MOLECULAR AND CELLULAR BIOLOGY, Dec. 2005, p. 10442–10453
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 23
SF-1 (Nuclear Receptor 5A1) Activity Is Activated by Cyclic AMP
via p300-Mediated Recruitment to Active Foci, Acetylation,
and Increased DNA Binding
Wei-Yi Chen,1,2Li-Jung Juan,3and Bon-chu Chung1*
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan1; Graduate Institute of Life Sciences,
National Defense Medical Center, Taipei, Taiwan2; and President Laboratory,
National Health Research Institutes, Miaoli, Taiwan3
Received 15 June 2005/Returned for modification 8 July 2005/Accepted 19 September 2005
Steroidogenic factor 1 (SF-1) is a nuclear receptor essential for steroidogenic gene expression, but how its
activity is regulated is unclear. Here we demonstrate that p300 plays an important role in regulating SF-1
function. SF-1 was acetylated in vitro and in vivo by p300 at the KQQKK motif in the Ftz-F1 (Fushi-tarazu
factor 1) box adjacent to its DNA-binding domain. Mutation of the KQQKK motif reduced the DNA-binding
activity and p300-dependent activation of SF-1. When stimulated with cyclic AMP (cAMP), adrenocortical Y1
cells expressed more p300, leading to additional SF-1 association with p300 and increased SF-1 acetylation and
DNA binding. It also increased SF-1 colocalization with p300 in nuclear foci. Collectively, these results indicate
that SF-1 transcriptional activity is regulated by p300 in response to the cAMP signaling pathway by way of
increased acetylation, DNA binding, and recruitment to nuclear foci.
Steroidogenic factor 1 (SF-1), also known as Ad4BP (adre-
nal 4 binding protein), is a member of the nuclear receptor
superfamily, designated NR5A1 (39). SF-1 plays a critical role
in the development, differentiation, and function of the hypo-
thalamus, pituitary, adrenal glands, and gonads (43). SF-1 con-
trols the expression of a variety of genes, such as steroidogenic
genes, Mu ¨llerian inhibitory substance, and the ?-subunit and
?-subunit of gonadotropins (37, 43). SF-1 exerts its transcrip-
tional activity through interaction with numerous proteins, in-
cluding coactivators, corepressors, and other transcription fac-
tors (2, 9–11, 24, 35, 36, 42).
SF-1 is structurally similar to steroid receptors; it contains a
zinc finger DNA-binding domain (DBD) and a C-terminal
ligand-binding domain but lacks the N-terminal A/B domain
(Fig. 1A). Members of the nuclear receptor 5 (NR5) subfamily,
including Drosophila melanogaster FTZ-F1 (NR5A3), silkworm
BmFTZ-F1, and mammalian LRH-1 (liver receptor homo-
logue 1) (NR5A2) and SF-1 (NR5A1), share a conserved 30-
amino-acid (aa) basic region, designated the Ftz-F1 (Fushi-
tarazu factor 1) box, adjacent to the C terminus of the DNA-
binding domain (52). This box facilitates recognition of the first
three bases of the DNA sequence PyCAAGGPyCPu (52). The
Ftz-F1 box together with its adjacent proline-rich sequence (aa
78 to 172), called the FP domain, is important for the trans-
activation function of SF-1 (30). It is also important for nuclear
localization as well as interaction with TFIIB and c-Jun (30).
SF-1 is modified by phosphorylation and SUMO conjugation
at the hinge domain. The phosphorylation site is mediated by
mitogen-activated protein kinase and required for maximal
transcriptional activity of SF-1 (17). SUMO conjugation re-
presses SF-1 activity by recruiting transcriptional repressors like
DP103 and/or by relocating SF-1 to nuclear speckles (7, 26, 28).
In addition to phosphorylation and SUMO conjugation,
SF-1 is also acetylated (25). Two well-characterized histone
acetyltransferase (HAT) proteins are in the p300/CBP (CREB-
binding protein) family and the PCAF/GCN5 (p300/CBP-as-
sociated factor/general control nonderepressed 5) family.
These HATs function as coactivators for transcription factors
(49), many of which are acetylated, like p53 (15), E2F1 (33),
c-Myb (50), EKLF (erythroid Kru ¨ppel-like factor) (57), MyoD
(44), GATA-1 (4), and androgen receptor (AR) (14). Acety-
lation modulates the functions of these transcription factors by
affecting DNA-binding activity, interaction with other proteins,
stability, and nuclear localization. For example, acetylated p53
binds DNA and activates transcription more efficiently than
unacetylated p53 (15, 32), probably in a promoter-specific
manner (18). SF-1 is acetylated by GCN5 in vitro (25). Acet-
ylation affects the transcriptional activity of SF-1. However, the
mechanism of SF-1 activation by acetylation is still unclear.
The subcellular localization of transcription factors is impor-
tant for gene activation. Activated transcription factors like
ligand-induced steroid receptors glucocorticoid receptor (19),
AR (51), mineralocorticoid receptor (13), and hypoxia-induc-
ible factor 1 (HIF-1) (46) are concentrated at specific regions
of the nuclei. These nuclear clusters partially overlap with
activated RNA polymerase II (Pol II) or nascent mRNA.
Many proteins in the transcription machinery are also at these
foci. These include transcriptional coactivators p300/CBP,
SRC-1 (steroid receptor coactivator 1) (48), components of
chromatin remodeling complexes (34), and RNA Pol II (46,
53). Thus, nuclear-cluster formation may be a process of gene
activation, in which activated transcription factors and coacti-
vators can be recruited to the active transcription sites.
Cyclic AMP (cAMP) is the intracellular molecule that con-
ducts the signal of extracellular tropic hormones to cAMP-
* Corresponding author. Mailing address: Institute of Molecular
Biology, 48, Academia Sinica, Nankang, Taipei 115, Taiwan. Phone:
886-2-2789 9215. Fax: 886-2-2782 6085. E-mail: email@example.com
dependent protein kinase A (PKA) and the downstream sig-
naling pathway. In adrenocortical cells, activation of the
cAMP-PKA pathway increases the expression of several SF-1-
regulated steroidogenic genes (5, 20, 29, 41), including
Cyp11a1, which encodes cytochrome P450scc, catalyzing the
first and rate-limiting step of steroidogenesis. However, the
mechanism by which the cAMP-PKA pathway activates SF-1-
mediated transcription is still elusive.
