![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, June 2003, p. 3763-3773, Vol. 23, No. 11
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.11.3763-3773.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Received 2 January 2003/ Returned for modification 5 February 2003/ Accepted 12 March 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although members of the homologous SRC family possess limited functional redundancy, accumulating evidence suggests that SRCs also play distinct roles in biological processes (4, 13, 25, 32, 49). These functional differences could result in part from the preferential recruitment of coactivators induced by ligands (6, 34). SRC-1 and SRC-2 have been reported to exhibit similar, but not identical, binding preferences to nine different NRs. For example, the androgen receptor binds well to SRC-2 but poorly to SRC-1 (11). The molecular basis of NR preferences for coactivators is linked to the structure of the ligand-binding domain of NRs and their concomitant specificity for individual NR boxes (9, 11, 31, 35, 48). For instance, the vitamin D receptor and estrogen receptor beta interact with different alpha-helical NR boxes, as in SRC-3 (27). The differences in affinity for NR box 2 or 3 of SRC-2 by glucocorticoid receptor (GR) and thyroid receptor (TR) ligand-binding domains provide additional evidence for underlying specific receptor-coactivator interactions (9). Furthermore, these contacts are sensitive to conformational changes induced by distinct ligands or DNA binding (26).
Despite all these reports, the precise assembly of coactivators and components of coactivator complexes required for physiological nuclear receptor function are not well understood. Since the functions of components of coactivator complexes appear to be distinct (21), it is likely that transcription factor-specific differences in configuration and content within the coactivator complex dictate acetyltransferase activities that bring about histone modification and further transcriptional regulation. An intriguing question involves the physiologic contexts under which coactivator utilization is determined by different NRs. We have approached this question with the hypothesis that specific NR-coactivator interactions exist in cells and generate distinct regulatory patterns in differing signaling contexts. We explored the hypothesis by studying coregulator assembly on a mouse mammary tumor virus (MMTV) promoter that is both progesterone and glucocorticoid responsive.
The actions of GR and PR have been extensively studied in an effort to understand how ligands for either receptor can elicit distinct biological activities when both steroid receptors act on the same common hormone responsive element (HRE) (2, 10, 23). One of the mechanisms by which GR or PR activity could be achieved is differential interactions with transcriptional coregulators, which further direct downstream events during transcription. A T47D cell line with stably integrated MMTV-chloramphenicol acetyltransferase (CAT) reporter (24) was used to test our hypothesis. Since all these analyses have been performed on a single promoter in a single cell line, cell-specific and promoter-specific effects can be minimized.
In this work, we demonstrate coactivator specificity for PR or GR as well as the effect that initial receptor-coactivator interactions have on the assembly of subsequent cofactors on the MMTV promoter. Upon steroid treatment, both PR and GR were recruited to the MMTV promoter and induced transcription of MMTV-CAT. PR selectively recruited SRC-1 and CBP, resulting in acetylation of K5 on histone H4. GR preferentially associated with SRC-2, which subsequently recruited pCAF, leading to specific histone modification of histone H3. Studies using RNA interference further substantiate an important role of SRC-1 in PR function and of SRC-2 in GR function. Taken together, this study elucidates differential mechanisms by which distinct nuclear receptor-coactivator interactions mediate diverse signaling events on the identical gene under physiological conditions in nontransfected cells.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Chromatin immunoprecipitation.
