Progesterone and glucocorticoid receptors recruit distinct coactivator complexes and promote distinct patterns of local chromatin modification

Abstract

It is well established that steroid receptor function requires interaction with coactivators. However, the mechanisms through which steroid receptors elicit precise assembly of coactivator complexes and the way the steroid activation signal is transduced remain elusive. Using a T47D cell line stably integrated with a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter, we demonstrate that specific steroid receptors exhibit preferential recruitment of SRC-1 family coactivators, which determines the subsequent recruitment of specific downstream coregulator molecules. Upon ligand treatment, progesterone receptor (PR) interacted preferentially with SRC-1, which recruited CBP and significantly enhanced acetylation at K5 of histone H4. In contrast, activated glucocorticoid receptor (GR) preferentially associated with SRC-2 (TIF-2/GRIP-1), which subsequently recruited pCAF and led to specific modification of histone H3, suggesting that specific coactivators recruit distinct histone acetyltransferases to modulate the transcription of steroid-responsive genes. Loss-of-function experiments further support the predicted roles of SRC-1 and SRC-2 in, respectively, PR- and GR-mediated transcription on the MMTV promoter. This study indicates that differential recruitment of coactivators by nuclear receptors determines the assembly of coactivator complexes on target promoters to mediate specific transcription signals.

FIG. 1.

Properties of T47D/MMTV-CAT as a system for in vivo studies. (A) Schematic structure of MMTV-CAT integrated into the T47D cells. The locations of PCR primers specific for the MMTV promoter region containing the HRE are indicated. (B) MMTV-CAT gene expression induced by progesterone (lane P) or dexamethasone (lane D) was analyzed by RT-PCR after 3 h of treatment. The levels of GAPDH serve as a reference for quantitation as well as control for loading. The semiquantitative results represent the relative levels of CAT mRNA expression and GAPDH levels relative to the those of uninduced control. Lanes −, controls without hormone. (C) Ligand-dependent recruitment of nuclear receptors to the MMTV promoter examined by ChIP. Soluble chromatin was prepared from T47D cells treated with progesterone (lane P), dexamethasone (lane D), or synthetic androgen (lane A) R1881 for 1 h. Receptor-bound DNA immunoprecipitated with antibody against PR, GR, or AR was amplified by the MMTV primers illustrated in Fig. 1A. The PCR products are labeled −MMTV. Primers specific for the GAPDH coding region were included in the same PCRs to amplify nonspecifically immunoprecipitated DNA as a loading control, indicated by asterisks. The material in the Input panel was amplified by using DNA extracted from diluted chromatin that was not immunoprecipitated. (D) Significant amounts of PR and GR are present in the T47D/CAT0 cells. Endogenous PR and GR were immunoprecipitated from the T47D/CAT0 cells and detected by Western blotting, as described in Materials and Methods.

FIG. 2.

Detection of PR/GR and SRC proteins on the MMTV promoter by ChIP. (A) Ligand- and receptor-mediated recruitment of SRC-1, SRC-2, and SRC-3 was determined by ChIP assays using the same batch of T47D/CAT0 cells treated with progesterone (lanes P) or dexamethasone (lanes D) or untreated cells (lanes −). The lower bands were amplified by using primers for the MMTV promoter, and the slower-migrating bands represent GAPDH control. (B) SRC antibody specificities were tested by using in vitro-translated SRC-1, -2, or -3 or T47D whole-cell lysate by immunoprecipitation-Western blotting with the indicated antibodies. About 20% of each sample was used for inputs. (C) Xenopus oocytes lysate expressed Flag-tagged SRC proteins were prepared in serial twofold dilutions for Western blot analysis using anti-Flag antibody (left panel). Equivalent amounts of each Xenopus lysate dilution were used in Western blotting with anti-SRC-1, -2, or -3. Similar serial dilutions of T47D cell lysate were prepared for Western blotting together with the blots containing Flag-tagged SRC proteins. The bands labeled with asterisks represent nonspecific reactions. Results of Western blotting were quantitated with NIH Image version 1.62. The relative levels of SRC proteins were calculated by normalization with quantitated Flag-SRC references. The relative level for SRC-3 was arbitrarily set as 100%.

FIG. 3.

Differential assembly of HATs in disparate NR signaling. ChIP analysis was performed to detect the occupancy of HATs, TRAP220, or polymerase II on the MMTV promoter after a 1-h treatment with progesterone (lanes P) or dexamethasone (lanes D). The faster-migrating bands represent products amplified from the MMTV promoter.

FIG. 4.

Diverse histone modifications associated with distinct hormone pathways. Hormone-dependent acetylation, phosphorylation, or methylation-demethylation of selective histone residues on the MMTV promoter was examined by ChIP assays with the indicated antibodies. The symbols are as described in the legends to Fig. 2A and 3.

FIG. 5.

Modulation of PR or GR activity by loss of SRC coactivator function. (A) RNA interference was carried out by introducing siRNA against individual SRCs (siSRC-1, -2, and -3) into the T47D/CAT0 cells for 3 days. Total protein was extracted with TRI reagent (Molecular Research Center, Inc). Western blotting analysis was performed to monitor the reduction of specific proteins with the indicated antibodies. The asterisk indicates a nonspecific band, used as a loading control in place of β-actin. (B) CAT activity was measured after RNA interference with the indicated siSRCs and treatment with progesterone or dexamethasone for 24 h. The uninduced or induced controls (lanes 1, 2, and 7) were treated with an unrelated siRNA against luciferase. The siSRCs labeled 1+3 and 2+3 represent the combinations of siRNA against SRC-1 and SRC-3 and SRC-2 and SRC-3, respectively. Triplicate results were normalized against results for hormone-treated controls (lanes 2 and 7), which are arbitrarily set as 100 for comparison. Analysis of variance of each individual group was done. Statistical results are mentioned in the text.

FIG. 6.

Effects of SRC silencing on cofactor recruitment and histone modification. (A) Reduction of SRC-1, -2, and -3 specifically eliminated recruitment of these proteins on the MMTV promoter. (B) The recruitment of receptors or HATs or acetylation of selective histone residues on the MMTV promoter was monitored by ChIP assays after a 1-h hormone treatment following RNA silencing of the indicated SRCs, as described in the legends to Fig. 2A, 3, and 4. Relative changes of PCR products were calculated based on the quantitated results from original images. Normalization was performed against GAPDH first and then against the adjusted input, followed by normalization against the untreated sample (−) that was arbitrarily set to 1.

FIG. 7.

Models of stepwise events in NR signaling. Progesterone activation signal is transmitted from PR recruitment of SRC-1/SRC-3, which further recruits CBP along with other coactivators (not included in the diagram) to induce specific histone modification, including acetylation at lysine 5 of histone H4. The cascade originating from dexamethasone induction is conveyed through GR's interaction with SRC-2 and SRC-3, which dictates recruitment of pCAF and other cofactors with various enzyme activities, leading to specific posttranslational modification of histone H3.