Department of Molecular Biology and Pharmacology Washington University School of Medicine, St Louis, MO 63110, USA
* Author for correspondence (e-mail: kornfeld{at}molecool.wustl.edu)
Accepted 23 December 2004
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SUMMARY |
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Key words: LIN-1, SUMO, ETS, Chromatin, Vulval development, Transcription
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Introduction |
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Genetic analysis indicates that lin-1 is a crucial target of the
RTK/Ras/ERK signaling pathway. lin-1(lf) mutations cause a strong
multivulva (Muv) phenotype; P3.p, P4.p and P8.p inappropriately adopt vulval
cell fates, and the resulting ectopic tissue forms a series of ventral
protrusions. Thus, lin-1 activity inhibits the 1° vulval cell
fate and/or promotes the 3° cell fate. The Muv phenotype caused by
lin-1(lf) mutations is epistatic to the Vul phenotype caused by
loss-of-function mutations in mpk-1 and other upstream signaling
genes, indicating that lin-1 functions downstream of MPK-1
(Ferguson et al., 1987;
Lackner et al., 1994
;
Wu and Han, 1994
).
The lin-1 gene encodes a 441 amino acid protein that contains a
conserved ETS DNA-binding domain (Beitel et
al., 1995). Mutations in the ETS domain that abrogate DNA binding
cause a strong Muv phenotype, demonstrating that DNA binding is necessary for
LIN-1 to inhibit the 1° vulval cell fate
(Miley et al., 2004
). LIN-1
contains two docking sites for ERK, the D domain and FQFP motif, and 17 S/TP
motifs that are potential ERK phosphorylation sites
(Fig. 1A)
(Fantz et al., 2001
;
Jacobs et al., 1999
;
Tan et al., 1998
). Mutations
of the FQFP motif that decrease phosphorylation of LIN-1 by ERK cause a
gain-of-function Vul phenotype (Jacobs et
al., 1998
). Thus, phosphorylation of LIN-1 by MPK-1 ERK prevents
LIN-1 from functioning as a constitutive inhibitor of the 1° cell fate.
The mechanisms that enable LIN-1 to inhibit vulval cell fates and
phosphorylation of LIN-1 to relieve this inhibition are not well defined.
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Materials and methods |
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To monitor activation of the LexA-dependent lacZ reporter, we
prepared lysates from at least six independent yeast transformants of
equivalent size and measured ß-galactosidase activity using the
Galacto-Light Plus System (Applied Biosystems). For
Fig. 5, yeast transformants
were grown in selective media at 30°C to an optical density of 1.0
before analysis.
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The plasmid pFastBac DUAL (Invitrogen) was modified to encode
GST:LIN-1(1-64), GST:LIN-1(1-64; 9-16A) or GST:LIN-1(1-64; K10A) with or
without 6xHis- and FLAG-tagged C. elegans SMO-1. Proteins were
expressed in Sf9 cells using the baculovirus system (Invitrogen, Bac-to-Bac
Baculovirus Expression Systems manual). Infected cells were lysed in buffer
containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM
NEM, 1.5 mM DTT, 1.5 mM PMSF and complete protease inhibitor cocktail (Roche).
The GST:LIN-1 fusion proteins were purified using glutathione sepharose as
described (Jacobs et al.,
1998), separated by SDS-PAGE and immunoblotted with
-GST
antibody (Santa Cruz Biotechnology) or
-FLAG M2 antibody.
Cell culture and reporter gene assays
The 293 human embryonic kidney (HEK) cell line (ATCC CRL-1573) was
transfected using Ca2+ phosphate precipitation
(Sambrook et al., 1989) so
that each well received 100 ng L8G5-luciferase reporter plasmid (kindly
provided by Dr Khochbin) (Lemercier et
al., 2000
), 200 ng LexA-VP16 expression plasmid
(Lemercier et al., 2000
), 100
ng CMV-ß-galactosidase expression plasmid and 200 ng GAL4 DNA-binding
domain (G4) fusion protein expression plasmid. Cells were harvested after
19 hours, and luciferase activity was measured according to the
manufacturer's techniques (Promega).
To generate SMO-1:LIN-1 fusion proteins that would be resistant to isopeptidase cleavage, we designed G4 and LA plasmids (Figs 3, 5) that expressed SMO-1 residues 1-88; this fragment lacks the C-terminal di-glycine isopeptidase cleavage site. Fusion proteins were confirmed to be the predicted size for the intact protein by western blotting.
