SNF2-Related CBP Activator Protein (SRCAP) Functions as a Coactivator of Steroid Receptor-Mediated Transcription through Synergistic Interactions with CARM-1 and GRIP-1
M. Alexandra Monroy,
Natalie M. Schott,
Linda Cox,
J. Don Chen,
Mary Ruh and
John C. Chrivia
Department of Pharmacological and Physiological Science (M.A.M., N.M.S., L.C., M.R., J.C.C.), St. Louis University School of Medicine, St. Louis, Missouri 63104; and the Department of Pharmacology (J.D.C.), University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
Address all correspondence and requests for reprints to: John C. Chrivia, Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri 63122. E-mail: Chrivia{at}slu.edu.
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ABSTRACT
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SRCAP (SNF2-related CBP activator protein) is a 350-kDa protein that shares homology with the SNF2 family of proteins whose members function in various aspects of transcriptional regulation. In various cell types, SRCAP is found in distinct multiprotein complexes that include proteins found in SWI/SNF chromatin remodeling complexes. SRCAP was identified by its ability to bind to CBP and was found to potentiate the ability of CBP to activate transcription. Studies in our laboratory have demonstrated that SRCAP functions as a coactivator for CREB-mediated transcription of a number of promoters, including that of the phosphoenolpyruvate carboxykinase gene. Our current studies demonstrate that SRCAP enhances phosphoenolpyruvate carboxykinase promoter transcription induced by glucocorticoids. SRCAP also enhances glucocorticoid receptor-mediated transcription of a simple promoter containing only two glucocorticoid response elements, indicating that SRCAP functions as a glucocorticoid receptor coactivator. In similar studies, SRCAP was also found to serve as a coactivator for the androgen receptor. SRCAP exhibits synergistic activation with nuclear receptor coactivators and functionally interacts in vivo with glucocorticoid receptor-interacting protein-1 and coactivator-associated arginine methyltransferase-1. We propose that SRCAP, by virtue of its ability to interact with CBP, functions as a coactivator to regulate transcription initiated by several signaling pathways.
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INTRODUCTION
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THE GLUCOCORTICOID RECEPTOR (GR) is a member of the steroid receptor family of transcriptional factors and contains structural features common to this family (1). Studies to understand how the GR modulates transcription have identified a number of proteins which contact GR. These include various coactivators and corepressors, the human and yeast switch/sucrose nonfermenting (SWI/SNF) remodeling complexes, the yeast SAGA and Nu4A histone acetyltransferase (HAT) complexes, and the components of a vitamin D receptor-interacting complex, DRIP (2, 3, 4, 5).
GR interacts with the p160 coactivators [GR-interacting protein (GRIP)-1/TIF2, SRC-1/NCoA, and pCIP/RAC3/ACTR/AIB1/Tram1] described by a number of laboratories (6, 7, 8, 9, 10, 11). Interaction between the p160 coactivators and nuclear receptors occurs through contact of LBD of the nuclear receptors and a conserved cluster of amino acids (LXXLL motif) found within the p160 coactivators (12). The LBD of several nuclear receptors including GR has been crystallized and indicate that the binding of ligand induces a change in the secondary structure such that the activation function (AF)-2 helix is aligned for correct contact with coactivators (13, 14).
GRs can be recruited to larger complexes by interaction with the p160 coactivators. For example, GRIP-1, in addition to binding GR, can bind a second coactivator cAMP response element (CREB)-binding protein (CBP) that has HAT activity. This assembled GR/GRIP-1/CBP complex can be further stabilized by CBP interacting with GR through contact between the C-terminal domain of CBP and the AF-1 domain of GR (15). Formation of even higher order complexes have been identified involving the histone methyltransferase CARM (coactivator-associated arginine methyltransferase)-1, e.g. a complex between CBP/GRIP-1/CARM-1 has been shown to occur in vivo. Consistent with the formation of this complex, CBP, GRIP-1, and CARM-1 have been shown to function together to synergistically activate nuclear receptor-mediated transcription (16). How the combination of these factors leads to synergistic activation is not completely understood but appears to require the methyltransferase activity of CARM-1.
