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J. Biol. Chem., Vol. 276, Issue 43, 40146-40155, October 26, 2001
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From the Departments of
Received for publication, July 17, 2001
We examined the phosphorylation and acetylation
of histone H3 in ovarian granulosa cells stimulated to differentiate by
follicle-stimulating hormone (FSH). We found that protein kinase A
(PKA) mediates H3 phosphorylation on serine 10, based on inhibition
exclusively by PKA inhibitors. FSH-stimulated H3 phosphorylation in
granulosa cells is not downstream of mitogen-activated protein
kinase/extracellular signal-regulated kinase, ribosomal S6
kinase-2, mitogen- and stress-activated protein kinase-1, p38 MAPK,
phosphatidylinositol-3 kinase, or protein kinase C. Transcriptional
activation-associated H3 phosphorylation on serine 10 and acetylation
of lysine 14 leads to activation of serum glucocorticoid kinase,
inhibin Maturation of ovarian follicles to a preovulatory stage requires
follicle-stimulating hormone
(FSH)1 production by the
pituitary gland. The FSH receptor is a member of the G protein-coupled
seven-transmembrane receptor family and is coupled to adenylyl cyclase
(1). It is expressed exclusively on ovarian granulosa cells in female
mammals (2). Most of the actions of FSH are mediated by cAMP formation
and activation of protein kinase A (PKA), based on the ability of
cell-permeable cAMP analogs to mimic the known differentiation
responses to FSH in granulosa cells (2) and on the ability of the
PKA inhibitors H892
(3) and KT57202 to inhibit granulosa cell
differentiation. The downstream
consequences of FSH are well established and include, for example, the
induction of receptors for luteinizing hormone (LH) and prolactin,
induction of enzymes associated with the increased steroidogenic
capacity of granulosa cells including P450 aromatase and cholesterol
side chain cleavage, induction of proteins associated with PKA
signaling including RII We recently reported that histones H1 and H3 are also downstream
targets of FSH and PKA in granulosa cells (14). Under conditions in
which FSH stimulates granulosa cell differentiation and not proliferation (2, 15), histone H3 is transiently phosphorylated exclusively on serine 10 (14). The addition of factors that do not
promote granulosa cell differentiation (2), such as epidermal growth
factor (EGF), does not lead to histone H3 phosphorylation (14). This
phosphorylation event appears to represent an early marker of
differentiation and is distinct from the well established marker of
chromosome condensation during mitosis (16-18) in granulosa cells as
these cells undergo differentiation and not proliferation in response
to FSH in serum-free cultures.
While histone H3 phosphorylation on serine 10 has traditionally been
viewed as a marker for mitosis (16-18), there were also reports in the
early 1980s that histone H3 was phosphorylated in response to
differentiative stimuli such as nerve growth factor in PC-12 cells and
cAMP/isoproterenol in C6 glioma cells (19, 20). Ten years later, the
rapid phosphorylation of a small fraction of histone H3 on serine 10 was reported in quiescent C3H 10T1/2 mouse fibroblasts in response to
phorbol esters, okadaic acid, EGF, and protein synthesis inhibitors
coincident with the induction of immediate early genes such as
c-fos and c-jun (21, 22).
Recent reports have identified two related histone H3 kinases as likely
mediators in these pathways. Both ribosomal S6 kinase-2 (RSK-2), which
is downstream of p42/p44 MAPK/ERK, and mitogen- and stress-activated
protein kinase-1 (MSK-1), which is downstream of both MAPK/ERK and p38
MAPK (23), phosphorylate histone H3 in vitro (24-26).
EGF-stimulated histone H3 phosphorylation was restored after the
addition of the RSK-2 gene to a fibroblast cell line derived from
patients with Coffin-Lowry syndrome with mutations in the RSK-2
gene (24). In quiescent C3H 10T1/2 mouse fibroblasts,
anisomycin- and phorbol ester-stimulated histone H3 phosphorylation was
inhibited by the p38 MAPK inhibitor SB203580 (27) and the MAPK/ERK
kinase (MEK) inhibitor PD98059 (28, 29), respectively. Both phorbol
ester- and anisomycin-stimulated histone H3 phosphorylation were
inhibited by H89, based on the ability of this kinase inhibitor to
inhibit MSK-1 (25) rather than its well recognized inhibition of PKA
(30, 31). Similarly in JB6 epidermal C1 cells, ultraviolet B-induced
histone H3 phosphorylation at serine 10 was inhibited by the MEK
inhibitor PD98059 or by the p38 MAPK inhibitor SB202190 (32). These
reports thus establish a prominent role for RSK-2 and MSK-1 downstream
of the p38 MAPK and/or ERK/MAPK pathways in growth factor- and phorbol
ester-stimulated histone H3 phosphorylation.
As FSH stimulates the MAPK/ERK pathway in granulosa cells in a
PKA-dependent manner (9), we hypothesized that, as in
fibroblasts and epidermal cells, histone H3 phosphorylation in
granulosa cells might be mediated by either RSK-2 and/or MSK-1
activated by a MAPK pathway downstream of PKA. We additionally sought
direct evidence that histone H3 phosphorylation is functionally linked to the induction of characteristic FSH-responsive immediate early genes. Finally, based on abundant evidence that histone acetylation is
associated with transcriptional activation (33, 34), we sought to
determine whether acetylation of histone H3 on lysine 14 and/or lysine
9 is stimulated by FSH.
Materials--
Ovine FSH (oFSH-19) was kindly provided by Dr.
A. F. Parlow of the National Hormone and Pituitary Agency of the
National Institute of Diabetes and Digestive and Kidney Diseases
(Torrence, CA). The following were purchased: H89, AG1478, GF109203X,
KT5720, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), phorbol myristic acid (PMA), and okadaic acid from LC Laboratories (San Diego, CA); PD98059,
SB203580, myristoylated PKA inhibitor (PKI) 14-22 amide, and
wortmannin from Calbiochem; anti-histone H3 acetylated on lysines 9 and/or 14 (anti-H3AcLys-9/Lys-14), anti-histone H3 acetylated on lysine 14 (anti-H3AcLys-14) or lysine 9 (anti-H3AcLys-9),
anti-histone H3 phosphorylated on serine 10 (anti-H3PhSer-10),
anti-histone H3 both phosphorylated on serine 10 and acetylated on
lysine 14 (anti-H3PhSer-10/AcLys-14), anti-CREB phosphorylated
on serine 133, and anti-CREB antibodies from Upstate Biotechnology
(Lake Placid, NY); anti-histone H3, anti-MAPK/ERK phosphorylated on threonine 202 and tyrosine 204, anti-p38 MAPK phosphorylated on threonine 180 and tyrosine 182, anti-MSK-1 phosphorylated on serine 376, and anti-RSK phosphorylated on serine 381 (which detects RSK-1 and
RSK-2) antibodies from New England Biolabs/Cell Signaling (Beverly,
MA); anti-p38 MAPK antibody from Santa Cruz Biotechnology (Santa Cruz,
CA); anti-MAPK/ERK antibody from Zymed Laboratories Inc. (San Francisco, CA); anti-HSP27 antibody from
StressGen (Victoria, British Columbia); trichostatin A (TSA) from Wako
(Osaka, Japan); fluorescein isothiocyanate (FITC) from Molecular
Probes, Inc. (Eugene, OR); and anti-hemagglutinin peptide antibody and
H3 purified from calf thymus from Roche Molecular Biochemicals. All
other chemicals were from sources described previously (14, 35, 36).