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HT115(DE3) E. coli transformed with a plasmid that expresses
double-stranded RNA from the smo-1, ubc-9 or mep-1 gene were
produced by Fraser et al. (Fraser et al.,
2000) and Kamath et al.
(Kamath et al., 2003
), and
distributed by MRC geneservice. Generally, genomic fragments were PCR
amplified using the indicated primers and cloned into the L4440 control
plasmid (Timmons and Fire,
1998
) between copies of the bacteriophage T7 promoter
(smo-1, 5'-GAGAAACCGAGTATCTCAGTGGA-3' and
5'-GCGATGCGTTTAATTAAGTTTTG-3'; ubc-9,
5'-CTTATCGATCGGATTTCTGTTTG-3' and
5'-CTACCACGAAGCAAGCATCTACT-3'; mep-1,
5'-CCTCTTCTGGAACGCTTGTC-3' and
5'-CTGGTTCTCTTGTGCGTTCA-3'). Cells were grown overnight at
37°C in LB media containing 50 µg/ml ampicillin, diluted 1:100 in
2xYT media containing 50 µg/ml ampicillin, grown at 37°C for 6
hours and seeded onto a Petri dish containing NGM agar, 50 µg/ml ampicillin
and 100 µM IPTG. The following day (day 1), L4 hermaphrodites were placed
on the Petri dish. These hermaphrodites were transferred to a new Petri dish
daily, and progeny were scored for the Muv phenotype. We defined limited and
extensive exposure to RNAi as progeny laid on day 1 and day 2 plates or day 3
and day 4 plates, respectively. We determined the number of descendants of
P3.p, P4.p and P8.p for hermaphrodites at the `Christmas tree' stage of vulval
development based on cell position and morphology using DIC microscopy for
limited exposure smo-1 RNAi and extensive exposure mep-1
RNAi.
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Results |
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To define regions of LIN-1 that are necessary and sufficient to bind UBC-9,
we analyzed fragments of LIN-1 containing amino acids 1-64, 65-145 and
146-252. LA:LIN-1(1-64) and LA:LIN-1(146-252) were sufficient to mediate
robust binding to UBC-9, indicating that LIN-1 contains two separable binding
sites for UBC-9 (Fig. 1B, lines
2, 9). We noted that the LIN-1(1-64) fragment contains the sequence
VK10KE that matches the KxE consensus sumoylation motif. To
determine if this motif is necessary for LIN-1 to bind UBC-9, we mutated
residues 9-16 to alanine. The binding of UBC-9 to the LA:LIN-1(1-64; 9-16A)
mutant was reduced 28-fold relative to the binding of LA:LIN-1(1-64)
(Fig. 1B, line 3). This motif
was further characterized by mutating each of the four residues individually.
A substitution of the predicted SUMO acceptor lysine (K10A) or the
highly-conserved glutamic acid (E12A) dramatically reduced binding of UBC-9
(Fig. 1B, lines 5, 7). A
substitution of the moderately conserved valine (V9A) partially decreased
binding of UBC-9, whereas a substitution of the non-conserved lysine (K11A)
had no significant effect (Fig.
1B, lines 4, 6). These results demonstrate a correlation between
the function of each residue in the
KxE consensus sumoylation motif in
promoting sumoylation (Sampson et al.,
2001
) and the function of each residue in the VK10KE
motif in promoting binding of UBC-9. In particular, residues predicted to be
crucial for sumoylation were crucial for the binding of UBC-9.
We noted that the LIN-1(146-252) fragment that was sufficient to bind UBC-9 contains the sequence VK169DE that matches the consensus sumoylation motif. A 25 amino acid segment of LIN-1 that contains this motif, LIN-1(156-180), also bound robustly to UBC-9 (Fig. 1B, line 10). To determine if this motif is necessary for binding, we mutated the entire motif (168-171A) or the predicted SUMO acceptor lysine (K169A). The binding of UBC-9 to the LA:LIN-1(156-180; 168-171A) mutant was decreased by 60-fold relative to the binding of LA:LIN-1(156-180) (Fig. 1B, line 11). Mutation of the predicted SUMO acceptor lysine also significantly reduced binding of UBC-9 (Fig. 1B, line 12).
