From the Schools of Biochemistry and Molecular
Biology and ¶ Biomedical Sciences, Faculty of Biological Sciences,
University of Leeds, Leeds LS2 9JT, United Kingdom and the
§ Wellcome Laboratory for Molecular Pharmacology, Department
of Pharmacology, University College London, Gower Street,
London WC1E 6BT, United Kingdom
Received for publication, December 22, 2000, and in revised form, February 2, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many aspects of neurogenesis and neuronal
differentiation are controlled by basic helix-loop-helix (bHLH)
proteins. One such factor is SHARP-1, initially identified on the basis
of its sequence similarity to hairy. Unlike
hairy, and atypically for bHLHs, SHARP-1 is expressed late
in development, suggestive of a role in terminal aspects of
differentiation. Nevertheless, the role of SHARP-1 and the identity of
its target genes remain unknown. During the course of a one-hybrid
screen for transcription factors that bind to regulatory domains of the
M1 muscarinic acetylcholine receptor gene, we isolated the
bHLH transcription factor SHARP-1. In this study, we investigated the
functional role of SHARP-1 in regulating transcription. Fusion proteins
of SHARP-1 tethered to the gal4 DNA binding domain repress both basal
and activated transcription when recruited to either a
TATA-containing or a TATAless promoter. Furthermore, we identified two
independent repression domains that operate via distinct
mechanisms. Repression by a domain in the C terminus is
sensitive to the histone deacetylase inhibitor trichostatin A,
whereas repression by the bHLH domain is insensitive to TSA.
Furthermore, overexpression of SHARP-1 represses transcription from the
M1 promoter. This study represents the first report to assign a function to, and to identify a target gene for, the bHLH transcription factor SHARP-1.
Transcription factors of the basic helix-loop-helix
(bHLH)1 family play an
important role in neuronal determination and early differentiation in
all phyla that have been examined. Numerous vertebrate bHLHs (for
reviews see Refs. 1-3) have been identified on the basis of homology
to their Drosophila counterparts (reviewed in Refs. 4-6).
Some, such as Mash-1 (mouse
achaete
scute homologue), are
transcriptional activators and act as positive regulators of
neurogenesis, whereas others, such as HES-1 (homologue of
Hairy and
Enhancer of
Split), are transcriptional
repressors and act as negative regulators of neurogenesis.
Structurally, bHLH proteins share a number of common features. The HLH
domain mediates homomeric or heteromeric dimerization (7), and the
adjacent basic region mediates DNA binding. Three groups of bHLH
proteins can be recognized, according to the target binding site they
recognize (8-10). Class A and Class C bHLHs function as
transcriptional activators and repressors, respectively, whereas class
B bHLH proteins can be either activators or repressors.
Recently, the cDNA for SHARP-1, a novel bHLH protein, was isolated
on the basis of its homology to Hairy and Enhancer of
Split (11). However, sequence alignment showed that SHARP-1 is
only distantly related to these proteins, exhibiting 37-42% sequence identity within the bHLH domain. Unlike most bHLH proteins, SHARP-1 is
not expressed in neuronal progenitor cells or early differentiating neurons but is restricted to a subset of neurons of the postnatal central nervous system (11), suggestive of a role in terminal neuronal
differentiation rather than in neural determination. Unlike all other
HAIRY/E(spl)/HES proteins, SHARP-1 lacks the hallmark WRPW
domain, which binds the co-repressor GROUCHO (or GROUCHO-like proteins)
and is required for both transcriptional repression and suppression of
neurogenesis. Absence of the WRPW motif suggests that SHARP-1 functions
by recruiting transcriptional machinery other than GROUCHO. All class B
bHLH proteins contain an arginine at position 13 in the basic
region, essential for these proteins to bind to class B sites. The
presence of an arginine at this position in SHARP-1 suggests that it
belongs in this group, but because this group contains both activators
and repressors, SHARP-1 function cannot be predicted on the basis of
protein sequence.
At present, almost nothing is known about the role of bHLH proteins in
differentiated neurons, and in common with many other bHLH proteins, no
target genes of SHARP-1 are known. In the present study, we ascribe a
transcriptional function to SHARP-1 and identify the M1
muscarinic acetylcholine receptor gene as a target gene. We show that
SHARP-1 is able to repress transcription of both TATA-containing and
TATAless promoters when recruited via a Gal4 DNA binding domain (DBD).
Repression occurs when SHARP-1 is bound either proximally or more
distally to the promoter. Furthermore, we show that repression by
SHARP-1 is bimodal. One mode of repression requires the bHLH domain and
is insensitive to the histone deacetylase inhibitor, TSA, whereas the
other is mediated via the C-terminal domain and represses transcription
through a TSA-sensitive mechanism. We also show that overexpression of
SHARP-1 represses transcription of a reporter construct containing the
M1 promoter. These results show that within the
HAIRY-related/HES family, SHARP-1 is unique in its combination of
presumed biological function and transcriptional mechanism.
