Repressor Element Silencing Transcription
Factor/Neuron-restrictive Silencing Factor (REST/NRSF) Can Act
as an Enhancer as Well as a Repressor of Corticotropin-releasing
Hormone Gene Transcription*
Kim A.
Seth
§ and
Joseph A.
Majzoub§¶
From the
Program in Neuroscience, Harvard Medical
School, and the § Division of Endocrinology, Children's
Hospital, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, August 24, 2000, and in revised form, January 11, 2001
 |
ABSTRACT |
The repressor element-1/neuron-restrictive
silencing element (RE-1/NRSE) mediates transcriptional repression by
the repressor element silencing transcription factor/neuron-restrictive
silencing factor (REST/NRSF) in many neuron-specific genes. REST/NRSF
is expressed most highly in non-neural tissues, where it is thought to
repress gene transcription, but is also found in developing neurons and
at low levels in the brain. Its null mutation in vivo results in embryonic lethality in mice. While the RE-1/NRSE-mediated repressive influence of REST/NRSF is well established, results in
transgenic studies have suggested that the action of the system is more
complex. Here, we report that transcription of the corticotropin releasing hormone (CRH) gene is regulated by REST/NRSF, in part through
the RE-1/NRSE. Expression of transfected Crh-luciferase constructs was down-regulated by REST/NRSF in a
RE-1/NRSE-dependent fashion in both muscle-derived L6 and
REST/NRSF co-transfected neuronal PC12 cells. Treatment of L6 cells
with trichostatin A revealed that REST/NRSF repression depends,
in part, on histone deacetylase activity in these cells. In another
neuronal cell line, NG108, REST/NRSF also repressed expression from
constructs containing an intact RE-1/NRSE. However, unexpectedly,
REST/NRSF up-regulated expression levels of constructs lacking an
intact RE-1/NRSE. These results suggest that REST/NRSF can act as both a repressor of Crh transcription, via the Crh
RE-1/NRSE, and an enhancer of Crh transcription, via a
mechanism independent of the Crh RE-1/NRSE.
 |
INTRODUCTION |
The 21-base pair (bp)1
repressor element -1, also called the neuron-restrictive silencing
element, (RE-1/NRSE), has been identified in over 30 genes, most of
which are expressed in neurons. The repressor element silencing
transcription factor (REST/NRSF) has been shown in a variety of genetic
contexts to repress transcriptional activity via binding to this
element (1-18). It was originally thought that the REST/NRSF-RE-1/NRSE
system served as a molecular switch that helped distinguish neural from
non-neural cell types, as the repression was thought to occur in
non-neural cells, which contain REST/NRSF, but not in neural cells,
which either lack or contain only relatively low levels of REST/NRSF
(6, 10, 13, 16). It has since become clear that REST/NRSF-RE-1/NRSE function is more complex. For example, multiple splice variants of
REST/NRSF have been identified, including at least one neural-specific form (19). Additionally, several groups have suggested that REST/NRSF
and/or the RE-1/NRSE may serve a dual function, as either repressor or
activator, depending on the spatial and temporal context of its
expression (1, 2, 19, 20).
REST/NRSF is thought to contain two main repressor domains (21).
Recently, several reports have suggested that REST/NRSF can repress, in
part, through association of a domain in its N-terminal with the
Sin3A-histone deacetylase 2 (HDAC2) complex (22-24).
Sin3A·HDAC2 is one of several histone deacetylase complexes thought
to down-regulate gene expression. Histone deacetylation is thought to
lead to a "closed" conformation of the chromatin surrounding a
gene, thereby restricting access of the RNA polymerase II complex and,
thus, repressing transcription (25, 26). Using trichostatin A (TSA), a
HDAC-specific inhibitor that has been shown to disrupt histone deacetylation activity in cell cultures (27), several groups have
demonstrated the importance of HDAC activity to REST/NRSF's repressive
function (22-24, 28). The nuclear receptor corepressor (N-CoR), which
can interact with Sin3A and other HDACs, including HDAC 3, 4, 5, and 7, has also been shown to mediate REST/NRSF repression in certain genetic
contexts (29). REST/NRSF's C-terminal zinc finger has been shown to
associate with at least one other factor, CoREST, to mediate a histone
deacetylase-independent repressor activity (22, 23, 30).
In this study, we evaluate the possible function of the
REST/NRSF-RE-1/NRSE system in the transcriptional regulation of the corticotropin releasing hormone (CRH) gene. CRH is a 41-amino acid
hypothalamic neuropeptide that plays a central, well characterized role
in development and homeostatic maintenance, modulating the hypothalamic-pituitary-adrenal axis in response to stress
(31-33). In this role, secreted CRH stimulates the production of
adrenocorticotropic hormone (ACTH) in the anterior pituitary gland,
which, in turn, stimulates the production of glucocorticoids in the
adrenal cortex. Glucocorticoids mediate a range of physiological
responses to stress (34). Abnormalities of CRH expression or function
have been suggested to play a role in many disorders, including Cushing disease, depression, and anorexia nervosa (35-37). It also may be
involved in modulating immune responses (38).
The RE-1/NRSE sequence found in the first intron of the CRH gene
corresponds very closely to the RE-1/NRSE element previously shown to
mediate transcriptional repression in other genes (14). Finding that
the RE-1/NRSE mediates an important regulatory function for
Crh transcription would be particularly interesting, as most in vitro studies to date on Crh transcriptional
regulation have relied on constructs of the Crh promoter
lacking the intron and, therefore, lacking the RE-1/NRSE element
(39-44).
In order to examine this possible function, we first show that
REST/NRSF binds to the Crh RE-1/NRSE and that this binding is both specific and saturable. We then demonstrate that
transcriptional repression of the CRH gene by REST/NRSF requires an
intact RE-1/NRSE, both in non-neuronal L6 rat myoblast cells, as well
as in the neuronal PC12 rat pheochromocytoma and NG108-15 rat
neuroblastoma-glioma cell types. Consistent with previous reports, we
show that the Crh transcriptional repression observed in L6
cells is TSA-sensitive, and therefore likely to be linked to HDAC
activity, but that this mechanism does not account for the full degree
of repression (22-24, 30). We also report the novel finding that
REST/NRSF can function as a RE-1/NRSE-independent enhancer of
Crh transcription in NG108 cells. Together, our results
suggest that REST/NRSF-RE-1/NRSE activity is quite complex, and both
genetic and cellular contexts appear to be critical for determining the
net effect on Crh transcriptional activity.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Rat myoblast L6 cells (American Type Culture
Collection (ATCC), Rockville, MD) were maintained in Dulbecco's
modified Eagle's medium (Sigma) containing 10% fetal calf serum (Life
Technologies, Inc.), and 1 mM sodium pyruvate (Sigma), rat
PC12 cells (kind gift of M. Greenberg, Harvard Medical School and
Children's Hospital, Boston, MA) in Dulbecco's modified Eagle's
medium containing 10% heat-inactivated horse serum (Life Technologies,
Inc.), 5% fetal calf serum, and 1% filter-sterilized
L-glutamine (Sigma), and rat NG108 cells (kind gift of Ian
C. Wood, Wellcome Laboratory for Molecular Pharmacology, University
College London and School of Biochemistry and Molecular Biology,
University of Leeds, United Kingdom) in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum. All cell cultures also
contained 1% penicillin/streptomycin/amphotericin B (Life
Technologies, Inc.). PC12 cells were grown on poly-L-lysine (Sigma)-treated plates and were passaged a maximum of five times before
discarding. Each experiment was conducted using cells of the same passage.
