (data not shown), and is responsible for all detectable
H3K36me2 until about the 40-cell stage, at which time another H3K36 HMT(s)
also becomes active. The current view is that C. elegans early
embryos inherit a large stockpile of maternal transcripts, initiate embryonic
transcription of at least some genes by the four-cell stage, and undergo a
`mid-blastula transition' from maternal to embryonic control of development at
about the 40-cell stage (Baugh et al.,
2003; Edgar et al.,
1994; Seydoux and Fire,
1994). Methylation of H3K36 catalyzed by the non-MES-4 HMT, which
becomes detectable by the 40-cell stage, is temporally correlated with
activation of the embryonic genome and may serve an essential role in that
process. This non-MES-4 HMT(s) is dependent on Pol II and thus may function
similarly to yeast Set2.
H3K36me2 marks may serve diverse roles
In the yeast S. cerevisiae, Set2 catalyzes all H3K36 methylation
and requires association with Pol II for this activity
(Strahl et al., 2002;
Kizer et al., 2005). H3K36me2
in S. cerevisiae is recognized by the Rpd3S complex, which
deacetylates nucleosomes within gene coding regions, to aid in suppressing
aberrant intragenic transcription initiation
(Carrozza et al., 2005;
Joshi and Struhl, 2005;
Keogh et al., 2005). H3K36me2
marks also may serve to distinguish actively transcribed sequences from
inactive genes and from regulatory sequences in yeast and higher eukaryotes
(Bannister et al., 2005;
Rao et al., 2005;
Sun et al., 2005).
Our studies suggest that MES-4 associates with and methylates chromatin
independently of Pol II. This in turn suggests that MES-4-catalyzed
methylation of H3K36 serves a role distinct from those described above. The
same may be true of the MES-4-related proteins in humans: NSD1, NSD2/MMSET and
NSD3. Significantly, all three have been implicated in causing or promoting
human cancers when mutated or overexpressed
(Schneider et al., 2002). Our
findings on C. elegans MES-4 invite speculation that the NSD family
HMTs may also operate independently of Pol II.
MES-4 and H3K36me2 on the left tip of the X chromosome
MES-4 and H3K36me2 both decorate autosomes in a banded pattern and are
excluded from all regions of the X chromosome except the left tip. The left
end of the X chromosome has emerged as being different from the remainder of
the X in several respects. The meiotic pairing center for the X chromosome is
located less than 2 Mb from the left end
(MacQueen et al., 2005). This
region binds to the zinc-finger protein HIM-8 and associates with the nuclear
envelope during meiotic prophase (Phillips
et al., 2005). The left end of the X is also enriched relative to
the rest of the X chromosome for AA/TT dinucleotides that are periodically
spaced along one face of the DNA helix, and that may influence DNA bending and
chromatin structure (Fire et al.,
2006). The X-chromosome left end also contains three copies of a
14 base pair perfect repeat that is distributed abundantly over the autosomes
(696 copies total), but found nowhere else on the X chromosome (I. Korf and J.
Bedell, personal communication). Tests to date have not revealed an
involvement of this 14-mer in MES-4 binding specificity (P. Poole, C.R.C. and
S.S., unpublished).
|
Among several models that might be postulated, we consider two to explain
the derepression of some X-linked genes in mes-4 mutants. (1) MES-4
normally activates the expression of an autosomally encoded repressor that
selectively represses genes on the X chromosome
(Fig. 8, upper right). The
expression of four autosomal genes is, in fact, downregulated >1.8-fold in
mes-4 mutants. However, these genes do not possess motifs or show
homologies that make them good candidates for serving as transcriptional
repressors. Furthermore, RNAi depletion of each gene did not result in
sterility (Gonczy et al.,
2000; Kamath et al.,
2003; Rual et al.,
2004; Sonnichsen et al.,
2005) (C.R.C. and S.S., unpublished). (2) An autosomal
concentration of MES-4 or its H3K36me2 mark repels a global repressor, thereby
concentrating repressor action on the X chromosomes
(Fig. 8, lower right). We
hypothesize that MES-2/MES-3/MES-6-catalyzed H3K27me3 concentrated on the X
chromosome acts to repel MES-4 (Fig.