Equivalent results for SDS-PAGE and acid urea gel blots were obtained
with anti-H3PhSer-10 and anti-H3PhSer-10/AcLys-14 antibodies obtained
from Upstate Biotechnology (and licensed from Dr. David Allis) and
directly from the Allis laboratory. Recognition by anti-H3PhSer-10
antibody of H3 phosphorylated on serine 10 was not affected by
acetylation of lysines 9 and/or 14 (16, 37); recognition by
anti-H3PhSer-10/Lys-14 antibody was selective for H3 phosphorylated on
serine 10 and acetylated on lysine 14 (37).
Production of TAT-PKI Fusion Protein--
An expression vector
containing PKI fused to the cell entry leader sequence TAT was
constructed by PCR. Oligonucleotides
5'-GCCGTTACCTCCCTGCTATGTGGATATTTG and
5'-CCGCTCGAGGCTTTCAGATTTTGCTGCTTCTC, which add the restriction sites KPN and XHO, respectively, were used to amplify PKI using Jurkat
cell cDNA as template. The fragment was cut with the above restriction enzymes and subcloned into the pTAT vector (38) generously
provided by Dr. Steven Dowdy. TAT-PKI protein was expressed in BL21 DE3
pLysS bacteria and purified by fast protein liquid chromatography. The
protein was labeled with FITC and determined to enter cells using a
fluorescence-activated cell sorter (Beckman Instruments). Expressed,
purified protein was tested for functionality by adding it to an
in vitro PKA assay. PKA activity in cell lysates was
stimulated 3.7-fold by the addition of cAMP. This stimulation was
inhibited 84% by 250 nM TAT-PKI, which is equivalent to
the 83% inhibition obtained using the PKI inhibitor peptide.
Granulosa Cell Culture, Western Blotting, and
Immunofluorescence--
Granulosa cells were isolated from the ovaries
of 26-day-old Sprague-Dawley rats that had been primed with
subcutaneous injections of 1.5 mg of estradiol-17 Chromatin Immunoprecipitation (ChIP) Assay--
Following
indicated treatments, granulosa cells were incubated (15 min at room
temperature) in 1% formaldehyde to cross-link DNA and proteins and
then sonicated in cell lysis buffer as described previously (36).
One-tenth of the total lysate was used for purification of total
genomic DNA. The rest of the lysate was incubated with anti-H3PhSer-10,
anti-H3AcLys-9/Lys-14, or anti-H3PhSer-10/AcLys-14 antibodies at
4 °C for 18 h. Following the collection of immunoprecipitates using protein A-agarose, DNA was extracted by phenol/chloroform extraction and ethanol precipitation, and PCR was performed using either total DNA or immunoprecipitated DNA in the presence of [ Immunolocalization of Phospho-histone H3 in FSH-treated Granulosa
Cells--
Phospho-histone H3 is undetectable in vehicle-treated
granulosa cells (Fig. 1C).
Treatment of cells with FSH for 1 h results in the detection of
phospho-histone H3 in small foci in the nuclei of most but not all
granulosa cells (compare Fig. 1, B and D). Overexposure results in complete nuclear staining, as seen in the cell
nucleus detected in Fig. 1D, lower portion.
FSH Stimulates Histone H3, MAPK/ERK, and RSK-2 Phosphorylation in a
Time-dependent Manner--
Recent evidence in fibroblasts
has identified both RSK-2, downstream of p42/p44 MAPK/ERK, and MSK-1,
downstream of both p42/p44 MAPK/ERK and p38 MAPK, as histone H3 kinases
(24, 25). We first compared the rates of phosphorylation/activation of
the p42/p44 MAPK/ERK pathway, histone H3 phosphorylation on serine 10, and CREB phosphorylation on serine 133. Increased phosphorylation of
CREB, an established PKA target (46), is first detected by Western blot
at 1 min and increases further at 10 min after FSH addition; increased
phosphorylation of histone H3 and MAPK/ERK is detected by 10 min, and
increased RSK-2 phosphorylation is detected at 20 min post-FSH (Fig.
2). Histone H3 phosphorylation peaks at
1 h. By 2 h, MAPK/ERK and RSK-2 phosphorylations have returned to basal levels; CREB and histone H3 phosphorylations returned to basal levels by 4 h post-FSH. MSK-1 phosphorylation is
also detected in vehicle-treated cells but is not affected by FSH (not
shown). These results show that FSH promotes both MAPK/ERK and RSK-2
phosphorylation, consistent with our previous evidence that FSH
stimulates RSK phosphorylation in granulosa cells incubated with
32Pi (9).
FSH-stimulated Histone H3 Phosphorylation Is Inhibited by PKA
Inhibitors but Is Not Blocked by MEK, p38 MAPK, EGF Receptor Tyrosine
Kinase, PI-3 Kinase, Protein Kinase C, or RSK-2 Inhibitors--
Based
on evidence in fibroblasts and epidermal cells that histone H3
phosphorylation is blocked by inhibitors of the p38 MAPK and/or the
MAPK/ERK pathways, we tested inhibitors of these and other pathways on
FSH-stimulated histone H3 phosphorylation. Cells were pretreated with
inhibitors for 30, 60, or 90 min, as
indicated in the legend for Fig. 3, and then treated for 1 h with FSH, the adenylyl cyclase activator forskolin, the protein
kinase C activator PMA, or the cell-permeable cAMP analog CPT-cAMP.