RT-PCR--
Total RNA was prepared from cultured L6 cells,
PC12 cells treated with 50 ng/ml NGF (Sigma) for 48 h, and
NG108-15 cells treated with 10 µM forskolin, 100 µM 3-isobutylmethylxanthine (IBMX) (Sigma) for 6 h,
using TRI Reagent (Sigma) according to manufacturer's protocol. The
recovery of RNA was quantified spectrophotometrically. RNA was digested
using DNA-freeTM (Ambion), according to the
manufacturer's protocol, to eliminate contaminating endogenous DNA.
Single-stranded cDNA was synthesized using Superscript
IITM reverse transcriptase (RT) (Life Technologies, Inc.)
according to the manufacturer's protocol, using random hexamers
[(pd(N)6] (Roche Molecular Biochemicals) as primers.
Following an initial 3 min at 96 °C, each PCR cycle (MJ Research)
consisted of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a 5-min extension at 72 °C. PCR was also conducted
on identical samples lacking RT treatment, to control for any remaining
endogenous DNA contamination. PCR reactions were performed on 1/10 of
the RT reaction, in 1 × PCR buffer (Roche Molecular Biochemicals) using 2 units of Taq enzyme (Roche Molecular Biochemicals),
0.4 mM dNTP, and 0.2 µM each primer. The PCR
products were separated and visualized in an ethidium bromide
containing 1.5% TBE-agarose gel, using a 100-bp DNA ladder (Roche
Molecular Biochemicals). The sense (S) and antisense (AS)
REST/NRSF-specific and actin-specific (control) primers used were as
follows: rNRSF-S, GACAGGTTCACAACGGGCC; rNRSF-AS, CCCTTCGGCACTTCGCCGCT;
actin-S, ATTCCTATGTGGGCGACGAG; and actin-AS, TGGATAGCAACGTACATGGC (45).
The primers, rNRSF-S and rNRSF-AS, were intron-spanning, such that
RT-PCR utilization of mRNA versus endogenous DNA
templates was distinguishable. The primers were specific for
full-length REST/NRSF, as distinguished from splice variants such as REST4.
Electrophoretic Mobility Shift Assays
(EMSAs)--
Nuclear extracts from L6 cells were prepared according to
Schreiber et al. (46) from ~106 cells per
extract; Buffer A contained a final concentration of 1.0 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotenin, 0.5 µg/ml leupeptin, and 0.7 µg/ml pepstatin, and Buffer C was the same except for phenylmethylsulfonyl fluoride, which was at 1.0 mM. Final nuclear extract protein
concentration was quantitated by spectrophotometry using a standard
curve (BCA Protein Assay Reagent, Pierce). All oligonucleotide probes
used were double stranded. They were synthesized as complementary
single strands (BIOSOURCE/Keystone) which were
then annealed by equimolar mixing, boiling for 5 min and slow cooling
to room temperature. Radiolabeled probe was made by 5' phosphorylation
of 1 pmol of the double-stranded (ds) RE-1/NRSE probe with 50 pmol of
[
-32P]ATP (product number BLU-2A, PerkinElmer Life
Sciences) by the T4 polynucleotide kinase forward reaction lasting 30 min at 37 °C, in a total volume of 50 µl. Labeled RE-1/NRSE probe
was purified by centrifugation through a Sephadex G-50 QuickSpin column
(or G-25 column, for the CRE probe) (Roche Molecular Biochemicals). Final counts for different experiments were in the range of 6 × 104 to 3 × 105 cpm/µl, and between 11 and 14 fmol of labeled probe were used per lane. Both the RE-1/NRSE
probe and the various competitor probes (except for the unrelated CRE
probe) contained EcoRI 5' overhangs for possible use in
subcloning. In addition to the 21-bp core of the RE-1/NRSE, we included
9 bp of 5'-flanking DNA and 7 bp of 3'-flanking DNA; this 37-bp
sequence was doubled in tandem. For the imperfectly conserved potential
RE-1/NRSE (p(RE-1)) sequences, we noted the outside limits of
conservation (i.e. the most 3' and most 5' conserved bases
of the RE-1/NRSE) and counted out both 5' and 3' to include an
equivalent amount of flanking DNA; some of these probes are therefore
longer and some are shorter than the RE-1/NRSE probe. The sequences of
the probes used were as follows (only sense strand shown):
[
-32P]RE-1 probe,
AATTCGTACCTAGCTTCAGCACCGCGGACAGCGTCACCGAAGTACCTAGCTTCAGCACCGCGGACAGAGTCACCGAAG; RE-1MUT (with mutation underlined in bold),
AATTCGTACCTAGCTTCAGCACCGCTTACAGCGTCACCGAAGTACCTAGCTTCAGCACCGCTTACAGAGTCACCGAAG; p(RE-1)A,
AATTCGAGCAGAGGCAGCACGCAATCGAGCTGTCAAGAGAGCGTCAGCTTAGAGCAGAGGCAGCACGCAATCGAGCTGTCAAGAGAGCGTCAGCTTAG; p(RE-1)B,
AATTCGCTGCTGTGGTGAGCCCCGGAGCCAGCTGCCCATGTGCTGCTGTGGTGAGCCCCGGAGCCAGCTGCCCATGTG; p(RE-1)C,
AATTCTGCAGATGCCTCAGCGCTCGCTCGACAGCCGCGCGGAGTGCAGATGCCTCAGCGCTCGCTCGACAGCCGCGCGGAG; p(RE-1)D,
AATTCTGTATTTCTGTGTCGTAACAAAACAGCGTTATTTGTTGTATTTCTGTGTCGTAACAAAACAGCGTTATTTGTG; Crh CRE, GCTCGTTGACGTCACCAAGA. Binding buffer (1 ×) was: 20 mM HEPES (pH 8), 0.1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 2.5 mM MgCl2,
250 mM KCl. Poly(dI-dC) (Sigma) was used in binding reaction at a concentration of 0.125 µg/ml. Polyclonal REST antibody (kind gift of Gail Mandel, Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, State University of New York, Stony
Brook, NY) was used at a 1:100 dilution, ~416 ng/reaction. The
control cAMP-response element-binding protein antibody is described in Waeber et al. (47). Binding buffer,
poly(dI-dC), and double-distilled (dd) H2O were combined
with nuclear extracts in 1.5-ml Eppendorf tubes and put on ice for 5 min. Competitor oligonucleotides and/or antibodies were added, and the
tubes were placed at 4 °C for 10 min.