LIN-1 is covalently modified by SUMO-1
Because SUMO and the sumoylation enzymes are highly conserved from S.
cerevisiae to H. sapiens, we monitored sumoylation of LIN-1 in
yeast and cultured cells. We co-expressed LIN-1 and yeast SUMO1/Smt3 with a
His- and FLAG-tag (HF-SUMO) in yeast cells and purified proteins covalently
modified by HF-SUMO by metal affinity chromatography. Western blotting
revealed species of LA:LIN-1(1-252) with retarded mobility in cells that
express HF-SUMO but not control cells lacking HF-SUMO
(Fig. 2A, lane 4 versus lane
3). The calculated molecular weight of these proteins suggests that LIN-1 was
covalently modified by multiple SUMO proteins. These results demonstrate that
LIN-1 is sumoylated in yeast.
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smo-1 and ubc-9 negatively regulate vulval cell fates and function at the level of lin-1
If sumoylation is important for LIN-1 function, then mutations that reduce
sumoylation might affect cell fate determination and result in a Vul or Muv
phenotype. C. elegans contains a single gene that encodes SUMO,
designated smo-1. We used two methods to reduce the function of
smo-1. First, we analyzed the smo-1(ok359) null allele that
contains a deletion of the entire smo-1 locus. smo-1(ok359)
homozygous mutants were sterile. To analyze vulval development, we derived
smo-1(ok359) homozygotes from smo-1(ok359)/+ hermaphrodites.
These mutants displayed a completely penetrant protruding vulva (Pvl)
phenotype. Broday et al. (Broday et al.,
2004) have attributed the Pvl phenotype to the presence of an
abnormal vulE cell and impaired formation of the uterine-seam cell and
demonstrated that the LIM domain transcription factor LIN-11 is sumoylated. In
addition, we observed that nine percent of smo-1(ok359) mutants
displayed a Muv phenotype, defined as one or more ventral protrusions
displaced from the position of the vulva when viewed with a dissecting
microscope (Table 1, line 2).
Broday et al. (Broday et al.,
2004
) observed a similar defect. Second, we used RNA interference
(RNAi) to reduce the levels of smo-1 RNA by feeding hermaphrodites
E. coli that express double-stranded smo-1 RNA. Consistent
with a previous report (Fraser et al.,
2000
), extensive exposure to smo-1(RNAi) resulted in a
highly penetrant embryonic lethal phenotype; the few surviving adults
displayed a Pvl phenotype. However, limited exposure to smo-1(RNAi)
allowed most animals to survive to adulthood, and 9% of these adult
hermaphrodites displayed a Muv phenotype
(Table 1, line 7). To
characterize the cellular basis for this Muv phenotype, we used DIC microscopy
to examine smo-1(RNAi) hermaphrodites. P3.p, P4.p and P8.p generated
three or more descendants, indicating that the cell adopted a partial vulval
fate, with a frequency of 0%, 18% and 18%, respectively (n=11). These
results indicate that smo-1 has multiple functions during development
and is necessary for embryonic viability, fertility, vulval morphogenesis and
inhibition of vulval cell fates.
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Loss-of-function mutations of smo-1 and lin-1 both cause
a Muv phenotype. To analyze the interaction between these genes, we generated
a partial loss-of-function lin-1 mutation. The lin-1(e1275
R175Opal) mutation causes a Muv phenotype with a penetrance of 91% at
20°C (Beitel et al., 1995).
The lin-1(e1275) mRNA contains a premature stop codon and is likely
to have a short half-life because of nonsense-mediated decay. In a double
mutant with smg-1(r861), a gene that is necessary for
nonsense-mediated decay, the lin-1(e1275) mRNA appears to be
stabilized and the penetrance of the Muv phenotype is reduced to 2%
(Table 1, line 12). If
smo-1 is necessary for the sumoylation and function of LIN-1, then
these mutants are predicted to be highly sensitive to a reduction of
smo-1 activity. smo-1(RNAi) caused the penetrance of the Muv
phenotype to increase to 78% in this smg-1(r861); lin-1(e1275)
genetic background (Table 1,
line 13). These results demonstrate a strong interaction between partial
loss-of-function mutations in smo-1 and lin-1.
We previously described gain-of-function mutations of lin-1
(Jacobs et al., 1998). The
strongest gain-of-function mutation is lin-1(n1790gf
R352Opal) (Fig. 1A).
