Ectopic Brd4 expression inhibits cell growth. (A) Reduced colony formation by NIH 3T3 cells expressing ectopic Brd4. Cells (10⁵) were transfected with an empty vector, pcxn (Control) or pcxn2-Brd4 and selected in G418 for 2 weeks (upper panels). Colonies were stained with methylene blue. The graph shows the number of colonies per plate in cells transfected with the control or Brd4. Also shown are the results of immunoblot analysis of Brd4 protein expression in control (pcxn2) or pcxn2-Brd4 transfectants (lower right panels). YY1 was used as a control for protein loading throughout this work. (B) TET-inducible Brd4 expression. TET-inducible cells were treated with DOX at the indicated doses for the indicated hours, and Brd4 expression was detected by immunoblot analysis. (C) TET-induced inhibition of cell growth. Parental or TET-inducible cells were treated with DOX (2 μg/ml) or left untreated, and cell numbers were counted on the indicated days (left panels). Inducible Brd4 expression was monitored by immunoblot analysis (right panels).

Cell cycle analysis. (A) Reduced S-phase cells after transient Brd4 transfection. NIH 3T3 cells (2 × 10⁶) were transfected with 30 μg of GFP-Brd4, Brd4-GFP, or control pEGFP-Cl (GFP only) along with 3 μg of pCMX-IL-2R (left panels). Twenty-four hours after transfection, cells were labeled with BrdU (10 μM) for 30 min and then sorted by anti-IL-2R antibody (GFP⁺ cells, ∼95%). Nuclei were double stained with fluorescein isothiocyanate-conjugated anti-BrdU antibody and PI and analyzed by flow cytometry. Also shown are the results of immunoblot detection of GFP-Brd4 with anti-GFP antibody (right panels). (B) The parental and TET-inducible cells were synchronized by serum starvation, and Brd4 was induced by DOX 24 h prior to release. Cells were harvested at 0 and 16 h after release, stained with PI, and analyzed by flow cytometry. The panels at the right show the percentage of the S-phase population. Values represent the averages from three independent experiments plus standard deviations. Panels at the bottom show the results of immunoblot detection of Brd4 expression.

Brd4-RFC interaction in vivo. (A) Coimmunoprecipitation of endogenous Brd4 and RFC-140. Extracts (500 μg of protein) from NIH 3T3 cells were immunoprecipitated (IP) with the antibodies indicated above the panels. Precipitates were tested for RFC-140 and Brd4 by immunoblot analysis (Blot) using the indicated antibodies. Input represents 10% of the extracts used for precipitation. (B) Extracts from cells transfected with a Flag-Brd4 or empty vector were immunoprecipitated with anti-Flag antibody, and precipitates were blotted for RFC-140 or Flag-Brd4 (left panels). Extracts from cells transfected with Flag-Brd4 were immunoprecipitated with anti-RFC-140 antibody and blotted with anti-Flag antibody or anti-RFC-140 antibody (right panels). Input represents 10% of extracts. (C) Coimmunoprecipitation of RFC subunits. Extracts from cells transfected with Flag-Brd4 or the empty Flag vector were precipitated with anti-Flag antibody and blotted with antibodies to the indicated subunits of RFC. Input represents 10% of extracts. (D) Lack of Brd4-PCNA interaction. Extracts were immunoprecipitated with the antibodies indicated above the panels and blotted for Brd4, RFC-140, or PCNA.

Brd4-RFC interaction in vitro. (A) Either full-length Brd4 (lanes 1 to 4) or D1 (lanes 5 to 8) (both 0.5 μg) was added to in vitro transcribed-translated RFC. ³⁵S-labeled RFC was immunoprecipitated with preimmune sera (Pr) (lanes 2 and 6), Brd4 antisera (lanes 3 and 7), or p37 antisera (lanes 4 and 8). Precipitated materials were resolved by SDS-12%PAGE. LO, load on, represents 10% of the aliquots used in the immunoprecipitation. (B) Full-length Brd4 (0.4 μg) was added to in vitro transcribed-translated RFC-p140, p40, p38, p37, and p36. Material shown in lanes 1, 4, and 7 was immunoprecipitated with either preimmune sera (lanes 2, 5, and 8) or anti-Brd4 antisera (lanes 3, 6, and 9). Fifty percent of the immunoprecipitated products was subjected to SDS-12%PAGE.