Reporter Plasmid Construction--
The plasmid pBM2389 +417/+166
M1 was generated as follows. A PCR product generated by
using as template pGL3 +166/+603 M1 and the primers
M1 235 (12) and RV3 (Promega) was cloned into pGem-T easy
(Promega). The fragment was excised with EcoRI and cloned
into pBM2389 (13). The SHARP-1 coding region was generated by PCR with
the primers SHARP-1.-11s and SHARP-1.762a (numbers are relative to the
translation start site of SHARP-1) containing NcoI linkers
and cloned in frame into the NcoI site of pCS2+MT (14) to
give pMT SHARP-1. To generate pMT G4 SHARP-1, a PCR product obtained
using the primers SHARP-1.4s and SHARP-1.763a, consisting of the
SHARP-1 coding region flanked by EcoRI linkers, was cloned
into the EcoRI site of pMT G4 (15). PCR products with
EcoRI linkers were generated using the sense primer
SHARP-1.4s, and the antisense primers SHARP-1.519a, SHARP-1.306a, or
SHARP-1.147a (numbers are relative to the translation start site of
SHARP-1) were similarly used to generate pMT G4 NbHO-SHARP-1 (residues 1-173), pMT G4 NbH-SHARP-1 (residues 1-102), and pMT G4 N-SHARP-1 (residues 1-49), respectively. PCR products containing SHARP-1 fragments between positions 520 and 762, 307 and 519, and 127 and 306 with EcoRI linkers were cloned into pMT G4 to generate pMT
G4 C-SHARP-1 (residues 174-253), pMT G4 O-SHARP-1 (residues 103-173),
and pMT G4 bH-SHARP-1 (residues 43-102), respectively. To clone
pSHARP-1 myc, a PCR fragment containing the open reading frame of SHARP-1 was generated using a sense primer containing a
BamHI linker and an antisense primer containing an
EcoRI linker and cloned into pCS2+MT (14). The reporter
plasmid pGL3-372/+602 M1 has been reported previously
(12). The reporter plasmids pTRE UAS TATA, pGL3 UAS TRE TATA, and pGL3
UAS TRE Inr have been described previously (15).
Yeast One-hybrid Screening--
pBM2389 +417/+166 M1
was transformed into the yeast strain SFY526 (16). This yeast strain
was then transformed with DNA from an adult rat brain cDNA yeast
expression library (CLONTECH), using the protocol
of Schiestl and Gietz (17), and transformants were grown on complete
supplement mixture Reverse Transcription-PCR--
RNA was extracted from cell
lines and brain tissue using Tri reagent (Sigma) and
reverse-transcribed using oligo(dT) and Moloney murine leukemia
virus reverse transcriptase (Promega). Oligonucleotides used to
amplify the SHARP-1 gene were as follows: SHARP-1.125s, 5'-AGGATACCTACAAATTACCGC; and SHARP-1.441a, 5'-CGCGAGGTATTGCAAGAC. Numbers are relative to the translation start site of SHARP-1. The
oligonucleotides used to amplify the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene were as follows:
hprt 231s, 5'-CCTGCTGGATTACATTAAAGCACTG; and hprt
567a, 5'-CCTGAAGTACTCATTATAGTCAAGG. Aliquots of the
reaction mixture were electrophoresed on a 2% agarose gel.
Cell Culture--
Cell lines were cultured in 5%
CO2 at 37 °C in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% fetal calf serum, 6 g/liter penicillin, 10 g/liter streptomycin, and 2 mM
L-glutamine.
DNA Transfections--
Qiagen column-purified DNA was
transfected into cells using TfxTM 50 (Promega) according to the
manufacturer's instructions. Briefly, cells were plated onto 10-mm
wells to a density of 50%. For IMR32 and 3T3 cells, 750 ng of plasmid
(for amounts of individual plasmids see the figure legends), 3 ng of
pRL-CMV, and 1.5 µl of TfxTM 50 were mixed and made up to a
total volume of 200 µl with Opti-MEM (Life Technologies, Inc.) and
applied to cells for 3-4 h. For Neuro 2a cells, 250 ng of plasmid (for
amounts of individual plasmids see the figure legends), 1 ng of
pRL-CMV, and 0.5 µl of TfxTM 50 were used. Cells were harvested
24 h later into 60 µl Passive lysis buffer (Promega), of which
30 µl were used in the Promega DLRTM assay system. Luminescence was
measured using a Mediators PhL 1.8 luminometer, firefly luciferase was
normalized to Renilla luciferase, and the results were
expressed as a percentage of normalized expression in the presense of
pMT G4. For transfections in the presence of TSA (Wako Chemical), cells
were treated with the indicated concentrations of TSA for 24 h
prior to transfection and fed with media containing TSA for
24 h.
Immunoprecipitation Assay--
Neuro 2a cells were plated onto
10-cm plates to a density of 50%. Cells were incubated for 3-4 h with
10 µg of DNA and 22.5 µl of TfxTM 50 in a final volume of
4.8 ml. Cells were harvested after 2 days into 1 ml of 1×
phosphate-buffered saline containing 0.5% Nonidet P-40 and protease
inhibitors Block (Roche Molecular Biochemicals), sonicated for 90 s, and centrifuged at maximum speed for 10 min at 4 °C. The
supernatant was precleared for 2 h with 80 µl of protein
G-Sepharose. For each immunoprecipitation, half of the total sample was
incubated with 3 µl of Gal4 DBD antiserum (Santa Cruz Biotechnology)
overnight. Beads were added, and samples were incubated for an
additional 2 h. Samples were washed four times with 20 mM Tris, pH 8.0, 1 mM EDTA, 100 mM
NaCl, 0.5 mM Nonidet P-40, 10% glycerol, and 0.1% SDS.
Proteins were eluted with 15 µl of loading dye. Samples were run on a
10% SDS-polyacrylamide gel electrophoresis gel and blotted onto a
Hybond C+ nylon membrane (Amersham Pharmacia Biotech). The membrane was
subjected to Western blot analysis using a 1/1000 dilution of
c-myc antiserum (Santa Cruz Biotechnology).
SHARP-1 Binds to the M1 Promoter--
In our previous
studies, we have shown that transcription of the M1
muscarinic acetylcholine receptor gene is regulated by several domains
within the first exon (12, 18). In particular, the region between +166
and +412 (relative to the transcription start site) appears to contain
both enhancer and repressor elements. To identify transcription factors
that bind to this region of the M1 gene we used the yeast
one-hybrid approach (19, 20). Using the +166/+417 domain as bait
to screen an adult rat brain cDNA library, we isolated two
independent positive clones (Fig. 1a, colonies
3 and 10). Digestion of the isolated clones
showed that they both contain an insert of 1.5 kilobases (Fig.
