(A) Knock-down by siRNA. MCF-7 cells were transfected with 2ERE-TK-LUC and 20 (+) or 40 (++) pmole of either the CCAR1 siRNA duplex or non-specific (NS) siRNA duplex, as indicated. 48 hr after transfection, cells were treated with 100 nM E2 or untreated and harvested after an additional 24 hr for luciferase assays and immunoblot analysis with CCAR1 antibody 435A. Results shown are mean and SD of triplicate points.
(B) MCF-7 cells were transfected with the indicated siRNA (20 pmole) and treated or untreated with E2 as in (A). Total RNA was prepared, and real-time qRT-PCR was performed. CCAR1 and pS2 mRNA levels are normalized to β-actin mRNA levels, and results shown are mean and SD from triplicate reactions.
(C) Statistical correlation of endogenous CCAR1 and pS2 mRNA levels. MCF-7tTS/shCCAR1 6-4 cells were cultured in the absence or presence of doxycyclin (Dox) for 48 hr, treated with E2 for 24 hr, and collected for the preparation of total RNAs. Real-time qRT-PCR analyses were performed on RNA from 11 independent experiments. The protein levels of CCAR1 and actin were determined by immunoblot; the CCAR1 antibody was 435A.
(D) CCAR1 is required for E2-dependent growth of MCF-7 cells. MCF-7tTS cell lines in 12 well plates were cultured in the absence or presence of 10 nM E2 and 1 μg/ml Dox, as indicated. Viable cells were counted by trypan blue staining. Results shown are mean and SD of triplicate wells in a single experiment and are representative of three independent experiments.

(A) Immunoblot analyses of A549 cells infected with a lentivirus expressing a non-specific (NS) or CCAR1 shRNA. The CCAR1 antibody used was 435A.
(B) A549 cells expressing a NS or CCAR1 shRNA were treated with 100 nM DEX or ethanol vehicle for 2 hr. The indicated mRNA levels were determined by qRT-PCR as in Figure 2B.
(C) Occupancy of the IGFBP1 promoter by Mediator (MED1) and Pol II. A549 cells infected with lentivirus encoding a non-specific (NS) or CCAR1 shRNA were treated with 100 nM DEX for 2 hr. ChIP assays were performed as in Figure 3A. The CCAR1 antibody used was 270A.
(D) The proposed role of CCAR1 in coordinating p160 coactivator and Mediator complexes. CCAR1 associates with DNA-bound NRs either directly or through interaction with CoCoA. CCAR1 recruits Mediator, which helps to recruit and activate RNA polymerase II and its basal transcription factors.

(A) CCAR1 and CoCoA are recruited to the endogenous p21 promoter. Crosslinked, sheared chromatin from MCF-7 cells treated with or without 10 μM etoposide (Eto) for 2 hr was immunoprecipitated with the indicated antibodies. Real-time PCR analyses were performed using primers for the p21 promoter (nucleotide -2290 to -2074 relative to transcription start site) or pS2 promoter. The results are shown as percentage of input and are the mean and SD from triplicate PCR reactions. Results shown are from a single experiment and are representative of 3 independent experiments.
(B) CCAR1 and CoCoA are required for p53-mediated transcription. U2OS cells were transfected with the indicated siRNA (40 pmole) and treated or untreated with 10 μM etoposide (Eto) for 18 hr. Total RNA was prepared, and real-time qRT-PCR was performed to determine p21, CCAR1, CoCoA, and β-actin mRNA levels. Results shown are normalized to β-actin mRNA levels, are mean and SD from triplicate PCR reactions, and are representative of 3 independent experiments. NS, non-specific.