One factor that interacts with CBP is the protein SRCAP (SNF2-related-CBP activator protein), which binds to CBP and potentiates its transcriptional activity (17). The structure of SRCAP is very homologous to proteins related to the chromatin remodeling protein SNF-2. SNF2-related proteins have several functions, including remodeling chromatin, regulation of transcriptional activation, DNA repair, and recombination (18, 19, 20). SRCAP shares homology with a Drosophila ATPase termed domino, which is present in large protein complexes in embryo extracts. Domino has gene expression regulatory effects and is localized to distinct sites on larval polytene chromosomes (21). In mammalian cells, SRCAP is most homologous to p400, an SNF2 related component of a complex essential for E1A-mediated transformation (22). Like p400, SRCAP was shown to be part of a large complex that includes NCoR1, BRG1, BAF 170, BAF 155, BAF 47 and the histone deacetylases, HDAC3 and KAP-1 (23). We have previously shown that SRCAP acts as a coactivator for CREB-mediated transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene and other reporters (24). In this study, we show that SRCAP also functions as a coactivator for GR-mediated transcription. SRCAP exhibits synergistic activation with nuclear receptor coactivators, and functionally interacts in vivo with GRIP-1 and CARM-1. SRCAP was also found to function as a coactivator for AR-mediated transcription, suggesting that it may function as a general coactivator for other steroid hormone receptors that utilize CBP, GRIP-1, and CARM-1 to activate transcription.
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RESULTS
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SRCAP Functions as a Coactivator for the GR
The observation that SRCAP interacts with CBP and enhances its ability to activate CREB-mediated transcription suggested the hypothesis that SRCAP may serve as a coactivator for other transcription factors that also interact with CBP including the steroid hormone nuclear receptors. To test this hypothesis, HeLa cells that express the glucocorticoid receptor were transiently transfected with a reporter gene containing two copies of a glucocorticoid receptor-binding site [glucocorticoid response element (GRE)-chloromphenicol acetyltransferase (CAT)] and a plasmid expressing SRCAP. As expected, dexamethasone (Dex) activated transcription of the GRE-reporter gene (Fig. 1
), and this activation could be blocked by the antagonist RU486. Cotransfection of the plasmid expressing SRCAP further enhanced the ability of Dex to activate transcription of the GRE-CAT reporter gene 4- to 5-fold. This additional activation was completely blocked by treatment with excess RU486, indicating that SRCAP mediates transcriptional activation through GR. Consistent with this conclusion, in the absence of Dex, SRCAP did not activate transcription of the GRE-CAT reporter.

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Fig. 1. SRCAP Activates GR-Mediated Transcription
HeLa cells were transfected with 200 ng of the reporter gene GRE-CAT and, where indicated, 1000 ng of plasmid expressing SRCAP. Each transfection was adjusted to contain the same amount of DNA by addition of equal molar amounts of the pcDNA 3.1 vector and various amounts of the plasmid pGem 7. Fifteen hours post transfection, the cells were washed and incubated for 24 h in serum-free DMEM containing where indicated 100 nM Dex and 10 µM RU486. The cells were then harvested and CAT enzymatic activity measured. The results are reported relative to the CAT activity observed in cells treated with Dex and are the means and SE of three to five independent experiments done in triplicate.
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To test whether SRCAP can also function as a coactivator when the GR binding site is located within the context of a native promoter, transient transfections were repeated in a human hepatoma cell line (HepG2 cells) using a PEPCK-CAT reporter gene (25). The PEPCK promoter contains multiple cis-acting elements that are required for maximal induction by glucocorticoids including two GREs and a cAMP response element (26, 27). To determine whether maximal induction of the PEPCK promoter can be further enhanced by SRCAP, these experiments were carried out in the presence of catalytic subunit of protein kinase A (PKA). Shown in Fig. 2
, transcription of the PEPCK-CAT reporter gene was activated about 5-fold in cells transfected with the plasmid expressing PKA and then treated with Dex as compared with control cells which were only transfected with the plasmid expressing PKA (Fig. 2
, lanes 1 and 3). Transcription of the PEPCK-CAT reporter gene was also activated 5-fold in cells transfected with a combination of the plasmid expressing PKA and the plasmid expressing SRCAP (lane 4). This activation is consistent with previous experiments that demonstrate that SRCAP serves as a coactivator for CREB-mediated transcription (24). Transfection of cells with both plasmids expressing PKA and SRCAP, followed by treatment with Dex, resulted in a 20-fold synergistic activation of transcription (lane 4). RU486 reduced this activation to the level achieved by SRCAP and PKA in the absence of Dex treatment indicating it was mediated by GR. Taken together, the results of Figs. 1
and 2
indicate that SRCAP functions as a coactivator for GR-mediated transcription. This suggests SRCAP may interact with other proteins that modulate GR function, such as GR coactivators.

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Fig. 2. SRCAP Activates Dex-Mediated Transcription of the PEPCK Promoter
HepG2 cells were transiently transfected with 200 ng of the PEPCK-CAT reporter gene, 100 ng of the plasmid expressing PKA, and where indicated, 1000 ng of the plasmid expressing SRCAP or the control plasmid pcDNA 3.1. Fifteen hours post transfection, the cells were washed and incubated for 24 h in serum-free DMEM containing where indicated 100 nM Dex and 10 µM RU486. The cells were harvested and CAT enzymatic activity measured. The results are reported relative to the CAT activity observed in cells transfected with PKA and are the means and SE of three to five independent experiments done in triplicate and the results of two experiments for the last two conditions (treatment with RU486).
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SRCAP Functions with CARM-1 and GRIP-1 to Synergistically Activate GR-Mediated Transcription
To test whether SRCAP interacts with known coactivators of GR-mediated transcription, a CV-1 cell transient transfection assay was used. CV-1 cells have little or no nuclear receptors and have been used as a sensitive assay to demonstrate synergy of function between various coactivators of nuclear receptor-mediated transcription (16). For these studies, CV-1 cells were transfected with the GRE-CAT reporter gene and with various combinations of the plasmids expressing GR, CBP, CARM-1, GRIP-1, or SRCAP. Similar to studies reported in other laboratories, GRIP-1 and CARM-1 were found to act together to synergistically activate transcription (Fig. 3
, compare lanes 1 and 6). However, in contrast to other reports, additional CBP only slightly enhanced transcriptional activation by GRIP-1 and CARM-1 (lane 3). The source of these differences with the previously published reports is unclear but may reflect variation in CBP levels occurring in different passages of CV-1 cells.

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Fig. 3. SRCAP Functions Synergistically with CARM-1 and GRIP-1 to Activate GR-Mediated Transcription
CV-1 cells were transfected with 200 ng of the reporter gene GRE-CAT, 10 ng of the plasmid expressing GR, and 1000 ng of the plasmids expressing the indicated proteins. Each transfection was adjusted to contain the same amount of DNA by addition of equal molar amounts of the pcDNA 3.1 vector and various amounts of the plasmid pGem 7. Fifteen hours post transfection, the cells were washed and incubated for 24 h in serum-free DMEM containing 100 nM Dex. The CAT enzymatic activity relative to that achieved by GR alone in the presence of Dex is reported and each value is the mean and SE from three experiments done in triplicate. As shown, the combination of SRCAP, GRIP, and CARM-1 stimulated GR-mediated transcription 300-fold.
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Maximal transcriptional activation of the GRE-CAT reporter gene required cotransfection of the plasmids expressing CARM-1, GRIP-1, and SRCAP (lane 11). Greatly reduced rates of transcription were observed when only two of these three plasmids were used, e.g. the combination of SRCAP and GRIP-1 (lane 9) or the combination SRCAP and CARM-1 (lane 10). Interestingly, CBP could partially replace CARM-1 to function with GRIP-1 and SRCAP (lane 14). However, CBP could not replace GRIP-1 to function with CARM-1 and SRCAP (lane 13). Shown in lane 12, the ability of SRCAP to function with CARM-1 and GRIP-1 to activate transcription was dependent on GR.
Multiple Regions of SRCAP Contribute to Synergistic Activation of GR-Mediated Transcription
Multiple domains that modulate transcriptional activity have been described for a number of proteins including CBP/p300 and the p160 coactivators. To assay for domains of SRCAP that might participate in the activation of transcription, CV-1 cells were transfected with CARM-1 and GRIP-1 and suboptimal amounts of plasmid expressing full length SRCAP. Shown in Fig. 4
, a dose-response curve demonstrated that a sharp threshold exists for the amount of SRCAP needed to synergistically activate transcription with CARM-1 and GRIP-1. At 1000 ng of transfected SRCAP plasmid, transcription was activated about 6-fold over that achieved by GRIP-1 and CARM-1 alone (compare lanes 1 and 2). This activation increased to about 11-fold at 1500 ng of transfected SRCAP plasmid (lane 3) and increased to almost 90-fold at 2000 ng of transfected SRCAP plasmid (lane 4).