The lin-1(n1790) mutation causes a weak vulvaless phenotype and
partially suppresses the Muv phenotype caused by activated let-60
ras; the LIN-1(1-351) protein lacks the FQFP MAPK docking site and is
partially resistant to negative regulation by MPK-1
(Jacobs et al., 1999
). The
lin-1(n1790gf) allele also causes a low penetrance Muv
phenotype because the lin-1 mRNA contains a premature stop codon and
is subject to nonsense-mediated decay. If smo-1 is necessary for the
sumoylation and function of LIN-1(1-351), then the double mutant is predicted
to lack functional LIN-1 and display a strong Muv phenotype. The
smo-1(ok359); lin-1(n1790gf) double mutants displayed a Muv
phenotype that was 82% penetrant, significantly greater than the Muv phenotype
of ok359 and n1790 single mutants
(Table 1, line 4).
lin-1(n1790gf) hermaphrodites fed smo-1(RNAi)
likewise displayed a highly penetrant Muv phenotype
(Table 1, line 15). These data
support the model that smo-1 functions at the level of lin-1
and that sumoylation of LIN-1 is necessary for inhibition of vulval cell
fates.
To investigate the function of ubc-9, we fed hermaphrodites E. coli that expressed double-stranded ubc-9 RNA. Wild-type hermaphrodites exposed to ubc-9(RNAi) occasionally displayed a Muv phenotype, although the penetrance was only 0.4% (Table 1, line 16). ubc-9(RNAi) caused a significant Muv phenotype of 12% and 27% in smg-1(r861); lin-1(e1275) and lin-1(n1790) hermaphrodites, respectively (Table 1, lines 17, 18). These results indicate that ubc-9 functions to repress vulval cell fates and interacts genetically with lin-1.
Sumoylation of LIN-1 promotes transcriptional repression
To investigate the mechanism by which sumoylation of LIN-1 inhibits vulval
cell fates, we monitored the transcriptional activity of LIN-1 in 293 human
embryonic kidney cells. We used a reporter plasmid that contains eight
LexA-binding sites and five GAL4-binding sites upstream of an E1A promoter
that regulates expression of luciferase
(Fig. 3A). A LexA DNA-binding
domain:VP16 (LexA:VP16) fusion protein was used to robustly activate this
reporter (Fig. 3B, lines 1, 2).
The ability of fusion proteins containing the GAL4 DNA binding domain (G4) to
activate or repress transcription was monitored. G4:LIN-1(1-64) repressed
transcription sevenfold relative to G4 alone
(Fig. 3B, lines 2, 3). Thus,
LIN-1 residues 1 to 64 are sufficient to repress transcription. Substitutions
of the entire consensus sumoylation motif (9-16A) or the SUMO acceptor lysine
(K10A) resulted in LIN-1 mutants that failed to repress transcription
(Fig. 3B, lines 5, 7). Thus,
the VK10KE consensus sumoylation motif is necessary for
transcriptional repression by LIN-1(1-64). The same assay system was used to
show that the VK169DE consensus sumoylation motif is necessary for
transcriptional repression mediated by LIN-1(156-180) (data not shown).
The VK10KE motif might be necessary for transcriptional repression because it mediates sumoylation of LIN-1 or because it has an additional activity. To distinguish between these models, we determined if sumoylation of LIN-1 is sufficient to mediate transcriptional repression. C. elegans SMO-1(1-88) was fused to LIN-1(1-64) or the sumoylation-defective LIN-1 mutants. Addition of SMO-1 to the sumoylation-defective LIN-1 mutants restored transcriptional repression; G4:SMO-1:LIN-1(1-64, 9-16A) repressed transcription 13-fold relative to G4:LIN-1(1-64, 9-16A) and G4:SMO-1:LIN-1(1-64, K10A) repressed transcription sixfold relative to G4:LIN-1(1-64, K10A) (Fig. 3B, lines 6, 8). Translational fusion of SMO-1 to LIN-1(1-64) resulted in a twofold repression relative to LIN-1(1-64) (Fig. 3B, line 4). Interestingly, SMO-1 fused to G4 in the absence of LIN-1 repressed transcription by fourfold relative to G4 alone (Fig. 3B, line 9). These results demonstrate that SMO-1 is sufficient to restore transcriptional repression to LIN-1 mutants that lack the VK10KE motif, indicating that sumoylation of this motif mediates transcriptional repression.