1b). Sequencing of the inserts in these clones showed that
they were identical and contained the entire open reading frame of the
previously identified transcription factor SHARP-1 (11). Growth of
yeast on plates containing 10 mM 3-amino-1,2,4-triazole was
dependent upon the binding of SHARP-1 to the M1 sequence,
because no growth was seen in the absence of either bait or SHARP-1
(Fig. 1a).
SHARP-1 Has High Homology to SHARP-2, Stra13, and
DEC1--
SHARP-1 was originally isolated during the course of a
search for mammalian bHLH proteins expressed in differentiated neurons (11). Although SHARP-1 was isolated by homology to hairy and Enhancer of Split, sequence alignment with these proteins
shows that they are quite distantly related, sharing only 37-42%
homology within the bHLH domain (11).
As a first step toward identification of a function for SHARP-1, we
carried out a data base search for proteins with homology to SHARP-1
and identified three proteins: SHARP-2, Stra13, and DEC1 (Fig.
2). SHARP-2 is a bHLH protein isolated in
the same screen as that used to identify SHARP-1 (11), and its function is also unknown. Stra13 was isolated as a retinoic acid-inducible gene
in mouse P19 embryonic carcinoma cells and has been shown to be able to
repress the thymidine kinase promoter when fused to Gal4 DBD (21).
Finally, DEC1 is a protein that was cloned by subtractive hybridization
to identify mRNAs expressed in cAMP-differentiated human embryo
chondrocytes (22). Again, no function for DEC1 has been reported.
Inspection of amino acid sequences shows that SHARP-2, Stra13, and DEC1
contain 411 or 412 amino acids, of which 366 are conserved, showing a
sequence identity between them of 89%, suggesting that they are, in
fact, rat, mouse, and human homologues. SHARP-1 is more divergent and
contains only 253 amino acids. The highest sequence identity is seen in
the bHLH domain and in helices 3 and 4 (also called Orange domain (23,
24)), whereas within the C-terminal domain only two stretches of 8 and 11 amino acids are conserved.
SHARP-1 Homodimerizes--
All bHLH proteins dimerize to bind DNA
(25). Because SHARP-1 was identified in the present study using a yeast
one-hybrid screen, it seemed likely that SHARP-1 could either
homodimerize or heterodimerize with a yeast partner. To distinguish
between these possibilities, we carried out an immunoprecipitation
assay using differentially tagged recombinant SHARP-1. Neuro 2a cells were transfected with either a combination of myc-tagged
SHARP-1 (pMT SHARP-1) and myc-tagged Gal4 DBD (pMT G4) (Fig.
3, lanes 1 and 3)
or a combination of myc-tagged SHARP-1 (pMT SHARP-1) and a
myc-tagged fusion of Gal4 DBD and SHARP-1 (pMT G4 SHARP-1) (Fig. 3, lanes 2 and 4). Cell extracts were
immunoprecipitated with Gal4 DBD antiserum and subjected to
polyacrylamide gel electrophoresis, and the results were visualized by
Western blot analysis using c-myc antiserum. An antibody to
Gal4 was able to co-immunoprecipitate SHARP-1 only in the presence of a
GAL4-SHARP-1 fusion protein (Fig. 3, compare lanes 4 and
3), demonstrating that SHARP-1 is able to homodimerize.
Expression of SHARP-1 in Different Cell Lines--
It has been
shown previously that expression of SHARP-1 is largely
restricted to differentiated neurons in the postnatal central nervous system, predominantly in the cerebellum and hippocampus, although it is also detectable at a reduced level in heart, muscle, and
lung (11). We examined expression of SHARP-1 in different cell lines
and cerebellum using reverse transcription-PCR. As seen in Fig.
4a, SHARP-1 is highly
expressed in IMR32 cells, a human neuroblastoma cell line that also
expresses M1, Neuro 2a cells, and NB4 1A3, two mouse
M1 non-expressing neuroblastoma cell lines. Low levels of
expression were detected in the 3T3 fibroblast cell line. PCR was
carried out using hprt primers as a cDNA loading control (Fig.
4b).
SHARP-1 Acts as Transcriptional Repressor--
bHLH proteins can
act as transcriptional activators or repressors (reviewed in Ref. 1).
Because the transcriptional function of SHARP-1 is unknown, we assessed
its ability to (a) regulate transcription from both TATA-containing and
TATAless promoters, (b) regulate transcription when bound
either proximally or distally, and (c) regulate basal and activated
transcription. IMR32, 3T3, and Neuro 2a cells were transfected with
plasmids expressing SHARP-1 fused to Gal4 DBD with each of the reporter
genes showed in Fig. 5. pTRE UAS TATA
contains seven TRE and five Gal4 binding sites 21 base pairs upstream
of the E1b TATA box. pGL3 UAS TRE TATA contains five Gal4 binding sites
(placed 350 base pairs from the TATA box) and seven TRE upstream of the
TATA box. In pGL3 TRE UAS Inr, the TATA box from pGL3 TRE UAS TATA, was
replaced by the adenovirus major late promoter initiator. Expression
values of all reporter constructs were normalized to expression in the presence of Gal4 DBD alone. SHARP-1 was able to repress transcription of a TATA-containing promoter when bound proximally to the
transcription start site in all cell lines (Fig. 5a,
left). SHARP-1 was also able to repress activated
transcription by Tet-VP16 (activation domain of the herpes simplex
virus transcriptional activator VP-16 fused to the binding domain of
the tetracycline-responsive factor) in all cell lines (Fig.
5a, right). In addition, SHARP-1 was able to
repress both basal and activated transcription from a TATA-containing promoter when bound distally to the transcription start site (Fig. 5b). We also tested the ability of SHARP-1 to regulate
transcription from a TATAless promoter. As can be seen in Fig.