RFC-140 overexpression alleviates Brd4 inhibition of G₁/S transition. (A) Partial restoration of cell growth by RFC-140. TET-inducible Brd4 cells (2 × 10⁶) were transfected with 30 μg of pcDNA-RFC-140 or empty vector along with 3 μg of pCMX-IL-2R. Transfected cells were separated by sorting and induced by treatment with DOX for 2 days. The graph indicates relative cell recovery, with the cell yield from untreated cells taken to be 100%. Values are the means from three independent experiments. The mean cell recoveries shown in lanes 1 to 4 were 6.3 × 10⁶, 3.2 × 10⁶, 7.2 × 10⁶, and 6.1 × 10⁶, respectively. The panels below the graph show the results of immunoblot detection of RFC-140 and Brd4. (B) Counterregulation of S-phase entry by RFC-140 and Brd4. Cells (2 × 10⁶) were transfected with 15 μg of pcxn2-Brd4 and/or 15 μg of pcDNA-RFC-140 along with 3 μg of pcMX-IL-2R. Cells were allowed to proceed to S phase for 24 h andwere labeled with 10 μM BrdU for 30 min and then sorted as described in the legend to Fig. 2A. Cells were stained with fluorescein isothiocyanate-conjugated anti-BrdU antibody and PI and were analyzed by flow cytometry. The panels at the bottom show the results of immunoblot detection of Brd4 and RFC-140 in transfected cells.

Brd4 domains required for the interaction with RFC and inhibition of S-phase progression. (A) Diagram of Brd4 deletion constructs. BD, bromodomain; ET, ET domain. Solid boxes represent the presence of indicated domains, and diagonal lines represent the absence of indicated domains. (B) Subcellular localization of GFP-Brd4. NIH 3T3 cells (10⁵) were transfected with 2 μg of the indicated constructs, and GFP localization was visualized by microscopy 24 h after transfection. (C) HeLa cells synchronized at G₁ were transfected with empty pCMV-Flag vector (Cont, control), pCMV-Flag full-length Brd4, or the indicated deletion constructs and harvested at 16 h. Extracts were precipitated with anti-Flag antibody and analyzed for RFC-140 and Flag-Brd4 by immunoblot analysis. Input represents Flag-Brd4 in 8% of the extracts. (D) Inhibition ofBrdU uptake by ectopically expressed Brd4 deletion constructs. Serum-starved NIH 3T3 cells were transfected with 4 μg of EGFP-Brd4 by Lipofectamine-Plus 4 h prior to release. Cells were allowed to proceed to S phase in the presence of BrdU (10 μM) for 16 h and were fixed and stained for BrdU (diagram at top left). FBS, fetal bovine serum. The number of cells with GFP-Brd4 (green) with or without BrdU incorporation (red) was counted from five independent fields. All cells were detected by Hoechst DNA stain (blue). A total of ∼150 cells was counted in each field. The table at top right indicates the percentage of BrdU-positive cells in the total number of GFP-positive cells. (E) Influence of recombinant Brd4 deletion constructs on the elongation of a singly primed DNA template by the DNA polymerase δ holoenzyme. DNA elongation reactions were carried out with 1 fmol (lanes 1 to 7) and 10 fmol (lanes 8 to 10) of RFC. Thirty-five, 70, 175, and 175 ng of D3 was added to lanes 2, 3, 4 and 9, respectively, and 35, 70, 175, and 175 ng of D1 was added to lanes 5, 6, 7 and 10, respectively. The DNA synthesized in each reaction (in picomoles) was as follows: 9.81 in lane 1, 13.7 in lane 2, 9.82 in lane 3, 9.51 in lane 4, 8.01 in lane 5, 5.01 in lane 6, 2.61 in lane 7, 17.0 in lane 8, 16.4 in lane 9, and 12.2 in lane 10. In the absence of RFC, no DNA synthesis was observed (data not shown).

Cell cycle-regulated interaction between Brd4 and RFC-140. (A) NIH 3T3 cells synchronized by serum starvation were released and harvested at the indicated times and analyzed by immunoblot analysis. Cell synchronization was confirmed by flow cytometry analysis (bottom). (B) A model for Brd4 action. Levels of Brd4 and RFC-140 increase during G₁, and Brd4 interacts efficiently with RFC during G₁ to S. This interaction may influence the activity of RFC in supporting DNA replication.