Like most acetylated transcription factors, SF-1 is acetylated
not just by GCN5; in the current report, we show that p300 can
also acetylate and activate SF-1. We demonstrate that p300
acetylates the KQQKK sequence at the Ftz-F1 box of SF-1.
This acetylation correlates with DNA binding and p300-poten-
tiated transcriptional activation of SF-1. In addition, SF-1 is
also recruited to RNA polymerase II-associated nuclear clus-
ters by p300. Acetylation, association with these nuclear clus-
ters, and DNA binding of SF-1 were increased after cAMP
stimulation, which also increases the amount of p300. These re-
sults suggest a novel mechanism of the cAMP signaling pathway
to stimulate SF-1 activity through the increase of p300 level.
MATERIALS AND METHODS
Cell culture, plasmids, and recombinant proteins. Mouse fibroblast NIH 3T3
and human kidney 293T cell lines were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum. Mouse adreno-
cortical tumor Y1 cells were maintained in DMEM–F-12 supplemented with
10% fetal bovine serum. The following constructs have previously been de-
scribed: phscc2.3k-LacZ, pGEX-SF-1-FP (30), pGal4-SF-1 (7), pET-p300 HAT
(40), pGEX5X-PCAT-HAT (56), pCMV-p300-myc WT and pCMV-p300-myc
DY (23), pcDNA3.1-SF-1-HA (36), and pcDNA3-FLAG-GCN5 (55). Mutated
SF-1 constructs (mt1, mt2, dmt, mt3, and mt4 [see Fig. 4]) were generated by
PCR-based site-directed mutagenesis in pcDNA3-SF-1-HA. The plasmid
pcDNA5-3xFLAG-SF-1 was generated by inserting the coding sequence of SF-1,
amplified by PCR from pcDNA3-SF-1-HA, into XhoI/XbaI sites of pcDNA5/TO
(Invitrogen). The resulting plasmid was then inserted with the coding sequence
of the Flag tag repeating three times at BamHI/XhoI sites. The sequences of
constructs were verified by direct sequencing. The recombinant proteins GST-
FP, His-p300 HAT, and GST-PCAF HAT (where GST is glutathione S-trans-
ferase) were overexpressed in Escherichia coli strain BL21(DE3) pLys and pu-
rified as described previously (22). Purified histone H3 protein was purchased
from Roche Molecular Biochemicals (Mannheim, Germany).
In vitro acetylation assay. Two micrograms of each substrate (histone H3,
GST-SF-1-FP, and GST) was added to HAT buffer (50 mM Tris-HCl [pH 8.0],
10% glycerol, 0.1 mM EDTA, and 1 mM dithiothreitol) containing 5 ?Ci/ml of
[3H]acetyl coenzyme A (10 Ci/mmol) with or without 200 ng of purified HAT
proteins. Reaction mixtures were incubated at 30°C for 30 min, stopped by
adding 1 volume of 2? sample buffer and heating to 100°C for 3 min, and then
separated by 12% polyacrylamide gel electrophoresis. The gel was stained with
Coomassie brilliant blue, immersed into Amplify solution (Amersham Bio-
sciences) for 20 min, dried, and exposed to X-ray film. Quantitation was per-
formed using Image Gauge version 3.2 software with a FujiFilm LAS-1000plus
In vivo acetylation assay. Ten micrograms of each of the expression plasmids
for SF-1-HA, wild-type p300 (p300 WT), or HAT-defective p300 (p300 DY) was
cotransfected into 293T cells. Forty-six hours after transfection, the medium was
FIG. 1. SF-1 is acetylated by p300 in vitro and in vivo. (A) Schematic representation of SF-1 and GST-FP. Residue numbers in SF-1 are
indicated. F, Ftz-F1 box; P, proline-rich domain; and LBD, ligand-binding domain. (B) In vitro acetylation of GST-FP. Purified histone H3,
GST-FP, or GST was incubated with GST, recombinant p300 HAT, or PCAF HAT in the presence of [3H]acetyl coenzyme A. The acetylated
proteins, detected by fluorography, are shown in the top gel; the bottom gel shows proteins stained by Coomassie brilliant blue. The asterisks
denote nonspecific signals probably arising from impurities in the commercial histone H3 extract. (C) SF-1 is acetylated by p300 in vivo. Expression
plasmid for SF-1-HA was cotransfected with c-Myc-tagged p300 WT or p300 DY plasmid into 293T cells, followed by labeling with [3H]acetate.
The acetyl-SF-1-HA proteins (top) were visualized by fluorography after immunoprecipitation (IP) with an anti-HA antibody and gel separation.
SF-1-HA and p300 were detected by immunoblotting (IB) with an anti-SF-1 (middle) or anti-c-Myc (bottom) antibody, respectively.
VOL. 25, 2005 cAMP-INDUCED SF-1 ACETYLATION MEDIATED BY p30010443
replaced with DMEM containing 1 mCi/ml sodium [3H]acetate (5 Ci/mmol) and
100 ng/ml trichostatin A (TSA) for 2 h. Whole-cell extracts were prepared in IPH
buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, and
1? Complete protease inhibitor cocktail) and immunoprecipitated with 2 ?g rat
anti-hemagglutinin (HA) antibody (clone 3F10; Roche). The immunoprecipi-
tates were separated by 10% polyacrylamide gel electrophoresis followed by
fluorography at ?70°C for 10 days. The nonradiolabeled parallel group of trans-
fectants was analyzed by immunoblotting with a polyclonal antiserum against
full-length SF-1 (7) and visualized by chemiluminescence.
For3H-incorporation of SF-1 in response to cAMP, Y1 cells were transfected
with 3 ?g expression plasmids for wild-type or KQQKK mutated 3xFLAG-SF-1.
After 24 h, cells were labeled with 1 mCi/ml sodium [3H]acetate in the absence
or presence of 1 mM 8-Br-cAMP, a cell-permeable cAMP analog, for 6 h. After
immunoprecipitation using 2 ?g mouse anti-FLAG antibody (Sigma), the im-
munoprecipitates were analyzed as described above.
Transcriptional-activity assay. For transfection into NIH 3T3 cells in 35-mm
culture dishes, 200 ng of SF-1-HA plasmid and SF-1-dependent reporter gene
phscc2.3k-LacZ were cotransfected with increasing amounts (0.2, 0.5, and 1 ?g)
of p300 WT or p300 DY as indicated. After 48 h, ?-galactosidase activities of
whole-cell extracts were assayed as described previously (21). Equal volumes of
whole-cell extracts were subjected to 10% polyacrylamide gel electrophoresis
and immunoblotted with an SF-1 polyclonal antiserum. The specific activities of
SF-1 were calculated from ?-galactosidase activities normalized with the levels of
transfected SF-1-HA protein.