5c, SHARP-1 can repress basal and activated transcription
from a TATAless promoter. We therefore conclude that SHARP-1 acts as a
repressor of both basal and activated transcription of both
TATA-containing and TATAless promoters. In the case of a
TATA-containing promoter, repression is evident when SHARP-1 is bound
either proximally or distally to the promoter, although the degree of
repression is more marked when SHARP-1 is tethered proximally.
SHARP-1 Represses Transcription through Two Independent
Domains--
To map the domain(s) responsible for the repression
function of SHARP-1, we generated deletion mutants of the Gal4-SHARP-1 fusion protein. The ability of these fusion proteins to repress transcription was analyzed using the reporter gene driven by a TATA-containing promoter with five Gal4 binding sites proximal to the
transcription start site (pTRE UAS TATA) in Neuro 2a cells (Fig.
6). Results were normalized to expression
of the reporter gene in the presence of Gal4 DBD alone. Western blot
analysis showed that all constructs were expressed at similar levels
(data not shown). Deletion of the C-terminal domain of SHARP-1 (to give pMT G4 NbHO-SHARP-1) slightly relieved repression by SHARP-1, but the
C-terminal domain (residues 174-253) of SHARP-1 fused to Gal4 DBD (pMT
G4 C-SHARP-1) was able to repress transcription as robustly as
full-length SHARP-1. Therefore, it would appear that SHARP-1 must
contain at least two independent repression domains, one in the
C-terminal domain and another in the remaining fragment. To map the
second repression domain of SHARP-1, more deletion mutants were
examined. Deletion of the Orange domain and C-terminal domain to give
pMT G4 NbH-SHARP-1 still gave robust repression, but further deletion
of the bHLH domain to leave only the N-terminal domain (pMT G4
N-SHARP-1) led to relief of most of the repression activity, suggesting
that the bHLH domain also mediates repression. This was confirmed by
analysis of two further constructs. Fusion of the Orange domain and
flanking sequence (residues 103-173) and the Gal4 DBD (pMT G4
O-SHARP-1) showed some degree of repression, but fusion of
the bHLH domain (residues 43-102) and Gal4 DBD (pMT G4 bH-SHARP-1)
indicated that the bulk of repression in this second region was
mediated by the bHLH domain. In summary, we identified two independent
repression domains in SHARP-1, one in the bHLH domain and the other in
the C terminus.
SHARP-1 Represses Transcription through Two Different
Mechanisms--
Recent studies have shown that many transcriptional
repressors exert their action through recruitment of histone
deacetylase activity (see Ref. 26 for review). To test whether SHARP-1
represses transcription through such a mechanism, we treated Neuro 2a
cells with the histone deacetylase inhibitor TSA (27) and examined the
effect on SHARP-1-mediated repression (Fig.
7). For each concentration of TSA used,
expression values of the reporter gene were normalized to those
obtained in the presence of Gal4 DBD alone, and results were expressed
as fold over untreated cells. Repression by full-length SHARP-1 is
partially relieved by TSA, because expression of the reporter gene was
derepressed 4-fold in the presence of 100 nM TSA. Deletion
of the C-terminal domain of SHARP-1 (to give pMT G4 NbHLHO-SHARP-1)
showed that repression mediated by the bHLH domains was much less
sensitive to TSA, resulting in a 1.6-fold derepression by 100 nM TSA. However, repression by the C-terminal domain alone
was relieved by 6.5-fold with 100 nM TSA, and 2-fold derepression could be seen in the presence of 10 nM TSA.
These results show that the C-terminal domain of SHARP-1 represses
transcription via a mechanism that is likely to involve histone
deacetylase activity but that the bHLH domain represses transcription
in a histone deacetylase-independent manner.
SHARP-1 Controls the Levels of M1 Expression--
To
test the functional effect of SHARP-1 on M1 expression,
IMR32, 3T3, and Neuro 2a cells were transfected with a reporter vector
containing the region of the M1 gene between bHLH proteins are key players that regulate many aspects of
development and differentiation in all tissues and phyla. To date, no
target genes or function of SHARP-1 has been identified. SHARP-1 is
unusual in two respects. First, SHARP-1 is related to, but distinct
from, HAIRY/E(spl)/HES bHLH proteins. Second, expression of SHARP-1
appears to be restricted to postnatal neurons of the central nervous
system, rather than neural progenitors, implying a role in late
neuronal differentiation rather than neurogenesis. These features
suggested that SHARP-1 may affect transcriptional regulation and target
promoters distinct from those used by other members of the
HAIRY/E(spl)/HES bHLH family.
SHARP-1 contains an Arg in position 13 of the basic region present in
all class B bHLH proteins. This residue is essential for class B
proteins to bind the consensus sequence CA(C/T)GTG (9). Sequence
analysis of the region between +166 and +417 of the M1 gene
does not indicate the presence of any known recognition consensus motif
(class A, B, or C) for bHLH (8-10), suggesting that SHARP-1 may
recognize a novel binding site. Gel electrophoresis mobility shift
assays have failed to demonstrate an ability of Stra13 to bind to
either an E-box or N-box (21). Because SHARP-1 and SHARP-2/Stra13/DEC1
are all highly conserved within their basic regions or presumptive DNA
binding domain, it is possible that both SHARP-1 and SHARP-2 recognize
a common binding site, distinct from class A, B, or C sites.
By recruiting SHARP-1 to heterologous promoter constructs through the
Gal4 DBD, we have shown that SHARP-1 acts as a transcriptional repressor and furthermore that SHARP-1 is able to repress both basal
and activated transcription (Fig. 5).
It is well documented that arrangement of basal promoter elements can
profoundly influence the response of a promoter to different factors.