-32P-Labeled
RE-1/NRSE probe was then added and the reaction was incubated for 15 min at 4 °C. The reactions were then loaded onto a 5%
polyacrylamide gel (Proto-Gel, Minneapolis, MN) and run at 175 mV for
roughly 3 h at 4 °C. The gels were then vacuum-heat dried and
bands were visualized by exposure to autoradiographic film (Kodak) for
3 to 6 h at
80 °C.
Plasmids--
All primary reporter plasmids were cloned
into the pXP2 luciferase backbone (ATTC) (48). All cloning
transformations were performed using Subcloning Efficiency DH5
cells
according to manufacturer's protocol (Life Technologies, Inc.). Source
constructs for Crh sequence were: CRH5.0, containing 5 kb of
5' promoter sequence (B57 mouse) cloned into Bluescript
(SKII+) (Stratagene);
Xho-Kpn-Kpn, containing the
full-length CRH gene plus ~6-kb flanking sequence (B57 mouse) (49).
Full-length constructs (Intron(+)WT,
Intron(+)MUT) were made as follows: we excised a fragment
containing roughly 5 kb of 5' Crh sequence from CRH 5.0 by
cutting at a BamHI site 5 kb upstream from the promoter and at the unique SalI site at position 409 in the promoter. A
3' contiguous 712-bp SalI-KpnI fragment was then
excised from the Xho-Kpn-Kpn
construct, cutting at the same SalI site and a
KpnI site at position 1121. A third contiguous fragment was
created by using forward primer starting at (5' position 1097)
CGCACCAGTTGGAGCTTTGCAGGT (3'), and reverse primer, (5' position 1292)
TTTTTTTTAGATCTTAGGGGCGCTCTCTGAAGC (3'), to amplify the sequence
flanking the KpnI (1117) site (see underlined, bold below)
and up to the translational start site (1292) by PCR (same reagents as
above, except using 30 pmol of each primer on 616 ng of template DNA),
starting with 96 °C for 2 min and then 24 cycles of: 60 °C, 1 min; 72 °C, 2 min; 96 °C, 30 s; followed by a final
extension at 72 °C for 4 min. Another forward primer was used to
introduce the GG/TT mutation (italics, bold) in the RE-1/NRSE at
position 1138-39: (5' position 1110) CTTTGCAGG TAC
CTAGCTTCAGCACCGCTTACAGCGTCACCG (3'). We attached a BglII site at the 5' end of the reverse
primer, with 8 Ts extending to allow for eventual cutting by
BglII and cloning into the BglII site of pXP2.
The resulting PCR fragments were digested with KpnI and
BglII. We then performed a double-ligation of the 712-bp
SalI-Kpn and 171-bp
KpnI-BglII fragments into
SalI/BglII-digested pXP2. The process was
repeated, substituting the GG/TT KpnI-BglII fragment. Finally, the resulting two vectors were digested with BamHI and SalI to allow for insertion of the 5-kb
BamHI-SalI fragment to result in the final
full-length constructs, Intron(+)WT and Intron(+)MUT. Intron-less constructs were made by cutting
the Intron(+)WT construct at the SbfI site at
position 541 and the BglII site at position 1310 to excise
the intron. Forward and reverse strands of a linker containing the
Crh RE-1/NRSE (including flanking sequence, as used in the
EMSAs) element and containing a 5' SbfI half-site were
synthesized (BIOSOURCE/Keystone): (forward) GGAGGCATCCTGAGATCTGTACCTAGCTTCAGCACCGCGGACAGCGTCACCGAA; (reverse)
GATCTTCGGTGACGCTGTCCGCGGTGCTGAAGCTAGGTACAGATCTCAGGATGCCTCCTGCA. As before, the strands were combined, boiled, and slow-annealed (see
EMSA methods). The resulting linker thus contained a Crh RE-1/NRSE + flanker DNA cassette (in italics), preceded by a
BglII site (5') and followed by a BglII half-site
(3'). The complete SbfI-BglII linker was cloned into the
SbfI/BglII-digested Intron(+)WT construct (intron thus removed), thus replacing the intron with the
SbfI-BglII, RE-1/NRSE-containing, linker to make
the Intron(
)RE1(+) construct. We made the Intron(
)RE1(
) construct
by digesting this new vector with BglII to excise the
RE-1/NRSE element, and then religating. The
Intron(
)RE1MUT construct was created by following the
same procedure used to make Intron(
)RE1(+), simply substituting a
GG/TT mutated linker for the one detailed above. All constructs were
purified using well established miniprep (50) or maxiprep (Qiagen)
procedures and were sequenced across the full promoter and transcribed
regions, including all cloning junctions, to verify correct sequence.
DNA fragments were isolated from agarose gels using the Qiaex II and
QIAquick gel extraction kits (Qiagen).
Luciferase Assays--
We transfected all cell lines in 24-well
plates, using FuGene6 reagent according to the manufacturer's
instructions (Roche Molecular Biochemicals). FuGene:DNA ratio was kept
at 3:1 in all experiments. 48 h after transfection, cells were
lysed using 1 × Passive Lysis Buffer (Promega). We controlled for
total DNA in all co-transfection experiments, replacing REEX-1 with an
empty vector, and also controlled for transfection efficiency by
co-transfecting a second, thymidine kinase- or
cytomegalovirus-driven, reporter in all experiments. Null vector (pXP2)
values were subtracted from all measurements. In some experiments, 100 nM TSA was applied for 24 h prior to lysis. Each well
in the L6 transfections contained 0.5 µg of pRL-TK (Promega) with 0.5 µg of firefly reporter vector (full-length constructs) or 0.2 µg of
pRL-TK with 0.2 µg of firefly reporter (intron-less
constructs). Signal measurement was done using the Dual Luciferase
Assay kit, according to manufacturer's instructions (Promega). PC12
and NG108 transfections were also conducted in 24-well plates. PC12
cells were transfected with 10 ng of cytomegalovirus-CAT (Invitrogen)
and 0.2 µg of firefly reporter, along with 0.2 µg of either
pcDNA1.1/amp (Invitrogen) or REEX-1 (REST expression cassette in
pcDNA1/amp backbone, kindly provided by Gail Mandel). NGF was
applied to PC12 cells at a final concentration of 50 ng/ml, for 48 h prior to lysis by 1 × Passive Lysis Buffer. CAT assays were
performed using the Promega kit, according to manufacturer's protocol
(14C-labeled chloramphenicol from PerkinElmer Life
Sciences). NG108 cells were transfected identically to the PC12 cells,
both initially using CAT and then using
-galactosidase, to normalize
transfection efficiency. Results reported herein used
-galactosidase, but no difference was seen when CAT was used. In
individual experiments, we used either 0.1 g or 0.01 µg of
cytomegalovirus-
-galactosidase (CLONTECH). After
confirming that results were identical to using pcDNA1.1/amp, in
some experiments we used pBluescript to equalize total DNA in all
wells. NG108 cells were treated with 10 µM
forskolin, 100 µM IBMX for 6 h prior to lysis
by 1 × Passive Lysis Buffer.