LIN-1 binds MEP-1, and the interaction is mediated by two consensus sumoylation motifs
To characterize the mechanisms by which sumoylation of LIN-1 mediates
transcriptional repression, we analyzed proteins that were identified in the
two-hybrid screen using LIN-1(1-252) as bait and have been implicated in
transcriptional regulation. One-hundred and twenty-three out of 233 cDNAs
identified encode MEP-1. MEP-1 is a zinc finger protein that associates with
C. elegans LET-418/CHD-4 and HDA-1, homologs of the vertebrate
Mi-2ß and HDAC-1, respectively (Fig.
4A) (Belfiore et al.,
2002; Unhavaithaya et al.,
2002
). These proteins are core components of the NuRD
transcriptional repression complex.
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To investigate MEP-1 binding to LIN-1 residues 146-252, we analyzed the LIN-1(156-180) fragment that contains the VK169DE sumoylation motif. MEP-1 strongly interacted with LIN-1(156-180) (Fig. 4E). Mutations of the entire motif (168-171A) or the predicted SUMO acceptor lysine (K169A) markedly reduced binding of MEP-1 to LIN-1 (Fig. 4E). These studies demonstrate that a 64 amino acid fragment of LIN-1 containing the consensus sumoylation motif VK10KE and a 25 amino acid fragment of LIN-1 containing the consensus sumoylation motif VK169DE are sufficient to bind MEP-1, and for both LIN-1 fragments the SUMO acceptor lysine is necessary for binding.
Sumoylation of LIN-1 promotes binding of MEP-1
The KxE motifs of LIN-1 may directly interact with MEP-1, or
post-translational modification of these motifs by SUMO may promote the
binding of MEP-1. To investigate these possibilities, we expressed His-tagged
MEP-1 in baculovirus-infected Sf9 cells and partially purified the protein
using metal affinity chromatography. GST:LIN-1(1-64) was expressed in E.
coli and purified by glutathione affinity chromatography. His:MEP-1 did
not detectably interact with GST:LIN-1(1-64) in a GST pull-down assay. Because
bacterially expressed LIN-1 is not sumoylated, these data suggest that
sumoylation of LIN-1 is necessary for the interaction with MEP-1.
We reasoned that if sumoylation of the LIN-1 KxE motifs mediates MEP-1
binding, then the addition of SUMO to a LIN-1 mutant that lacks the
KxE
motif might restore binding of MEP-1. We generated a translational fusion of
the C. elegans SUMO-1 homolog, SMO-1, and the LIN-1(1-64; 9-16A)
mutant that lacks the
KxE motif and measured its interaction with MEP-1
in yeast. The interaction of MEP-1 with LA:SMO-1:LIN-1(1-64; 9-16A) was
increased by eightfold relative to the interaction with LA:LIN-1(1-64; 9-16A)
(Fig. 5, lines 2, 3). If
sumoylation of LIN-1 mediates the interaction with MEP-1, then MEP-1 might
display binding to SUMO in the absence of LIN-1. Consistent with this
prediction, MEP-1 displayed a threefold greater interaction with LA:SMO-1 than
LA alone (Fig. 5, lines 4, 5).
These findings indicate that the
KxE motif promotes binding by mediating
sumoylation of LIN-1 and not by directly interacting with MEP-1.
mep-1 inhibits vulval cell fates and acts at the level of lin-1
To test the model that the interaction of LIN-1 and MEP-1 is important for
lin-1 function in vivo, we used genetic analysis to characterize the
function of mep-1 during vulval development. The activity of the
mep-1 gene was reduced by feeding wild-type hermaphrodites bacteria
that express double-stranded mep-1 RNA. Limited exposure of wild-type
hermaphrodites to mep-1(RNAi) caused 6% of hermaphrodites to display
a Muv phenotype (Table 2, line
2), whereas extensive exposure to mep-1(RNAi) caused a 58% Muv
phenotype (n=326). To characterize how mep-1 RNAi affects
Pn.p cell fates, we examined hermaphrodites using DIC microscopy. P3.p, P4.p
and P8.p generated three or more descendants, indicating that the cell adopted
a partial vulval fate, with frequencies of 10%, 60% and 50%, respectively
(n=10) (Fig. 6). These
results indicate that mep-1 inhibits vulval cell fates in P3.p, P4.p
and P8.p.
