1. CoCoA, a Nuclear Receptor Coactivator which Acts through an N-Terminal Activation Domain of p160 Coactivators 
Jeong Hoon Kim, Hongwei Li, Michael R Stallcup 
Molecular Cell 
Volume 12, Issue 6, Pages (December 2003)
DOI: /S (03)

2. Figure 1
Contribution of the bHLH-PAS Domain of GRIP1 to Coactivator Activity
CV-1 cells were transfected with plasmids as indicated and grown in medium containing 100 nM E2 for ER or 20 nM DHT for AR. The luciferase activity results shown are representative of five independent experiments. Reporter plasmids (200 ng): MMTV(ERE)-LUC for ER, MMTV-LUC for AR. Expression vectors: pHE0 encoding ERα, 2 ng for (A) and 0.02 ng for (C); pSV-AR0, 5 ng for (B) and 0.5 ng for (D); pSG5.HA-GRIP1 or pSG5.HA-GRIP1(563–1462), 200 ng; pSG5.HA-CARM1, 200 ng; pCMV-p300, 200 ng.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Identification, Sequence, Secondary Structure, and Expression of CoCoA
(A) Functional domains of GRIP1 include the basic-helix-loop-helix (bHLH) region, Per-Arnt-Sim (PAS) A and B regions, three NR binding motifs (NR Boxes), and activation domains (AD1 and AD2). The N-terminal GRIP1 fragment used as bait in the yeast two-hybrid screen is also indicated (bait).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Interaction of CoCoA Coiled-Coil Domain with bHLH-PAS Domain of GRIP1 In Vivo and In Vitro
(A) For mammalian two-hybrid assays CV-1 cells were transfected with 200 ng of GK1-LUC reporter plasmid (controlled by Gal4 response elements) and 200 ng of each indicated expression plasmid. Luciferase activity results shown are representative of three independent experiments.
(B) Coimmunoprecipitation of V5-epitope tagged CoCoA and HA-epitope tagged GRIP1 or its deletion mutants was performed with COS-7 cells in 100 mm dishes transfected with 2.5 μg each of pcDNA3.1-CoCoA.V5 and pSG5.HA-GRIP1, pSG5.HA-GRIP1N (amino acids 5–479), or pSG5.HA-GRIP1ΔN (amino acids 563–1462). Cell extracts were prepared 48 hr after transfection and immunoprecipitated with anti-V5 monoclonal antibody or control normal mouse IgG. Immunoprecipitated V5-CoCoA (ii and iv) and coprecipitated HA-GRIP1 proteins (i and iii) were detected by immunoblot analysis with antibodies against the epitope tags. A portion of the original cell extract was also examined for HA-GRIP1 and V5-CoCoA expression (5% input).
(C) To test binding of full-length (FL) CoCoA or its fragments to GST-fused GRIP1 bHLH-PAS domain (GRIP1N) in vitro, pSG5.HA vectors (2.5 μg) encoding HA-epitope-tagged CoCoA fragments were transfected into COS-7 cells. Cell extracts were prepared 48 hr after transfection and incubated with glutathione-Sepharose beads bound with bacterially expressed GST-GRIP1N (amino acids 5–479) or GST. The bound proteins were analyzed by immunoblot with anti-HA antibody. A portion of the original cell extracts was also tested for HA-CoCoA expression (10% input).
(D) CV-1 cells were transfected with 200 ng of GK1-LUC reporter plasmid and 200 ng of the indicated expression plasmids. Luciferase activity results shown are representative of three independent experiments.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Coactivator Function of CoCoA with NR Depends on the Presence of GRIP1 with an Intact bHLH-PAS Domain
(A) CV-1 cells were transfected with MMTV(ERE)-LUC reporter plasmid (200 ng), pHE0 encoding human ER (2 ng), pSG5.HA-GRIP1 (100 ng), pSG5.HA-CARM1 (200 ng), and variable amounts (50, 100, 200, and 400 ng) of pSG5.HA-CoCoA, as indicated, and grown in medium containing or lacking E2. Luciferase activity results shown are representative of five independent experiments.
(B) COS-7 cells were transfected with 2 μg each of pHEG0, pSG5.HA-CoCoA, and pSG5.HA-GRIP1, as indicated, and grown with E2. Cell extracts were immunoprecipitated (IP) with anti-ERα antibody or control normal rabbit IgG. Immunoprecipitated ERα (bottom panels) and coprecipitated HA-CoCoA (top panels) were detected by immunoblot (IB) analysis with anti-ERα antibody and anti-HA antibody.
(C) CV-1 cells were transfected with 200 ng of MMTV(ERE)-LUC, 2 ng of ER expression vector, 100 ng of pSG5.HA-GRIP1 or pSG5.HA-GRIP1(563–1462), and 200 ng of pSG5.HA-CoCoA, as indicated. Luciferase activity results shown are representative of three independent experiments.