Chromatin immunoprecipitation (ChIP) analyses were performed by following a modified procedure based on previously described protocols (3, 5, 41). The T47D/CAT0 cell line was grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% charcoal-dextran stripped serum for 3 days. Cells with 90% confluence (
1 x 108) were treated with dexamethasone (10-8 M), progesterone (10-8 M), or R1881 (10-9 M; NEN Life Sciences Products, Boston, Mass.) for 1 h. Cells were washed once with phosphate-buffered saline (PBS), pH 7.4, and fixed with formaldehyde (1% final concentration) for 10 min at room temperature; cross-linking was terminated upon addition of glycine (0.125 M final concentration). Cells were then rinsed twice with cold PBS, collected, and swollen on ice in 20 volumes of nuclei preparation buffer [5 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH 8.0)-85 mM KCl-0.5% Nonidet P-40, supplemented with 1x protease inhibitor cocktail (Roche, Mannheim, Germany)] for 30 min. Nuclei were collected by microcentrifugation and resuspended in 300 µl of ChIP lysis buffer (50 mM Tris-HCl [pH 8.1], 1% sodium dodecyl sulfate [SDS], 10 mM EDTA, 1x protease inhibitor cocktail), followed by incubation on ice for 20 min. Samples were then sonicated on ice four to six times for 10 s each (duty output, 30%; duty cycle, 90%), followed by centrifugation for 10 min. The chromatin solution was diluted 10-fold with dilution buffer (20 mM Tris-HCl [pH 8.1], 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1x protease inhibitor cocktail). The chromatin preparation was precleared with 80 µl of salmon sperm DNA-protein A/G-agarose (Upstate Biotechnology, Lake Placid, N.Y.) and 20 µl of preimmune serum. Chromatin complexes were then incubated with 5 to 10 µl of specific antibodies and rotated at 4°C overnight. Immune complexes were collected with 40 µl of protein A/G-agarose with agitation for 1 h at 4°C. A supernatant fraction (20 µl) from reaction mixtures lacking primary antibodies was saved as an input control and processed in parallel with the eluted immunoprecipitates beginning at the cross-link reversal step. Immunoprecipitates were sequentially washed for 5 to 10 min in wash buffer I (20 mM Tris-HCl [pH 8.1], 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 150 mM NaCl), wash buffer II (20 mM Tris-HCl [pH 8.1], 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 500 mM NaCl), wash buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]), and TE buffer (three times). Washed beads were extracted with 50 µl of elution buffer (1% SDS, 100 mM NaHCO3) three times. The elution was combined in one tube, and the protein-DNA cross-linking was reversed by incubation at 65°C for 4 h. Each sample was treated with 15 µg of proteinase K (Gibco BRL, Grand island, N.Y.) in proteinase K buffer (50 mM Tris-HCl [pH 8.5], 1% SDS, 10 mM EDTA) for 2 h at 45°C. The DNA was purified with the QIAquick PCR purification kit (Qiagen, Valencia, Calif.) and eluted in 50 µl of elution buffer. Total input samples were eluted in 100 µl of elution buffer and diluted 1:10 before PCR analysis. Each PCR mixture contained 6 µl of immunoprecipitate or input, 0.5 µM each primer, 0.4 mM deoxynucleoside triphosphate mixture, 1x Titanium Taq PCR buffer (Clontech, Palo Alto, Calif.), and 1x Titanium TaqDNA polymerase (Clontech) in a total volume of 25 µl. The primers for the MMTV promoter were as follows: forward, 5'-TAT GGT TAC AAA CTG TTC TTA AAA CGA GGA TG-3'; reverse, 5'-GCA AGT TTA CTC AAA AAT CAG CAC TCT TT-3'. PCR was performed for 26 to 29 cycles with 1 min of denaturing at 94°C, annealing at 62°C, and extension at 68°C. PCR results were analyzed by agarose gel (1.5%) electrophoresis.
Cell culture and transient transfection of siRNA. T47D-CAT0 (human breast cancer) cells were routinely maintained in DMEM supplemented with 10% fetal bovine serum and 0.2 mg of Geneticin/ml (G418; Sigma). Prior to transfections, 3 x 105 cells per well of a six-well multiplate were grown overnight until reaching 50 to 60% confluence. Cells were then washed with phenol red-free DMEM three times and supplemented with 1 ml of phenol red-free DMEM containing 5% dextran-coated charcoal-stripped fetal bovine serum. For each well, 15 µl of TransIT-TKO transfection reagent (Mirus, Madison, Wis.) was premixed with 200 µl of Opti-MEMI (Gibco BRL) serum-free medium for 15 min. Synthesized small interfering RNA (siRNA) (Dharmacon Research, Inc., Lafayette, Colo.) duplexes sufficient for 100 nM final concentrations per well were added to the diluted TransIT-TKO reagent and incubated at room temperature for 15 min. The transfection reagent-siRNA mixture was added dropwise to the cells with gentle rocking and incubated for 48 to 72 h. For optimal results, a combination of two SRC-1 siRNAs at 50 nM each was utilized, namely, siRNA 1a (5'-AAC ACG ACG AAA UAG CCA UAC-3') and siRNA 1b (5'-AAG UGA UGA CUC GUG GCA CUG-3'). The siRNA sequence for SRC-2 is 5'-AAG UCA GAU GUA UCC UCU ACA-3'. The siRNA sequence for SRC-3 is the same as previously described (40). Transfection efficiency was monitored by fluorescence-labeled luciferase siRNA (Dharmacon Research, Inc.), which also served as a negative control.
In vitro mRNA synthesis and microinjection of Xenopus oocytes.