Although a 1-h treatment with FSH is optimal for H3 phosphorylation,
MAPK/ERK phosphorylation is declining or has declined to basal levels
by this time (Fig. 2). Neither the MEK inhibitor PD98059 (28, 29) nor
the p38 MAPK inhibitor SB203580 (27), separately or together, inhibits
histone H3 phosphorylation (Fig. 3A). Similarly histone H3
phosphorylation is not altered by the RSK-2/protein kinase C inhibitor
GF109203X (47), although this agent does inhibit the modest stimulation
by PMA of histone H3 phosphorylation (Fig. 3B, lanes
5 and 21). PD98059 inhibits all detectable MAPK/ERK phosphorylation stimulated by FSH and forskolin as well as FSH- and
forskolin-stimulated RSK-2 phosphorylation (Fig. 3A). The p38 MAPK inhibitor SB203580, which has previously been shown to inhibit
FSH-stimulated p38 signaling to HSP27 (10), also abolishes RSK-2
phosphorylation (Fig. 3A), suggesting that in granulosa cells, as in some other cells (48-50), RSK-2 is downstream not only of
MAPK/ERK but also of p38 MAPK. Histone H3 phosphorylation is also
unaffected by the EGF receptor tyrosine kinase inhibitor AG1478 (51),
which consistently abolishes MAPK/ERK and RSK-2 phosphorylation (Fig.
3B). Likewise, H3 phosphorylation is unaffected by the PI-3
kinase inhibitor wortmannin (52) (Fig. 3B), which effectively reduces PI-3 kinase-dependent protein kinase B
(AKT) phosphorylation.3 The increased MAPK/ERK
phosphorylation seen in wortmannin-treated cells could reflect
inhibition by wortmannin of the inhibitory PI-3
kinase-dependent Raf phosphorylation (53).
Only the PKA inhibitors H89 (30, 31) (Fig. 3B) and KT5720
(54) (Fig. 3C) inhibit histone H3 phosphorylation stimulated by FSH, forskolin, or CPT-cAMP. H89 also inhibits MAPK/ERK and RSK-2
phosphorylation, which is consistent with evidence that FSH signals to
MAPK/ERK via PKA (9). The finding that H89 only partially inhibits CREB
phosphorylation (Fig. 3B, lanes 6-9
versus lanes 1-4) concurs with a recent report
(11). To confirm the PKA dependence of histone H3 phosphorylation,
cells were also pretreated without or with the PKA inhibitor protein
PKI linked to the cell-permeabilizing TAT peptide (38, 55) and then
treated for 1 h with vehicle or FSH. Results (Fig. 3D)
show that histone H3 phosphorylation is reduced ~4-fold in cells
treated with TAT-PKI. Similarly, in cells pretreated with a
myristoylated PKI peptide, FSH-stimulated H3 phosphorylation is ablated
(Fig. 3E). These results indicate that histone H3
phosphorylation in granulosa cells in response to FSH treatment is not
downstream of MAPK/ERK, RSK-2, MSK-1, p38 MAPK, PI-3 kinase, or protein
kinase C. Rather, histone H3 phosphorylation appears to be mediated
directly by PKA. In vitro phosphorylation experiments
confirm the ability of recombinant PKA catalytic subunit to directly
phosphorylate purified histone H3 on serine 10 (Fig.
3F).
Effect of FSH on Histone H3 Acetylation--
We next sought to
determine whether the acetylation of histone H3 is linked to its
phosphorylation. The results shown in Fig. 2 show strong reactivity to
anti-histone H3 acetylated on either lysines 9 and/or 14 (anti-H3AcLys-9/Lys-14) antibody in granulosa cells in the absence of
FSH treatment (lane 1). This result suggests that a portion
of histone H3 is already acetylated in untreated cells. Treatment of
granulosa cells with FSH over a 24-h time course does not cause a
detectable change in this bulk acetylation of histone H3 (Fig. 2).
Similarly, neither forskolin, PMA, CPT-cAMP, nor the addition of
protein kinase inhibitors exerts a detectable modulation of histone H3
acetylated on lysines 9/14 (Fig. 3, A-C). To determine
whether a selective deacetylation event precedes histone H3
phosphorylation on serine 10, cells were pretreated with vehicle or
with the histone deacetylase inhibitor TSA. Results show that whereas
pretreatment of cells for 1 h with TSA promotes the expected
increase in the bulk acetylation of histone H3 (Fig. 4A, lanes 6-10
versus lanes 1-5), the FSH-stimulated
phosphorylation of histone H3 on serine 10 is unaffected. Consistent
with the characteristics of the phospho-H3 antibody (37), and in
contrast to another phospho-H3 antibody (56), this result shows that increased acetylation of H3 does not appear to impede the ability of
the anti-H3PhSer-10 antibody to detect phosphorylated H3. This result
also suggests that acetylation does not predispose histone H3 to
phosphorylation. We next sought to determine whether the fraction of
histone H3 that becomes phosphorylated (on serine 10) also becomes
acetylated (on lysine 14) in response to FSH, using an antibody
reactive only with histone H3 both phosphorylated on serine 10 and
acetylated on lysine 14 (anti-H3PhSer-10/AcLys-14) (37). Results show
that, as is true of H3 phosphorylation, this dual modification of
histone H3 is detected by 10 min (Fig. 4B) and increases
with time after FSH treatment of cells (Fig. 4C). Moreover,
the increase in the relative signals detected by the two antibodies
over time is not significantly different at any time point (Fig.
4C). Similar results were obtained when granulosa cell
acid-soluble histones were separated on acid urea gels (Fig. 4D). These results suggest either that FSH stimulates the
phosphorylation of histone H3 molecules that are already acetylated on
lysine 14 or that both modifications of H3 occur concurrently. That FSH most likely stimulates the apparently concurrent phosphorylation and
acetylation of histone H3 is suggested by results showing that FSH also
produces a modest increase by 10 min in the acetylation of histone H3
on lysine 14 (Fig. 4E) but not on lysine 9 (Fig. 4F). Consistent with the results shown in Fig. 3, the PKA
inhibitor H89 abolishes the signal detected by the
anti-H3PhSer-10/AcLys-14 antibody (Fig. 4G). Taken together,
these results indicate that FSH stimulates the phosphorylation of
serine 10 via PKA as well as the acetylation on lysine 14 of a small
fraction of histone H3.
