Bromodomain-containing Protein 4 (BRD4) Regulates RNA Polymerase II Serine 2 Phosphorylation in Human CD4+ T Cells

Weishi Zhang,Celine Prakash,Calvin Sum,Yue Gong,Yinghui Li,Jeffrey J.T Kwok,Nina Thiessen,Sven Pettersson,Steven J.M Jones,Stefan Knapp,Henry Yang,Keh-Chuang Chin

doi:10.1074/jbc.m112.413047

Abstract

Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser-2-phosphorylated Pol II (Pol II Ser-2) at both enhancers and promoters of active genes. Disruption of bromodomain-histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser-2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited diacetylated H4 (lysine 5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells.

Highlights

bromodomain-containing protein 4 (BRD4) interacts with positive transcription elongation factor b (P-TEFb), which regulates polymerase II (Pol II) elongation
BRD4 Binding Is Associated with Active Genes across the Genome—To establish whether BRD4 regulates transcription elongation in a genome-wide manner, we performed ChIP followed by DNA sequencing in primary human CD4ϩ T cells to identify 33,544 BRD4 binding sites
Inactive loci IL13 and IL5 lacked occupancy by BRD4 (Fig. 1B). To determine whether these findings were indicative of a broader trend in transcriptional regulation, we examined the relationship between BRD4 sites and levels of gene expression on a global scale

Summary

Background

BRD4 interacts with P-TEFb, which regulates Pol II elongation. Results: Disruption of BRD4 binding by JQ1 resulted in reduced Pol II Ser-2 in CD4ϩ T cells. Conclusion: BRD4 affects Pol II Ser-2 phosphorylation at a subset of lineage-specific active genes in primary human CD4ϩ T cells. Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4؉ T cells to reveal that BRD4 co-localizes with Ser-2-phosphorylated Pol II (Pol II Ser-2) at both enhancers and promoters of active genes. Disruption of bromodomain-histone acetylation interactions by JQ1, a smallmolecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser-2, and reduced expression of lineage-specific genes in primary human CD4؉ T cells. BRD4 has been implicated in the recruitment of P-TEFb and transcription elongation [8, 10], but the

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION