DBC1 interacts with CCAR1 and functions as an ERα co-activator. (A) Nuclear extracts from HeLa S cells stably expressing FLAG-CCAR1 were immunoprecipitated with anti-FLAG M2 agarose. Bound proteins were eluted with FLAG peptide, resolved on 4%–12% gradient Bis-Tris gels, silver-stained, and analyzed by mass spectrometry. The identified peptides of DBC1 are listed. (B) Schematic representation of DBC1 and CCAR1 structures. NLS, nuclear localization signal; LZ, leucine zipper motif; SAP, SAP domain; Nudix, Nudix hydrolase domain. (C) Endogenous interaction between DBC1 and CCAR1. MCF-7 cell lysates were immunoprecipitated with anti-CCAR1 antibody or normal rabbit IgG, followed by immunoblot with the indicated antibodies. (D) CV-1 cells were transfected with pHE0 encoding ERα (2 ng) and MMTV(ERE)-LUC reporter in combination with various amounts (200, 400 and 800 ng) of pSG5.HA-DBC1 and grown in medium containing or lacking 100 nM estradiol (E2) before conducting luciferase assays on cell extracts. Data are means ± SD (n = 3). (E) Synergy between CCAR1 and DBC1. Transient transfections were performed as described in D. Expression vectors: pSG5.HA-CCAR1 (200 ng), pSG5.HA-DBC1 (200 and 400 ng). Data are means ± SD (n = 3).

Recruitment of DBC1 to selective ERα target genes and requirement of DBC1 for optimal recruitment of ERα to the target promoters. (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. qPCR analyses were performed using primers specific for the indicated promoters. The results are shown as percentage of input and are means ± SD (n = 3). Numbers above the bars indicate fold increase in the promoter occupancy with E2 treatment. (B) ChIP and ReIP assays, using the indicated antibodies, were performed as described in A. (C and D) MCF-7 cells infected with lentiviruses encoding a NS or DBC1 shRNA were treated with 100 nM E2 for 45 min. Protein levels were monitored by immunoblot using the indicated antibodies. ChIP assays using ERα antibody were performed as described in A.

DBC1 reverses SIRT1-mediated repression of ERα. (A) CV-1 cells were transfected with pHE0 (2 ng) and MMTV(ERE)-LUC reporter (200 ng) in combination with various amounts (10, 25 and 50 ng) of expression vectors for FLAG-tagged nuclear sirtuins and grown in medium containing or lacking 100 nM E2. Luciferase activity was measured 48 h after transfection. Expression levels of transfected sirtuins were analyzed by immunoblot with anti-FLAG antibody. (B) CV-1 cells were transfected with pHE0, MMTV(ERE)-LUC reporter, and FLAG-SIRT1 (25 and 50 ng) as in A and grown in medium containing or lacking 100 nM E2 and 5 µM EX-527 as indicated before measuring luciferase activity. Expression levels of transfected SIRT1 were analyzed by immunoblot with anti-FLAG antibody. (C) DBC1 rescues SIRT1-mediated repression of ERα. CV-1 cells were transfected with MMTV(ERE)-LUC reporter and pHE0 as in A in combination with expression vectors for FLAG-SIRT1 (50 ng) and HA-DBC1 or HA-DBC1ΔLZ (300 and 600 ng) as indicated before measuring luciferase activity. Expression levels of transfected SIRT1 and DBC1 were analyzed by immunoblot with anti-FLAG and anti-HA antibody, respectively. (D) Transient transfections using MMTV(ERE)-LUC and expression vectors for ERα, FLAG-SIRT1, HA-DBC1 or HA-DBC1ΔLZ and HA-CCAR1 (200 ng) were performed as described in C. Expression levels of transfected CCAR1 and DBC1 were analyzed by immunoblot with anti-HA antibody.

DBC1 is required for ERα function and estrogen-dependent growth of MCF-7 cells. (A) MCF-7 cells were transfected with 40 pmol of DBC1 siRNA#1 (siDBC1) or NS siRNA duplex. After 48 h transfection, cells were treated with 100 nM E2 or untreated and harvested after an additional 24 h. Total RNA was examined by real-time qRT–PCR analysis with primers specific for the indicated mRNAs. Results shown were normalized to β-actin mRNA levels and are means ± SD (n = 3). (B and C) MCF-7 cells infected with lentiviruses encoding a NS or CCAR1 shRNA were transfected with siDBC1#1 or siNS and treated or untreated with E2 as in A. Protein levels were monitored by immunoblot using the indicated antibodies. The indicated mRNA levels were determined by real time qRT–PCR as in A. (D) MCF-7 cells infected with lentiviruses encoding a NS or DBC1 shRNA were cultured in the absence or presence of 10 nM E2 for 8 days. Cell viability was determined by MTT assay. Data are means ± SD (n = 6). P-value was determined by Student’s t-test.

DBC1 inhibits SIRT1-mediated deacetylation of ERα. (A) 293 T cells were transfected with expression vectors as indicated. After 36 h transfection, the cells were treated with 0.5 µM TSA (Sigma) and 100 nM E2 for 12 h. Cell extracts were immunoprecipitated with anti-ERα antibody, and levels of acetylated ERα were determined by immunoblot using anti-acetyl lysine antibody. Input and immunoprecipitated proteins were analyzed by immunoblot with the indicated antibodies. (B) Acetylated ERα was incubated with purified SIRT1 and DBC1 in the presence of 1 mM NAD+ as indicated. Immunoblots were performed as described in A. (C) 293 T cells infected with lentiviruses encoding a NS or DBC1 shRNA were transfected with expression vectors as indicated and treated with TSA and E2 as described in A. Immunoprecipitation and immunoblots were performed as described in A. (D) Competition between DBC1 and SIRT1 for ERα binding. 293 T cells were transfected with expression plasmids as indicated and treated with E2 as described in A. FLAG-SIRT1 immunoprecipitates were analyzed by immunoblot using antibodies as indicated.

SIRT1 represses the co-activator synergy between DBC1 and CCAR1. (A) The LZ motif of DBC1 is required for DBC1-CCAR1 interaction. In vitro translated HA-DBC1 (WT or ΔLZ mutant) was incubated with GST or GST-CCAR1, and bound proteins were analyzed by immunoblot with anti-HA antibody. (B) SIRT1 blocks the interaction between DBC1 and CCAR1. 293 T cells were transfected with expression plasmids as indicated. HA-CCAR1 immunoprecipitates were analyzed by immunoblot using antibodies as indicated. (C) Transient transfections using MMTV(ERE)-LUC and expression vectors for ERα, HA-CCAR1, HA-DBC1 and FLAG-SIRT1 (WT or H363Y mutant, 25 and 50 ng) were performed as described in Figure 1E. (D) The proposed reciprocal roles of DBC1 and SIRT1 in ERα-mediated transcription and co-activator synergy. DBC1 has a dual function in regulating ERα activity: first as a co-activator by cooperating synergistically with CCAR1, and second as a negative regulator of SIRT1 by inhibiting SIRT1-mediated ERα deacetylation, and thus increasing DNA binding and transcriptional activity of ERα. SIRT1 also has a dual role in regulating ERα activity. In addition to deacetylating ERα, SIRT1 disrupts the interaction between ERα co-activators, DBC1 and CCAR1, by competing with CCAR1 for DBC1 binding.