(A) Schematic representation of CCAR1 structure. SAP, SAP domain; PABP, poly A binding protein homology region.
(B) MCF-7 cell nuclear extracts were incubated with immobilized GST-CoCoA AD. The bound proteins were analyzed by immunoblot; the CCAR1 antibody was 435A.
(C) HA-tagged CCAR1 synthesized in vitro was incubated with equal amounts of GST or GST-CoCoA protein bound to beads. Bound proteins were analyzed by immunoblot with anti-HA antibody.
(D) GST pull-down assays were performed as described in (C) using in vitro translated HA-CoCoA or its fragments and GST or GST-CCAR1 1-660 bound to beads.
(E) MCF-7 cells in 12-well plates were transfected with luciferase reporter plasmids (200 ng) containing estrogen-responsive elements (ERE) in combination with variable amounts (300, 600, and 900 ng) of pSG5.HAb-CCAR1 and grown in medium containing or lacking 100 nM estradiol (E2) before conducting luciferase assays on cell extracts. Results shown are mean and SD of triplicate points.
(F) Transient transfections using ERE-reporters and pHE0 encoding ER (2 ng) in CV-1 cells were performed as described in (E).
(G) CV-1 cells were transfected with plasmids as indicated below and grown in medium containing 100 nM dexamethasone (DEX) for GR or 100 nM triiodothyronine (T3) for TR. Reporter plasmids (200 ng): MMTV-LUC for GR, MMTV(TRE)-LUC for TR. Expression vectors: pKSX (GR) (10 ng), pCMX-TR (10 ng), pSG5.HAb-CCAR1 (300, 600, and 900 ng). Results shown are mean and SD of triplicate points.

(A) ChIP assay. Crosslinked, sheared chromatin from MCF-7 cells treated with or without 100 nM E2 (45 min) was immunoprecipitated with the indicated antibodies. The CCAR1 antibody used was an equal mixture of N306 and C1128. qPCR analyses were performed using primers for the pS2 promoter. The results are shown as percentage of input and are the mean and SD from triplicate reactions. Numbers above the bars indicate fold increase in pS2 promoter occupancy with E2 treatment.
(B and C) ChIP and ReIP assays, using the indicated antibodies, were performed as described in (A) and (Kim et al., 2003). The CCAR1 antibody used was 435A.
(D and E) CCAR1 is required for recruitment of Mediator and Pol II to the pS2 promoter. Specific siRNAs targeting CCAR1 or control non-specific (NS) siRNAs were transfected into MCF-7 cells 72 hr before E2 stimulation. ChIP assays were performed as in (A). Protein levels were monitored by immunoblot using the indicated antibodies. The CCAR1 antibody used was 435A. * indicates p<0.05 and ** indicates p<0.01 with Student's t-test on triplicate PCR reactions from a single experiment. p values from paired, two-tailed t-tests for ChIP results from n independent experiments comparing RNAi against CCAR1 or against non-specific (NS) targets were: for CCAR1 recruitment to pS2 promoter in the presence of E2, p = 0.00016 (n = 9); for MED1 recruitment, p = 0.0011 (n = 9); for MED6 recruitment, p = 0.034 (n = 6); for pol II recruitment, p = 0.037 (n = 5).
(F) Cyclic and sequential recruitment of ER and its coactivators. ChIP assays were performed as in Supplemental Figure S5, except that MCF-7 cells were treated with E2 for the indicated times. The CCAR1 antibody used was 435A.

(A) HA-tagged p53 synthesized in vitro was incubated with equal amounts of GST, GST-CCAR1, or GST-CoCoA protein bound to beads. Bound proteins were analyzed by immunoblot with anti-HA antibody.
(B) Coactivator activity of CCAR1 and CoCoA for p53. SAOS2 cells (p53-null) in 12-well dishes were transfected with pSG5.HA-p53 (2 ng) and pG13-LUC (200 ng) in combination with variable amounts (200, 400, and 800 ng) of pSG5.HAb-CCAR1 or pSG5.HA-CoCoA. Cell extracts were assayed for luciferase activity. Results shown are mean and SD of triplicate points.
(C) CCAR1 cooperates with CoCoA to enhance p53 activity. SAOS2 cells were transfected with pG13-LUC (200 ng), pSG5.HA-p53 (2 ng), pSG5.HAb-CCAR1 (200 ng), and pSG5.HA-CoCoA (200 ng), and luciferase activity of cell extracts was determined.
(D) Synergistic enhancement of p53 activity under low-p53 conditions, which facilitate multiple-coactivator synergy. SAOS2 cells were transfected with pG13-LUC (200 ng), pSG5.HA-p53 (0.2 ng), pSG5.HAb-CCAR1 (200 ng), pSG5.HA-CoCoA (200 ng), and pCMV-p300 (200 ng).