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Fig. 4. Multiple Domains of SRCAP Activate GR-Mediated Transcription
CV-1 cells were transfected with 200 ng of the reporter gene GRE-CAT and 10 ng of the plasmid expressing GR, 1000 ng of the plasmids expressing GRIP-1 and CARM-1, and 1000 ng of plasmid (pSRCAP 1-1186, pSRCAP 1275-2971, or pSRCAP 1380-1670) expressing the indicated proteins. In the case of SRCAPL FL 1X refers to 1000 ng, 1.5x to 1500 ng, and 2x to 2000 ng. Each transfection was adjusted to contain the same amount of DNA by addition of equal molar amounts of the pcDNA 3.1 vector and various amounts of the plasmid pGEM7. Fifteen hours post transfection, the cells were washed and incubated for 24 h in serum-free DMEM containing 100 nM Dex. The CAT enzymatic activity relative to that achieved by GR alone in the presence of Dex is reported and each value is the mean and SE from three experiments done in triplicate.
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A suboptimal amount of plasmid (1000 ng) expressing full-length SRCAP was cotransfected into CV-1 cells along with plasmids expressing several SRCAP peptides to determine whether they contained domains that could cooperate with full-length SRCAP to activate transcription (Figs. 4
and 5
). This included plasmids expressing the amino terminal end of SRCAP (amino acids 11186) or the carboxyl-terminal end of SRCAP (amino acids 12752971) or the CBP interaction domain of SRCAP (amino acids 13801670). As shown, although these plasmids by themselves only weakly activated transcription, they cooperated with full-length SRCAP to strongly activate transcription. This indicates that these regions of SRCAP contain domains that can participate in activation of transcription. This result is consistent with our previous data, which indicated these same regions of SRCAP can function independently to activate transcription when expressed as Gal-SRCAP chimeras (24).

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Fig. 5. A Schematic of SRCAP
Domains identified within the 2971 amino acid SRCAP are indicated including two highly charged domains, the CBP interaction domain (amino acids 13801670), and the position of the regions that make up the conserved ATPase domains (I, IaVI). The position of putative nuclear receptor interaction domains, leucine zippers, and A/T hook DNA binding domains are indicated. The positions of the proteins encoded by pSRCAP 11186, pSRCAP 12752971, pSRCAP Gal-12752309, and VP16-SRCAP 13801729 chimera are indicated.
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SRCAP Interaction with GRIP-1 Is Enhanced by CBP
A complex consisting of CBP, GRIP-1, and CARM-1 has been demonstrated in CV-1 cells (16). This suggested the hypothesis that SRCAP might function synergistically with CARM-1 and GRIP-1 to activate GR-mediated transcription by formation of a higher order complex consisting of SRCAP linked to CARM-1 through formation of a SRCAP/CBP/GRIP-1/CARM-1 complex. To test this hypothesis, various mammalian two-hybrid assays were performed. For these studies, the ability of a Gal-SRCAP 12752309 chimera, which contains the CBP interaction domain of SRCAP, was tested for its ability to interact with a viral protein (VP) 16-GRIP-1 chimera. As shown, 500 ng of the plasmid expressing the VP16-GRIP-1 chimera had no effect on the ability of the Gal-SRCAP 12752309 chimera to activate transcription (Fig. 6
). Transfection of 500 ng of the plasmid expressing CBP was found to activate transcription of the Gal-SRCAP 12752309 chimera about 3-fold. However, if the plasmids expressing VP16-GRIP-1 chimera and CBP were cotransfected, transcription was activated greater than 20-fold. This enhancement required the GRIP-1 portion of the chimera because transfection of a plasmid expressing only the VP16 moiety did not effect activity of the Gal-SRCAP 12752309 chimera in the absence or presence of CBP. Collectively, these observations are consistent with the model that SRCAP contacts GRIP-1 by bridging through CBP.

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Fig. 6. The Ability of a Gal-SRCAP Chimera to Interact with a VP16-GRIP Chimera Is Enhanced by CBP
HeLa cells were transiently transfected with plasmids indicated: 1000 ng of pGal-SRCAP 1275-2309, 500 ng of pCBP, pVP16-GRIP, and pVP16 and 300 ng of the pGal-CAT reporter gene containing binding sites for Gal 4. The Gal-SRCAP chimera (pGal-SRCAP 1275-2309) contains the CBP interaction domain of SRCAP. The pVP16-GRIP-1 plasmid encodes amino acids 51462 of GRIP-1 fused to VP16 and the pCBP plasmid encodes full-length CBP. Each transfection was adjusted to contain the same amount of DNA by addition of equal molar amounts of either the pSG5 or pcDNA 3.1 vectors and various amounts of the plasmid pGem 7. Fifteen hours post transfection, the cells were washed and incubated for 24 h in serum-free DMEM containing 100 nM Dex. The CAT enzymatic activity relative to that achieved by the Gal-SRCAP 1275-2309 chimera is reported and each value is the mean and SE from two to five experiments performed in triplicate.
