The bromodomain protein, BRD4, has been identified recently as a therapeutic target in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease; its loss is a prognostic signature for metastatic breast cancer. BRD4 also contributes to regulation of both cell cycle and transcription of oncogenes, HIV, and human papilloma virus (HPV). Despite its role in a broad range of biological processes, the precise molecular mechanism of BRD4 function remains unknown. We report that BRD4 is an atypical kinase that binds to the carboxyl-terminal domain (CTD) of RNA polymerase II and directly phosphorylates its serine 2 (Ser2) sites both in vitro and in vivo under conditions where other CTD kinases are inactive. Phosphorylation of the CTD Ser2 is inhibited in vivo by a BRD4 inhibitor that blocks its binding to chromatin. Our finding that BRD4 is an RNA polymerase II CTD Ser2 kinase implicates it as a regulator of eukaryotic transcription.B RD4 is a BET family protein that was identified originally as a ubiquitously expressed chromatin adapter that maintains epigenetic memory and regulates cell cycle progression (1). More recently, it has been characterized as a key determinant in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease (2-7). It suppresses tumor metastasis in mice, and its expression is a prognostic signature of breast cancer survival (8). BRD4 has been proposed to be a structural scaffold that regulates transcription indirectly by recruiting the elongation factor, PTEFb, to the transcription preinitiation complex (9).Productive transcription depends on the phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II). The Pol II CTD consists of consecutive repeats of the heptad Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , which vary in number between 26 in yeast and 52 in mammals (10). Phosphorylation of the CTD residues serine 5 (Ser5) and serine 2 (Ser2) is necessary for the recruitment of RNA capping and splicing factors, respectively (11). The order, pattern, and temporal separation of these phosphorylation events ensure an orderly transition from initiation to productive transcription elongation (11-13). CTD Ser5 residues are phosphorylated primarily by the CDK7 kinase component of TFIIH (14), whereas the subsequent Ser2 phosphorylation, currently attributed primarily to the CDK9 subunit of PTEFb and/or CDK12/13, releases Pol II from an early elongation block (15, 16). PTEFb nuclear localization and activation depends on BRD4, which is also known to interact with a variety of other factors, including the mediator complex (1,17). Despite the preponderance of evidence indicating a vital role for BRD4 in transcription, its precise molecular function is unknown.Here, we show that BRD4 is an atypical protein kinase that exhibits both auto-and transphosphorylation. Furthermore, BRD4 directly and specifically phosphorylates the Pol II CTD at the Ser2 position, and it is di...

Recent therapeutic successes have renewed interest in drug combinations, but experimental screening approaches are costly and often identify only small numbers of synergistic combinations. The DREAM consortium launched an open challenge to foster the development of in silico methods to computationally rank 91 compound pairs, from the most synergistic to the most antagonistic, based on gene-expression profiles of human B cells treated with individual compounds at multiple time points and concentrations. Using scoring metrics based on experimental dose-response curves, we assessed 32 methods (31 community-generated approaches and SynGen), four of which performed significantly better than random guessing. We highlight similarities between the methods. Although the accuracy of predictions was not optimal, we find that computational prediction of compound-pair activity is possible, and that community challenges can be useful to advance the field of in silico compound-synergy prediction.

SUMMARY We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using ChIP-Seq, versus TOP1 activity using TOP1-Seq, a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation, but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.

Bromodomain protein 4 (BRD4) is a chromatin-binding protein implicated in cancer and autoimmune diseases that functions as a scaffold for transcription factors at promoters and super-enhancers. Whereas chromatin de-compaction and transcriptional activation of target genes are associated with BRD4 binding, the mechanism(s) involved are unknown. We report that BRD4 is a novel histone acetyltransferase (HAT) that acetylates histones H3 and H4 with a pattern distinct from other HAT’s. Both mouse and human BRD4 demonstrate intrinsic HAT activity. Importantly, BRD4 acetylates H3K122, a residue critical for nucleosome stability, resulting in nucleosome eviction and chromatin de-compaction. Nucleosome clearance by BRD4 occurs genome-wide, including at its targets MYC, FOS and AURKB (Aurora B kinase), resulting in increased transcription. Since BRD4 regulates transcription, these findings lead to a model where BRD4 actively links chromatin structure and transcription: It mediates chromatin de-compaction by acetylating and evicting nucleosomes of target genes, thereby activating their transcription.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous largescale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.Cancer forms and progresses through a series of critical transitions-from pre-malignant to malignant states, from locally contained to metastatic disease, and from treatment-responsive to treatment-resistant tumors (Figure 1). Although specifics differ across tumor types and patients, all transitions involve complex dynamic interactions between diverse pre-malignant, malignant, and non-malignant cells (e.g., stroma cells and immune cells), often organized in specific patterns within the tumor