The evolutionarily conserved Kruppel-associated box present in the
N-terminal regions of most Kruppel-class zinc finger proteins specifically silences the activity of promoters whose initiation is
dependent on the presence of a TATA box (28), whereas
initiator-containing promoters are relatively unaffected. Similarly,
Oct2 isoforms repress transcription only from TATA-containing
promoters (29). Here, we show that SHARP-1 is more promiscuous and can
repress transcription driven from both TATA-containing and TATAless
promoters (Fig. 5). In addition to promoter type, the relative position of its DNA binding site to the promoter can also influence repressor action. This can be seen in the case of the neuron-restrictive silencer
factor (20, 30), which represses transcription when recruited
distally but can activate transcription when recruited proximally
(31).2 Here again, SHARP-1
appears to act as a more global repressor and is capable of repressing
transcription from a TATA-containing promoter when recruited either
proximally or distally. Nevertheless, repression is more marked when
SHARP-1 is tethered proximal to the TATA box (Fig. 5).
Transcriptional repression can take many forms (32). Several repressors
function by deacetylating the N-terminal tails of histones that are
thought to render chromatin inaccessible to the transcriptional
machinery. Such is the case of the Mad family of bHLH-ZIP
proteins, whose members bind to mSIN3A, an adapter molecule that links
histone deacetylases to DNA-bound transcription factors (33, 34). Other
repressors appear to interact directly with the transcriptional
machinery itself, for example Kruppel, which at high
concentrations homodimerizes and becomes a potent repressor by
interacting directly with TFIIE (35, 36). Others such as retinoblastoma
can interact both with general transcription factors or with
histone deacetylase (37-40). Repression by SHARP-1 appears to be
bimodal. One mode of repression is mediated via the bHLH domain and is
TSA-insensitive, whereas the other mode is mediated by the C-terminal
domain and is TSA-sensitive (probably due to recruitment of
HDAC). Interestingly, Stra13 has been shown to repress
expression of the c-myc gene through an HDAC- independent pathway and to negatively autoregulate its own promoter through an
HDAC-dependent mechanism (41). The region responsible for the HDAC-independent repression of Stra13 has not been mapped, but
glutathione S-transferase pull-down studies showed that
residues 111-343 are required for interaction with HDAC-1, Sin3, and
NcoR (41), whereas functional analysis of mutated Stra13 demonstrated that residues 147-354 were required for Stra13 repression of
VP16-activated transcription. As shown in Fig. 2, this region contains
the C-terminal domain and part of the Orange domain of
SHARP-2/Stra13/DEC1. We have mapped the domain responsible for the
TSA-sensitive component of SHARP-1 repression to the C-terminal domain
(residues 174-253). Within this region, SHARP-1 and Stra13 display
pockets of homology, principally within two stretches of 8 (residues
205-211) and 15 amino acids (residues 221-236), suggesting that these
may mediate the interaction with HDAC. The HDAC-independent repression
domain of SHARP-2/Stra 13/DEC1 has not been reported, but the
GROUCHO-independent mode of repression by HAIRY/E(spl) requires the
bHLH and Orange domains (23). Although SHARP-1 and SHARP-2 are almost
identical in their bHLH domains (95% over 59 residues), they diverge
more markedly from HAIRY (37% over 59 residues). It remains to be seen whether the same domains or mechanisms are involved in mediating HDAC-independent repression by SHARP-1, SHARP-2/Stra13/DEC1, and HAIRY/E(spl) proteins.
Bimodal repression is not uncommon. Repression by the
neuron-restrictive silencer factor (20, 30) is mediated by an
N-terminal domain that recruits Sin3/HDAC-1 (15, 42-44) and a
C-terminal domain that recruits a novel corepressor
Co-REST (45). Repression by retinoblastoma is also
mediated via an HDAC-1-dependent and HDAC-1-independent
mechanism (37, 38-40). In the latter case, some promoters such as the
adenovirus major late promoter are repressed by the
HDAC-1-dependent arm, whereas transcription activated by
the SV40 enhancer or driven by the thymidine kinase promoter is
repressed by the HDAC-insensitive arm. It may be that the breadth and
selectivity of repressor action of bimodal repressors such as SHARP-1
is enhanced by their ability to bring one or both repression mechanisms
to bear upon different promoters.
Although a function for SHARP-1 is unknown, its presence in terminally
differentiated neurons suggests that it may be involved in regulating
gene expression in differentiated neurons. Consistent with this
hypothesis is our finding that SHARP-1 can bind to elements within the
5' untranslated region of the M1 muscarinic receptor gene
(Fig. 1) (12, 18). To date, the gene structures for three of the family
of muscarinic receptor genes have been identified: the rat
M4 (47, 48), the chicken M2 (49), and the rat
M1 genes (12). Although the expression patterns of the
M1 and M4 genes are very similar, transcription
of the two genes appears to be controlled by different mechanisms.
Previously, we and others have shown that the M4 gene
contains a constitutively active core promoter that is silenced, at
least in non-neuronal cells, by the transcriptional repressor
REST/neuron-restrictive silencer factor (50, 51). On the other
hand, we have previously shown that transcription of the M1
gene is controlled by several elements within the 5' untranslated
region acting both in a positive manner and in a negative manner (18).