The Flag-tagged full-length SRC-1, SRC-2, and SRC-3 cDNAs cloned in pSP64 [poly(A)] vector (28) were linearized with a unique restriction enzyme cutting 3' to the poly(A) site. The digested DNA was purified with phenol-chloroform extraction and ethanol precipitation. The in vitro synthesis of mRNA was then carried out with an SP6 Message Machine kit from Ambion (Austin, Tex.), following the manufacturer's instructions. The preparation and microinjection of Xenopus stage VI oocytes were performed essentially as previously described (28). Diluted mRNA solutions containing approximately 100 ng/µl were injected into the cytoplasm of stage VI oocytes (25 nl/oocyte). The injected oocytes were incubated for 24 h to allow the synthesis of proteins. An equivalent amount of injected or uninjected oocytes was harvested for subsequent studies.
Immunoprecipitation and Western blotting analysis. Cells were washed once with PBS and lysed in ice cold immunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and 1x protease inhibitor cocktail) for 30 min, and the debris was cleared by centrifugation at 13,400 x g for 10 min at 4°C. The in vitro translated proteins or lysate was incubated with 0.5 µg of desired antibodies overnight at 4°C. After incubation with 40 µl of protein A/G-agarose for 1 h, the immune complexes were collected and washed extensively with immunoprecipitation assay buffer. For Western blot analysis, the samples were resolved by SDS-7% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Hercules, Calif.). The primary antibodies were diluted in blocking buffer (50 mM Tris-HCl, 150 mM NaCl [pH 7.5], 0.1% Tween 20, 5% skim milk) and added to the membranes for overnight rotation at 4°C. The membrane was washed four times in 1x PBS (Gibco BRL) and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. All blots were visualized with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, Ill.).
RNA preparation and RT-PCR. Total RNA was extracted with TRI reagent (Molecular Research Center, Inc., Cincinnati, Ohio) following the manufacturer's instruction. The reverse transcriptase (RT)-PCR analysis was performed with the Access RT-PCR system (Promega, Madison, Wis.). The primers for the CAT reporter gene were as follows: forward, 5'-GTG AGC TGG TGA TAT GGG ATA GTG TT-3'; reverse, 5'-CAT ATT GGC CAC GTT TAA ATC AAA A-3'. The PCR conditions were the same as described in the previous section except that 24 cycles of reaction were carried out.
CAT assay. The reporter activity of MMTV-CAT was quantitatively measured by enzyme-linked immunosorbent assay. In accordance with the instructions with the CAT enzyme-linked immunosorbent assay kit (Roche), diluted cell lysates were added to the anti-CAT-coated microtiter plates and incubated at 37°C for 1 h. After extensive washes, incubation of digoxigenin-conjugated anti-CAT was followed by anti-digoxigenin-peroxidase and substrate. The photometric detection was carried out with a microtiter plate reader at 405 nm with a reference wavelength of approximately 490 nm. The results were normalized with respect to total protein concentration. Total protein concentration was measured with Bio-Rad protein assay dye reagent.
Statistical analysis and quantitative analysis. Statistical analysis of multiple comparisons was performed with SPSS (Statistical Package for the Social Sciences software (SPSS, Inc.). Analysis of variance was carried out by both Tukey and Dunnett methods. The significance level was set at 0.05. The results described in the text are consistent with both Tukey and Dunnett analyses. Data quantitation was performed with NIH Image software version 1.62.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
To confirm the expression of SRCs in T47D cells and to understand whether the preferential recruitment of SRC-1 and SRC-2 by activated NR is a consequence of dramatically different levels of these proteins, we estimated endogenous SRC levels by comparison with Flag-tagged SRC proteins expressed in Xenopus oocytes. Serial dilutions of equivalent amounts of Flag-tagged SRC-1, -2, and -3 normalized according to Western blotting with anti-Flag antibody were probed with individual SRC antibodies to provide references for the estimation of SRC antibody efficiency (Fig. 2C). Western blotting analyses of endogenous SRC proteins were performed simultaneously with the reference Flag-tagged SRC proteins. The normalized results shown in Fig. 2C demonstrated that SRC-2 and SRC-1 levels were 89.5 and 52.6% of that of SRC-3, respectively. These results suggest that endogenous SRC-1, -2, and -3 levels are comparable. It seems highly unlikely that the slight differences between SRC-1 and SRC-2 levels could contribute to NR selectivity.