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SRCAP Forms a Complex in Vivo with CARM-1
A reverse hybrid experiment was performed using plasmids expressing a Gal-CARM-1 chimera and a VP16-SRCAP 13801729 chimera (Fig. 7
). As shown in the left panel, a control plasmid expressing only VP16 had only a small effect (2-fold) on the transcription mediated by the Gal-CARM-1 chimera, whereas the plasmid expressing the VP16-SRCAP 13801729 chimera increased about 6-fold transcription mediated by Gal-CARM-1 chimera. This increase in transcription was higher than the increase in transcription mediated by direct interaction of Gal-CARM-1 with a VP16-GRIP-1 chimera (right panel). Therefore, the CARM1-SRCAP interaction is at least as good as the known CARM1-GRIP1 interaction, as determined by this method.

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Fig. 7. A SRCAP-VP16 Chimera Interacts with a Gal-CARM-1 Chimera
HeLa cells were transiently transfected with 1000 ng of the plasmids indicated: pGal-CARM-1, which encodes full-length CARM-1 fused to the DNA binding domain of Gal 4; pGal 1147, which encodes the Gal 4 DNA binding domain, VP16-GRIP; VP16-SRCAP 1380-1729 of SRCAP, which expresses the CBP binding domain of SRCAP fused to the VP16 activation domain; pVP16, which expresses the VP16 activation domain. Each transfection was adjusted to contain the same amount of DNA by addition of equal molar amounts of either pSG5 or pcDNA 3.1 vectors and various amounts of the plasmid pGem 7. The CAT enzymatic activity relative to that achieved by the Gal-CARM-1 is reported and each value is the mean and SE from three experiments performed in triplicate.
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To confirm the conclusions of the mammalian two-hybrid assays, coimmunoprecipitation studies were carried out to determine if CARM-1 can complex with full-length SRCAP. For these studies, COS-7 cells were cotransfected with the plasmid expressing HA-tagged CARM-1 and Flag-tagged SRCAP (Fig. 8
). Flag-tagged SRCAP protein in extracts from these cells was immunoprecipitated using an anti-Flag antibody and the presence of associated HA-tagged CARM-1 was detected by Western blot using an anti-HA antibody. Shown in Fig. 8A
lanes 4 and 5, HA-tagged CARM-1 coimmunoprecipitated with Flag-tagged SRCAP. This interaction was specific because CARM-1 is not immunoprecipated directly by the anti-Flag antibody (lane 6). The specificity is further demonstrated in Fig. 8B
, where an antibody against adenoviral protein E1A (M73) also does not pull down the SRCAP-CARM-1 complex. Also shown in Fig. 8A
, lane 3, and as previously reported by other groups, CARM-1 was found to coimmunoprecipitate with CBP.

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Fig. 8. SRCAP Forms a Complex with CARM-1
COS-7 cells were mock transfected or transfected with plasmids expressing the indicated plasmids. Forty-eight hours post transfection, whole extracts were made and Flag-tagged proteins immunoprecipitated in (A) using the anti-FLAG antibody and as indicated in (B) immunoprecipitated using the anti-Flag antibody (FL) or an antibody against the adenoviral protein E1A(M73) and analyzed by Western Blot using anti-HA antibodies. *, Position of CARM-1. The lower band in all lanes is heavy chain IgG.
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SRCAP Activates Transcription Mediated by the Androgen Receptor (AR)
To determine whether SRCAP is capable of functioning as a coactivator for nuclear receptors other than GR the ability of SRCAP to enhance AR-mediated transcription was tested. CV-1 cells were cotransfected with the mouse mammary tumor virus (MMTV)-luciferase reporter gene and either the plasmid expressing AR (pAR), or a combination of pAR and pSRCAP plasmids. Cells were subsequently treated with 100 nM 5
dihydrotestosterone (DHT) and transcriptional activation was measured. Compared with control cells (not treated with 5
DHT) SRCAP enhanced about 16-fold the 5
DHT-dependent activation of the MMTV promoter (Fig. 9
).

