Abstract Epigenetic reprogramming, particularly histone deacetylation (HDAC), and reactive oxygen species (ROS) play key roles in melanoma progression and the development of drug resistance. While various histone deacetylase inhibitors (HDACi) are already in clinical use for hematological cancers, recent studies indicate their potential effectiveness in melanoma models as well. Given the increasing interest in enhancing the therapeutic efficacy of HDACi, we investigated the molecular mechanisms underlying their association with ROS formation in melanomas. Uveal melanoma (UM), a rare and aggressive eye cancer, has been identified in the TCGA dataset as expressing the highest levels of the superoxide-producing enzyme NADPH oxidase 5 (NOX5) among all human cancers. Our study demonstrates that HDACi treatment increases NOX5 expression in UM cell lines. This upregulation is accompanied by elevated extracellular ROS, which is suppressed when NOX5 is silenced. The use of actinomycin D and cycloheximide significantly reduced HDACi-induced NOX5 expression, suggesting regulation at both transcriptional and translational levels. HDACi treatment also led to accumulation of acetylated histones H3 and H4. Since histone acetylation (particularly H4K5/8/12Ac) recruits BRD4 to facilitate transcription, we evaluated the effect of BRD4 knockdown or exposure to BRD4 inhibitors on NOX5 levels in UM cells. Both approaches reduced NOX5 expression induced by HDACi. Furthermore, dual silencing of the transcription factors Sp1 and Sp3 markedly attenuated both basal and HDACi-induced expression of NOX5. ChIP assays revealed that HDACi treatment increases chromatin-associated acetylated histone H3, phosphorylated RNA polymerase II (p-Ser2), BRD4, Sp1, and Sp3 at the NOX5 promoter region. Collectively, these findings suggest that HDACi-mediated upregulation of NOX5 in UM cells occurs, at least in part, via transcriptional activation of the NOX5 promoter. This process is driven by HDACi-induced histone hyperacetylation, which enhances the recruitment of BRD4, Sp1, and Sp3 to the promoter region, thereby facilitating RNA polymerase II-dependent transcription. Given that HDAC inhibition elevates both NOX5 expression and ROS production, co-targeting HDACs and NOX5 may offer a novel and effective therapeutic strategy in the treatment of UM. Citation Format: Smitha Antony, Ballachanda N. Devaiah, Mariam M. Konaté, Yongzhong Wu, Guojian Jiang, Jennifer L. Meitzler, Jiamo Lu, Becky A. Diebold, David J. Mallick, Krishnendu K. Roy, Dinah S. Singer, James H. Doroshow. HDAC inhibitors induce NOX5 expression in human uveal melanoma cells in a Brd4-Sp1/Sp3-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4491.

Addiction to oncogene-rewired transcriptional networks is a therapeutic vulnerability in cancer cells, underscoring a need to better understand mechanisms that relay oncogene signals to the transcriptional machinery. Here, using human and mouse T cell acute lymphoblastic leukemia (T-ALL) models, we identify an essential requirement for the endosomal sorting complex required for transport protein CHMP5 in T-ALL epigenetic and transcriptional programming. CHMP5 is highly expressed in T-ALL cells where it mediates recruitment of the coactivator BRD4 and the histone acetyl transferase p300 to enhancers and super-enhancers that enable transcription of T-ALL genes. Consequently, CHMP5 depletion causes severe downregulation of critical T-ALL genes, mitigates chemoresistance and impairs T-ALL initiation by oncogenic NOTCH1 in vivo. Altogether, our findings uncover a non-oncogene dependency on CHMP5 that enables T-ALL initiation and maintenance.

BRD4, a pleiotropic regulator of chromatin structure and transcription, plays critical roles in cancer and immune responses. Unlike other transcriptional regulators, BRD4 largely remains bound to chromosomes during early mitosis. Here we report that BRD4 also regulates mitosis through its direct interaction with and phosphorylation of Aurora B kinase, an essential regulator of mitosis. BRD4 binding to Aurora B inhibits its kinase activity, preventing autophosphorylation and phosphorylation of the key mitotic targets histone H3 and MCAK, the mitotic centromere associated kinesin. This inhibition is relieved during metaphase when JNK is activated and phosphorylates BRD4, triggering its transient release from chromatin. Importantly, Aurora B activity during mitosis inversely correlates with BRD4 binding and directly correlates with JNK activation and BRD4 release. Our findings thus reveal a regulatory mechanism whereby Aurora B activity is directly controlled by BRD4, which in turn is regulated by JNK.