Stable transfection. Y1 cells were transfected with pcDNA3-SF-1-HA or
pcDNA3-SF-1-HA mt1 1 day before subculture in 100-mm culture dishes sup-
plemented with 500 ?g/ml G418 sulfate. Individual clones were generated after
2 weeks of selection. For each construct, 40 to 60 clones were picked, expanded,
and tested for SF-1-HA expression by immunoblotting with antibody against the
HA tag. Two clones expressing the highest amounts of SF-1-HA were further
expanded for subsequent experiments.
ChIP assay. A chromatin immunoprecipitation (ChIP) assay was performed as
described previously, with some modifications (3). Briefly, Y1 SF-1-HA stable
clones were grown to 80 to 90% confluence in 100-mm culture dishes treated
with or without 1 mM 8-Br-cAMP for 6 h. Cells were cross-linked with 1%
formaldehyde for 10 min, and the reaction was stopped with 0.125 M glycine for
5 min at room temperature. Nuclei were collected and sonicated in lysis buffer
(1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1], 0.5 mM
phenylmethylsulfonyl fluoride, and 1? Complete protease inhibitor cocktail
[Roche]). Soluble chromatin was precleared with bovine serum albumin-sheared
herring sperm DNA pretreated Staph A cells and immunoprecipitated with
antibodies against the HA tag (Santa Cruz), acetylated histone H3 (Upstate),
and immunoglobulin G (IgG) control or with no antibody (for input) for 16 h at
4°C. Immunoprecipitates were recovered using Staph A cells. DNA was ex-
tracted from immunoprecipitates by phenol-chloroform extraction and ethanol
precipitation. Extracted DNA was analyzed by PCR using primers spanning the
proximal (nucleotide positions ?71 to ?209) or distal (?873 to ?1141) region
of mouse Cyp11a1. Following 30 cycles of amplification, PCR products were run
on a 1.5% agarose gel and analyzed by ethidium bromide staining.
Electrophoretic mobility shift assay. Electrophoretic mobility shift assays were
performed as described previously, with some modifications (16). SF-1-HA con-
taining nuclear extracts was prepared from transfected 293T cells and incubated
with32P-labeled oligonucleotide containing the SF-1-binding sequence on ice for
30 min. Subsequently, samples were separated by 5% native acrylamide gel
electrophoresis. Competition for binding was performed by adding a 100-fold
excess of unlabeled DNA, and supershift was detected by 1 ?g rat anti-HA
antibody. The intensity of each band was quantified using an image reader as
Immunostaining and confocal microscopy. Y1 cells were subcultured on cov-
erslips at a density of 1 ? 104cells/well in 6-well plates. Before immunostaining,
cells were treated with 1 mM 8-Br-cAMP for 6 h or transfected with the expres-
sion plasmids as indicated for 24 h by using Lipofectamine Plus according to the
manufacturer’s protocol. Treated or transfected cells were fixed in methanol at
?20°C (or in 4% paraformaldehyde for green fluorescent protein fusion pro-
teins) for 3 min, permeabilized with 0.2% Triton X-100–phosphate-buffered
saline, and blocked in 2% blocking reagent (Roche). Subsequently, the cells were
immunostained with antibody against SF-1, p300 (clone RW128, no cross-reac-
tion with CBP; Upstate), RNA Pol II (C21, recognizing both hyper- and hypo-
phosphorylated Pol II; Santa Cruz), c-Myc, or FLAG as indicated in blocking
buffer at 4°C overnight. The cells were washed with 0.2% Triton X-100–phos-
phate-buffered saline and stained with Alexa 488- or Alexa 546-conjugated sec-
ondary antibodies (Molecular Probes) in blocking buffer at room temperature
for 1 h. After three extensive washes, the coverslips were mounted on glass slides
in 50% glycerol–phosphate-buffered saline. Fluorescent cells were examined with
a Zeiss LSM 510 confocal microscope.
Measurement of colocalization. The percentage of protein colocalization was
determined by using LSM software release 3.2 (Carl Zeiss). Briefly, single-cell
images were obtained from dual-immunolabeled Y1 cells treated with or without
1 mM 8-Br-cAMP by using confocal laser scanning microscopy. Optical sections
from the middles of cells were used for the generation of scatter plots. The
thresholds for both red and green fluorescent signals were determined after
reducing the backgrounds of cell images to the lowest setting, and the same
condition was applied to all images. Eighteen cells were randomly selected and
analyzed in four separate immunostaining experiments in the absence or pres-
ence of 1 mM 8-Br-cAMP treatment. The percentage of SF-1 or Pol II colocal-
ization with p300 was calculated by dividing pixels that were colocalized with
p300 in area 3 by the total pixels in areas 2 and 3, with error bars representing
Coimmunoprecipitation assay. Y1 cells were grown in 10-cm-diameter plates
for 24 h and transfected with 3 ?g expression plasmids for 3xFLAG-SF-1. After
24 h, cells were treated with or without 1 mM 8-Br-cAMP for 6 h. Whole-cell
extracts were prepared in IPH buffer containing 240 mM NaCl. Equal amounts
of whole-cell extracts were used for coimmunoprecipitation with 2 ?g anti-p300
antibody (N-15; Santa Cruz) for 1 h at 4°C. The immunoprecipitates and 10%
input of whole-cell extracts were separated by 7% polyacrylamide gel electro-
phoresis, followed by immunoblotting with antibodies against p300, SF-1, or Pol
II, and visualized by chemiluminescence.
SF-1 is acetylated by p300 in vitro and in vivo. SF-1 is a zinc
finger protein. Many zinc finger proteins, like GATA-1, EKLF,
and AR, are acetylated around the zinc finger region (4, 14,
57). Given that SF-1-mediated transcription can be potentiated
by p300/CBP (36), we reasoned SF-1 might be acetylated by
these HATs as well. To test this hypothesis, the FP domain of
SF-1, which contains the Ftz-F1 box and the Pro-rich sequence,
was fused with GST to be a substrate for the in vitro acetylation
assay (Fig. 1A). The acetylation enzymes tested were the HAT
domains from p300 (aa 1195 to 1673) and PCAF (aa 352 to
832). As shown in Fig. 1B, the HAT domains of p300 and
PCAF acetylated their known substrate, histone H3 (lanes 2
and 3), but not GST (lanes 7 and 8), showing that both HATs
were active and substrate specific. GST-FP was acetylated ef-
ficiently by p300 but poorly by PCAF (Fig. 1B, lanes 5 and 6).
