The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis

Abstract

Previous in vitro studies showed that the bromodomain binds to acetyllysines on histone tails, leading to the proposal that the domain is involved in deciphering the histone code. However, there is little in vivo evidence supporting the binding of bromodomains to acetylated chromatin in the native environment. Brd4 is a member of the BET family that carries two bromodomains. It associates with mitotic chromosomes, a feature characteristic of the family. Here, we studied the interaction of Brd4 with chromatin in living cells by photobleaching. Brd4 was mobile and interacted with chromatin with a rapid "on and off" mode of binding. This interaction required both bromodomains. Indicating a preferential interaction with acetylated chromatin, Brd4 became less mobile upon increased chromatin acetylation caused by a histone deacetylase inhibitor. Providing biochemical support, salt solubility of Brd4 was markedly reduced upon increased histone acetylation. This change also required both bromodomains. In peptide binding assays, Brd4 avidly bound to di- and tetraacetylated histone H4 and diacetylated H3, but weakly or not at all to mono- and unacetylated H3 and H4. By contrast, it did not bind to unacetylated H4 or H3. Further, Brd4 colocalized with acetylated H4 and H3 in noncentromeric regions of mitotic chromosomes. This colocalization also required both bromodomains. These observations indicate that Brd4 specifically recognizes acetylated histone codes, and this recognition is passed onto the chromatin of newly divided cells.

Fig. 2.

FLIP analysis of bromodomain point mutants. (A) Bromodomain sequence comparison. (B) Bromodomain point mutants tested in this study. (C) FLIP analysis of mutants tested without (control, Upper) or with (Lower) TSA treatment. T_1/2 of M5 and M6 was ≈120 s, whereas that of wild-type Brd4 and other mutants was ≈120 s. None of single point mutants showed a discernible difference in the FLIP pattern (data not shown).

Fig. 3.

Solubility of Brd4 in differential salt extraction. (A) Endogenous Brd4 was extracted with indicated concentrations of NaCl and detected by immunoblot. (B) Transfected GFP-Brd4 or GFP-Brd4 deletions were extracted and tested as in A. (C) Histone tail peptides tested for Brd4 binding (Top). Bound Brd4 was detected by immunoblot (Middle). TFIIB in unbound materials was detected to verify equal loading (Bottom).

Fig. 1.

FLIP analysis of GFP-Brd4. Murine P19 embryonal carcinoma cells expressing GFP-Brd4 or free GFP were photobleached in the circled area, and loss of fluorescence was monitored in the rectangle. (A) Distribution of GFP proteins before and after photobleaching. Bleaching of free GFP (Top) and GFP-Brd4 was performed with live (Middle) or fixed (Bottom) cells. (Bar indicates 3.6 μm.) (B) Quantification of fluorescence loss in A. Values represent the average of eight independent measurements ± SD. Arrows indicate the time (s) required for 50% fluorescence loss (T_1/2). (C) Diagram of Brd4 deletions. Solid blocks (BD1 and BD2) mark bromodomains; shaded blocks mark the ET domain. (D) Distribution of indicated GFP proteins in cells pretreated with 50 ng/ml TSA for 4 h. (E) FLIP patterns for indicated GFP proteins without (control) or with TSA treatment.

Fig. 4.

Chromosomal localization of Brd4 and Brd2. (A) Endogenous Brd4, Brd2, or acetylated H4 was visualized on mitotic chromosomes of P19 cells by indirect immunostaining. DNA was counterstained by Hoechst 33342. Arrows indicate centromeres where staining of Brd4, Brd2, or acetyl H4 was absent. (Bar indicates 8 μm.) (Upper Right) Enlargement of chromosome stained for acetyl H4 (red), Brd4 (green), and DNA (blue). Yellow arrow indicates a centromere. (B) Distributions of transfected GFP-Brd4 and deletions were analyzed on mitotic chromosomes prepared from cells treated with or without TSA.