FSH Stimulates the Association of Phosphorylated and
Acetylated Histone H3 with Promoter Sequences of Serum Glucocorticoid
Kinase, Inhibin
Similar to results following immunoprecipitation with anti-H3PhSer-10
antibody, immunoprecipitation with anti-H3AcLys-9/Lys-14 antibody
evokes ~2-fold increased amplification of SGK and inhibin
We additionally assessed whether there is a direct link between both
phosphorylation on serine 10 and acetylation on lysine 14 in histone H3
and promoter activation. Immunoprecipitation with
anti-H3PhSer-10/AcLys-14 antibody results in ~4-fold more amplified
PCR product corresponding to SGK, inhibin
These results demonstrate that although FSH treatment of granulosa
cells does not promote a detectable increase by Western blotting in
bulk levels of acetylated histone H3 (on lysines 9 and/or 14) (Figs. 2
and 3), in the ChIP assay, more promoter DNA characteristic of
FSH response genes is associated with the acetylated histone H3 after
FSH treatment of granulosa cells. These results therefore support our
evidence (presented in Fig. 4E) showing that FSH stimulated
a modest increase in H3 acetylation on lysine 14, and these results are
consistent with the idea that only a small fraction of total cellular
histone H3 is acetylated at specific loci in response to FSH treatment
of granulosa cells. In contrast to bulk histone H3 acetylation levels,
increased levels of phosphorylated histone H3 as well as of histone H3
both phosphorylated on serine 10 and acetylated on lysine 14 are
readily detected by Western blotting in FSH-treated granulosa cells and
are associated with increased promoter amounts of FSH-responsive genes.
These results thus provide direct evidence that both phosphorylation
(on serine 10) and acetylation (on lysine 14) in histone H3 are
associated with FSH-responsive promoters.
FSH stimulates the differentiation of granulosa cells to a
preovulatory phenotype (2). In the intact ovary, FSH also stimulates granulosa cell proliferation (59, 60), although this response to FSH is
lost in serum-free granulosa cell cultures (2, 15), presumably because
of the loss of the contribution of growth factors by adjacent thecal
cells (61). It is generally believed that most of the cellular pathways
downstream of the FSH receptor that lead to both granulosa cell
proliferation and differentiation are mediated, directly or indirectly,
by PKA (2, 4, 62). It is interesting that granulosa cells appear to
express the same signaling pathways that in other cells are activated
by growth factors or phorbol esters and are often inhibited by PKA (63, 64), yet in granulosa cells, these pathways are activated by PKA. A
prime example is the MAPK/ERK pathway, which in granulosa cells is
activated downstream of PKA (9, 10, 65). Thus, granulosa cells must
have evolved unique routes for PKA to modulate these conserved
signaling pathways.
Consistent with the hypothesis that FSH uniquely signals predominately
via PKA, we have shown in this report that histone H3 phosphorylation
in granulosa cells appears to be mediated directly by PKA.
In other cellular models, histone H3 phosphorylation is downstream of
the p38 MAPK and/or MAPK/ERK pathways and mediated by RSK-2 or MSK-1
(24, 25, 32, 66). However in granulosa cells, inhibition of these
pathways (by PD98059, SB203580, GF109203X, or AG1478) does not diminish
FSH-stimulated histone H3 phosphorylation. Therefore in granulosa
cells, histone H3 phosphorylation is not a consequence of the
activation of the MAPK pathways. Our results support the hypothesis
that the catalytic subunit of PKA directly phosphorylates histone H3,
based on stimulation of histone H3 phosphorylation by cell-permeable
cAMP analogs as well as by the adenylyl cyclase activator forskolin and
on inhibition by the ATP competitive inhibitors H89 and KT5720.
Although H89 also inhibits MSK-1 (25), the absence of FSH-stimulated
MSK-1 phosphorylation coupled with the lack of inhibition of
FSH-stimulated histone H3 phosphorylation by inhibitors upstream of
MSK-1 indicates that the effect of H89 in granulosa cells is on PKA and
not on MSK-1. In support of these data, histone H3 phosphorylation was
inhibited on the introduction into granulosa cells of the specific PKA
inhibitor protein PKI using the TAT transducing peptide as well as
inhibited by myristoylated PKI. We and others have shown that histone
H3 is readily phosphorylated in vitro by the catalytic
subunit of PKA purified from mammalian sources (14, 67, 68). We show herein that recombinant purified PKA catalytic subunit also directly phosphorylates histone H3 on serine 10, as detected with the
anti-H3PhSer-10 antibody. Moreover, the catalytic subunit of PKA is
known to translocate into the nucleus of granulosa cells in response to
FSH treatment to phosphorylate nuclear substrates such as CREB (3, 14). Although we have been unsuccessful in our attempts to cross-link histone H3 and the catalytic subunit of PKA, the inability to demonstrate the physical association of these two proteins most likely
reflects the transient nature of this catalytic reaction.
Histones are ubiquitous proteins that organize DNA into nucleosomes and
chromatin. Nucleosomes consist of an inner core of two copies each of
the four core histones, H2A, H2B, H3, and H4, surrounded by 146 base
pairs of double-stranded DNA (69). H1 histones bind to the outer
surface of the DNA that surrounds the core histones and to the
stretches of linker DNA that connect histones (69). There is compelling
evidence that chromatin remodeling involves the modification of core
histones in nucleosomes in activated target genes (33, 70-72). These
core histone modifications generally consist of the covalent addition
of an acetyl or phospho group to lysines or serines, respectively,
primarily in the N-terminal tails of the core histones H3 and H4.
Acetylation neutralizes the positive charge of the histone, and
phosphorylation adds a negative charge to histone, thereby decreasing
the affinity of histone for DNA (33). The predicted result of these
histone modifications is the destabilization of nucleosomes and
chromatin structure, resulting in the access of select promoter regions to transcription factors and co-activators (72, 73). In support of
these suppositions, mutational analysis of yeast Gcn5, one of the well
known nuclear histone acetyltransferases (HATs), indicates a direct
role for histone acetylation in the transcriptional activation of
target genes in vivo (74, 75). It has been similarly shown that the substitution in Gcn5 of arginine 164 for alanine, a residue close to serine 10 in the structure of the ternary Gcn5-CoA-histone H3
complex, reduces promoter activity of a set of
Gcn5-dependent genes; mutation of serine 10 to alanine in
histone H3 impairs transcription of the same set of genes (76).