-Galactosidase was measured
according to manufacturer's instructions, using the Luminescent
-galactosidase Detection Kit II (CLONTECH). All
firefly luciferase, Renilla luciferase, and
-galactosidase readouts
were conducted on an signal integrating Berthold LB 9501 luminometer.
Data Analysis--
Luciferase expression data are displayed as
the means (after normalization to CAT or
-galactosidase expression),
with standard errors shown in the positive direction. Statistical
significance was evaluated using the Student's t test, with
the Bon Feronni correction for multiple comparisons, where appropriate.
 |
RESULTS |
A Highly Conserved Intronic Element Sharing Close Similarity to an
Established Regulatory Element--
The RE-1/NRSE noted by Shoenherr
et al. (14) in the first intron of the CRH gene shares
considerable similarity, varying by only one nucleotide in human, to a
consensus sequence derived from the comparison of RE-1/NRSE elements
found in 19 different genes, many of which have been shown to be
regulated by REST/NRSF in vitro (14). The RE-1/NRSE element
is also highly conserved among the CRH genes of several species,
including mouse, the focus of the present study (Fig.
1). In all mammalian species, the
Crh RE-1/NRSE is located in the gene's single intron
(position 1127 in mouse), ~140 bp upstream from the beginning of the
second exon, which encodes prepro-CRH. Moreover, the 21-bp element is
more highly conserved than the rest of the intron, as well. Outside of
the RE-1/NRSE, the Crh intron is roughly 75% conserved
(90% rat/mouse; ~73% human/rat, mouse; 76% human/sheep; ~67%
sheep/rat, mouse). The RE-1/NRSE element is 90-95% identical among
all species examined to date, including Xenopus (14). This
high degree of conservation, combined with the non-coding, intronic
position of the element suggests that it might mediate an important
regulatory function in Crh transcription.

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Fig. 1.
The murine Crh RE-1/NRSE is
very similar to a consensus RE-1/NRSE (14), and it is highly conserved
among at least five species. The high level of similarity and
conservation, along with its intronic position, suggests that the
Crh RE-1/NRSE is functionally important for Crh
transcriptional regulation (modified from Schoenherr et al.
(14)).
|
|
REST/NRSF Is Expressed in Non-neuronal L6 Muscle Cells but Not in
More Neuronal PC12 or NG108-15 Cells--
We performed RT-PCR on L6,
NGF-treated PC12, and forskolin/IBMX-treated NG108 cells to determine
their REST/NRSF expression profiles. REST/NRSF expression was only
observed in the L6 cells (Fig. 2). We
chose these cell types because they have been used extensively in
previous studies of REST/NRSF-RE-1/NRSE function and are thus
relatively well characterized for this system, with non-neural L6 cells
containing high levels of REST/NRSF, and neuronal PC12 and NG108 cells
having virtually none (6, 16, 51). Consistent with the hypothesis that
REST/NRSF may repress Crh transcription, we confirmed by
Northern blot that Crh expression and REST/NRSF levels are
reciprocal, with Crh expression absent in L6 cells and
present in PC12 cells (data not shown). The latter finding confirms a
prior report of CRH expression in PC12 cells (52).

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Fig. 2.
REST/NRSF mRNA is expressed in L6 muscle
cells but not in neuronal PC12 or NG108-15 cells. RT-PCR of RNA
extracted from L6, NGF-treated PC12, and forskolin/IBMX-treated
NG108-15 cells was performed using an intron-spanning primer pair
specific for full-length REST/NRSF, as described under "Experimental
Procedures." After 35 cycles, REST/NRSF cDNA was observed in L6
(lane 1, middle arrow), but not PC12 (lane 2) or
NG108 (lane 3), cells. No signal was observed in the
RT-lacking controls (lanes 4-6). Actin was used as an
internal control for RNA degradation (bottom arrow).
Lane M is a 100-bp DNA ladder. Top arrow shows
position of gel wells.
|
|
REST/NRSF Binds Specifically to the Crh RE-1/NRSE--
To
establish REST/NRSF binding to the Crh RE-1/NRSE, to
evaluate the specificity of this binding, and to test its sensitivity to sequence perturbation, we conducted a series of EMSAs. We obtained nuclear extracts from L6 rat myoblast cells and used several different oligonucleotide probes ("Experimental Procedures"). Using the Crh RE-1/NRSE probe, we observed binding activity (Fig.
3A, lane 2). This shift was
specifically eliminated by a REST-specific polyclonal antibody, but not
by a cAMP-response element-binding protein-specific antibody (Fig.
3A, compare lanes 3 and 4), and was
competed away by a 33-fold molar excess of cold RE-1/NRSE oligonucleotide (Fig. 3A, lane 5). Along with the highly
conserved RE-1/NRSE sequence, we also tested a mutant form of the
element (RE1MUT), containing a GG- to -TT (GG/TT)
mutation at its center that has previously been shown to disrupt
REST/NRSF binding (10), as an unlabeled competitor (Fig. 3A, lane
11). Additionally, we tested several other potential RE-1/NRSE
elements, p(RE-1)A-D, which were found elsewhere in the
murine CRH gene and showed some sequence similarity to the RE-1/NRSE
(Fig. 3B), for their ability to compete with the conserved
RE-1/NRSE for REST/NRSF binding (Fig. 3A, lanes 6-9). In
multiple experiments (n = 3), only the intact RE-1/NRSE
was observed to compete for REST/NRSF binding, indicating both that
REST/NRSF binding to the Crh RE-1/NRSE is greatly disrupted
by the GG/TT mutation and that the several p(RE-1)s are not likely to
be important for REST/NRSF function.

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Fig. 3.
REST/NRSF binding to the intact
Crh RE-1/NRSE is both specific and saturable.
A, incubation of L6 nuclear extract with 11-14 fmol of
-32P-labeled RE-1/NRSE probe results in a clear shift
(lane 2). REST antibody (Ab) treatment
specifically abolishes the REST/NRSF-RE-1/NRSE shift (lane
3). Binding is competed by a 33-fold molar excess of intact
RE-1/NRSE (lanes 5 and 10) but not by an
identical amount of p(RE-1)s (lanes 6-9) or the GG/TT
mutant RE-1/NRSE (lane 11). The two panels represent
separate experiments. 6 µg of nuclear extract were used in each lane.
B, potential RE-1/NRSE elements, p(RE-1)A-D,
that demonstrate some limited sequence similarity to the intronic
RE-1/NRSE at position 1127 were identified throughout the mouse CRH
gene and used in the EMSAs in panel A.
|
|
The Crh RE-1/NRSE Is Functionally Important for Crh Transcriptional
Regulation--
Having demonstrated sequence-specific, saturable
binding of REST/NRSF to the Crh RE-1/NRSE, we next wished to
test the possible functional significance of the observed
REST/NRSF-RE-1/NRSE association. We made five different luciferase
reporter constructs to establish the importance of the Crh
RE-1/NRSE and its flanking DNA in Crh transcriptional
regulation (Fig. 4). All constructs
contain 5 kb of upstream 5'-flanking sequence, in addition to the
promoter and first, non-coding, exon of the mouse CRH gene. Two of the constructs contain the intron as well, with either an intact
(Intron(+)WT) or a GG/TT-mutated (Intron(+)MUT)
RE-1/NRSE site. An additional three constructs contain no intron and
either an intact RE-1/NRSE (Intron(
)RE1(+)), a GG/TT-mutated
RE-1/NRSE (Intron(-)RE1MUT), or no RE-1/NRSE at all
(Intron(
)RE1(-)) (Fig. 4). All constructs were cloned into a pXP2
firefly luciferase reporter backbone. We transiently transfected L6,
PC12, and NG108 cells with these constructs, either alone or with a
co-transfected, cytomegalovirus-driven, REST/NRSF expression vector,
REEX-1 (21). Each condition was tested multiple times, with either
three or, more commonly, four wells per condition.