Within this fragment, we have identified a polypyrimidine/polypurine
tract (from +412 to +485) and a conserved region across species (from
+504 to +602) that act in concert to enhance expression in
M1-expressing cells (18). Interestingly, this gene is
expressed by differentiated neurons, and its expression increases in
the first postnatal weeks (46, 52) with an ontogenic profile similar to
SHARP-1 (11). We show that overexpression of SHARP-1 represses
expression of a reporter gene driven by the M1 promoter in
M1-expressing cells, suggesting that SHARP-1 can modulate
levels of M1 expression. By advancing our knowledge
of the molecular mechanisms employed by SHARP-1 and identifying a
potential target gene, these studies provide a platform for
understanding the role of SHARP-1 in regulating neuronal gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
His/
Leu/
Trp (Bio 101, Vista, CA), containing 5 mM 3-amino-1,2,4-triazole (Sigma) to select for
interactions. Library candidates were tested for their ability to
specifically activate the M1-containing reporter plasmid by
retransforming back into SFY526. Library plasmids producing interacting
proteins were sequenced for identification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
SHARP-1 binds to the M1 promoter.
a, an adult rat brain cDNA library was screened
using the region of the M1 gene between +166 and +417 as
bait and the yeast strain SFY526. Candidate clones were retransformed
into SFY526-pBM2389 +417/+166 M1 and plated onto
Trp/
His/
Leu plus 10 mM 3-amino-1,2,4-triazole
(3AT). Two positive clones were isolated (number 3 and 10),
which failed to grow with either empty reporter vector, pBM2389, or
with empty expression vector, pGAD10, showing that growth was dependent
upon the interaction of the expressed library protein with the
M1 bait. b, digestion of the isolated library
clones with BglII showed that both clones contain a cDNA
of 1.5 kDa. Sequencing of the two clones showed that they both
encompass the entire SHARP-1 open reading frame and also 130 base pairs
of the 5' untranslated region.
View larger version (62K):
[in a new window]
Fig. 2.
SHARP-1 is closely related to
SHARP-2/Stra13/DEC1. Shown is a sequence alignment of
SHARP-1 with other bHLH proteins identified in a data base search for
proteins related to SHARP-1. SHARP-2 (11), Stra13 (21), and DEC1 (22)
share 89% sequence identity between them and appear to be homologues
derived from rat, mouse, and human, respectively. SHARP-1 is highly
conserved within the bHLH domain with SHARP-2/Stra13/DEC1 (95%
sequence identity) and within helix III and IV, also called Orange
domain (60% sequence identity). Outside of these domains, SHARP-1
diverges from the other three proteins, apart from two stretches of 8 and 15 amino acids, respectively, within the C-terminal domain.
View larger version (27K):
[in a new window]
Fig. 3.
SHARP-1 homodimerizes. Neuro2a cells
were transfected with either myc-tagged Gal4DBD (pMT G4) and
myc-tagged SHARP-1 (pMT SHARP-1) (lane 1) or
myc-tagged Gal4 SHARP-1 (pMT G4 SHARP-1) and
myc-tagged SHARP-1 (pMT SHARP-1) (lane 2). Cells
were harvested after 2 days, and cell extracts were immunoprecipitated
with Gal4 DBD antibody and analyzed by Western blotting with
c-myc antibody (Ab). SHARP-1 was not
immunoprecipitated in the presence of the Gal4 DBD (lane 3),
but G4-SHARP-1 was immunoprecipitated (lane 4), showing
that SHARP-1 is able to homodimerize. IP,
immunoprecipitation.
View larger version (94K):
[in a new window]
Fig. 4.
Expression of SHARP-1 in different cell
lines. a, PCR was performed using a rat SHARP-1 sense primer
within the basic domain (SHARP-1.125s) and an antisense primer within
helix 3 (SHARP-1.441a) on cDNA derived from IMR32 (lane
1), 3T3 (lane 2), Neuro 2a (lane 3), NB4 1A3
(lane 4), rat cerebellum (lane 5), and mouse
cerebellum (lane 6). Lane 7 shows the water
negative control, and the two outer lines correspond to a
1-kilobase+ DNA marker ladder (Life Technologies, Inc). PCR
products were electrophoresed through a 2% MetaPhor agarose gel.
b, PCR was performed on the same cDNA samples as above,
using primers for hypoxanthine-guanine phosphoribosyl transferase gene
hprt 231 s and hprt 567a to verify that
equivalent amounts of cDNA had been assayed.
View larger version (24K):
[in a new window]
Fig. 5.
SHARP-1 acts as transcriptional
repressor. IMR32, 3T3, and Neuro 2a cells were transfected with
vectors expressing Gal4 DBD alone or fused to SHARP-1 together with or
without a vector expressing Tet-VP16 and the reporter plasmid shown in
each figure. Equal amounts of Gal4 DBD fusion, Tet-VP16/stuffer, and
reporter plasmids were used. Values are the averages of three
independent transfections, each performed in triplicate, normalized to
cotransfected CMV-Renilla, and expressed as a percentage of the values
obtained with Gal4 alone for each cell line in the absence or presence
of Tet-VP16 (open bars). a shows repression by
proximal recruitment of SHARP-1 to a TATA-containing promoter, whereas
b shows the effect when distally recruited. c
shows repression of a TATA-less promoter. In each case the left
hand panel shows repression of basal promoter activity, whereas
the right panel shows repression of VP16-activated
transcription.
View larger version (26K):
[in a new window]
Fig. 6.
SHARP-1 represses transcription through two
independent domains. Neuro 2a cells were transfected with vectors
expressing Gal4 DBD alone or fused to different deletion mutants of
SHARP-1 along with a reporter gene driven by a TATA-containing
promoter with five Gal4 binding sites proximal to the transcription
start site (pTRE UAS TATA). Equal amounts of Gal4 DBD fusion and
reporter plasmids were used. Values are the averages of three
independent transfections, each performed in triplicate, normalized to
cotransfected CMV-Renilla, and expressed as a percentage of the values
obtained with Gal4 alone for each cell line.
View larger version (28K):
[in a new window]
Fig. 7.