Bromodomain 4 (BRD4), a key regulator with pleiotropic functions, plays crucial roles in cancers and cellular stress responses. It exhibits dual functionality: chromatin-bound BRD4 regulates remodeling through its histone acetyltransferase (HAT) activity, while promoter-associated BRD4 regulates transcription through its kinase activity. Notably, chromatin-bound BRD4 lacks kinase activity, and RNA polymerase II (RNA Pol II)-bound BRD4 exhibits no HAT activity. This study unveils one mechanism underlying BRD4's functional switch. In response to diverse stimuli, c-Jun N-terminal kinase (JNK)-mediated phosphorylation of human BRD4 at Thr1186 and Thr1212 triggers its transient release from chromatin, disrupting its HAT activity and potentiating its kinase activity. Released BRD4 directly interacts with and phosphorylates RNA Pol II, PTEFb, and c-Myc, thereby promoting transcription of target genes involved in immune and inflammatory responses. JNK-mediated BRD4 functional switching induces CD8 expression in thymocytes and epithelial-to-mesenchymal transition (EMT) in prostate cancer cells. These findings elucidate the mechanism by which BRD4 transitions from a chromatin regulator to a transcriptional activator.

The MYC proto-oncogene and BRD4, a BET family protein, are two cardinal proteins that have a broad influence in cell biology and disease. Both proteins are expressed ubiquitously in mammalian cells and play central roles in controlling growth, development, stress responses and metabolic function. As chromatin and transcriptional regulators, they play a critical role in regulating the expression of a burgeoning array of genes, maintaining chromatin architecture and genome stability. Consequently, impairment of their function or regulation leads to many diseases, with cancer being the most predominant. Interestingly, accumulating evidence indicates that regulation of the expression and functions of MYC are tightly intertwined with BRD4 at both transcriptional and post-transcriptional levels. Here, we review the mechanisms by which MYC and BRD4 are regulated, their functions in governing various molecular mechanisms and the consequences of their dysregulation that lead to disease. We present a perspective of how the regulatory mechanisms for the two proteins could be entwined at multiple points in a BRD4-MYC nexus that leads to the modulation of their functions and disease upon dysregulation.

The bromodomain and extra terminal (BET) protein family has four members, BRD2, BRD3, BRD4 and BRDT. Through their extensive molecular functions, they regulate chromatin architecture and transcription. Their deregulation leads to a wide array of diseases, ranging from cancer, viral infections and metabolic diseases to mental disability. BRD4 is the most studied member of the BET family being structurally and functionally unique with an extended C-terminal tail, a defined dimerization motif and intrinsic kinase as well as HAT activities. Despite the development of several pan BET inhibitors as potential therapeutic agents, specific targeting of individual BET proteins remains challenging sincemost drugs target the structurally conserved bromodomains. There is thus a need to understand the additional roles and mechanisms of individual BET members to develop more effective therapeutics. The main objective of this Research Topic was to put together a collection of articles to better understand biochemical activities and functions of BET proteins. In all, six articles by experts specializing in different aspects of BET protein biology were published under this Research Topic. These articles are summarized below and document the wide range of biological processes and diseases regulated by BET proteins. We hope that this collection will serve both as a primer to the field and a snapshot of future directions. Cornelia de Lange Syndrome (CdLS) is a geneticmental disability traditionally linked with the mutations in core (SMC1A, SMC3 and RAD21) and regulatory (NIPBL) subunits of cohesin with up to 70% of cases associated with NIPBL mutations. The sister chromatid cohesion function of cohesin is not affected in CdLS, instead the etiological basis of CdLS has been linked with transcriptional dysregulation. The review by García-Gutiérrez and Mario García-Domínguez discusses the roles of chromatin factors, and their potential mechanisms of action, in CdLS, with an emphasis on BRD4. BRD4 has been recently identified as one of the proteins mutated in CdLS-like phenotypes, a finding that was explained by showing an interaction between the BRD4 and NIPBL. This review discusses the cohesin complex, NIPBL OPEN ACCESS

The TFIID component, TAF7, has been extensively characterized as essential for transcription and is critical for cell proliferation and differentiation. Here, we report that TAF7 is a previously unknown RNA chaperone that contributes to the regulation of protein synthesis. Mechanistically, TAF7 binds RNAs in the nucleus and delivers them to cytoplasmic polysomes. A broad spectrum of target RNA species, including the HIV-1 transactivation response element, binds TAF7 through consensus CUG motifs within the 3′ untranslated region. Export to the cytoplasm depends on a TAF7 nuclear export signal and occurs by an exportin 1–dependent pathway. Notably, disrupting either TAF7’s RNA binding or its export from the nucleus results in retention of target messenger RNAs in the nucleus and reduced levels of the protein products of TAF7-target RNAs. Thus, TAF7, an essential transcription factor, plays a key role in the regulation of RNA translation, thereby potentially connecting these processes.

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4's biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimenta-tion coefficient of X6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503-548) disrupts BRD4's dimeriza-tion. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifu-gation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra -terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4.