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Fig. 4.
Constructs used for luciferase transfection
assays. Two intron-containing and three intron-lacking constructs
were made by inserting mouse Crh DNA into the pXP2 firefly
luciferase expression vector. All constructs contain 5 kb of upstream
sequence and the first, untranslated exon of the CRH gene. The
RE-1/NRSE element is either intact [Intron(+)WT,
Intron( )RE1(+)], mutated [Intron(+)MUT,
Intron( )RE1MUT], or absent altogether
[Intron( )RE1( )].
|
|
We first tested the importance of the RE-1/NRSE element,
itself, in regulating Crh transcriptional activity. If the
RE-1/NRSE were mediating transcriptional repression by REST/NRSF,
disrupting the RE-1/NRSE in L6 cells would be expected to lead to
transcriptional de-repression, since these cells contain high levels of
endogenous RE-1/NRSE-binding REST/NRSF activity (6, 16). A marked
de-repression was indeed observed in these cells upon disruption of the
RE-1/NRSE sequence (Fig. 5A).
With an intact RE-1/NRSE, both the intron-containing (Fig. 5A,
left panel) and the intron-lacking (Fig. 5A, right
panel) constructs were essentially silent. In all cases, with the
RE-1/NRSE either mutated or deleted altogether, the expression levels
were markedly up-regulated. This de-repression ranged from 18- to over 60-fold. In addition, intron-lacking constructs in general were more
active (Fig. 5A, note different ordinate
scales).

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Fig. 5.
REST/NRSF represses Crh
transcription through a RE-1/NRSE-dependent mechanism
that is partially HDAC dependent. A, in L6 cells,
disruption or deletion of the RE-1/NRSE results in a significant
de-repression (p < .0001, using the Bon Feronni
t test correction for multiple comparisons), both in the
presence (left panel) and absence (right panel)
of the intron. The intron appears to lower the overall level of
expression in these cells (note different ordinate scales of
the left and right panels) (n = 4 replicates per condition). B, in PC12 cells treated with 50 ng/ml NGF, expression is significantly repressed by the introduction of
REST/NRSF when the RE-1/NRSE is intact (bar 2,
p < .05; bar 6, p < .00005) but not when the RE-1/NRSE is either disrupted (bars
4 and 8) or deleted (bar 10). As with the L6
cells, the presence of the intron appears to result in an overall
lowering of expression levels (note different ordinate
scales of the left and right panels). The
intron also appears to limit the degree of RE-1/NRSE-mediated
transcriptional repression by REST/NRSF (n = 4 replicates per condition). C, 100 nM TSA
treatment partially relieves RE-1/NRSE-mediated repression in L6 cells,
regardless of intron context. TSA treatment led to significant partial
de-repression of Crh-luciferase construct expression
when the RE-1 was intact (bars 1 and 2,
p < .004; and bars 5 and 6,
p < .002) but not when the RE-1 was disrupted
(bars 3 and 4 and bars 7 and
8). This RE-1 dependence was observed both with and without
the intron present and suggests that RE-1 mediated REST repression in
L6 cells occurs, in part, through a HDAC-dependent
mechanism (n = 3 replicates per condition).
|
|
REST/NRSF represses Crh transcription via the RE-1/NRSE--
In
order to test for the function of the REST/NRSF protein itself, we
transiently transfected PC12 and NG108 cells (which lack REST/NRSF, see
Fig. 2) with the above constructs, both with and without
co-transfection of the REST expression vector. Undifferentiated PC12
cells did not express any of the Crh constructs used (data not shown). NGF treatment has previously been shown to enhance endogenous CRH production in these cells and to result in a more neuronal phenotype (52, 53). We therefore tested NGF-treated PC12 cells
and found that they did express the Crh constructs. All
subsequent PC12 transfection experiments were consequently conducted
using NGF-treated cells. When transfected alone (without REST/NRSF),
expression of intron-containing constructs (Fig. 5B, left
panel) tended to be less than that of constructs lacking the
intron (Fig. 5B, right panel; note different ordinate
scales). In the absence of added REST/NRSF, disruption of the
RE-1/NRSE resulted in a minimal increase in expression levels (Fig.
5B) compared with the over 20-fold increase observed in L6
cells, consistent with the undetectable amount of REST/NRSF mRNA we
observed in the NGF-treated PC12 cells. In the setting of an intact
RE-1/NRSE sequence, co-transfection of REST/NRSF appeared to lead to
greater suppression in the absence of the intron (86% repression,
p < .00005, for the Intron(
)RE1(+) construct) than
in the presence of the intron (35% repression, p < .047, for the Intron(+)WT construct) (Fig. 5B).
In the presence of the intron, disruption of the RE-1/NRSE element (Intron(+)MUT) eliminated REST/NRSF-induced
transcriptional repression. Disruption or deletion of the RE-1/NRSE
element in the intron-lacking constructs (Intron(
)RE1(
) and
Intron(
)RE1MUT) (Fig. 5B, bars 7-10) also
eliminated the significant REST/NRSF-mediated repression observed with
an intact RE-1/NRSE (Fig. 5B, bars 5 and 6).
These results suggest that REST/NRSF itself functions as a repressor of
Crh transcription and, furthermore, that this repression is
mediated by the intronic RE-1/NRSE. As noted above, the presence of the
intron, independent of the RE-1/NRSE, seemed to reduce both the basal
levels of construct activity and the amount of repression seen upon
REST/NRSF introduction in PC12 cells. To confirm that this result was
not peculiar to one particular line of PC12 cells, which are known to
drift genetically upon passaging, two other independent PC12 lines were
also similarly tested and yielded essentially identical results (data
not shown).
REST/NRSF-RE-1/NRSE-mediated Repression Occurs in Part through a
HDAC-dependent Mechanism--
In order to evaluate
deacetylation as a possible mechanism for REST/NRSF repression of the
CRH gene, we treated L6 cells with 100 nM TSA for 24 h
and looked for disruption of the previously observed RE-1/NRSE-mediated
repression. As shown in Fig. 5C, TSA treatment led to
significant de-repression only for the constructs containing an intact
RE-1/NRSE (bars 1 and 2, 5 and 6).
Intron(+)WT expression was de-repressed by roughly 14-fold
while Intron(+)MUT expression remained unchanged.