Differential assembly of HATs mediated by disparate receptor-SRC interactions. Multiple components of transcription coactivator complexes are important for transcriptional activation mediated by nuclear receptors (32, 37, 41). However, the composition of coactivator complexes in each NR signaling may not be the same due to relative affinity between the receptor and cofactors, competition between cofactors or coactivator complexes for common interaction motifs (47), or cell-specific cofactor levels (40). Having demonstrated a distinct pattern of SRC coactivator recruitment by two different NRs, we then examined the outcome of this differential utilization of SRCs on the recruitment of other coregulators. In a proof-of-principle experiment, we compared PR- and GR-mediated recruitment of additional coactivators on the MMTV promoter by using antibodies against several HATs (CBP, p300, and pCAF), TRAP220 (an important component of mediator complex), and BRG-1. Remarkably, CBP was significantly recruited in the presence of progesterone while minimally recruited by dexamethasone (Fig. 3). The observation that PR, SRC-1, and CBP were recruited concomitantly to the promoter coincides with a report where a large multicomponent complex was identified in T47D cells, the members of which include PR, SRC-1, and CBP (50). To our surprise, pCAF appeared to be recruited to the MMTV promoter in the presence of dexamethasone but not in the presence of progesterone. There were no significant differences between PR- and GR-mediated recruitment of TRAP220, polymerase II (Fig. 3), and BRG-1 (data not shown). However, p300 was recruited in response to stimulation with both steroids, but slightly higher levels of occupancy were repeatedly observed after dexamethasone induction. Furthermore, SRCs and HAT cofactors were recruited to the MMTV promoter within a similar time frame (data not shown). Based on the data in Fig. 2 and 3, it seems that PR, SRC-1, SRC-3, and CBP are major components in progesterone-dependent transcription, while GR, SRC-2, SRC-3, and pCAF constitute complexes for dexamethasone induction on the MMTV promoter. Thus, hormone stimulations of different NRs induce differential coactivator utilization within the same MMTV promoter context, and a different subset of HATs are recruited in each case for transcriptional activation, endowing potential downstream specificity to different ligand signals.
|
|
Transcription signal cascades determined by specific SRC-NR interactions. We next wished to determine whether the differential recruitment of CBP versus pCAF by PR versus GR and distinct patterns of histone modification are the downstream effect of differential recruitment of SRC-1 or SRC-2. Therefore, we performed RNA interference of SRCs followed by ChIP to examine the potential changes that knockdown of either coactivator would produce. To eliminate the nonspecific effects of transfection reagent on cell growth, the control groups (Fig. 6A and B) were treated with siRNA against luciferase, which is absent in the T47D/CAT0 cells. Reduction of SRC-1, -2, or -3 levels in T47D cells by siRNA appeared to eliminate the recruitment of these coactivators to the MMTV promoter (Fig. 6A), while these siRNA had no effect on PR or GR recruitment (Fig. 6B), confirming the expected specificity of individual siRNA. The recruitment of CBP and acetylation of histone H4 lysine 5 with progesterone stimulation decreased significantly in the absence of SRC-1 and were even further decreased when both SRC-1 and SRC-3 were reduced by RNA interference (Fig. 6B). As expected, recruitment of CBP and acetylation at lysine 5 of histone H4 were not affected by the reduction of the SRC-2 level and were only marginally reduced by loss of both SRC-2 and SRC-3. In contrast, reduction of pCAF recruitment and acetylation of lysine 14 on histone H3 after dexamethasone treatment were associated with reduction of SRC-2 expression and were further decreased by reducing both SRC-2 and SRC-3. In controls, reduction of SRC-1 had little effect on either pCAF recruitment or acetylation of histone H3 at lysine 14. When both SRC-1 and SRC-3 expression were inhibited, reduction of pCAF recruitment was not proportional to the decrease in acetylation at lysine 14 on histone H3, indicating involvement of another HAT, possibly GCN5. GCN5 has been shown to acetylate lysine 14 of histone H3 as well. These observations suggest that initial NR-SRC interactions influence the way by which transcription signal is transmitted. Our results also substantiated an order of sequential recruitment: PR/GR to SRCs to other HATs, in agreement with previous reports (29, 42).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although SRC-1 and SRC-2 have been reported to interact with both PR and GR in overexpression transfection experiments (11, 14, 19, 32, 45), several indirect lines of evidence have suggested the existence of coactivator preferences. First, on the basis of chromatography and coimmunoprecipitation approaches, a large multicomponent complex containing PR, SRC-1, and CBP was identified in T47D cells (50). Second, in an attempt to isolate steady-state coregulator complexes, both SRC-1 and SRC-2 were identified in a preformed complex. However, upon progesterone induction, more SRC-1 than SRC-2 appeared to comigrate with PR (33). Third, an SRC-1 mutant devoid of activation domains but containing HAT and receptor interaction domains displayed a strong dominant-negative repressor activity for certain nuclear receptors (18, 36), but the dominant-negative activity of this mutant SRC-1 is less for GR (14). Finally, a preferential interaction between GR and SRC-2 has been suggested in several studies (9, 14, 16, 25, 48). In this study, we have provided in vivo evidence that PR prefers SRC-1 and GR prefers SRC-2 on MMTV promoter. Our data substantiate the observations mentioned above. Furthermore, our results suggest a potential mechanism by which different NRs mediate transcription signaling by preferential recruitment of a subset of cofactors.