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Fig. 9. SRCAP Activates AR-Mediated Transcription
CV-1 cells were transfected with 250 ng of the reporter gene MMTV-luciferase and 100 ng of the plasmid expressing the AR and where indicated 1000 ng of plasmid expressing SRCAP or equal molar amounts of the control vector pcDNA 3.1. Each was adjusted to contain the same amounts of DNA by the addition of the pGem7 plasmid. Fifteen hours post transfection, the cells were washed and incubated for 24 h in serum free DMEM containing where indicated 10 nM 5 DHT. The cells were then harvested and luciferase enzymatic activity measured. The results are reported relative to the luciferase activity observed in untreated cells and are the means and SE of three to five independent experiments done in triplicate.
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DISCUSSION
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In the present studies, we have found that SRCAP functions as a coactivator for GR-mediated transcription. This coactivator function could be demonstrated when a GR binding site was located within the context of a simple artificial promoter gene that contained only GRE elements. The coactivator function could also be seen with the native PEPCK promoter, which has the GR binding site located among the binding sites for a number of additional transcription factors. The coactivator function of SRCAP at both these promoters occurred only in the presence of agonist and was blocked by the antiglucocorticoid RU486 indicating that SRCAP acts as a GR coactivator.
SRCAP was also found to cooperate synergistically with the GR coactivators CARM-1 and GRIP-1 to strongly activate transcription. This strong activation of transcription was greatly reduced if either GRIP-1 or CARM-1 was omitted from the transfection. This indicates that distinct functions associated with each of these proteins cooperate with SRCAP to activate GR-mediated transcription. Experiments by Lee et al. (16) have demonstrated that CBP/p300, CARM-1, and GRIP-1 cooperate synergistically to maximally activate transcription mediated by AR, TR, and ER. However, the specific requirements for individual coactivators appears to have some flexibility because replacement of p300 or CARM-1 with p/CAF resulted in only a modest reduction in activation (16).
In a mammalian two-hybrid assay, we found that a Gal-SRCAP 12752309 chimera weakly interacted with a VP16-GRIP-1 chimera. This interaction was greatly increased in the presence of additional exogenous CBP, and is consistent with the model that SRCAP interaction with GRIP-1 occurs through CBP. However, interaction of theVP16-GRIP-1 chimera with SRCAP can occur in the absence of additional exogenous CBP, if higher levels of pVP16-GRIP-1 plasmid (1000 ng) is used in mammalian two-hybrid assay (data not shown). One explanation (one we favor) for these results is that a critical concentration of GRIP-1 and CBP is needed to drive formation of a SRCAP/GRIP-1/CBP complex. This critical concentration may be reached using lower amounts of GRIP-1 (500 ng) and higher amounts of CBP (500 ng) in the transfection (Fig. 6
) or it may also be reached by using a higher amount of GRIP-1 (1000 ng) and a lower amount of CBP (the endogenous CBP) in the transfection. Alternatively, the ability of the higher amounts of the pVP16-GRIP-1 plasmid (1000 ng) to activate transcription of the Gal-SRCAP 12752309 chimera in the absence of exogenous CBP may occur by GRIP-1 contacting SRCAP through a second CBP-independent mechanism.
Interaction of a VP16-CARM-1 chimera with the Gal-SRCAP 12752309 chimera was also observed but was much weaker (3-fold) than occurred with the VP16-GRIP-1 chimera (data not shown). To test whether this weak interaction might be a consequence of steric hindrance, a reverse hybrid assay was performed using Gal-CARM-1 and VP16-SRCAP chimeras. The reverse two-hybrid assay indicated that CARM-1 and SRCAP could form a complex in vivo. This interaction was confirmed in our immunoprecipitation studies that indicate CARM-1 can interact with full-length SRCAP. The specific components that make up the SRCAP/CARM-1 complex have not been conclusively identified. However, recent studies indicate that a CBP/GRIP-1/CARM-1 complex can form (16) and our studies indicate that a SRCAP/CBP/GRIP-1 complex can be formed. This suggests that the SRCAP/CARM-1 complex also contains CBP and GRIP-1. This model is further supported by our observations that SRCAP functions synergistically with CBP, GRIP-1, and CARM-1 to activate GR-mediated transcription.
In previous studies, we have found that the SRCAP peptides containing N- and C-terminal deletions could function as coactivators for CREB-mediated transcription. The present studies indicate that these same SRCAP peptides can function as coactivators for GR-mediated transcription. Because deletion of either N or C terminal domains results in SRCAP mutant molecules lacking an intact ATPase domain, these findings indicated that SRCAP can activate both GR and CREB-mediated transcription through a distinct mechanism that is independent of abilities associated with its ATPase function (24). The finding that SRCAP activates transcription via a distinct mechanism is not surprising given the observation that many coactivators appear to be able to activate transcription via multiple mechanisms. For example, the HAT activity of p300 is not needed for it to serve as a coactivator for some nuclear receptors (16). In transient assays, the methyltransferase activity of CARM-1 is necessary but not sufficient for activation of transcription (28). Multiple functions have also been reported for the SNF2 family member, Cockayne syndrome (CS). Mutation of the ATPase domain of CS prevents it from remodeling chromatin but does not inhibit the ability of CS to function as a topoisomerase (29).