Similarly, Intron(
)RE1(+) expression was boosted by almost 4-fold
while Intron(
)RE1(
) expression was not de-repressed, and actually
fell slightly. These results suggest that the HDAC-mediated repression
relieved by TSA treatment depends on the presence of an intact
RE-1/NRSE, irrespective of the presence or absence of the intron. This,
in turn, suggests that REST/NRSF represses CRH gene expression, in
part, through a HDAC-dependent mechanism. The
TSA-dependent de-repression, however, does not account for
the full degree of de-repression observed when the RE-1/NRSE is
disrupted. For example, TSA treatment of Intron(+)WT does
not lead to the fully de-repressed level resulting from disruption of
the RE-1/NRSE in Intron(+)MUT (Fig. 5C, compare the de-repression between bars 1 and 2 with that
between bars 1 and 3). This is not surprising, as
REST/NRSF has been previously shown to associate through its C-terminal
zinc finger, with a protein called Co-REST to mediate repression by an
HDAC-independent mechanism (22, 23, 30).
REST/NRSF Can Act as an Enhancer of Crh
Transcription--
NG108 cells can be differentiated to a more
neuronal phenotype by treating with 10 µM forskolin and
100 µM IBMX (54, 55). We therefore used
forskolin/IBMX-treated NG108 cells to study the function of
REST/NRSF-RE-1/NRSE in the setting of another neuronal phenotype (Fig.
6). As in PC12 cells, co-transfected REST/NRSF significantly repressed expression only when an intact RE-1/NRSE was in place, regardless of whether the remainder of the
intron was present (Fig. 6, A and B). As with L6
and PC12 cells, repression appeared to be greater (70% repression,
p < .005) in the RE-1/NRSE-containing construct
lacking the intron (Fig. 6B, bars 1 and 2) than
in the construct containing the intron (28% repression,
p < .004) (Fig. 6A, bars 1 and
2). However, when the RE-1/NRSE was either disrupted
(Intron(+)MUT and Intron(
)RE1MUT) or deleted
altogether (Intron(
) RE1(
)), rather than a lack of repression, we
observed a significant 1.2-2.5-fold up-regulation of reporter activity
over baseline (compare bars 3 and 4 in Fig. 6A; bars 3 and 4 in Fig.
6B; bars 5 and 6 in Fig.
6B). The presence of the intron appeared to facilitate,
rather than mute, this enhancer effect of REST/NRSF on transcription
(note different ordinate scales in Fig. 6, A and
B), although it is not clear whether this was a specific or
nonspecific (spacer or conformational) effect. The up-regulation of
transcriptional activity was seen consistently in three independent
experiments (data not shown), with both the intron-containing and the
intron-lacking constructs. Similar enhancer and repressor effects were
also seen in cells untreated with forskolin/IBMX, indicating that PKA
stimulation in this context does not change the fundamental properties
of the system (data not shown). It appears that REST/NRSF can act as an
activator of Crh transcription, independent of the
Crh RE-1/NRSE, in addition to acting as a
RE-1/NRSE-dependent repressor of Crh
transcription. This enhancer function is unmasked by the disruption of
the repression-mediating RE-1/NRSE element. A dual role for REST/NRSF,
as an enhancer as well as repressor of gene transcription, has been
previously suggested (1, 2, 20). In this case, the observed effect on
Crh transcription could be a direct effect of REST/NRSF or
an indirect effect of REST/NRSF, if, for example, another REST/NRSF
target is involved in regulating Crh transcription. The
enhancer effect of REST/NRSF seen in NG108 cells is not simply due to
their being transfected with this factor, as Crh
transcription is not enhanced in L6 cells following similar
transfection with REST/NRSF (data not shown).

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Fig. 6.
In NG108 cells, REST/NRSF represses
transcription from the Crh promoter constructs
containing intact RE-1/NRSEs, while enhancing transcription from
constructs lacking intact RE-1/NRSEs. A, with the
intron intact, introduction of REST/NRSF results in a significant
repression of the construct containing an intact RE-1/NRSE (bar
2, p < .004), while expression of the mutant
construct is significantly boosted ~2.5-fold (bar 4,
p < .003) (n = 4 replicates per
condition). B, without the intron, REST/NRSF introduction
leads to a significant repression of the construct containing an intact
RE-1/NRSE (bar 2, p < .005), and a
significant 1.25-1.65-fold increase in expression from the constructs
with a disrupted RE-1/NRSE (bar 4, p < .003; and bar 6, p < .034). The intron
appears to reduce RE-1/NRSE mediated repression by REST/NRSF, while
facilitating the REST/NRSF induced enhancement of transcriptional
activity. (Note different ordinate scales in A
and B; n = 4 replicates per
condition.)
|
|
 |
DISCUSSION |
In L6 and PC12 cells, the REST/NRSF-RE-1/NRSE system
appears to function in Crh regulation much as it has been
previously shown to function in other genetic contexts. NG108 cells
reveal another potential mode of CRH gene regulation by REST/NRSF. It has become clear, from previous studies, that genetic context is
extremely important in determining the function of the
REST/NRSF-RE-1/NRSE system (1, 4-10, 12, 17, 18, 20). A possible
explanation for at least a part of this variability is that REST/NRSF
may act through independent mechanisms, associating with different co-factors, such as the Sin3A complex, the nuclear receptor corepressor (N-CoR), and CoREST, to repress expression of different
REST/NRSF-dependent genes (29). Results from the knockout
of REST/NRSF in mouse strongly suggest that tissue/cell-type, as well
as genetic context, is important for determining the effect of
REST/NRSF on gene expression (2). Our results underscore the fact that
the cellular context is critical for understanding and defining the
function(s) of the system. Not only is the distinction between neuronal
and non-neuronal cell types important, but perhaps equally important is
the distinction between different neuronal cell types. Furthermore,
various forms of REST/NRSF are expressed in many neurons within the
brain (19), suggesting more than a repressive function for REST/NRSF.
This is particularly relevant for a gene such as Crh, which
is differentially regulated, at times in opposite directions, in
different brain regions and cell types (56, 57).
The intron appears to be important for Crh transcriptional
regulation in more than one way. The 21-bp RE-1/NRSE element, itself, exerts a clear influence on CRH gene transcription. In L6 cells, which
contain a high level of REST/NRSF protein and do not express CRH, the
element mediates a potent transcriptional repression of the gene. This
repression is substantially relieved by either mutating or deleting the
RE-1/NRSE. In NGF-treated PC12 cells, which express the CRH gene,
addition of REST/NRSF results in transcriptional repression of
Crh construct expression when an intact RE-1/NRSE is
present, but not when the RE-1/NRSE has been mutated or deleted. Treatment of L6 cells with 100 nM TSA confirms that
REST/NRSF may repress partially through a HDAC-dependent
mechanism. In both cell types, the remainder of the intron
(i.e. excluding the RE-1/NRSE) appears to confer a
generalized down-regulation of basal expression levels. It is possible
that this is due to the presence of an as yet unidentified regulatory
element in the intron or simply to a nonspecific, possibly
conformational, effect of the intron.