The molecular details of PR and GR preferences for different coactivators in vivo are not clear. One possibility is NR box binding specificity, as suggested above. A small region of SRC-2 (amino acids 730 to 1121) retains steroid receptor binding, transactivation, and coactivator activities. However, this region of SRC-2 exhibits relatively low homology with SRC-1 (16), suggesting a potential molecular basis for functional differences between SRC-1 and SRC-2. In addition, allosteric effects upon DNA binding may trigger different receptor conformations (26, 51), which in turn could be a determinant for the binding of coactivators. In an effort to determine whether or not the coactivator preferences are observed in solution or evolve only on the MMTV promoter, we have performed coimmunoprecipitation studies of PR and GR with SRCs in the presence or absence of ligands. Since only GR/SRC-2 interaction was observed in solution (data not shown), it is possible that allosteric regulation of coactivator interactions on the MMTV promoter may influence the PR/SRC-1 preference.
Importantly, we have observed differential recruitment of CBP and pCAF on the MMTV promoter in response to progesterone or dexamethasone stimulation, whereas p300 is recruited in both cases. The differential recruitment of CBP or pCAF appears to correlate with the preferential targeting by SRC-1 or SRC-2. Although the function of SRC coactivators' recruitment of CBP/p300 is well documented, such specificity has not been shown before. Only specificity of HAT requirement for different transcription factors has been indicated in previous studies. For example, inactivation of the HAT domains of CBP has no influence on the coactivation of RAR. On the other hand, CREB (CRE-binding protein) function needs CBP-HAT activity and not P/CAF-HAT activity (21). Although SRC-1 has been shown to interact with p300, CBP, and pCAF (18, 19, 43, 45), only the recruitment of CBP/p300 was clearly observed upon progesterone treatment. It is interesting that pCAF associates with the GR/SRC-2 complex specifically in the presence of dexamethasone. The ability of pCAF to mediate GR function was recently tested by examining the effects of cotransfected pCAF on GR transactivation properties (14). The requirement of pCAF in RAR-mediated activation was also established (21). We show here that SRC-2 is required for pCAF recruitment, either directly or through an interaction with p300, as reduction of SRC-2 by RNA interference limited the recruitment of pCAF (Fig. 6). It is unlikely that CBP/p300 interacts directly with GR/PR (19), since reports from different groups reveal little or no direct binding to these receptors (7, 43).
The state of chromatin modification has long been recognized to influence gene expression. Our results indicate that differentially assembled coactivator complexes lead to specific patterns of targeted histone modification. The acetylation of specific lysine residues on histone H3 or H4 was correlated with the recruitment of distinct HAT activity that could be altered by knockdown of either SRC-1 or SRC-2 (Fig. 6). To date, the manner by which different HATs discriminate lysines in vivo is not fully understood. CBP and p300 have been shown to acetylate histone H4 at lysine 5, 8, 12, and 16 in vitro (20). However, we have observed significant acetylation of lysine 5 and 8 on histone H4 by CBP/p300 only after progesterone stimulation. This suggests that the actual target of p300/CBP acetylation could be redirected in a different context. It may also reflect the diversity of histone modification in vivo, since acetylation at lysine 12 on histone H4 was considered to be an inactivating modification in certain cellular contexts (17). The acetylation of K14 on histone H3 after dexamethasone induction is consistent with the recruitment of pCAF (39). However, GCN5, which bears homology to human pCAF, also displays a preference for K14 of histone H3. It is possible that GCN5 also could be recruited to the MMTV promoter after GR recruitment, as demonstrated for pCAF. Histone modification associated with acetylation of K14 (phosphorylation of serine 10 and hypomethylation of lysine 9) is consistent with the histone code pattern observed by others (17). Histone H3 phosphorylation at Ser10 has recently been elucidated as a novel regulatory mechanism involving histone kinases such as Rsk-2 and Msk-1 (8). These histone codes and the recently reported in vitro binding of a HAT bromodomain with acetylated lysines within H3 and H4 peptides have been implicated in a targeting step for events following histone modification.