How the SRCAP peptide corresponding to amino acids 11186 activates transcription is not known. This region contains one putative receptor interaction domain (LEELL), and although usually more than one of these domains is required for binding, Christiaens et al. (30) indicate a portion of GRIP containing a single receptor interaction motif binds the AR. This region of SRCAP also contains the N-terminal half of the ATPase domain of SRCAP and a charged domain which may provide an interaction domain for contact with other proteins involved in transcriptional activation. In support of this notion Johnston et al. (17) found this region of SRCAP when expressed as a Gal-chimera has the ability to activate transcription of a reporter gene containing the binding site for Gal 4. The SRCAP peptide corresponding to amino acids 12752971 gave the strongest transcriptional activation which was equivalent to activity achieved with full-length SRCAP (Fig. 4
, lane 8). This region contains several domains which may be responsible for activation of transcription. It contains a leucine zipper motif, three putative nuclear receptor interaction motifs (LPPLL and LAALL used by coactivators and LXXIIXXI used by corepressors). It also contains a charged domain located adjacent to the CBP interaction domain. The SRCAP peptide (amino acids 13801670) corresponding to the CBP interaction domain also strongly activates transcription. This finding was surprising, since in our previous studies this fragment of SRCAP was found to act as a dominant negative inhibitor of CREB-mediated transcription. One possible explanation for these conflicting observations is that amino acids 13801670 do not contain sufficient information for activation of CREB-mediated transcription but do contain sufficient information for activation of GR-mediated transcription. This might occur if the nature of the interaction of this domain with CBP is dependent on the type of transcription factor bound to CBP. Context-dependent consequences have been reported by Xu et al. (31), who demonstrated that the retinoic acid receptor stimulated the methylation of CBP by CARM-1, whereas CBP associated with CREB did not stimulate the methylation of CBP.
We have also found that SRCAP functions as a coactivator for AR. Because AR similar to GR utilizes CARM-1, GRIP-1, and CBP to activate transcription, SRCAP may modulate AR-mediated transcription by interactions with these proteins (16). The ability of SRCAP to function as a coactivator of AR- and GR-mediated transcription suggests that it might also function as a coactivator for other steroid receptors that interact with CARM-1 and GRIP-1. Consistent with this hypothesis we have found that SRCAP also serves a coactivator for ER (Chrivia, J., unpublished data).
In summary, the ability of a CBP/GRIP-1/CARM-1 complex to associate and to interact with various promoters after stimulation by ligand has been well documented. Our data suggest that, in addition to these well-characterized interactions, SRCAP participates in the regulation of GR-mediated transcription (and perhaps AR-mediated transcription) by interaction with the CBP/GRIP-1/CARM-1 complex.
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MATERIALS AND METHODS
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Plasmids
The pSRCAP plasmid expressing amino acids 12971 of SRCAP was generated by subcloning the 9121-bp SRCAP cDNA into the pcDNA 3.1 Myc/His plasmid (Invitrogen, Carlsbad, CA) digested with the restriction enzymes NheI and BamHI. The pSRCAP-1X Flag and pSRCAP-3X Flag plasmids were generated by digesting the pSRCAP plasmid with BamHI and ligating into that site either an oligonucleotide expressing one or three copies of the Flag epitope. The pSRCAP 11186, pSRCAP 12752971, pSRCAP 13801670, pGal-SRCAP 12752309, and pVP16-SRCAP 13801729 were previously described (24). The PEPCK-CAT (pPL32) reporter gene was a gift from Dr. Daryl Granner (Vanderbilt University, Nashville, TN). The GRE-CAT reporter gene was obtained from Dr. Steve Nordeen (University of Colorado, Denver, CO). It consists of two GRE sites in a minimal TATA box containing the E1B promoter. The pPKA plasmid expressing the catalytic subunit of PKA was obtained from Dr. Mike Uhler (University of Michigan, Ann Arbor, MI). The pSG5, pSG5-HA-CARM1, and the pSG5-HA-GRIP expression vectors were obtained from Dr. Michael Stallcup (University of Southern California, Los Angeles, CA). The pGal-CARM-1 plasmid was obtained by subcloning the EcoRI fragment of pSG5-HA-CARM-1 into EcoRI site of pcDNA Gal 1147. This latter plasmid was derived by subcloning a cDNA corresponding to amino acids 1147 of Gal 4 into the NheI and EcoRI site of the pcDNA 3.1 Myc/His plasmid. The pCBP-Flag tagged plasmid was described in Ref. 32 . The plasmid expressing GR was a gift of Dr. Ron Evans at The Salk Institute (La Jolla, CA). The pMMTV-luciferase and pAR plasmids have been previously described (33).