NG108 cells provide another opportunity to study REST/NRSF action in a
neuronal cell type. Consistent with the results obtained in L6 and PC12
cells, the addition of REST/NRSF to these cells leads to a repression
of Crh construct expression when an intact RE-1/NRSE is
present, but not when it is either disrupted or deleted. Unlike in PC12
cells, however, disruption or deletion of the RE-1/NRSE element in
NG108 cells not only restores Crh construct expression to
basal levels but actually leads to a significant enhancement of
expression. The intron appears to facilitate this enhancement in NG108
cells, while reducing the amount of RE-1/NRSE-mediated repression by
REST/NRSF in both PC12 and NG108 cells.
Our results suggest that, in the context of the CRH gene, REST/NRSF may
exert both a RE-1/NRSE-dependent repressive effect and a
RE-1/NRSE-independent enhancer effect simultaneously, and it is the
balance of these effects that determines REST/NRSF's final impact on
transcriptional activity. It is not yet clear whether the
RE-1/NRSE-independent activity of REST/NRSF on Crh transcription is direct, acting on some novel element within the gene,
or indirect, acting on another gene or factor involved in Crh transcriptional regulation. It is strictly possible that
the novel enhancer effect of REST/NRSF is limited to NG108-15 cells. However, it is also possible that it extends to other neuronal cell
types and possibly even to non-neuronal cell types. Whether any such
observations have physiological correlates can ultimately only be
evaluated satisfactorily in vivo. However, these results suggest that, depending on the genetic and cellular contexts, the net
effect of REST/NRSF function can be very different from what its
RE-1/NRSE-mediated repressor function alone would suggest.
 |
ACKNOWLEDGEMENTS |
We thank Ian Wood, Wellcome Laboratory for
Molecular Pharmacology, Leeds, UK, for the generous gift of NG108-15
cells, and Michael Greenberg, Harvard Medical School, Boston, for the
generous gift of PC12 cells. We thank Gail Mandel, Howard Hughes
Medical Institute and SUNY Stony Brook, NY, for the generous gift of
REST antibody and the REEX-1 REST expression vector and Joel Habener, Harvard Medical School, Boston, for the generous gift of cAMP-response element-binding protein antibody. We also thank Lou Muglia, Washington University, St. Louis, for providing the Crh DNA source
plasmids, CRH 5.0 and Xho-Kpn-Kpn,
Frederick Grant, Children's Hospital, Boston, for helpful discussions
and advice throughout, Gael McGill, Harvard Medical School, for
technical advice and assistance, and Nina Irwin, Children's Hospital,
Boston, for valuable technical advice and training.
 |
FOOTNOTES |
*
This work was supported in part by grants from the National
Institutes of Health (to J. M. and K. S.).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: Div. of
Endocrinology, Children's Hospital, Harvard Medical School, Boston,
Massachusetts 02115. Tel.: 617-355-6421; Fax: 617-734-0062; E-mail:
joseph.majzoub@tch.harvard.edu.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M007745200
 |
ABBREVIATIONS |
The abbreviations used are:
bp, base pair(s);
RE-1/NRSE, repressor element-1 neuron-restrictive silencing
element;
REST/NRSF, repressor element silencing transcription
factor/neuron-restrictive silencing factor;
HDAC, histone
deacetylase;
TSA, trichostatin A;
CRH, corticotropin releasing hormone;
ACTH, adrenocorticotropic hormone;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
NGF, nerve growth factor;
IBMX, 3-isobutylmethylxanthine;
EMSA, electrophoretic mobility shift
assay;
kb, kilobase(s);
CAT, chloramphenicol acetyltransferase.
 |
REFERENCES |
1.
|
Bessis, A.,
Champtiaux, N.,
Chatelin, L.,
and Changeux, J. P.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5906-5911[Abstract/Free Full Text]
|
2.
|
Chen, Z. F.,
Paquette, A. J.,
and Anderson, D. J.
(1998)
Nature Genet.
20,
136-142[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Ishiguro, H.,
Kim, K. T.,
Joh, T. H.,
and Kim, K. S.
(1993)
J. Biol. Chem.
268,
17987-17994[Abstract/Free Full Text]
|
4.
|
Kallunki, P.,
Edelman, G. M.,
and Jones, F. S.
(1997)
J. Cell Biol.
138,
1343-1354[Abstract/Free Full Text]
|
5.
|
Kallunki, P.,
Jenkinson, S.,
Edelman, G. M.,
and Jones, F. S.
(1995)
J. Biol. Chem.
270,
21291-21298[Abstract/Free Full Text]
|
6.
|
Kraner, S. D.,
Chong, J. A.,
Tsay, H. J.,
and Mandel, G.
(1992)
Neuron
9,
37-44[Medline]
[Order article via Infotrieve]
|
7.
|
Li, L.,
Suzuki, T.,
Mori, N.,
and Greengard, P.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
1460-1464[Abstract]
|
8.
|
Lonnerberg, P.,
Schoenherr, C. J.,
Anderson, D. J.,
and Ibanez, C. F.
(1996)
J. Biol. Chem.
271,
33358-33365[Abstract/Free Full Text]
|
9.
|
Mieda, M.,
Haga, T.,
and Saffen, D. W.
(1997)
J. Biol. Chem.
272,
5854-5860[Abstract/Free Full Text]
|
10.
|
Mori, N.,
Schoenherr, C.,
Vandenbergh, D. J.,
and Anderson, D. J.
(1992)
Neuron
9,
45-54[Medline]
[Order article via Infotrieve]
|
11.
|
Myers, S. J.,
Peters, J.,
Huang, Y.,
Comer, M. B.,
Barthel, F.,
and Dingledine, R.
(1998)
J. Neurosci.
18,
6723-6739[Abstract/Free Full Text]
|
12.
|
Schoch, S.,
Cibelli, G.,
and Thiel, G.
(1996)
J. Biol. Chem.
271,
3317-3323[Abstract/Free Full Text]
|
13.
|
Schoenherr, C. J.,
and Anderson, D. J.
(1995)
Science
267,
1360-1363[Medline]
[Order article via Infotrieve]
|
14.
|
Schoenherr, C. J.,
Paquette, A. J.,
and Anderson, D. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9881-9886[Abstract/Free Full Text]
|
15.
|
Wood, I. C.,
Roopra, A.,
and Buckley, N. J.
(1996)
J. Biol. Chem.
271,
14221-14225[Abstract/Free Full Text]
|
16.
|
Chong, J. A.,
Tapia-Ramirez, J.,
Kim, S.,
Toledo-Aral, 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]
|
17.
|
Mori, N.,
Stein, R.,
Sigmund, O.,
and Anderson, D. J.
(1990)
Neuron
4,
583-594[Medline]
[Order article via Infotrieve]
|
18.
|
Maue, R. A.,
Kraner, S. D.,
Goodman, R. H.,
and Mandel, G.
(1990)
Neuron
4,
223-231[Medline]
[Order article via Infotrieve]
|
19.
|
Palm, K.,
Belluardo, N.,
Metsis, M.,
and Timmusk, T.
