The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3

Abstract

Bromodomain protein 4 (Brd4) plays critical roles in development, cancer progression, and virus-host pathogenesis. To gain mechanistic insight into the various biological functions of Brd4, we performed a proteomic analysis to identify and characterize Brd4-associated cellular proteins. We found that the extraterminal (ET) domain, whose function has to date not been determined, interacts with NSD3, JMJD6, CHD4, GLTSCR1, and ATAD5. These ET-domain interactions were also conserved for Brd2 and Brd3, the other human BET proteins tested. We demonstrated that GLTSCR1, NSD3, and JMJD6 impart a pTEFb-independent transcriptional activation function on Brd4. NSD3 as well as JMJD6 is recruited to regulated genes in a Brd4-dependent manner. Moreover, we found that depletion of Brd4 or NSD3 reduces H3K36 methylation, demonstrating that the Brd4/NSD3 complex regulates this specific histone modification. Our results indicate that the Brd4 ET domain through the recruitment of the specific effectors regulates transcriptional activity. In particular, we show that one of these effectors, NSD3, regulates transcription by modifying the chromatin microenvironment at Brd4 target genes. Our study thus identifies the ET domain as a second important transcriptional regulatory domain for Brd4 in addition to the carboxyl-terminal domain (CTD) that interacts with pTEFb.

Fig. 1.

Human Brd4 associates with multiple epigenetic and transcription regulators. (A) Schematic of the Brd4 fragments covering various domains used for the proteomic analysis. Full-length protein or the indicated fragments were expressed as N-terminally Flag-HA-tagged proteins in 293T cells, large-scale HA IPs were performed with whole-cell lysates, and immunoprecipitates were analyzed by LC-MS/MS. (B) Interaction maps. “Interaction from MS” was identified via mass spectrometry and CompPASS analysis.

Fig. 2.

Validation of interactions with human Brd4. (A) The indicated Brd4 fragments stably expressed in 293T cells were subjected to small-scale HA IPs. Equal amounts of input (IN) samples along with 50% of IPs were separated by SDS-PAGE and immunoblotted with antibodies specific to HA, ATAD5, NSD3, JMJD6, CHD4, and actin. (B) 293T cells were transiently transfected with Flag-GLTSCR1, Flag-ATAD5, Flag-NSD3, Flag-JMJD6, or Flag-CHD4, and cell lysates were harvested 48 h posttransfection. Proteins were immunoprecipitated with anti-Flag antibody and visualized as described in panel A with antibodies to Flag epitope, Brd4, or actin. (C) Endogenous IPs were performed using whole-cell lysates from 293T cells with antibodies to CHD4 or JMJD6. Bound proteins were captured using protein A beads, eluted, and analyzed by Western blotting with anti-CHD4, anti-JMJD6, anti-Brd4, or actin.

Fig. 3.

ET domain-mediated protein-protein interaction is conserved across the human BET proteins. (A) Amino acid sequence alignment of human BET proteins, Brd2, Brd3, Brd4, and Brd6, demonstrating high sequence similarity. (B) 293T cells were transfected with GFP-tagged constructs expressing the ET domains of Brd4, Brd3, and Brd2 (35). At 48 h posttransfection, anti-GFP immunoprecipitations were performed with whole-cell extract. Equal amounts of input (IN) samples along with 50% of IPs were separated by SDS-PAGE and immunoblotted with antibodies specific to GFP, NSD3, JMJD6, CHD4, and actin. The symbols *1, *2, and *3 denote the GFP band, the Brd4 band, and the Brd2/Brd3 bands, respectively.

Fig. 4.

The Brd4 ET domain associates with several multiprotein complexes. HA immunoprecipitations were performed with lysates from 293T cells stably expressing HA-GLTSCR1, HA-ATAD5, HA-NSD3, HA-JMJD6, or HA-CHD4, and the bound proteins were subjected to mass spectrometry and CompPASS analysis (MS or IP).

Fig. 5.

Effect of siRNA-mediated knockdown of Brd4 ET interactors on Brd4-dependent E2-mediated transcriptional activation of the BPV1 LCR. C33A/E2TA/BPV1LCRc2 cells were transfected with the indicated siRNA duplexes. (A) Knockdown of NSD3, JMJD6, CHD4, and CDK9 protein levels shown by resolving equal amounts of lysates by SDS-PAGE and immunoblotting with the indicated antibodies or the control, actin. (B) Effect of knocking down Brd4 ET interactors on BPV1 LCR reporter activity was quantified by measuring luciferase activity 72 h posttransfection and normalizing the relative luciferase units (RLU) to total protein concentrations from three replicate experiments. Equal amounts of lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies to determine knockdown levels of the proteins targeted by siRNA or the control, actin.

Fig. 6.

GLTSCR1, NSD3, and JMJD6 contribute to transcriptional activation of cellular target genes by Brd4. C33A cells were treated with siRNA to Brd4, GLTSCR1, NSD3, JMJD6, or CDK9, and total RNA was extracted 72 h posttransfection from duplicate experiments. Reverse transcription was then performed to obtain cDNA followed by RQ-PCR. Quantities determined were normalized to the housekeeping gene GAPDH and then compared to siControl sample to calculate relative mRNA levels.

Fig. 7.

The Brd4 ET domain-associated NSD3 is recruited to Brd4 target genes, CCND1, DCPS, and PIM2. (A to C) 293T cells stably expressing Flag-HA-NSD3 were treated with siRNA to NSD3, siNSD3, or with the control siRNA, siControl, for 72 h. ChIP was performed with anti-IgG (IgG), anti-Brd4 (Brd4), or anti-HA (NSD3) as indicated on the x axis. Immunoprecipitated DNA was quantified by real-time PCR in duplicates with primers specific to the promoter or coding regions of CCND1, PIM2, and DCPS genes, and results are presented as enrichment relative to input DNA. Each ChIP was repeated twice with reproducible results. Error bars represent standard deviations from means. (D to F) 293T/Flag-HA-NSD3 cells were treated with siRNA to Brd4, siBrd4, or with the control siRNA, siControl, for 72 h, and ChIP analysis was performed as indicated in panels A to C.

Fig. 8.

Knockdown of Brd4 or NSD3 reduces H3K36 trimethylation levels within the gene body of CCND1. (A) Schematic showing locations of primers used for ChIP/RQ-PCR analysis within the CCND1 locus. Nucleotide positions (bp) are depicted with respect to transcription start site (arrow). (B) HeLa cells were treated with siRNA to Brd4 (siBrd4), NSD3 (siNSD3), or the control siRNA (siControl) for 72 h. Chromatin extracts from these cells were subjected to immunoprecipitation with anti-H3 antibody (H3), anti-H3K36me3 antibody (H3K36me3), or the negative-control anti-IgG antibody (IgG). Immunoprecipitated DNA was quantified by RQ-PCR in duplicates with primers specific to the promoter or coding regions of the CCND1 gene. The results from this analysis are presented as percentages of input DNA. The experiment was repeated three times with reproducible profiles. Error bars represent standard deviations from means.