This result suggests that the FP domain of SF-1 is preferen-
tially acetylated by p300 rather than by PCAF.
To examine whether SF-1 is acetylated by p300 in vivo, we
expressed both SF-1-HA and p300 in 293T cells and incubated
the cells with [3H]acetate before immunoprecipitating SF-1-
HA. In the absence of overexpressed p300, the [3H]acetyl
group was incorporated into SF-1, indicating that SF-1 was
acetylated at a basal level (Fig. 1C, top, lane 2). This basal level
of acetylation was enhanced by p300 WT, as revealed by the
increased amount of acetyl-SF-1 (Fig. 1C, lane 3). Thus, p300
can acetylate SF-1 in vivo. A HAT-defective p300 (p300 DY),
however, could not acetylate SF-1 to the same extent as p300
WT could (Fig. 1C, lane 4 versus lane 3), implying the impor-
tance of the HAT activity of p300 in SF-1 acetylation. Our
control experiment showed that equivalent amounts of SF-
1-HA were expressed and immunoprecipitated from each lane
(Fig. 1C, middle), indicating that the increased level of acetyl-
SF-1 was due to increased acetylation, not due to an increased
amount of SF-1. These results indicate that p300 transfers
acetyl groups to SF-1 in vivo and in vitro and that the acety-
lation site lies in the FP domain of SF-1.
10444CHEN ET AL.MOL. CELL. BIOL.
Mutation of the KQQKK sequence in the Ftz-F1 box abol-
ishes p300-promoted acetylation of SF-1. In order to map the
p300 acetylation site of SF-1, we searched and identified two
potential acetylation sequences in SF-1: one in the middle
of the DNA-binding domain, similar to acetylation sequence
KXXKXXXK in histone H3, and the other at the C terminus
of the Ftz-F1 box, similar to p53 acetylation sequence KXKK
(15). Both sequences are conserved in SF-1 proteins from
several species (Fig. 2A). To examine whether these two sites
could be acetylated, we replaced the Lys residues of both
potential acetylation sites with Arg to form mt1, mt2, and dmt
(Fig. 2A). In vivo acetylation assays demonstrated that the
acetylation level of SF-1 mt1, in which the KQQKK motif in
the Ftz-F1 box was mutated, was much lower than that of the
wild-type SF-1 even in the presence of p300 (Fig. 2B, lane 2
versus lane 1). The lack of p300-specific acetylation in SF-1
mt1 indicated that p300 specifically acetylated this KQQKK
sequence. The acetylation level of SF-1 mt2 was unchanged
(Fig. 2B, lane 3), thus indicating that the Lys residues in
KXXKXXXK are not p300 substrates. SF-1 dmt, mutated at
both sites, also exhibited a low acetylation level (Fig. 2B,
lane 4). Collectively, these results indicate that the KQQKK
motif in the Ftz-F1 box of SF-1 is acetylated by p300.
The HAT activity of p300 is required for full activation of
SF-1. Since SF-1 is acetylated by p300, we next investigated
FIG. 2. The KQQKK sequence at the Ftz-F1 box of SF-1 is acety-
lated by p300 in vivo. (A) Potential acetylation sites of SF-1. The
amino acid sequences of two potential acetylation sites of SF-1 are
shown, and the conserved Lys (K) residues are marked. In mutated
SF-1 (mt1, mt2, and dmt), the K residues were replaced with Arg (R).
F, Ftz-F1 box; P, proline-rich domain; and LBD, ligand-binding do-
main. (B) Mutation of KQQKK sequence abolishes p300-enhanced
acetylation of SF-1. The incorporation of [3H]acetate into wild-type
(WT) and mutated SF-1 following expression and immunoprecipita-
tion (IP) of SF-1-HA are shown; the amounts of immunoprecipitated
SF-1 and p300 expression were determined by SF-1 and c-Myc immu-
noblotting (IB), respectively.
FIG. 3. p300 HAT activity is required for full activation of SF-1.
Expression plasmids for SF-1-HA (0.2 ?g) and increasing amounts of
p300 WT (0.1, 0.5, and 1 ?g) or p300 DY were cotransfected with 0.3
?g SF-1-dependent reporter gene phscc-2.3K-LacZ into NIH 3T3
cells. (A) The specific activity of each transfectant consisted of the
?-galactosidase (?-gal) activity normalized with the SF-1 expression
level. The specific activity of SF-1-HA alone was set to 1. All values
represent the results of at least three separate transfection experi-
ments, with error bars representing standard deviations. ?, P of ?0.05,
and ??, P of ?0.01, compared with wild-type SF-1-HA transfection
only, by Student’s t test. (B) Immunoblotting (IB) for expression of
SF-1-HA (top) and c-Myc-tagged p300 (bottom; transfection with 1?g
plasmid) of cell lysate from panel A. WT, wild type. Triangles and
trapezoids denote increasing amounts of p300.
VOL. 25, 2005cAMP-INDUCED SF-1 ACETYLATION MEDIATED BY p300 10445
whether this acetylation regulates its activity. SF-1 activity was
measured by its ability to activate an SF-1-dependent reporter
gene, phscc-2.3K-LacZ, which contains a 2.3-kb CYP11A1 pro-
moter linked to a reporter. NIH 3T3 cells were chosen because
they express less endogenous p300. The specific activity of SF-1
was calculated from the reporter gene activity normalized
against the protein level of SF-1, obtained after immunoblot-
ting of whole-cell lysate (Fig. 3B). As shown in Fig. 3A, the
specific activity of SF-1 was enhanced by p300 WT in a dose-
dependent manner. Although expressed at a level comparable
to that of p300 WT, the HAT-defective p300 (p300 DY) could
not potentiate SF-1 activity as efficiently. Since p300 DY was
also less effective in acetylating SF-1 (Fig. 1C), the ability of
p300 to acetylate SF-1 parallels that of p300 to potentiate SF-1.
These observations indicate that the HAT activity of p300 is
required for full activation of SF-1.
Acetylation-deficient SF-1 cannot be fully activated by p300.