(1998)
J. Neurosci.
18,
1280-1296[Abstract/Free Full Text]
|
20.
|
Kallunki, P.,
Edelman, G. M.,
and Jones, F. S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3233-3238[Abstract/Free Full Text]
|
21.
|
Tapia-Ramirez, J.,
Eggen, B. J.,
Peral-Rubio, M. J.,
Toledo-Aral, J. J.,
and Mandel, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1177-1182[Abstract/Free Full Text]
|
22.
|
Huang, Y.,
Myers, S. J.,
and Dingledine, R.
(1999)
Nat. Neurosci.
2,
867-872[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Naruse, Y.,
Aoki, T.,
Kojima, T.,
and Mori, N.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13691-13696[Abstract/Free Full Text]
|
24.
|
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[Abstract/Free Full Text]
|
25.
|
Pazin, M. J.,
and Kadonaga, J. T.
(1997)
Cell
89,
325-328[Medline]
[Order article via Infotrieve]
|
26.
|
Grunstein, M.
(1997)
Nature
398,
349-352
|
27.
|
Yoshida, M.,
Kijima, M.,
Akita, M.,
and Beppu, T.
(1990)
J. Biol. Chem.
265,
17174-17179[Abstract/Free Full Text]
|
28.
|
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[Abstract/Free Full Text]
|
29.
|
Jepsen, K.,
Hermanson, O.,
Onami, T. M.,
Gleiberman, A. S.,
Lunyak, V.,
McEvilly, R. J.,
Kurokawa, R.,
Kumar, V.,
Liu, F.,
Seto, E.,
Hedrick, S. M.,
Mandel, G.,
Glass, C. K.,
Rose, D. W.,
and Rosenfeld, M. G.
(2000)
Cell
102,
753-763[Medline]
[Order article via Infotrieve]
|
30.
|
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[Abstract/Free Full Text]
|
31.
|
Vale, W.,
Spiess, J.,
Rivier, C.,
and Rivier, J.
(1981)
Science
213,
1394-1397[Medline]
[Order article via Infotrieve]
|
32.
|
Owens, M. J.,
and Nemeroff, C. B.
(1991)
Pharmacol. Rev.
43,
425-473[Medline]
[Order article via Infotrieve]
|
33.
|
Muglia, L.,
Jacobson, L.,
Dikkes, P.,
and Majzoub, J. A.
(1995)
Nature
373,
427-432[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Munck, A.,
Guyre, P. M.,
and Holbrook, N. J.
(1984)
Endocr. Rev.
5,
25-44[Abstract]
|
35.
|
Gold, P. W.,
Loriaux, D. L.,
Roy, A.,
Kling, M. A.,
Calabrese, J. R.,
Kellner, C. H.,
Nieman, L. K.,
Post, R. M.,
Pickar, D.,
Gallucci, W.,
Avgerinos, P.,
Paul, S.,
Oldfield, E. H.,
Cutler, G. B.,
and Chrousos, G. P.
(1986)
N. Engl. J. Med.
314,
1329-1335[Abstract]
|
36.
|
Gold, P. W.,
Goodwin, F. K.,
and Chrousos, G. P.
(1988)
N. Engl. J. Med.
319,
413-420[Abstract]
|
37.
|
Gold, P. W.,
Gwirtsman, H.,
Avgerinos, P. C.,
Nieman, L. K.,
Gallucci, W. T.,
Kaye, W.,
Jimerson, D.,
Ebert, M.,
Rittmaster, R.,
Loriaux, D. L.,
and Chrousos, G. P.
(1986)
N. Engl. J. Med.
314,
1335-1342[Abstract]
|
38.
|
Karalis, K. P.,
Kontopoulos, E.,
Muglia, L. J.,
and Majzoub, J. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7093-7097[Abstract/Free Full Text]
|
39.
|
Scatena, C. D.,
and Adler, S.
(1998)
Mol. Endocrinol.
12,
1228-1240[Abstract/Free Full Text]
|
40.
|
Seasholtz, A. F.,
Thompson, R. C.,
and Douglass, J. O.
(1988)
Mol. Endocrinol.
2,
1311-1319[Abstract]
|
41.
|
Guardiola-Diaz, H. M.,
Boswell, C.,
and Seasholtz, A. F.
(1994)
J. Biol. Chem.
269,
14784-14791[Abstract/Free Full Text]
|
42.
|
Van, L. P.,
Spengler, D. H.,
and Holsboer, F.
(1990)
Endocrinology
127,
1412-1418[Abstract]
|
43.
|
Spengler, D.,
Rupprecht, R.,
Van, L. P.,
and Holsboer, F.
(1992)
Mol Endocrinol
6,
1931-1941[Abstract]
|
44.
|
Malkoski, S. P.,
and Dorin, R. I.
(1999)
Mol Endocrinol
13,
1629-1644[Abstract/Free Full Text]
|
45.
|
Shimojo, M.,
Paquette, A. J.,
Anderson, D. J.,
and Hersh, L. B.
(1999)
Mol. Cell. Biol.
19,
6788-6795[Abstract/Free Full Text]
|
46.
|
Schreiber, E.,
Matthias, P.,
Muller, M. M.,
and Schaffner, W.
(1989)
Nucleic Acids Res.
17,
6419[Medline]
[Order article via Infotrieve]
|
47.
|
Waeber, G.,
Meyer, T. E.,
LeSieur, M.,
Hermann, H. L.,
Gerard, N.,
and Habener, J. F.
(1991)
Mol. Endocrinol.
5,
1418-1430[Abstract]
|
48.
|
Nordeen, S. K.
(1988)
BioTechniques
6,
454-458[Medline]
[Order article via Infotrieve]
|
49.
|
Muglia, L. J.,
Jenkins, N. A.,
Gilbert, D. J.,
Copeland, N. G.,
and Majzoub, J. A.
(1994)
J. Clin. Invest.
93,
2066-2072[Medline]
[Order article via Infotrieve]
|
50.
|
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
51.
|
Mu, W.,
and Burt, D. R.
(1999)
Brain Res. Mol. Brain Res.
67,
137-147[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Venihaki, M.,
Gravanis, A.,
and Margioris, A. N.
(1997)
Endocrinology
138,
698-704[Abstract/Free Full Text]
|
53.
|
Greene, L. A.,
and Tischler, A. S.
(1976)
Proc. Natl. Acad. Sci. U. S. A.
73,
2424-2428[Abstract]
|
54.
|
Bergsbaken, C. L.,
Sommers, S. L.,
and Law, P. Y.
(1993)
J. Pharmacol. Exp. Ther.
264,
1474-1483[Abstract]
|
55.
|
Tojima, T.,
Yamane, Y.,
Takahashi, M.,
and Ito, E.
(2000)
Neurosci. Res.
37,
153-161[CrossRef][Medline]
[Order article via Infotrieve]
|
56.
|
Makino, S.,
Gold, P. W.,
and Schulkin, J.
(1994)
Brain Res.
640,
105-112[CrossRef][Medline]
[Order article via Infotrieve]
|
57.
|
Robinson, B. G.,
Emanuel, R. L.,
Frim, D. M.,
and Majzoub, J. A.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
5244-5248[Abstract]
|
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