Recent evidence from yeast and EGF-treated C3H 10T1/2
fibroblasts indicates that acetylation on lysine 14 rapidly follows
phosphorylation on serine 10 in histone H3 (37, 76). Consistent with
these results, in vitro studies show that several HATs,
including p300 and p300/CREB-binding protein (CBP)-associated factor
(PCAF) and Gcn5, display increased HAT activity toward a histone H3
peptide phosphorylated on serine 10 versus the
unphosphorylated or mutated peptide (37, 76). However, these authors
did not detect global modifications in histone H3 acetylation (by
Western blotting with anti-H3AcLys-9/Lys-14 antibody) (37), suggesting
that these modifications are restricted to a small portion of growth
factor-responsive genes in the genome. While our data show that FSH
stimulates both the phosphorylation (on serine 10) and acetylation (on
lysine 14) of histone H3 in primary rat granulosa cells and that both modifications occur on at least a pool of the same H3 molecules, phosphorylation and acetylation occurred in a statistically
simultaneous manner, although in some experiments,
phosphorylation appeared to precede acetylation (Fig.
4C). Additional studies are needed to resolve the precise
timing of H3 phosphorylation versus acetylation in response
to FSH in granulosa cells. It has been proposed that transcriptional
activation requires both the serine 10 phosphorylation and acetylation
on lysine 14 of histone H3, whereas mitotic activity requires only
histone H3 phosphorylation (73). As granulosa cells exhibit
differentiation and proliferation responses to FSH in the absence and
presence, respectively (2), of growth factors, this cell offers an
ideal model to explore this hypothesis.
We detected increased representation of c-fos and SGK
promoter DNA in the phosphorylated, acetylated, and dual-phosphorylated and acetylated histone H3-chromatin pools in response to FSH treatment of granulosa cells in ChIP assays. Similarly in C3H 10T1/2 fibroblasts, EGF-stimulated phosphoacetylation of H3 is associated with
c-fos activation (37, 56). However, the activation of
c-fos in granulosa cells most likely reflects signaling to
the c-fos gene via PKA and CREB rather than the more
commonly used MAPK/ERK pathway (57, 58) as the c-fos
promoter contains a functional cAMP response element (77) that can bind
phospho-CREB, leading to the recruitment of CBP and perhaps other
co-activators to activate the c-fos gene.
Transcriptional activation of the SGK gene in response to FSH/PKA
requires an Sp1/Sp3 binding site (78). Sp1 sites are also required both
for FSH/PKA induction of the LH receptor gene (79) and the cholesterol
side chain cleavage CYP11A gene (80) and for LH/PKA induction of the
progesterone receptor gene (81), but the mechanism by which PKA
modulates Sp1-directed gene activation is not known. Because we have
shown that both phosphorylation and acetylation of histone H3 are
linked to the rapid activation of the SGK gene, based on our evidence
that FSH increases the amount of SGK promoter DNA in immunoprecipitates
of phosphorylated, acetylated, and dual-phosphorylated and acetylated
histone H3, we anticipate that co-activators with HAT activity, such as
CBP or PCAF, may complex with Sp1 at the SGK promoter. These findings also raise the possibility that some of the transcriptional effects of
PKA may reflect H3 phosphorylation and chromatin reorganization rather
than, or in addition to, direct phosphorylation of transcription factors.
We also detected increased representation of inhibin PKA-dependent activation of both CYP11A and StAR genes
requires not only SF-1 but also Sp1 (80, 97). SF-1 and Sp1 have been
shown to bind to nearby sites on the CYP11A promoter (80) and to
physically interact, as demonstrated both in yeast two-hybrid and gel
mobility shift assays (80, 97). As SF-1 can recruit and interact with
CBP (86, 87) and CBP can enhance transcription of CYP11A reporter
constructs (87) and recruit PCAF (89), the role of PKA in the
activation of the CYP11A promoter and possibly the StAR promoter, like
that of inhibin We hypothesize that the phosphorylation on serine 10 and rapid
acetylation on lysine 14 of histone H3 constitutes a necessary step in
the transcriptional activation of FSH responsive genes leading to
granulosa cell differentiation and that the kinase responsible for this
phosphorylation is PKA, as depicted in the model shown in Fig.
6. Perhaps it is histone H3, in its
phosphorylated and acetylated conformation, that functions as a
scaffold to mediate the assembly of the multiprotein complex that leads
to transcription (25, 73). Future studies are planned to evaluate the
components of the multimeric complex associated with phospho-histone H3
at the promoters of the FSH responsive genes in granulosa
cells.
It is also entirely possible that PKA functions to promote histone H3
phosphorylation, acetylation, and resulting gene activation in the many
other physiological models in which hormone actions are mediated by
PKA. Thus, the actions of PKA in granulosa cells to modulate chromatin
structure may be duplicated in target cells for such hormones as
thyroid-stimulating hormone, LH, epinephrine, and vasopressin, for
instance. PKA-mediated histone H3 phosphorylation could prove to be a
universal mechanism to regulate gene activation.
We thank Drs. Carl Peters, Teresa
Woodruff, and Kelly Mayo for intellectual input and Meredith Gonzales
for assistance in immunofluorescence studies. We gratefully acknowledge
the gifts of HSP27 antibody from Dr. Michael Welsh, University of
Michigan, Ann Arbor, Michigan, pTAT vector from Dr. Steven
Dowdy, Washington University School of Medicine, St. Louis, Missouri,
and recombinant PKA catalytic subunit from Dr. Susan Taylor, University
of California at San Diego, La Jolla, California.
*
This work was funded by Grants P01 HD21921 (to
M. H. D. and J. L. J.), GM40922 (to C. D. A), and HD36408 (to
D. W. C.) from the National Institutes of Health and by U. S. Army
USAMRMC Grant DAMD 17-00-1-0386 (to L. M. S.). Preliminary results
were presented at the 13th Ovarian Workshop, 2000, in Madison,
Wisconsin.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.
§
These authors contributed equally to this work.
**
Present address: Wyeth Ayerst Women's Health Research Institute,
145 King of Prussia Rd., Radnor, PA 19087.
¶¶
To whom correspondence should be addressed:
Dept. of Cell and Molecular Biology, Northwestern University Medical
School, 303 East Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8940;
Fax: 312-503-0566; E-mail: mhd@northwestern.edu.
Published, JBC Papers in Press, August 9, 2001, DOI 10.1074/jbc.M106710200
2
D. W. Carr and M. Hunzicker-Dunn,
unpublished data.