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Discussion |
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LIN-1 is sumoylated
Here, we present evidence indicating that LIN-1 is sumoylated. First, LIN-1
contains two KxE consensus sumoylation motifs, VK10KE and
VK169DE. Second, UBC-9, the homolog of the S. cerevisiae
Ubc9 SUMO conjugating enzyme, binds both of the LIN-1 consensus sumoyation
motifs. These results suggest that UBC-9 conjugates SUMO to K10 and
K169 of LIN-1. Third, biochemical studies demonstrated that LIN-1
is covalently modified by one or more SUMO moieties, and the consensus
sumoylation motif is required for sumoylation. LIN-1 has not been previously
reported to be sumoylated, and these findings reveal a new mechanism of LIN-1
regulation.
Sumoylation of LIN-1 promotes inhibition of the 1° vulval cell fate
The function of LIN-1 sumoylation was investigated in animals by reducing
the activity of smo-1 using a deletion allele and RNAi and by
reducing the activity of ubc-9 using RNAi. Because smo-1 was
essential for embryonic viability and fertility, vulval development was
examined in adult hermaphrodites with a partial reduction of smo-1
activity. A reduction of smo-1 function caused a Muv phenotype,
demonstrating that smo-1 inhibits Pn.p cells from adopting vulval
cell fates. The smo-1 Muv phenotype was partially penetrant; this
might be a result of residual smo-1 activity, or smo-1 might
not always be necessary to inhibit vulval cell fates. The smo-1(lf)
Muv phenotype was not suppressed by a probable null mutation of mek-2
or a partial loss-of-function mutation of mpk-1. These mpk-1
and mek-2 mutations strongly suppress more highly penetrant Muv
phenotypes caused by synthetic multivulva genes or upstream genes in the Ras
signaling pathway (Kornfeld et al.,
1995; Lackner et al.,
1994
). Thus, smo-1 probably functions downstream of
mek-2 and mpk-1 if these genes act in a linear signaling
pathway. Furthermore, reducing the activity of smo-1 and
ubc-9 diminished the activity of a constitutively active LIN-1
mutant, indicating that smo-1 and ubc-9 are necessary for
LIN-1 to inhibit vulval cell fates. Together, the biochemical studies showing
that LIN-1 is sumoylated and the genetic studies showing that SMO-1 and UBC-9
are necessary for LIN-1-mediated inhibition of vulval cell fates support the
model that sumoylated LIN-1 inhibits vulval cell fates.
Sumoylation of LIN-1 mediates transcriptional repression
A diverse group of transcription factors are post-translationally modified
by SUMO. For most of these proteins, including Sp3, Myb, Jun, Elk1, p300,
C/EBP and CtBP, sumoylation promotes transcriptional repression
(Bies et al., 2002;
Dahle et al., 2003
;
Gill, 2003
;
Girdwood et al., 2003
;
Kim et al., 2002
;
Lin et al., 2003
;
Muller et al., 2000
;
Ross et al., 2002
;
Sapetschnig et al., 2002
;
Subramanian et al., 2003
;
Yang et al., 2003
). However,
for a few proteins, including HSF1, sumoylation promotes transcriptional
activation (Hong et al.,
2001
). To characterize how sumoylation affects LIN-1, we monitored
the transcriptional activity of LIN-1 in cultured cells. A fragment of LIN-1
containing a consensus sumoylation motif caused transcriptional repression.
The consensus sumoylation motif was necessary for transcriptional repression,
and fusion of SUMO to the mutant LIN-1 was sufficient to restore repression.
These findings demonstrate that sumoylation of LIN-1 mediated this
transcriptional repression activity.
Previous studies of lin-1 did not distinguish between the models
that lin-1 inhibits vulval cell fates by activating transcription of
genes that promote the 3° non-vulval cell fate or repressing transcription
of genes that promote the 1° vulval cell fate. Based on the results that
sumoylation of LIN-1 mediates transcriptional repression and inhibition of
vulval cell fates, we infer that LIN-1 inhibits the 1° vulval cell fate by
repressing target gene transcription. Therefore, lin-1 target genes
promote the 1° vulval cell fate. Together, these findings suggest that in
the six Pn.p cells during larval development, LIN-1 is sumoylated and
represses transcription of target genes that promote the 1° fate. When the
anchor cell activates the RTK/Ras/ERK pathway in P6.p, MPK-1 ERK
phosphorylates LIN-1 and relieves the LIN-1-mediated transcriptional
repression, and genes that promote the 1° fate are now transcribed in
P6.p. Phosphorylation may disrupt sumoylation of LIN-1 and cause LIN-1 to
activate transcription of genes that promote the 1° vulval cell fate, as
phosphorylation of human Elk1 by ERK activates transcription
(Treisman, 1994;
Yang et al., 2003
).