We next examined the role of p300 acetylation in transcription
by comparing the transcriptional activities of wild-type and
acetylation-deficient SF-1 in the presence or absence of p300.
p300 enhanced the activities of wild-type SF-1 and mt2 but not
of mt1 and dmt (Fig. 4A and B). Since mt1 and dmt were not
acetylated by p300, the lack of p300 acetylation correlates with
the loss of p300-enhanced transcriptional activity. This result
indicates the importance of the KQQKK motif in p300-poten-
tiated transcription activity of SF-1.
It has been proposed that acetylation of histone H3 neutral-
izes the positive charge of Lys, leading to a disruption of charge
interactions between histone H3 and DNA, chromatin remod-
eling, and transcription activation (8). In order to assess
whether SF-1 acetylation follows a similar mechanism, we re-
placed Lys of the KQQKK motif with neutral amino acids Gln
and Ala to form SF-1 mt3 and mt4, respectively (Fig. 4A). It
was expected that higher transcriptional activities would have
been seen for mt3 and mt4 had charge neutralization had an
effect on transcriptional activity. On the contrary, the basal
transcriptional activities of mt3 and mt4, similar to those for
mt1, were significantly lower than that of wild-type SF-1
(Fig. 4C). SF-1 mt2, whose mutation site is not in the KQQKK
motif, had activity similar to that of wild-type SF-1. Therefore,
simple charge neutralization cannot explain the function of
acetylation in regulating SF-1 activity.
FIG. 4. The KQQKK sequence is required for p300-potentiated activation and basal activity of SF-1. (A) Schematic representation of SF-1
constructs. The amino acid sequences of two potential acetylation sites are shown. Lys (K) residues were replaced with Arg (R), Gln (Q), or Ala
(A) as indicated. (B) Lys-to-Arg mutation at the KQQKK motif of SF-1 (mt1) diminishes its p300-potentiated transcriptional activity. The specific
activities of SF-1 were determined in the presence (p300 WT) or absence (Vector) of p300 in NIH 3T3 cells. (C) Mutation of K to Q or A at the
KQQKK motif of SF-1 reduces its basal transcriptional activity. The specific activities of wild-type and mutated SF-1 expressed in NIH 3T3 cells
are shown. ?, P of ?0.05, and ??, P of ?0.01, compared with wild-type SF-1-HA transfection, by Student’s t test. (D) The KQQKK sequence is
not required for full activation of SF-1 when linked to the DBD of Gal4 protein. Y1 cells were transfected with the 5?Gal4-tk-Luc reporter gene
and expression plasmids for Gal4-DBD (G-DBD), wild-type Gal4-SF-1 (G-SF-1 WT), or mutated Gal4-SF-1 (G-SF-1 mt1). The transactivation
activity (n-fold) of each protein relative to that of the G-DBD control is shown as mean ? standard deviation on the y axis. F, Ftz-F1 box; P,
proline-rich domain; LBD, ligand-binding domain; WT, wild type; and ?-gal, ?-galactosidase.
10446CHEN ET AL.MOL. CELL. BIOL.
Since the Ftz-F1 box helps specific DNA recognition
through the DBD (52), we next examined whether abolishment
of acetylation by mutation would affect SF-1 function if the
ability to bind DNA was retained. The DBD of SF-1 was
replaced by the Gal4 DBD, which would bind the Gal4 recog-
nition site, and the function of the resultant G-SF-1, which
contains either the wild-type or mutated Ftz-F1 box, was
tested. Both wild-type and mutated G-SF-1 could activate the
reporter gene through the Gal4 recognition sequence, and the
presence of p300 could potentiate both activities (Fig. 4D).
This result indicates that acetylation-deficient SF-1 can still
activate transcription as long as it can bind DNA through other
FIG. 5. Mutation of the acetylation site in the Ftz-F1 box of SF-1 results in decreased binding to DNA. (A) ChIP. Soluble chromatin was
prepared from stable Y1 cell clones expressing SF-1-HA (clones no. 18 and no. 55) and SF-1-HA mt1 (clones no. 11 and no. 23) and
immunoprecipitated with antibodies against HA, acetyl-H3 (AcH3), or IgG control. DNA in the extracts was further analyzed by PCR using
primers that cover the regions of nucleotide positions ?71 to ?209 or ?873 to ?1141 of the Cyp11a1 gene, as indicated. Protein expression levels
of SF-1 in stable clones were determined by immunoblotting (IB) with antiserum against SF-1 (bottom). (B) Wild-type SF-1-HA (SF-1-HA WT)
or mutated SF-1-HA (SF-1-HA mt1) was coexpressed with p300 in 293T cells prior to DNA binding and electrophoretic mobility shift assays. The
DNA-SF-1 complex was formed with increasing amounts of nuclear (Nu.) extract (0.25, 0.5, 1, 2, and 4 ?g). Com, competition with 100? unlabeled
oligonucleotide; Ab, supershift with anti-HA antibody. The immunoblot with anti-HA antibody showing both WT and mt1 expressed at the same
level is shown at bottom. The triangle and trapezoid denote increasing amounts of nuclear extracts. (C) The relative binding abilities of SF-1-HA
WT and SF-1-HA mt1 are presented as the ratios of bound probe/total probe versus the amounts of nuclear extract.
VOL. 25, 2005 cAMP-INDUCED SF-1 ACETYLATION MEDIATED BY p30010447
DBDs. Thus, the acetylation of the Ftz-F1 box probably affects
only the DNA-binding ability of SF-1.
Mutation of the KQQKK motif in SF-1 results in reduced
DNA binding. We next examined whether acetylation of the
Ftz-F1 box modulates the DNA-binding activity of SF-1. To
compare DNA-binding activities of wild-type and acetylation-
deficient SF-1 in vivo, we established mouse adrenocortical
tumor Y1 cell clones that overexpress similar amounts of wild-
type and mt1 SF-1-HA proteins (Fig. 5A, bottom). Two clones
expressing wild-type SF-1-HA (no. 18 and no. 55) and two
clones expressing mt1 (no. 11 and no. 23) were tested. After
ChIP using antibody against the HA tag, SF-1-HA bound to
the region of nucleotide positions ?71 to ?209 of Cyp11a1,
which contains an SF-1-binding site. SF-1-HA mt1, however,
bound to this region with a reduced efficiency (Fig. 5A, left).
As a control, acetylated histone H3 from all four clones of cells
bound to this region of DNA equally well, and IgG did not pull
down chromatin. No proteins bound to intron 1 of Cyp11a1
(?873 to ?1141), indicating the specificity of the assay (Fig.