3
E. T. Maizels, L. Salvador, and M. Hunzicker-Dunn, manuscript in preparation.
4
O. K. Parke-Sarge, personal communication.
The abbreviations used are:
FSH, follicle-stimulating hormone;
TSA, trichostatin A;
FITC, fluorescein
isothiocyanate;
PKA, protein kinase A;
PKI, PKA inhibitor;
HSP27, heat
shock protein 27;
PI-3 kinase, phosphatidylinositol-3 kinase;
RSK-2, ribosomal S6 kinase-2;
MSK-1, mitogen- and stress-activated protein
kinase-1;
MAPK/ERK, mitogen-activated protein kinase/extracellular
signal-regulated kinase;
MEK, MAPK/ERK kinase;
SGK, serum
glucocorticoid kinase;
HAT, histone acetylase;
StAR, steroidogenic
acute regulatory protein;
EGF, epidermal growth factor;
CPT-cAMP, 8-(4-chlorophenylthio)-cAMP;
CREB, cAMP-response element-binding
protein;
CBP, p300/CREB-binding protein;
PCAF, CBP-associated factor;
ChIP, chromatin immunoprecipitation assay;
PMA, phorbol
myristic acid;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
SF-1, steroidogenic factor-1;
LH, luteinizing hormone;
Ph, phosphorylated;
Ac, acetylated.
Follicle-stimulating Hormone Stimulates Protein Kinase A-mediated
Histone H3 Phosphorylation and Acetylation Leading to Select Gene
Activation in Ovarian Granulosa Cells*
§,
,§**,,,,,¶¶
Cell and Molecular
Biology and ¶ Medicine, Division of Endocrinology,
Metabolism, and Molecular Medicine, Northwestern University Medical
School, Chicago, Illinois 60611, Veterans Affairs Medical Center and Oregon
Health Sciences University, Portland, Oregon 97201, and
§§ Biochemistry and Molecular Genetics,
University of Virginia, Charlottesville, Virginia 22908
ABSTRACT
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DISCUSSION
REFERENCES
, and c-fos genes. We propose that
phosphorylation of histone H3 on serine 10 by PKA in coordination with
acetylation of H3 on lysine 14 results in reorganization of the
promoters of select FSH responsive genes into a more accessible
configuration for activation. The unique role of PKA as the
physiological histone H3 kinase is consistent with the central
role of PKA in initiating granulosa cell differentiation.
INTRODUCTION
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DISCUSSION
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(2, 4) and AKAP80 (5), and expression of the
hormone inhibin (6). However, gene and/or protein induction for these
responses to FSH is generally delayed by at least 24 h (2-4, 7).
The more immediate responses to FSH, which lead to the induction of
immediate early genes such as c-fos and serum glucocorticoid
kinase (SGK) (3, 8), are less well understood. Further elucidation of
the FSH signaling pathways that lead to the induction of immediate
early genes would be useful to understand how FSH initiates granulosa
cell differentiation. We have previously shown that FSH (via PKA)
promotes activation of the p42/p44 mitogen-activated protein
kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (9).
FSH also activates the p38 MAPK pathway (10) and downstream
phosphorylation of the heat shock protein (HSP) 27, leading to
granulosa cell rounding (10).
We3 and others (11)
have recently identified phosphatidylinositol 3-kinase (PI-3 kinase) as
a downstream target of FSH and cAMP. The transcription factor
cAMP-response element-binding protein (CREB) is also phosphorylated in
response to FSH in a cAMP-dependent manner (3, 12, 13).
EXPERIMENTAL PROCEDURES
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on days 23-25 to
promote the growth of preantral follicles (5, 14). Cells were either
plated on 33-mm plastic dishes (Falcon) at a density of ~3 × 106 cells/dish (14) or on coverslips (for
immunofluorescence) and then treated with the indicated additions ~20
h after plating. For subsequent Western blotting, treatments were
terminated by aspirating medium, rinsing cells once with
phosphate-buffered saline, and collecting total cell extracts in 0.5 ml
of SDS sample buffer (39) followed by heat denaturation. Protein
concentrations were controlled by plating identical cell numbers per
plate in each experiment and then loading equal volumes of total cell
extract per gel lane. Ponceau S staining of the final blot was used to confirm equal protein loading. Granulosa cell proteins were separated by SDS-PAGE (10 or 12% acrylamide in running gel) (40) and transferred to Hybond-C Extra nitrocellulose (Amersham Pharmacia Biotech). Blots
were incubated with primary antibody overnight at 4 °C, and
antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). For acid urea gels, cells were lysed, and
acid-soluble histones were extracted from nuclei (37). Acid-soluble histones and H3 standard (Roche Molecular Biochemicals) were loaded in
sample buffer containing 6 M urea, 5% acetic acid, and
12.5 mg/ml protamine sulfate and then electrophoresed through a 1-cm stacking gel and 17.5-cm separating gel (15% acrylamide, 6 M urea, 5% acetic acid) for 24 h at 200 V (37).
Histones were transferred to polyvinylidene difluoride (Millipore
Corporation, Bedford, MA), stained with Ponceau S, and subjected to
Western blotting. For immunofluorescence, cells were treated as
indicated and then fixed with 3.7% formaldehyde and permeabilized with
1% Triton X-100 in phosphate-buffered saline, washed, and incubated
for 2 h at 37 °C with anti-H3PhSer-10 antibody (1:50 dilution)
in phosphate-buffered saline containing 5% normal goat serum.
Coverslips were washed and incubated for 1 h at 37° C with
FITC-conjugated goat anti-rabbit secondary antibody (Jackson
ImmunoResearch, West Grove, PA). Cells on coverslips were washed and
mounted on slides in diazabicyclo[2.2.2]octane antifading medium
(41). Slides were analyzed by a Zeiss Axiovert 100M confocal
microscope. For introduction of TAT-PKI, cells were plated in 1 ml of
Opti-MEM I medium (Life Technologies, Inc.) on 6-well plastic
dishes (Falcon). Approximately 20 h after plating, TAT-PKI was
added, and cells were incubated for 3 h. Opti-MEM I medium was
then aspirated off cells, serum-free Dulbecco's modified Eagle's
medium/Ham's F-12 medium (5) was added, and cells were treated as
indicated. Treatments were terminated as above, and total cell extracts
were collected in 0.1 ml of SDS sample buffer (39).