Repression by SHARP-1 is through two
independent mechanisms. Neuro 2a cells were incubated with 1, 50, or 100 nM TSA for 24 h before transfection. Cells were
transfected with vectors expressing Gal4 DBD alone or fused to
different deletion mutants of SHARP-1 along with a reporter vector
containing five Gal4 DBDs upstream of a TATA-containing promoter. Equal
amounts of Gal4 DBD fusion and reporter plasmids were used. Transfected
cells were fed with medium containing the indicated
concentration of TSA for 24 h before harvesting. Values are the
averages of three independent transfections, each performed in
triplicate and normalized to cotransfected CMV-Renilla. Results are
normalized to expression with Gal4 alone and expressed as fold over
cells untreated with TSA.
372 and +602 (relative to the transcription start site). This construct has been
shown before to be capable of driving expression in IMR32 cells, a
neuronal cell line that expresses the M1 gene (18). The
same construct does not drive expression in the
non-M1-expressing neuronal cell line Neuro 2a and drives
only low levels of expression in 3T3 cells (18). Overexpression of
SHARP-1 (pSHARP-1 myc) had no effect on expression of the
promoterless reporter vector pGL3 basic but reduced expression driven
by the M1 promoter by 55% in IMR32 cells (Fig.
8a). A similar effect was seen
in 3T3 cells (Fig. 8b). No effect was seen in Neuro 2a, the
neuronal cell line that does not express M1 (data not
shown). These results show that SHARP-1 is able to repress expression
of the M1 gene in an M1-expressing cell
line.
View larger version (18K):
[in a new window]
Fig. 8.
SHARP-1 represses M1
expression. IMR32 cells (a) and 3T3 cells
(b) were cotransfected with a reporter construct containing
the region of the M1 gene between 372 and +602 and either
pMT (open bars) or a vector expressing SHARP-1 (shaded
bars). Reporter and expression vectors were in a ratio of 1:5.
Values are the averages of three independent transfections, each
performed in triplicate, normalized to cotransfected CMV-Renilla, and
expressed as fold over luciferase activity obtained with the empty
reporter vector, pGL3 basic cotransfected with empty expression vector,
pMT.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Dr. Ian C. Wood for helpful comments and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: School of
Biochemistry and Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Tel.: 44 113 233 3020; Fax:
44 113 233 3019; E-mail: n.j.buckley@leeds.ac.uk.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M011619200
2 A. Roopra and N. J. Buckley, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: bHLH, basic helix-loop-helix; E(spl), Enhancer of Split; HES, hairy and Enhancer of Split; DBD, DNA binding domain; TSA, trichostatin A; PCR, polymerase chain reaction; hprt, hypoxanthine-guanine phosphoribosyl transferase; TRE, Tet-responsive element; HDAC, histone deacetylase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kageyama, R., Ishibashi, M., Takebayashi, K., and Tomita, K. (1997) Int. J. Biochem. Cell Biol. 29, 1389-1399[CrossRef][Medline] [Order article via Infotrieve] |
2. | Brunet, J. F., and Ghysen, A. (1999) Bioessays 21, 313-318[CrossRef][Medline] [Order article via Infotrieve] |
3. | Anderson, D. J. (1999) Curr. Opin. Neurobiol. 9, 517-524[CrossRef][Medline] [Order article via Infotrieve] |
4. | Campos-Ortega, J. A. (1995) Mol. Neurobiol. 10, 75-89[Medline] [Order article via Infotrieve] |
5. |
Fisher, A. L.,
and Caudy, M.
(1998)
Genes Dev.
12,
1931-1940 |
6. | Campuzano, S., and Modolell, J. (1992) Trends Genet. 8, 202-208[Medline] [Order article via Infotrieve] |
7. | Murre, C., McCaw, P. S., and Baltimore, D. (1989) Cell 56, 777-783[Medline] [Order article via Infotrieve] |
8. | Blackwell, T. K., and Weintraub, H. (1990) Science 250, 1104-1110[Medline] [Order article via Infotrieve] |
9. | Dang, C. V., Dolde, C., Gillison, M. L., and Kato, G. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 599-602[Abstract] |
10. | Ohsako, S., Hyer, J., Panganiban, G., Oliver, I., and Caudy, M. (1994) Genes Dev. 8, 2743-2755[Abstract] |
11. | Rossner, M. J., Dorr, J., Gass, P., Schwab, M. H., and Nave, K. A. (1997) Mol. Cell Neurosci. 10, 460-475[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Pepitoni, S.,
Wood, I. C.,
and Buckley, N. J.
(1997)
J. Biol. Chem.
272,
17112-17117 |
13. | Liu, J., Wilson, T. E., Milbrandt, J., and Johnston, M. (1993) Methods Enzymol. 5, 125-137[CrossRef] |
14. | Turner, E. E., Jenne, K. J., and Rosenfeld, M. G. (1994) Neuron 12, 205-218[Medline] [Order article via Infotrieve] |
15. |
Roopra, A.,
Sharling, L.,
Wood, I. C.,
Briggs, T.,
Bachfischer, U.,
Paquette, A. J.,
and Buckley, N. J.
(2000)
Mol. Cell. Biol.
20,
2147-2157 |
16. | Bartel, P. L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993) in Cellular Interactions in Development: A Practical Approach (Hartley, D. A., ed) , pp. 153-179, Oxford University Press, Oxford |
17. | Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339-346[Medline] [Order article via Infotrieve] |
18. | Wood, I. C., Garriga-Canut, M., Palmer, C. L., Pepitoni, S., and Buckley, N. J. (1999) Biochem. J. 340, 475-483[CrossRef][Medline] [Order article via Infotrieve] |
19. | Wang, R., and Reed, R. (1993) Nature 364, 121-126[CrossRef][Medline] [Order article via Infotrieve] |
20. | Chong, J. A., Tapia-Ramirez, J., Kim, S., Toledo-Arai, J. J., Zheng, Y., Boutros, M. C., Altshuller, Y. M., Frohman, M. A., Kraner, S. D., and Mandel, G. (1995) Cell 80, 949-957[Medline] [Order article via Infotrieve] |
21. |
Boudjelal, M.,
Taneja, R.,
Matsubara, S.,
Bouillet, P.,
Dolle, P.,
and Chambon, P.