5A, right). These results show that mutation of the acetylation
site reduces the DNA-binding ability of SF-1 in vivo.
To further confirm that the acetylation site is important for
DNA binding, electrophoretic mobility shift assays were per-
formed with nuclear extract from 293T cells cotransfected with
p300 and SF-1-HA wt or mt1 as a source of acetylated or
unacetylated SF-1. As shown in Fig. 5B, the DNA-SF-1 com-
plex was increased when the amount of nuclear extract was
increased (lanes 1 to 5). This DNA-SF-1 complex was specific,
as it disappeared when competed with 100-fold of unlabeled
oligonucleotide (Fig. 5B, lane 6) or supershifted with anti-HA
antibody (Fig. 5B, lane 7). Although expressed at the same
level as wild-type SF-1 (Fig. 5B, bottom), SF-1 mt1 bound to
DNA less efficiently (Fig. 5B, lanes 8 to 12). After quantitation
of the protein-DNA complexes, we found that SF-1 mt1 bound
FIG. 6. p300 recruits SF-1 to p300-RNA Pol II loci. (A) SF-1 partially colocalizes with p300 in the basal condition. Y1 cells grown on coverslips
were fixed and labeled with antibodies against SF-1 (red) and p300 (green). Immunofluorescence staining was analyzed by confocal laser scanning
microscopy. The area marked by a rectangle is enlarged in panel B. The arrows indicate yellow colors as a result of colocalization of SF-1 and p300.
(C) p300 foci contain RNA Pol II. Expression plasmid for c-Myc-tagged p300 was transfected into Y1 cells. Fixed cells were labeled with antibodies
against RNA Pol II (red) and c-Myc (green). An optical section of immunofluorescence staining is shown. (D) The HAT activity of p300 is not
required to recruit SF-1 to p300-foci. Y1 cells were transfected with expression plasmid for c-Myc-tagged p300 WT (top), p300 DY (middle), or
FLAG-tagged GCN5 (F-GCN5) (bottom). Fixed cells were labeled with antibodies against SF-1 (red), c-Myc (green), or FLAG (green). Optical
sections of immunofluorescence staining are shown.
10448CHEN ET AL.MOL. CELL. BIOL.
to DNA at a reduced efficiency (Fig. 5C). These results suggest
that mutation of the KQQKK sequence of SF-1 results in
reduced DNA-binding activity.
p300 recruits SF-1 to nuclear foci. Previously, we reported
that conjugation of SF-1 by SUMO changes its nuclear distri-
bution (7). It is interesting to ask whether p300 acetylation
would also modulate the distribution of SF-1. We performed
double-color immunostaining with antibodies against SF-1 and
p300 in Y1 cells. SF-1 and p300 were each located in discrete
nuclear domains of the nucleus (Fig. 6A); some of these signals
overlap when examined in the magnified image (Fig. 6B). p300
was located in transcriptionally active loci, as shown by colo-
calization of p300-c-Myc with Pol II (Fig. 6C). Therefore, SF-1
was partly activated in the basal condition. When Y1 cells were
transfected with p300-c-Myc, the spots of SF-1 and p300-c-Myc
colocalization were increased (Fig. 6D), indicating that SF-1
could be recruited by p300 to these transcriptionally active loci.
As described above, p300 DY that lost acetyltransferase
activity could still partially activate SF-1 activity (Fig. 3). We
then examined whether p300 DY retained the ability to recruit
SF-1 to p300 loci, and we found that it could (Fig. 6D). In line
with this observation, acetylation-deficient SF-1, mt1, was also
localized to these foci in the presence of p300 (data not
shown). Thus, HAT activity of p300 and acetylation of SF-1 are
not required for SF-1/p300 colocalization. We also checked
whether the other HAT enzyme, GCN5, could target SF-1 to
discrete nuclear loci. Both Flag-GCN5 and SF-1 signals were
FIG. 7. cAMP increases colocalization of SF-1 and p300. (A) Y1
cells were treated without (control [CTRL]) or with 1 mM 8-Br-cAMP
for 6 h. Fixed cells were labeled with antibodies against p300 and SF-1
(top) or RNA Pol II (bottom). After confocal laser scanning micros-
copy, the scatter diagrams for each staining were generated and are
shown. The thresholds for scoring p300, SF-1, or Pol II staining as
positive are shown as vertical or horizontal lines in each panel. Area 3
in each scatter plot represents pixels showing strong staining for both
p300 and SF-1 (Pol II). (B) Measurement of p300 colocalization with
SF-1 or Pol II. Eighteen cells were randomly selected and analyzed in
four separate immunostaining experiments in the absence (CTRL) or
presence of 1 mM 8-Br-cAMP treatment. The percentages of SF-1 or
Pol II colocalization with p300, calculated by computer software, are
FIG. 8. cAMP increases the acetylation and DNA-binding activity
of SF-1 in vivo. (A) Mutation of KQQKK sequence abolishes cAMP-
induced acetylation of SF-1. The expression plasmids for wild-type
(WT) 3xFLAG-SF-1 (3?F-SF-1) or mutated mt1 were transfected into
Y1 cells. After 24 h, cells were labeled with [3H]acetate in the presence
(?) or absence (?) of 1 mM 8-Br-cAMP for 6 h. The results for
acetyl-3xFLAG-SF-1 (top) following immunoprecipitation (IP) with
anti-FLAG antibody are shown; the amounts of immunoprecipitated
3xFLAG-SF-1 were determined by SF-1 immunoblotting (IB) (bot-
tom). (B) cAMP increases the DNA-binding activity of WT but not
acetylation-deficient SF-1. Stable Y1 cells expressing SF-1-HA (clone
no. 18) and SF-1-HA mt1 (clone no. 11) were treated with (?) or
without (?) 1 mM 8-Br-cAMP for 6 h. ChIP assays were performed
with antibodies against HA or acetyl-H3 (AcH3). The results of PCR
amplification using primers that cover the region of nucleotide posi-
tions ?71 to ?209 of the Cyp11a1 gene are shown. Numbers below
each lane are quantitations of the band intensities.
VOL. 25, 2005 cAMP-INDUCED SF-1 ACETYLATION MEDIATED BY p30010449
uniformly distributed in the nucleus (Fig. 6D), suggesting that
p300 but not GCN5 can direct SF-1 to discrete foci in the
Cyclic AMP increases the colocalization of SF-1 and p300.
