Journal: Molecular and Cellular Biology
Article Title: A Mammalian Bromodomain Protein, Brd4, Interacts with Replication Factor C and Inhibits Progression to S Phase
Figure Lengend Snippet: 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 (10 5 ) 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 G 1 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).
Article Snippet: Antibodies to PCNA, YY1 and green fluorescent protein (GFP) were obtained from Santa Cruz, Flag M2 was obtained from Sigma, bromodeoxyuridine (BrdU) was obtained from PharMingen, and second antibodies were obtained from Amersham.
Techniques: Inhibition, Construct, Transfection, Microscopy, Plasmid Preparation, Staining, BrdU Incorporation Assay, Recombinant, Synthesized, DNA Synthesis