8, left), as suggested by the observation that MES-4 spreads onto
the X chromosome in mes-2, mes-3 and mes-6 mutants
(Fong et al., 2002) (this
study), and that MES-4 and/or H3K36me2 concentrated on the autosomes repel an
unidentified repressor (`R' in the figure). The possibility that this
unidentified repressor is the MES-2/MES-3/MES-6 complex itself is unlikely, as
the pattern of H3K27me3 catalyzed by that complex is not visibly altered in
mes-4 mutants (see Fig. S2E,F in the supplementary material).
We postulate two types of X repression: a direct mechanism mediated by MES-2/MES-3/MES-6, and a novel, indirect mechanism mediated by MES-4. Loss of MES-2/MES-3/MES-6 would lead to the loss of repressive histone modifications and to at least some desilencing of the X chromosome. Loss of MES-4 would lead to insufficient levels of global repressor (model 1, Fig. 8) or promiscuous binding of the global repressor to autosomal regions, and, if the repressor is in limiting supply, to titration of repressor away from the X chromosome, causing at least some desilencing of the X (model 2, Fig. 8). One might expect promiscuous binding of the repressor to also cause widespread repression of autosomal loci, which we did not observe. If there is a limited supply of repressor and it is distributed over potentially six times more chromatin (10 autosomes in addition to the 2 Xs) in mes-4 mutants than in wild type, then the critical concentration of repressor needed for repression may not be achieved at most autosomal loci.
Our notion that MES-4 may have a role in repelling a global repressor from
the five autosomes, thereby focusing repressor action on the X chromosome, has
a precedent in van Leeuwen and Gottschling's proposed `gaining specificity by
preventing promiscuity' model for the function of the S. cerevisiae
H3K79 HMT Dot1 (van Leeuwen and
Gottschling, 2002). Dot1 methylates 90% of H3K79 residues in
the genome; notably, silent chromatin, which comprises 10% of the genome,
is hypomethylated at that residue. Loss of Dot1 function and H3K79 methylation
causes the SIR silencing proteins to spread from normally silent chromatin
into euchromatin, and causes loss of silencing. This illustrates how a
globally distributed histone modification can reduce nonspecific binding of
silencers, and focus their binding and silencing effects to discrete
domains.
MES-4 in the soma
Although MES-4 is not essential for the health and viability of somatic
tissue (Capowski et al., 1991),
recent studies of synMuv (for synthetic multivulva) genes have revealed a role
for MES-4 in somatic cells (Unhavaithaya
et al., 2002; Wang et al.,
2005; Cui et al.,
2006). Several synMuv class B mutants show a remarkable phenotype:
somatic cells display germline traits, including expression of the germline
marker PGL-1 and enhanced RNAi. Concomitant loss of mes-4 function
suppresses the `ectopic germline traits' and other synMuv phenotypes, and in
the case of mep-1 suppresses its larval lethality. This has led to a
model in which MES-4 participates in conferring germline identity on cells,
and the synMuv B regulators suppress or antagonize that function in somatic
cells, thus protecting their somatic fates
(Unhavaithaya et al., 2002;
Strome, 2005). MES-4 is not
alone in serving that proposed role; other mes genes and genes
encoding additional chromatin regulators show similar genetic interactions
with synMuv B mutants (Unhavaithaya et
al., 2002; Wang et al.,
2005; Cui et al.,
2006). The targets of MES-4 regulation in somatic cells, and the
mechanism by which MES-4 and synMuv B chromatin regulators antagonize each
other, remain to be determined.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3907/DC1
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ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Biostatistics, University of Washington,
Seattle, WA 98195, USA
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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