The activity of SF-1 to stimulate steroidogenic genes in Y1
cells is modulated by the cAMP signaling pathway. To deter-
mine how cAMP modulates SF-1 activity, we examined the
distribution of SF-1 and its pattern of colocalization with p300
in the nucleus following cAMP stimulation (Fig. 7). In the
absence of 8-Br-cAMP, many of the SF-1-positive signals
were devoid of p300 signals (Fig. 7A). In the presence of 8-Br-
cAMP, most of the SF-1-positive signals were also high in p300
signals. Quantification showed that the pixels of SF-1 and p300
colocalization increased from 47% to 78% after cAMP stimu-
lation (Fig. 7B). As a control, cAMP stimulation does not
affect the colocalization of p300 and Pol II. This result suggests
that cAMP might target SF-1 to p300-positive foci.
Cyclic AMP increases the acetylation and DNA-binding ac-
tivity of SF-1. Based on the result that cAMP stimulation
increases the colocalization of SF-1 and p300 in Y1 cells, we
next examined whether cAMP enhances the acetylation of
SF-1. As shown in Fig. 8A, a basal level of acetyl-SF-1 was
detected in Y1 cells expressing FLAG-tagged wild-type 3xF-
SF-1, and the acetylation signal was increased after cAMP
stimulation. In Y1 cells expressing the mutated SF-1 mt1, very
little basal acetylation was detected, and 8-Br-cAMP did not
enhance its acetylation. This result suggests cAMP stimulates
the acetylation of the KQQKK sequence of SF-1. To further
investigate the function of cAMP stimulation in DNA binding
in vivo, we performed a ChIP assay. Cyclic AMP also increased
the DNA-binding activity of wild-type SF-1-HA in vivo
(Fig. 8B) but had no effect on the binding of mutated SF-1-HA
mt1 to DNA. Collectively, these results suggest that cAMP
induced the increase of SF-1 acetylation and its binding to
DNA and that the KQQKK sequence is the target of this
Cyclic AMP increases the interaction between SF-1 and
p300. To analyze the interaction of SF-1 and p300 after cAMP
stimulation, 3xF-SF-1 expressed from Y1 cells was coimmuno-
precipitated with an anti-p300 antibody. As shown in Fig. 9,
both wild-type and mutated 3xF-SF-1 mt1 were efficiently co-
precipitated with endogenous p300 (lanes 1 and 3, top), and
more SF-1 proteins were coprecipitated after cAMP stimula-
tion (lanes 2 and 4 versus lanes 1 and 3, respectively). The
amount of p300 was increased by cAMP stimulation (Fig. 9,
lanes 2 and 4), while the protein level of Pol II was unchanged.
Thus, cAMP increases the amount of p300 and also enhances
the binding of SF-1 to p300.
In this report, we demonstrate that SF-1 is modified and
regulated by p300 through acetylation and localization to spe-
cific nuclear foci. Mutation of the KQQKK acetylation se-
quence in the Ftz-F1 box results in decreased DNA binding as
well as reduction of basal and p300-potentiated transcriptional
activity of SF-1. In addition, SF-1 acetylation, its localization to
p300-positive nuclear foci, and its binding to DNA can be
enhanced by cAMP signaling through an increased amount of
p300. These results indicate a novel mechanism for SF-1 acti-
vation in response to cAMP stimulation.
Acetylation modulates the function of the Ftz-F1 box. We
show that SF-1 is acetylated at the KQQKK motif of the Ftz-F1
box by p300. In addition, mutation of the acetylation sequence
results in a reduction of the DNA-binding activity of SF-1;
therefore, acetylation of the Ftz-F1 box correlates with in-
creased DNA binding.
How acetylation affects DNA binding is still not fully under-
stood. Our data show that this is unrelated to charge neutral-
ization (Fig. 4C). It appears that the site of acetylation in
relation to the DNA-binding domain may affect the ability of
proteins to bind DNA. Acetylation of DNA-binding domains
of YY1 (56) and HMGI(Y) (38) decreases DNA binding; on
the contrary, acetylation near the DNA-binding domain gen-
erally increases the DNA-binding activity of transcription fac-
tors such as p53 (15, 38), E2F1 (33), GATA-1 (4), and EKLF
(57). Acetylation of the Ftz-F1 box near the core DNA-binding
sequence of SF-1 also increases its DNA-binding activity. One
possible mechanism for the increased DNA-binding activity is
that acetylation modifies SF-1 conformation to favor DNA
binding, as has been hypothesized for p53 before (15). Further
investigation should be conducted to uncover the underlying
Acetylation of SF-1. We show that SF-1 is acetylated at the
KQQKK motif by p300. Aside from acetylation catalyzed by
p300, a basal level of acetylation was observed for wild-type
and mutated SF-1 (Fig. 1C and Fig. 2B). This implies that SF-1
FIG. 9. cAMP increases SF-1/p300 association in Y1 cells. The
expression plasmids for wild-type (WT) 3xFLAG-SF-1 (3xF-SF-1) or
mutated mt1 were transfected into Y1 cells. After 24 h, cells were
treated with (?) or without (?) 1 mM 8-Br-cAMP for 6 h. Results of
immunoblotting (IB) of SF-1 and p300 recovered from immunopre-
cipitates (IP) of cell lysates using anti-p300 antibody are shown. Im-
munoblotting of SF-1, p300, and Pol II in 10% of input cell lysate
(Input) are shown at the bottom.
10450 CHEN ET AL.MOL. CELL. BIOL.
has multiple acetylation sites for different HATs, such as CBP/
p300 and GCN5/PCAF. Indeed, SF-1 is acetylated by GCN5
(25). GCN5, however, probably did not acetylate the FP do-
main of SF-1 since its homologue, PCAF, acetylates FP poorly
in vitro (Fig. 1B).
In our study (Fig. 1C), we observed decreased but not absent
acetylation after transfection of p300 DY, which lacks acetyl-
transferase activity (23). This is probably because p300 DY can
still recruit SF-1 to nuclear foci (Fig. 6D), where p300 might
function as a scaffold to attract other HATs, like PCAF/GCN5
(6), which can in turn acetylate SF-1. Current investigations
demonstrate that SF-1 can be acetylated by both p300 (Fig. 1)
and PCAF/GCN5 (25) and that acetylation by both HAT pro-
teins correlates with transcriptional activation. This phenome-
non is similar to that of p53, which can be modified by p300
(15) and PCAF (32).