(1997)
Genes Dev.
11,
2052-2065 |
22. | Shen, M., Kawamoto, T., Yan, W., Nakamasu, K., Tamagami, M., Koyano, Y., Noshiro, M., and Kato, Y. (1997) Biochem. Biophys. Res. Commun. 236, 294-298[CrossRef][Medline] [Order article via Infotrieve] |
23. | Dawson, S. R., Turner, D. L., Weintraub, H., and Parkhurst, S. M. (1995) Mol. Cell. Biol. 15, 6923-6931[Abstract] |
24. |
Giebel, B.,
and Campos-Ortega, J. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6250-6254 |
25. | Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, L. Y., Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D., and Lassar, A. B. (1989) Cell 58, 537-544[Medline] [Order article via Infotrieve] |
26. | Ayer, D. E. (1999) Trends Cell Biol. 9, 193-198[CrossRef][Medline] [Order article via Infotrieve] |
27. | Yoshida, M., Horinouchi, S., and Beppu, T. (1995) Bioessays 17, 423-430[Medline] [Order article via Infotrieve] |
28. |
Pengue, G.,
and Lania, L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1015-1020 |
29. |
Liu, Y.-Z.,
Dawson, S. J.,
Gerstar, T.,
Friedl, E.,
Pengue, G.,
Matthias, P.,
Lania, L.,
and Latchman, D. S.
(1996)
J. Biol. Chem.
271,
20853-20860 |
30. | Schoenherr, C. J., and Anderson, D. J. (1995) Science 267, 1360-1363[Medline] [Order article via Infotrieve] |
31. |
Bessis, A.,
Champtiaux, N.,
Chatelin, L.,
and Changeux, J. P.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5906-5911 |
32. | Maldonado, E., Hampsey, M., and Reinberg, D. (1999) Cell 99, 455-458[Medline] [Order article via Infotrieve] |
33. | Ayer, D. E., Lawrence, Q. A., and Eisenman, R. N. (1995) Cell 80, 767-776[Medline] [Order article via Infotrieve] |
34. | Laherty, C. D., Yang, W. M., Sun, J. M., Davie, J. R., Seto, E., and Eisenman, R. N. (1997) Cell 89, 349-356[Medline] [Order article via Infotrieve] |
35. | Sauer, F., and Jackle, H. (1993) Nature 364, 454-457[CrossRef][Medline] [Order article via Infotrieve] |
36. | Sauer, F., Fondell, J. D., Ohkuma, Y., Roeder, R. G., and Jackle, H. (1995) Nature 375, 162-164[CrossRef][Medline] [Order article via Infotrieve] |
37. | Brehm, A., Miska, E. A., McCance, D. J., Reid, J. L., Bannister, A. J., and Kouzarides, T. (1998) Nature 391, 597-601[CrossRef][Medline] [Order article via Infotrieve] |
38. | Luo, R. X., Postigo, A. A., and Dean, D. C. (1998) Cell 92, 463-473[Medline] [Order article via Infotrieve] |
39. | Magnaghi-Jaulin, L., Groisman, R., Naguibneva, I., Robin, P., Lorain, S., Le Villain, J. P., Troalen, F., Trouche, D., and Harel-Bellan, A. (1998) Nature 391, 601-605[CrossRef][Medline] [Order article via Infotrieve] |
40. | Weintraub, S. J., Chow, K. N., Luo, R. X., Zhang, S. H., He, S., and Dean, D. C. (1995) Nature 375, 812-815[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Sun, H.,
and Taneja, R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4058-4063 |
42. |
Grimes, J. A.,
Nielsen, S. J.,
Battaglioli, E.,
Miska, E. A.,
Speh, J. C.,
Berry, D. L.,
Atouf, F.,
Holdener, B. C.,
Mandel, G.,
and Kouzarides, T.
(2000)
J. Biol. Chem.
275,
9461-9467 |
43. | Huang, Y., Myers, S. J., and Dingledine, R. (1999) Nat. Neurosci. 2, 867-872[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Naruse, Y.,
Aoki, T.,
Kojima, T.,
and Mori, N.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13691-13696 |
45. |
Andres, M. E.,
Burger, C.,
Peral-Rubio, M. J.,
Battaglioli, E.,
Anderson, M. E.,
Grimes, J.,
Dallman, J.,
Ballas, N.,
and Mandel, G.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9873-9878 |
46. | Hohmann, C. F., Potter, E. D., and Levey, A. I. (1995) J. Comp. Neurol. 358, 88-101[Medline] [Order article via Infotrieve] |
47. |
Wood, I. C.,
Roopra, A.,
Harrington, C.,
and Buckley, N. J.
(1995)
J. Biol. Chem.
270,
30933-30940 |
48. |
Mieda, M.,
Haga, T.,
and Saffen, D. W.
(1996)
J. Biol. Chem.
271,
5177-5182 |
49. |
Rosoff, M. L.,
Wei, J.,
and Nathanson, N. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14889-14894 |
50. |
Mieda, M.,
Haga, T.,
and Saffen, D. W.
(1997)
J. Biol. Chem.
272,
5854-5860 |
51. |
Wood, I. C.,
Roopra, A.,
and Buckley, N. J.
(1996)
J. Biol. Chem.
271,
14221-14225 |
52. | Wall, S. J., Yasuda, R. P., Li, M., Ciesla, W., and Wolfe, B. B. (1992) Brain Res. Dev. Brain Res. 66, 181-185[Medline] [Order article via Infotrieve] |