Tranfections
HeLa and CV-1 cells were maintained in DMEM, and HepG2 cells were maintained in MEM with Earles salts. Each was supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were seeded at 1 x 105 cells/35-cm dish 18 h before transfection. Each transfection used the indicated amounts of the PEPCK or GRE-CAT reporter plasmids and the indicated amounts of each additional plasmid. Each transfection was adjusted to contain equal molar amounts of the cytomegalovirus or simian virus 40 promoter using, respectively, the pcDNA3.1 Myc/His or pSG5 plasmids. The LipofectAMINE (Invitrogen, Carlsbad, CA) transfection method was used for HeLa cells, and Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) was used for HepG2 cells. Each was used according to the manufacturers directions. After overnight incubation, cells were washed and incubated overnight in serum-free medium, and then incubated an additional 24 h in the absence or presence of the indicated amount of Dex (Sigma, St. Louis, MO). Cells were harvested and assayed for CAT activity as described (24). CAT activity reported was normalized for variation of transfection efficiency between samples (the variation in the amount of plasmid taken up by cells in each sample) using a statistical approach where each experimental point was performed in triplicate in at least three separate experiments. We have used this approach since SRCAP regulates (presumably through regulation of CBP) the transcription of common reporter genes used as internal controls. For example, we have found SRCAP activates transcription of a CMV-ß-gal reporter gene. Similarly, luciferase activity was detected using the luciferase assay system from Promega (Madison, WI), according to the manufacturers protocol.
Coimmunoprecipitations
Cos-7 cells were grown in 100-mm diameter dishes seeded at 1 x 106 cells and transfected with Superfect (QIAGEN, Valencia, CA) according to the manufacturers protocol with combinations of SRCAP and coactivator expression plasmids pSRCAP-1X Flag or pSRCAP-3X Flag and pSG.HA-CARM1 as indicated. After transfection cells, were grown in DMEM with 10% fetal bovine serum for 40 h before harvest. Cells were harvested in p300 lysis buffer (34) containing a complete protease inhibitor mixture (Roche Molecular Biochemicals). Lysates were clarified by centrifugation and a portion of the supernatant was used for Western blot analysis of CARM-1 using monoclonal antibody against the HA epitope (Roche Molecular Biochemicals). The remaining supernatant was immunoprecipitated with anti-FLAG antibody (Sigma) overnight at 4 C. Immunoconjugates were precipitated by incubation with protein G agarose (Sigma), followed by centrifugation, and washed three times with NETN buffer [0.1% Nonidet P-40, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 100 mM NaCl]. Immunoprecipitates were resolved by SDS-PAGE and Western blotted with antibody against anti-HA epitope (Roche Molecular Biochemicals).
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FOOTNOTES
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This work was supported by United States Public Health Service Grant DK58262 (to J.C.C.) and DK52888 (to J.D.C.) from the NIH. J.D.C. is a Leukemia and Lymphoma Society Scholar.
Abbreviations: AF, Activation function; AR, androgen receptor; CARMI, coactivator-associated arginine methyltransferase; CAT, chloromphenicol acetyltransferase; CREB, cAMP response element binding protein; CBP, CREB binding protein; CS, Cockayne syndrome; Dex, dexamethasone; DHT, dihydrotestosterone; DRIP, vitamin D receptor-interacting complex; GR, glucocorticoid receptor; GRIP, GR-interacting protein; GRE, glucocorticoid response element; HAT, histone acetyltransferase; LBD, ligand binding domain; MMTV, mouse mammary tumor virus; pAR, plasmid expressing AR; PEPCK, phosphoenolpyruvate carboxykinase; PKA, protein kinase A; SRCAP, SNF2-related-CBP-activator protein; SWI/SNF, switch/sucrose nonfermenting; VP, viral protein.
Received for publication June 3, 2003.
Accepted for publication September 10, 2003.
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