Jacob et al. reported that TSA, an inhibitor of histone
deacetylase, induced the export of SF-1 from the nucleus to the
cytoplasm (25). We also examined the effect of TSA on SF-1 in
Y1 cells but obtained different results. We found that TSA
downregulated SF-1 expression in Y1 cells. This has been con-
firmed by promoter analysis, Western blot analysis, and immu-
nofluorescence detection (our unpublished data). We suspect
that Jacob et al. also failed to detect reasonable SF-1 expres-
sion after TSA treatment and that what they observed is prob-
ably overexposure of residual amounts of partially degraded
SF-1 located in both nucleus and cytoplasm.
Acetylation of steroid receptors. In addition to SF-1, other
steroid receptors, such as AR and estrogen receptor (ER), are
also substrates for acetylation (14, 54). The acetylation se-
quences of SF-1, AR, and ER are similar, either KXKK or
KXXKK, but different HATs acetylate these sites with differ-
ent efficiencies: ER is selectively acetylated by p300, while AR
is efficiently acetylated by both p300 and PCAF. Our data show
that KQQKK of SF-1 is preferentially acetylated by p300
rather than by PCAF in vitro. The mechanism governing sub-
strate specificity of HATs is still unclear.
Mutation of the acetylation site of AR reduces its ligand-
dependent transactivation function by lowering its ligand sen-
sitivity (14). In contrast, mutation of the acetylation site of ER
increases its ligand sensitivity (54). Unlike these two steroid
receptors, mutation of the acetylation site does not affect the
transactivation function of SF-1 (Fig. 4D). We show that
KQQKK acetylation correlates with increased DNA-binding
activity of SF-1 (Fig. 5). Thus, although these steroid receptors
can be similarly acetylated at similar sites, the effects of their
acetylation are vastly different. What controls this apparent
functional difference remains to be further investigated.
SF-1 localization to p300 foci in the nucleus. The cell nu-
cleus is compartmentalized, and various nuclear foci that exert
different biological actions have been described previously.
Activated transcription factors have been found in specific
nuclear foci (13, 19, 47), and p300/CBP are colocalized with
RNA Pol II in transcriptionally active domains (46, 53). We
found SF-1 in discrete nuclear sites by immunostaining with
two different anti-SF-1 antibodies (Fig. 6A; also data not
shown). This observation is different from the observation of
even distribution of exogenous SF-1 (12; also data not shown).
The reason for the diffuse distribution of exogenous SF-1 is
probably due to the effect of overexpression.
SF-1 constitutively activates basal expression of steroido-
genic genes, and SF-1 activity can be potentiated by p300. This
is consistent with the finding that SF-1 is partially localized to
p300 nuclear foci in Y1 cells, and SF-1 localization to p300
nuclear foci can be enhanced by overexpression of p300. These
results suggest that p300 potentiates the activity of SF-1 by two
means: acetylation and recruitment to specific nuclear foci.
To examine whether SF-1 localization to p300 nuclear foci
requires acetylation, we examined the location of the acetyla-
tion-deficient SF-1, mt1. SF-1 mt1 can interact with p300
(Fig. 9) and be recruited to specific nuclear foci together with
p300 (data not shown). Therefore, acetylation is not required
for recruitment to p300 foci. Consistent with this observation,
the HAT-defective p300 (p300 DY) also has the ability to form
nuclear foci and to recruit SF-1 into them. It appears that p300
first recruits SF-1 to specific nuclear foci and it then transfers
acetyl groups to SF-1, resulting in increased SF-1 binding to
DNA and final gene activation. This mode of action seems
different from that of GCN5, which does not recruit SF-1 to
specific nuclear foci (Fig. 6).
In addition to examining acetylation-deficient SF-1, we ex-
amined other mutated SF-1, including those defective in DNA
binding (G35E) (1), ligand binding (V349W) (31), intrastruc-
ture interaction (R314M) (27), and AF-2 domain (30); all of
these mutants were still localized to p300 nuclear foci (data not
shown). This observation suggests that these mutations may
not abolish the interaction of SF-1 with p300; thus, these mu-
tated SF-1 can be enriched in nuclear foci. These results are
compatible with the recent observation of HIF-1 being re-
cruited to nuclear foci by CBP (45). Only the transcriptionally
inert form of HIF-1, whose CBP-interacting domains were
completely disrupted, cannot interact with CBP and cannot be
recruited to nuclear foci (45). It is believed that p300/CBP foci
may also serve as storage, supply sites, or sites of transcrip-
tional initiation complex regeneration (53). Based on these
observations, we propose that SF-1 localization to p300 foci
may be prior to its binding to cognate DNA and interacting
with other coactivators, such as SRC-1. However, the mecha-
nism of recruiting transcription factors to p300/CBP foci re-
mains unclear and should be further investigated.
It is well known that activation of the cAMP-dependent
PKA signaling pathway can enhance SF-1-mediated transcrip-
tion of many steroidogenic genes (5, 20, 29, 41). Activation of
the PKA signaling pathway reorganizes SF-1/green fluorescent
protein fusion protein from a diffuse distribution pattern to
specific foci (12). In the current report, we further found that
colocalization of endogenous SF-1 with p300 was increased by
the addition of 8-Br-cAMP, whereas RNA polymerase II is
associated with p300 even in the absence of cAMP stimulation.
We found that cAMP increases the amount of p300 in Y1 cells,
leading to increased interaction of SF-1 and p300 and their
colocalization in specific nuclear foci. Furthermore, cAMP en-
hances the acetylation and DNA-binding ability of SF-1
(Fig. 8). These observations provide a direct link between the
cAMP signaling pathway and p300-mediated acetylation and
stimulation of SF-1. Collectively, our results suggest that SF-1
localization to p300 foci and acetylation by p300 might be a
novel regulatory mechanism underlying the activation of the
intracellular cAMP signaling pathway.
VOL. 25, 2005 cAMP-INDUCED SF-1 ACETYLATION MEDIATED BY p30010451
We thank Tso-Pang Yao, Yoshihiro Nakatani, Edward Seto,
Shigeaki Kato, and Dean Hum for pCMV-p300, pET-p300 HAT,
pGEX5X-PCAF-HAT, pcDNA3-FLAG-GCN5, and pcDNA3.1-SF-
1-HA plasmids, respectively. We also thank Ken Deen for editing the
This work was supported by grant NSC 91-2311-B-001-085 from the
National Science Council and by grant AS02IMB4PP from Academia
Sinica, Republic of China.
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