-32P]dCTP as described previously (36). PCR products
were separated on 6% nondenaturing polyacrylamide gels followed by
autoradiography. Primers used for PCR correspond to sequences within
the inhibin promoter region (36) (
160 to 141)
(5'-TTGGCGGGAFTGGGAGATAA-3') and (+68 to +49)
(5'-CTCTTGCCCTGACGACAGGG-3'); SGK promoter region (42) (+12 to 11)
(5'-TTAGCCAACAGTGAGCTCCGGCT-3') and (43) (285 to 265)
(5'-GCGGACGGAGGAGGCAGAGTGCAT-3'); c-fos promoter region (44)
(324 to 304) (5'-ACACAGGATGTCCATATTA-3') and (25 to 5)
(5'-TGGAGTAGTAGGCGCCTCAGC-3'); and progesterone receptor promoter
region (45) (376 to 359) (5'-GTGACATACACTCAGAGA-3') and (18 to
1) (5'-GGCTCCACAGCTTTCTAG-3').
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DISCUSSION
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Fig. 1.
Effect of FSH on the phosphorylation of
histone H3 in granulosa cells. Granulosa cells were
treated with vehicle or FSH for 1 h and then subjected to
immunofluorescence using anti-phospho-histone H3 antibody. Results are
representative of more than 5 separate experiments.
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Fig. 2.
Time course of FSH-stimulated phosphorylation
of histone H3, p42/44 MAPK/ERK, CREB, p90 RSK-2, and acetylated
histone H3. Granulosa cells were treated for the indicated times
with 50 ng/ml FSH followed by the preparation of total cell extracts,
as described under "Experimental Procedures." Following SDS-PAGE
and the transfer of proteins to nitrocellulose, blots were first
stained with Ponceau S to confirm equal protein loading per lane and
then probed with the indicated antibodies to phosphorylated
(Ph-) or acetylated (Ac-) proteins. Results are
representative of 2 separate experiments.
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Fig. 3.
Effect of protein kinase inhibitors on
FSH-stimulated histone H3 phosphorylation. A, granulosa
cells were pretreated with 50 µM PD98059 for 90 min, 10 µM SB203580 for 60 min, or both followed by treatment for
60 min with vehicle (Veh), 50 ng/ml FSH, or 10 µM forskolin (For), as indicated. For the rest
of the details, see the legend for Fig. 2. Results are representative
of 2 experiments. B, cells were pretreated with vehicle for
60 min, 10 µM H89 for 60 min, 250 nM AG1478
for 15 min, 100 nM wortmannin for 60 min, or 5 µM GF109203X for 30 min. Cells were then treated for 60 min with vehicle, 50 ng/ml FSH, 10 µM forskolin, 1 mM CPT-cAMP (cAMP), or 200 nM PMA.
Results are representative of 3 experiments. C, cells were
pretreated for 60 min with vehicle or 10 µM KT5720. For
the rest of the details, see the details for panel B. D, cells were pretreated for 3 h with vehicle
or 200 nM TAT-PKI and the medium was changed, followed by
treatment for 1 h with vehicle or FSH, as detailed under
"Experimental Procedures." Protein loading control is the HSP27
Western blot (Welsh antibody). Entry of hemagglutinin-tagged TAT-PKI is
confirmed by anti-hemagglutinin antibody (not shown). Results are
representative of 3 experiments. E, cells were pretreated
for 60 min with vehicle or 50 µM Myr-PKI amide followed
by treatment with vehicle or FSH for 10 min or 1 h. For the rest
of the details, see the legend for Fig. 2. Results are representative
of 2 separate experiments. F, histone H3 (5 µg, Roche
Molecular Biochemicals) was incubated for 38 min at 30 °C with the
indicated concentrations of recombinant (r) PKA catalytic
subunit (C) in a reaction mix containing 100 µM ATP, 10 mM MgCl2, and 50 mM TRIS-HCl, pH 7.5. Following SDS-PAGE of total sample and
transfer to nitrocellulose, blots were processed as described in the
legend for Fig. 2. Results are representative of 2 separate
experiments.
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Fig. 4.
Effect of FSH on the acetylation of histone
H3. A, cells were pretreated without or with the
histone deacetylase inhibitor TSA for 60 min. Cells were then treated
for 60 min with vehicle, 50 ng/ml FSH, 10 µM forskolin,
200 nM PMA, or 1 µM okadaic acid
(OA) followed by the preparation of total cell extracts for
SDS-PAGE and Western blotting. Results are representative of 2 experiments. In panels B, C, E and F, cells were treated for
the indicated times with FSH followed by the preparation of total cell
extracts. Blots were probed with the indicated antibodies.
C, the relative levels of phosphorylated and
phosphoacetylated H3 were quantitated with the Molecular
AnalystTM/PC Image Analysis software program, divided by
the densitometric signal for control protein load, and expressed
relative to the maximal signal, and plotted as a function of min of FSH
treatment. Results at each time point are means ± S.E. from 3 or
4 separate experiments and are not significantly different
(p > 0.01) by Student's t test (98). The
results shown in Fig. 2 and in panel E are from the same
samples; the Ph-CREB blot in Fig. 2 is duplicated in panel
E for reference. HSP27 antibody used in panel E
was obtained from Dr. Michael Welsh. Acetylated H3 relative to HSP27
signal is indicated. D, cells were treated with FSH for the
indicated times, and acid-soluble histones were extracted from nuclei
and electrophoresed in acid urea gels, as detailed under
"Experimental Procedures," and transferred to polyvinylidene
difluoride membranes. Blots were stained with Ponceau S and then probed
with the indicated antibodies. Results are representative of 4 separate
experiments. The H3 standard triplet does not correspond to multiple
modified forms of H3. G, cells were pretreated for 60 min
with vehicle or 10 µM H89 and then treated with the
indicated additions for 60 min followed by the preparation of total
cell extracts. Following SDS-PAGE and protein transfer to
nitrocellulose, blots were probed with antibody to histone H3
phosphorylated on serine 10 and acetylated on lysine 14 or to a control
protein HSP27 (StressGen). Results are representative of 2 experiments.
, and c-fos, but Not with the Progesterone
Receptor--
Finally we sought to determine whether the
phosphorylation of histone H3 in response to FSH promotes the
activation of genes specific for FSH but not those responsive to LH.
Granulosa cells were treated with vehicle or FSH for 1 h,
chromatin was cross-linked with formaldehyde, and histone H3 that was
either phosphorylated on serine 10, acetylated on lysines 9 and/or 14, or phosphorylated on serine 10 and acetylated on lysine 14 was
immunoprecipitated and protein-denatured. PCR was conducted on
immunoprecipitated DNA using primers specific for promoter sequences of
the FSH early response genes SGK and c-fos as well as for
the later response gene inhibin and for the progesterone receptor,
which is an LH early response gene. As a control, PCR was also
conducted on total genomic DNA. SGK and inhibin are established
downstream targets in granulosa cells of FSH and PKA (3, 7), whereas c-fos is generally thought to be downstream primarily of
MAPK/ERK and RSK-2 (57, 58). Results show that there is ~3-fold more amplified PCR product in anti-H3PhSer-10 immunoprecipitates
corresponding to SGK and inhibin promoter sequences and to a lesser
extent to c-fos from cells treated with FSH
versus vehicle (Fig.