Sumoylated LIN-1 binds MEP-1: a molecular mechanism for SUMO-mediated transcriptional repression
Although sumoylation has been shown to affect the activity of several
transcription factors, the mechanisms have not been well defined. The most
detailed descriptions of the mechanism of SUMO-mediated transcriptional
repression are the studies of Girdwood et al.
(Girdwood et al., 2003),
showing that sumoylated p300 interacts with HDAC6, and of Yang and Sharrocks
(Yang and Sharrocks, 2004
),
showing that sumoylated Elk1 interacts with HDAC-2. These studies indicate
that sumoylation mediates recruitment of chromatin remodeling enzymes.
However, these HDACs have not been shown to directly bind the SUMO moieties.
In our screen for proteins that interact with LIN-1, over 50% of the positives
were MEP-1. The Krüppel-type zinc-finger protein MEP-1 was identified as
a nuclear protein that associates with the MOG-1, MOG-4 and MOG-5 DEAH box
proteins, and the MOG-6 cyclophilin-like protein, suggesting that it functions
with these proteins to regulate the fem-3 RNA
(Belfiore et al., 2002
;
Belfiore et al., 2004
). In
addition, MEP-1 interacts with LET-418/CHD-4 and HDA-1, homologs of the Mi-2
and HDAC-1 core components of the NuRD complex, respectively
(Unhavaithaya et al., 2002
).
The NuRD complex possesses ATP-dependent nucleosome remodeling activity that
is dependent upon Mi-2 and histone deacetylase activity provided by HDAC-1 and
HDAC-2; both of these activities promote transcriptional silencing
(Tong et al., 1998
;
Wade et al., 1998
;
Xue et al., 1998
;
Zhang et al., 1998
).
mep-1 appears to have multiple functions during C.
elegans development, because it is necessary for larval viability,
gonadogenesis and oocyte production
(Belfiore et al., 2002;
Unhavaithaya et al., 2002
).
mep-1 mutants display abnormal gene expression in larvae, indicating
that mep-1 regulates gene expression. mep-1 mutants exhibit
a partially penetrant Muv phenotype
(Belfiore et al., 2002
); this
phenotype becomes highly penetrant in combination with a synMuv A allele
(Unhavaithaya et al.,
2002
).
Our studies have revealed that the LIN-1 interaction with MEP-1 required the VK10KE and VK169DE consensus sumoylation motifs. Translational fusion of SUMO to LIN-1 mutants lacking these motifs partially restored binding to MEP-1. These findings suggest that sumoylation of LIN-1 allows MEP-1 binding. If MEP-1 is associated with the NuRD complex, then sumoylation of LIN-1 might promote recruitment of the NuRD complex to lin-1 target genes, resulting in gene silencing.
The genetic analysis of mep-1 supports this model. Reducing the activity of mep-1 using RNAi caused a Muv phenotype. The mep-1(lf) Muv phenotype was not suppressed by a loss-of-function of mek-2 or mpk-1, indicating that mep-1 functions downstream or parallel to mek-2 and mpk-1. In addition, reducing mep-1 function diminished the activity of a constitutively-active LIN-1 mutant, indicating that MEP-1 is necessary for LIN-1 to inhibit vulval cell fates. Thus, smo-1, ubc-9 and mep-1 all displayed similar genetic properties and function at the level of lin-1 to inhibit vulval cell fates.
Based on our findings, we propose a model for the inhibition of vulval cell fates by LIN-1. Newly synthesized LIN-1 associates with the E2 SUMO-conjugating enzyme UBC-9 and becomes sumoylated at residues K and K to GGA motifs in target genes that promote the 1°10169. LIN-1 then binds vulval cell fate. The SUMO moieties of LIN-1 interact with MEP-1, leading to recruitment of the NuRD complex. This complex probably induces multiple changes in lin-1 target genes that promote silencing, including ATP-dependent nucleosome remodeling and histone deacetylation. Sumoylation of LIN-1, even if transient, can cause an enduring change in transcriptional activity by promoting covalent modifications of histones and chromatin restructuring. This may be a general mechanism for SUMO-mediated transcriptional repression, as MEP-1 might interact with the SUMO moieties of additional transcription factors.
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