(D) Transient transfections using pSG5.HA-GRIP1ΔAD1 or pSG5.HA-GRIP1ΔAD2 were performed as in (C).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
C-Terminal Activation Domain Is Required for CoCoA Coactivator Function
(A) CV-1 cells were transfected with GK1-LUC reporter plasmid (200 ng) and a plasmid encoding either Gal4 DBD or Gal4 DBD fused to various CoCoA fragments (200 ng), as indicated. In the schematic diagrams, numbers indicate CoCoA amino acid positions. Luciferase activity results shown are representative of three independent experiments.
(B) CV-1 cells were transfected with GK1-LUC (200 ng), plasmid encoding Gal4 DBD or Gal4 DBD fused to GRIP1N (amino acids 5–479) (200 ng), and 200 ng of pSG5.HA plasmid encoding full-length (FL) CoCoA or its fragments, as indicated. Luciferase activity results shown are representative of three independent experiments.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
CoCoA Is Recruited to Estrogen-Responsive Promoters in a Hormone-Dependent Manner
In vivo binding of ERα, GRIP1, and CoCoA to either the endogenous pS2 promoter or the transiently transfected MMTV (ERE) promoter was examined by ChIP (A–C) and reporter CoIP (D) assays.
(A) Crosslinked, sheared chromatin from MCF-7 cells grown with or without E2 (45 min) was immunoprecipitated with the indicated antibodies. The following volumes of the coprecipitated DNA were analyzed by PCR using primers to amplify the pS2 promoter (opposing pair of arrows in the diagram) or the β-actin coding region: 1 μl of 1:10 diluted samples (input, ER, and CoCoA); 1 or 2 μl of the undiluted samples (IgG or GRIP1). Results shown are representative of ten independent experiments.
(B) Real-time PCR analysis using primers for the pS2 promoter was performed with 1 μl of 1:10 diluted samples. The results are shown as percentage of input and are the mean and standard deviation from triplicate reactions.
(C) ChIP and ReIP assays were performed for the pS2 promoter in MCF-7 cells untreated or treated with E2 for 45 min. First (ChIP) and second (ReIP) chromatin immunoprecipitations were performed with the indicated antibodies. Results shown are representative of three independent experiments.
(D) Occupancy of the transiently transfected MMTV(ERE) promoter by ER and coactivators was examined by reporter CoIP assay as described in the Experimental Procedures. Transfected COS-7 cells were untreated or treated with E2 for 45 min before formaldehyde crosslinking. Chromatin immunoprecipitation was performed with the indicated antibodies; PCR analysis was performed with primers spanning the nucleosome B region of the MMTV promoter (pair of opposing arrows in the diagram). Results shown are representative of four independent experiments.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Requirement for Endogenous CoCoA for GRIP1 and ER Function, and Synergistic Enhancement of NR-Mediated Transcriptional Activation by Four Coactivators (GRIP1, CoCoA, CARM1, and p300)
(A) COS-7 cells in 12-well dishes were transfected using Lipofectamine 2000 with GK1-LUC reporter plasmid (200 ng), plasmids encoding Gal4 or Gal4-GRIP1N (amino acids 5–479) (200 ng), and 80 pmole of either the CoCoA siRNA duplex or scramble siRNA duplex. Luciferase activity was measured 72 hr after transfection. Results shown are representative of four independent experiments. (Inset) Total RNA from the siRNA-transfected cells was analyzed by reverse transcriptase-PCR analysis using primers for CoCoA or β-actin mRNA.
(B) MCF-7 cells were transfected using Lipofectamine 2000 with 2ERE-tk-LUC reporter (200 ng) and 40 (+) or 80 (++) pmole of either the CoCoA siRNA duplex or scramble siRNA duplex, as indicated. Forty-eight hours after transfection, cells were treated with E2 or untreated and harvested after an additional 24 hr for luciferase assays. Results shown are representative of three independent experiments. Reverse transcriptase-PCR analysis was also performed to confirm the reduction of CoCoA mRNA levels (data not shown).
(C) MCF-7 cells were transfected using Targefect-siRNA Transfection Kit with the indicated siRNA and treated or untreated with E2 as in (B). Total RNA was analyzed by reverse transcriptase-PCR to measure the levels of CoCoA, pS2, and β-actin mRNA. Results shown are representative of four independent experiments.
(D) CV-1 cells were transfected with MMTV(ERE)-LUC reporter plasmid (200 ng), pHE0 (0.002 ng), pSG5.HA-GRIP1 (100 ng), pSG5.HA-CARM1 (200 ng), pCMV-p300 (200 ng), and pSG5.HA-CoCoA (200 ng), as indicated. Transfected cells were grown in medium containing E2. Luciferase activity results shown are representative of four independent experiments.
(E) Model of the p160 transcription complex. The activated NR binds to the hormone response element (HRE) and recruits p160 coactivators to the promoter by directly binding to the NR interaction domain (NID) of the p160 coactivator. AD1 and AD2 in the C-terminal region of p160 coactivators transmit the transcriptional activation signals by recruiting downstream secondary coactivators p300/CBP and CARM1, respectively. AD3 activation domain in the N-terminal bHLH-PAS region of p160 coactivators also transmits the transcriptional activation signal by recruiting CoCoA. CoCoA mediates transcriptional activation through its C-terminal transcriptional activation domain by binding to currently unknown proteins or components of the transcription machinery.
Molecular Cell  , DOI: ( /S (03) )