5A, lanes 1-4,
lanes 9-12, and lanes 17-20). The response to
FSH for the SGK, inhibin , and c-fos promoters was
completely inhibited by pretreating cells with the PKA inhibitor H89
(compare lanes 1 and 2 with lanes 5 and 6, compare lanes 9 and 10 with
lanes 13 and 14, and compare lanes 17 and 18 with lanes 21 and 22). Promoter
DNA corresponding to the progesterone receptor was not detected in the
phosphorylated histone H3-chromatin pool of vehicle- or FSH-treated
cells (Fig. 5A, lanes 25 and 26),
although it was readily detected in total genomic DNA (lanes
27 and 28). This result is consistent with
evidence that FSH does not promote activation of the progesterone
receptor gene (4).4
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Fig. 5.
Association of histone H3 phosphorylation and
acetylation with activation of FSH-dependent early response
genes. ChIP assays were performed using DNA isolated from vehicle-
or FSH-treated cells. Briefly, granulosa cells were treated for 60 min
with vehicle or FSH or were pretreated for 60 min with 10 µM H89 prior to cell treatment, as indicated. Following
DNA and protein cross-linking with formaldehyde, an aliquot was removed
for purification of total DNA, and immunoprecipitations (IP)
were conducted using anti-histone H3 phosphorylated on serine
(A), anti-histone H3 acetylated on lysines 9 and 14 (B), or anti-histone H3 phosphorylated on serine 10 and
acetylated on lysine 14 (C). DNA was then extracted from
immunoprecipitates, and PCR was conducted on total DNA and
immunoprecipitated DNA with primers to the promoter regions of
indicated genes, as described for the ChIP assay under "Experimental
Procedures." Results are representative of at least 5 separate
experiments for each panel.
promoter DNA (Fig. 5B, lanes 1-4 and lanes
9-12) but not of the progesterone receptor promoter (lanes
13-16). However, in contrast to phospho-histone H3-associated
chromatin, the progesterone receptor promoter is readily detected in
the chromatin pool associated with histone H3 acetylated on lysines 9 and/or 14 (lanes 13-16). Only a minor increase in
c-fos promoter amplification is detected in response to FSH
in the anti-H3AcLys-9/Lys-14 immunoprecipitates (lanes
5-8).
, and c-fos promoter sequences in FSH versus vehicle-treated cells
(Fig. 5C).
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
promoter DNA
in the phosphorylated, acetylated, and dual-phosphorylated and
acetylated histone H3-chromatin pools in response to FSH treatment of
granulosa cells. Inhibin is generally considered to be a relatively
late FSH responsive gene (82) as maximal mRNA levels are not
reached until 12-24 h after FSH addition. However, inhibin mRNA is detectable as early as 4 h after FSH (83).
Interestingly in our ChIP assays, histone H3, which contains the dual
modifications of phosphorylation and acetylation, is already associated
with the inhibin promoter by a 1-h post-FSH treatment.
Perhaps the ChIP assay detects a reorganization of the promoter into a
relatively more accessible configuration for the entry of various
co-activators prior to the activation of transcription. Activation of
the inhibin gene is regulated by the nuclear transcription factor
steroidogenic factor-1 (SF-1) (36). Recent studies have shown that
SF-1, in conjunction with PKA, CREB, and CBP, synergize to strongly
activate the inhibin promoter (36). SF-1 and CREB constitutively
bind to adjacent sites on the inhibin promoter and physically
interact (36). CBP is known to be recruited by and to bind not only to phospho-CREB (84, 85) but also to SF-1 (86, 87) and to enhance
transcription of SF-1-regulated genes (87). Using the ChIP assay, it
was shown that PKA and SF-1 increased the histone H4 acetylation
associated with the inhibin promoter (36). The increased HAT
activity is believed to derive either from the recruitment of CBP (88,
89) or the recruitment by CBP of PCAF (90, 91). The role of PKA in
these events is believed to be primarily via its phosphorylation of
CREB (46), thereby leading to CBP recruitment (36, 85). Perhaps in
response to the translocation of the catalytic subunit of PKA to the
nucleus of granulosa cells in response to FSH, both CREB and histone H3
are phosphorylated, leading to the formation of a complex between
phospho-CREB and adjacent SF-1, recruitment of CBP and possibly PCAF,
acetylation of histones H3 and H4, engagement of basal transcription
machinery, and initiation of transcription. It is interesting
that PCAF has been shown to preferentially acetylate histone H3
(92). Since SF-1 also regulates the transcription of aromatase (13, 93, 94), CYP11A (86), high density lipoprotein receptor (95), and
steroidogenic acute regulatory protein (StAR) (96) genes, histone H3
may also be involved, along with CREB and PKA, in the induction
of these FSH-responsive genes.
, may also involve phosphorylation of histone H3
followed by its acetylation catalyzed by CBP or PCAF.
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Fig. 6.
Proposed model for FSH signaling to activate
histone H3. FSH via the catalytic (C) subunit of PKA
catalyzes both histone H3 and CREB phosphorylation as well as histone
H1 phosphorylation. Phosphorylated CREB, possibly in conjunction with
SF-1 and Sp1, recruits CBP and possibly other HATs such as PCAF,
inducing histone H3 acetylation and gene transactivation.
ACKNOWLEDGEMENTS
FOOTNOTES
Youngkyu Park is the recipient of an individual
National Research Service Award 5 F32 HD85602.
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ABSTRACT
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DISCUSSION
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J. Cottom, L. M. Salvador, E. T. Maizels, S. Reierstad, Y. Park, D. W. Carr, M. A. Davare, J. W. Hell, S. S. Palmer, P. Dent, et al. Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase J. Biol. Chem., February 21, 2003; 278(9): 7167 - 7179. [Abstract] [Full Text] [PDF]
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L. M. Salvador, E. Maizels, D. B. Hales, E. Miyamoto, H. Yamamoto, and M. Hunzicker-Dunn Acute Signaling by the LH Receptor Is Independent of Protein Kinase C Activation Endocrinology, August 1, 2002; 143(8): 2986 - 2994. [Abstract] [Full Text] [PDF]
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