In addition to coactivator specificities, our studies also suggest that SRC-3 is utilized similarly by either receptor. Elimination of SRC-3 along with either SRC-1 or SRC-2 resulted in a further reduction of PR- or GR-mediated CAT expression compared to knockdown of SRC-1 or SRC-2 alone. This suggests a role for SRC-3 in both pathways. ChIP assays indicate that SRC-3 also contributes to the recruitment of HAT and histone modification by SRC-1 or SRC-2. In addition, SRC-2 knockdown has a slight but reproducible influence on PR-mediated CAT expression (Fig. 6), indicating that it plays a minor role in PR function. However, such a contribution involving a limited amount of SRC-2 recruitment was not detectable by ChIP assays. Therefore, the reason for the moderate attenuation of the CAT activity after SRC-1 silencing (Fig. 6) could be explained by recruitment of SRC-3 and a small amount of SRC-2 to the MMTV promoter or to SRC coactivator-independent activation. Our observations are consistent with previous reports, which demonstrate the redundant functions of SRCs (38, 49).
Finally, we have revealed the roles of SRCs in mediating transcription signal cascades by RNA interference. Our suggestion that SRC-1 could be important in the progesterone pathway has been supported by the study of SRC-1 knockout mice in our lab (49). Therefore, we propose a model in which preferential SRC family coactivator-steroid receptor interactions influence differential recruitment of downstream components by each receptor (Fig. 7). Progesterone signaling is transduced from PR/SRC-1/CBP along with other coactivators to induce specific histone modification, including acetylation at lysine 5 of histone H4 on the MMTV promoter. The cascade originating from dexamethasone induction is conveyed primarily through a complex containing GR/SRC-2/pCAF and leads to specific posttranslational modification on histone H3. Our study provides conceptual proof of principle for a mechanism by which coactivator preferences for nuclear receptors can regulate the fates of downstream events in transcription signaling.
|
|
ACKNOWLEDGMENTS |
|---|
This work was supported by National Institutes of Health grants to B.W.O. (HD08818 and Atlas program).
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Allan, G. F., N. H. Ing, S. Y. Tsai, G. Srinivasan, N. L. Weigel, E. B. Thompson, M. J. Tsai, and B. W. O'Malley. 1991. Synergism between steroid response and promoter elements during cell-free transcription. J. Biol. Chem. 266:5905-5910.
3. Bhakat, K. K., and S. Mitra. 2000. Regulation of the human O(6)-methylguanine-DNA methyltransferase gene by transcriptional coactivators cAMP response element-binding protein-binding protein and p300. J. Biol. Chem. 275:34197-34204.
4. Borud, B., T. Hoang, M. Bakke, A. L. Jacob, J. Lund, and G. Mellgren. 2002. The nuclear receptor coactivators p300/CBP/cointegrator-associated protein (p/CIP) and transcription intermediary factor 2 (TIF2) differentially regulate PKA-stimulated transcriptional activity of steroidogenic factor 1. Mol. Endocrinol. 16:757-773.
5. Boyd, K. E., and P. J. Farnham. 1999. Coexamination of site-specific transcription factor binding and promoter activity in living cells. Mol. Cell. Biol. 19:8393-8399.
6. Bramlett, K. S., and T. P. Burris. 2002. Effects of selective estrogen receptor modulators (SERMs) on coactivator nuclear receptor (NR) box binding to estrogen receptors. Mol. Genet. Metab. 76:225-233.[CrossRef][Medline]
7. Chen, D., S. M. Huang, and M. R. Stallcup. 2000. Synergistic, p160 coactivator-dependent enhancement of estrogen receptor function by CARM1 and p300. J. Biol. Chem. 275:40810-40816.
8. Chen, H., M. Tini, and R. M. Evans. 2001. HATs on and beyond chromatin. Curr. Opin. Cell Biol. 13:218-224.[CrossRef][Medline]
9. Darimont, B. D., R. L. Wagner, J. W. Apriletti, M. R. Stallcup, P. J. Kushner, J. D. Baxter, R. J. Fletterick, and K. R. Yamamoto. 1998. Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev. 12:3343-3356.
10. Deroo, B. J., and T. K. Archer. 2001. Glucocorticoid receptor-mediated chromatin remodeling in vivo. Oncogene 20:3039-3046.[CrossRef][Medline]
11. Ding, X. F., C. M. Anderson, H. Ma, H. Hong, R. M. Uht, P. J. Kushner, and M. R. Stallcup. 1998. Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC- 1): multiple motifs with different binding specificities. Mol. Endocrinol. 12:302-313.
12. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.[CrossRef][Medline]
13. Gehin, M., M. Mark, C. Dennefeld, A. Dierich, H. Gronemeyer, and P. Chambon. 2002. The function of TIF2/GRIP1 in mouse reproduction is distinct from those of SRC-1 and p/CIP. Mol. Cell. Biol. 22:5923-5937.