The protooncogene MYC regulates a variety of cellular processes, including proliferation and metabolism. Maintaining MYC at homeostatic levels is critical to normal cell function; overexpression drives many cancers. MYC stability is regulated through phosphorylation: phosphorylation at Thr58 signals degradation while Ser62 phosphorylation leads to its stabilization and functional activation. The bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator with intrinsic kinase and histone acetyltransferase (HAT) activities that activates transcription of key protooncogenes, including MYC We report that BRD4 phosphorylates MYC at Thr58, leading to MYC ubiquitination and degradation, thereby regulating MYC target genes. Importantly, BRD4 degradation, but not inhibition, results in increased levels of MYC protein. Conversely, MYC inhibits BRD4's HAT activity, suggesting that MYC regulates its own transcription by limiting BRD4-mediated chromatin remodeling of its locus. The MYC stabilizing kinase, ERK1, regulates MYC levels directly and indirectly by inhibiting BRD4 kinase activity. These findings demonstrate that BRD4 negatively regulates MYC levels, which is counteracted by ERK1 activation.

Nat. Struct. Mol. Biol. 23, 540–548 (2016); published online 9 May 2016; corrected after print 22 July 2016 In the version of this article initially published, the blots in Figure 2c were inadvertently replaced with a duplicate of the blots in Figure 2b. The error has been corrected in the HTML and PDF versions of the article.

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 chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (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.

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 ≤ induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments.

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that plays a pivotal role in cancer and inflammatory diseases. BRD4 binds and stays associated with chromatin during mitosis, bookmarking early G1 genes and reactivating transcription after mitotic silencing. BRD4 plays an important role in transcription, both as a passive scaffold via its recruitment of vital transcription factors and as an active kinase that phosphorylates RNA polymerase II, directly and indirectly regulating transcription. Through its HAT activity, BRD4 contributes to the maintenance of chromatin structure and nucleosome clearance. This review summarizes the known functions of BRD4 and proposes a model in which BRD4 actively coordinates chromatin structure and transcription.

Bromodomain protein 4 (BRD4) is a master transcriptional and epigenetic regulator which plays a pivotal role in cancer development and immune diseases. As a chromatin binding protein that remains b...

The bromodomain protein BRD4 links cell cycle and transcription, bookmarking active genes during mitosis and serving as a scaffold for transcription factors. Our recent discovery that BRD4 is a RNA Polymerase II CTD kinase identifies a novel transcriptional function. Here we discuss our model in the context of current knowledge.

TAF7, a component of the TFIID complex, controls the first steps of transcription. It interacts with and regulates the enzymatic activities of transcription factors that regulate RNA polymerase II progression. Its diverse functions in transcription initiation are consistent with its essential role in cell proliferation.

The Major Histocompatibility Complex (MHC) class II transactivator (CIITA) mediates activated immune responses and its deficiency results in the Type II Bare Lymphocyte Syndrome. CIITA is a transcriptional co-activator that regulates γ-interferon-activated transcription of MHC class I and class II genes. It is also a functional homolog of TAF1, a component of the general transcription factor complex TFIID. TAF1 and CIITA both possess intrinsic acetyltransferase (AT) activity that is required for transcription initiation. In response to induction by γ-interferon, CIITA and it's AT activity bypass the requirement for TAF1 AT activity. TAF1 also has kinase activity that is essential for its function. However, no similar activity has been identified for CIITA thus far. Here we report that CIITA, like TAF1, is a serine-threonine kinase. Its substrate specificity parallels, but does not duplicate, that of TAF1 in phosphorylating the TFIID component TAF7, the RAP74 subunit of the general transcription factor TFIIF and histone H2B. Like TAF1, CIITA autophosphorylates, affecting its interaction with TAF7. Additionally, CIITA phosphorylates histone H2B at Ser36, a target of TAF1 that is required for transcription during cell cycle progression and stress response. However, unlike TAF1, CIITA also phosphorylates all the other histones. The identification of this novel kinase activity of CIITA further clarifies its role as a functional homolog of TAF1 which may operate during stress and γ-IFN activated MHC gene transcription.

CIITA is a transcriptional co-activator that regulates γ-interferon-activated transcription of Major Histocompatibility Complex (MHC) class I and class II genes. As such, it plays a critical role in immune responses: CIITA deficiency results in aberrant MHC gene expression and consequently in autoimmune diseases such as Type II bare lymphocyte syndrome. Although CIITA does not bind DNA directly, it regulates MHC transcription in two distinct ways– as a transcriptional activator and as a general transcription factor. As an activator, CIITA nucleates an enhanceosome consisting of the DNA binding transcription factors RFX, CREB and NF-Y. As a general transcription factor, CIITA functionally replaces the TFIID component, TAF1. Like TAF1, CIITA possesses acetyltransferase (AT) and kinase activities, both of which contribute to proper transcription of MHC class I and II genes. The substrate specificity and regulation of the CIITA AT and kinase activities also parallel those of TAF1. In addition, CIITA is tightly regulated by its various regulatory domains that undergo phosphorylation and influence its targeted localization. Thus, a complex picture of the mechanisms regulating CIITA function is emerging suggesting that CIITA has dual roles in transcriptional regulation which are summarized in this review.

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.