14. He, Y., D. Szapary, and S. S. Simons, Jr. 2002. Modulation of induction properties of glucocorticoid receptor-agonist and -antagonist complexes by coactivators involves binding to receptors but is independent of ability of coactivators to augment transactivation. J. Biol. Chem. 277:49256-49266.
15. Heery, D. M., E. Kalkhoven, S. Hoare, and M. G. Parker. 1997. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733-736.[CrossRef][Medline]
16. Hong, H., K. Kohli, M. J. Garabedian, and M. R. Stallcup. 1997. GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol. Cell. Biol. 17:2735-2744.[Abstract]
17. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science 293:1074-1080.
18. Kalkhoven, E., J. E. Valentine, D. M. Heery, and M. G. Parker. 1998. Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor. EMBO J. 17:232-243.[CrossRef][Medline]
19. Kamei, Y., L. Xu, T. Heinzel, J. Torchia, R. Kurokawa, B. Gloss, S. C. Lin, R. A. Heyman, D. W. Rose, C. K. Glass, and M. G. Rosenfeld. 1996. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403-414.[CrossRef][Medline]
20. Kimura, A., and M. Horikoshi. 1998. How do histone acetyltransferases select lysine residues in core histones? FEBS Lett. 431:131-133.[CrossRef][Medline]
21. Korzus, E., J. Torchia, D. W. Rose, L. Xu, R. Kurokawa, E. M. McInerney, T. M. Mullen, C. K. Glass, and M. G. Rosenfeld. 1998. Transcription factor-specific requirements for coactivators and their acetyltransferase functions. Science 279:703-707.
22. Kuo, M. H., J. E. Brownell, R. E. Sobel, T. A. Ranalli, R. G. Cook, D. G. Edmondson, S. Y. Roth, and C. D. Allis. 1996. Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. Nature 383:269-272.[CrossRef][Medline]
23. Kusk, P., S. John, G. Fragoso, J. Michelotti, and G. L. Hager. 1996. Characterization of an NF-1/CTF family member as a functional activator of the mouse mammary tumor virus long terminal repeat 5' enhancer. J. Biol. Chem. 271:31269-31276.
24. Le Bihan, S., V. Marsaud, C. Mercier-Bodard, E. E. Baulieu, S. Mader, J. H. White, and J. M. Renoir. 1998. Calcium/calmodulin kinase inhibitors and immunosuppressant macrolides rapamycin and FK506 inhibit progestin- and glucocorticosteroid receptor-mediated transcription in human breast cancer T47D cells. Mol. Endocrinol. 12:986-1001.
25. Leers, J., E. Treuter, and J. A. Gustafsson. 1998. Mechanistic principles in NR box-dependent interaction between nuclear hormone receptors and the coactivator TIF2. Mol. Cell. Biol. 18:6001-6013.
26. Lefstin, J. A., and K. R. Yamamoto. 1998. Allosteric effects of DNA on transcriptional regulators. Nature 392:885-888.[CrossRef][Medline]
27. Leo, C., H. Li, and J. D. Chen. 2000. Differential mechanisms of nuclear receptor regulation by receptor-associated coactivator 3. J. Biol. Chem. 275:5976-5982.
28. Li, J., B. W. O'Malley, and J. Wong. 2000. p300 requires its histone acetyltransferase activity and SRC-1 interaction domain to facilitate thyroid hormone receptor activation in chromatin. Mol. Cell. Biol. 20:2031-2042.
29. Liu, Z., J. Wong, S. Y. Tsai, M. J. Tsai, and B. W. O'Malley. 2001. Sequential recruitment of steroid receptor coactivator-1 (SRC-1) and p300 enhances progesterone receptor-dependent initiation and reinitiation of transcription from chromatin. Proc. Natl. Acad. Sci. USA 98:12426-12431.
30. Lo, W. S., R. C. Trievel, J. R. Rojas, L. Duggan, J. Y. Hsu, C. D. Allis, R. Marmorstein, and S. L. Berger. 2000. Phosphorylation of serine 10 in histone H3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14. Mol. Cell 5:917-926.[CrossRef][Medline]
31. McInerney, E. M., D. W. Rose, S. E. Flynn, S. Westin, T. M. Mullen, A. Krones, J. Inostroza, J. Torchia, R. T. Nolte, N. Assa-Munt, M. V. Milburn, C. K. Glass, and M. G. Rosenfeld. 1998. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev. 12:3357-3368.
32. McKenna, N. J., R. B. Lanz, and B. W. O'Malley. 1999. Nuclear receptor coregulators: cellular and molecular biology. Endocr. Rev. 20:321-344.
33. McKenna, N. J., Z. Nawaz, S. Y. Tsai, M. J. Tsai, and B. W. O'Malley. 1998. Distinct steady-state nuclear receptor coregulator complexes exist in vivo. Proc. Natl. Acad. Sci. USA 95:11697-11702.
34. Mukherjee, R., S. Sun, L. Santomenna, B. Miao, H. Walton, B. Liao, K. Locke, J. H. Zhang, S. H. Nguyen, L. T. Zhang, K. Murphy, H. O. Ross, M. X. Xia, C. Teleha, S. Y. Chen, B. Selling, R. Wynn, T. Burn, and P. R. Young. 2002. Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor alpha. J. Steroid Biochem. Mol. Biol. 81:217-225.[CrossRef][Medline]
35. Nolte, R. T., G. B. Wisely, S. Westin, J. E. Cobb, M. H. Lambert, R. Kurokawa, M. G. Rosenfeld, T. M. Willson, C. K. Glass, and M. V. Milburn. 1998. Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma. Nature 395:137-143.[CrossRef][Medline]
36. Onate, S. A., S. Y. Tsai, M. J. Tsai, and B. W. O'Malley. 1995. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354-1357.
37. Rosenfeld, M. G., and C. K. Glass. 2001. Coregulator codes of transcriptional regulation by nuclear receptors. J. Biol. Chem. 276:36865-36868.
38. Sadow, P. M., O. Chassande, K. Gauthier, J. Samarut, J. Xu, B. W. O'Malley, and R. E. Weiss. 2003. Specificity of thyroid hormone receptor subtype and steroid receptor coactivator (SRC)-1 on thyroid hormone action. Am. J. Physiol. Endocrinol. Metab. 284:E36-E46.
39. Schiltz, R. L., C. A. Mizzen, A. Vassilev, R. G. Cook, C. D. Allis, and Y. Nakatani. 1999. Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates. J. Biol. Chem. 274:1189-1192.
40. Shang, Y., and M. Brown. 2002. Molecular determinants for the tissue specificity of SERMs. Science 295:2465-2468.
41. Shang, Y., X. Hu, J. DiRenzo, M. A. Lazar, and M. Brown. 2000. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103:843-852.[CrossRef][Medline]
42. Sharma, D., and J. D. Fondell. 2002. Ordered recruitment of histone acetyltransferases and the TRAP/Mediator complex to thyroid hormone-responsive promoters in vivo. Proc. Natl. Acad. Sci. USA 99:7934-7939.
43. Sheppard, H. M., J. C. Harries, S. Hussain, C. Bevan, and D. M. Heery. 2001. Analysis of the steroid receptor coactivator 1 (SRC1)-CREB binding protein interaction interface and its importance for the function of SRC1. Mol. Cell. Biol. 21:39-50.
44. Smith, C. L., H. Htun, R. G. Wolford, and G. L. Hager. 1997. Differential activity of progesterone and glucocorticoid receptors on mouse mammary tumor virus templates differing in chromatin structure. J. Biol. Chem. 272:14227-14235.
45. Smith, C. L., S. A. Onate, M. J. Tsai, and B. W. O'Malley. 1996. CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription. Proc. Natl. Acad. Sci. USA 93:8884-8888.
46. Torchia, J., D. W. Rose, J. Inostroza, Y. Kamei, S. Westin, C. K. Glass, and M. G. Rosenfeld. 1997. The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387:677-684.[CrossRef][Medline]
47. Treuter, E., L. Johansson, J. S. Thomsen, A. Warnmark, J. Leers, M. Pelto-Huikko, M. Sjoberg, A. P. Wright, G. Spyrou, and J. A. Gustafsson. 1999. Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes. J. Biol. Chem. 274:6667-6677.
48. Voegel, J. J., M. J. Heine, M. Tini, V. Vivat, P. Chambon, and H. Gronemeyer. 1998. The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways. EMBO J. 17:507-519.[CrossRef][Medline]
49. Xu, J., Y. Qiu, F. J. DeMayo, S. Y. Tsai, M. J. Tsai, and B. W. O'Malley. 1998. Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 279:1922-1925.
50. Xu, Y., L. Klein-Hitpass, and M. K. Bagchi. 2000. E1A-mediated repression of progesterone receptor-dependent transactivation involves inhibition of the assembly of a multisubunit coactivation complex. Mol. Cell. Biol. 20:2138-2146.
51. Yi, P., M. D. Driscoll, J. Huang, S. Bhagat, R. Hilf, R. A. Bambara, and M. Muyan. 2002. The effects of estrogen-responsive element- and ligand-induced structural changes on the recruitment of cofactors and transcriptional responses by ER alpha and ER beta. Mol. Endocrinol. 16:674-693.
This article has been cited by other articles:
