RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

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.

MYC protein stability is negatively regulated by 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.

The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains.

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 sedimentation coefficient of ∼6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503-548) disrupts BRD4's dimerization. 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 ultracentrifugation, 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.

Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction.

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.

BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.

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.

Transcriptional coactivator CIITA, a functional homolog of TAF1, has kinase activity.

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.

The ESCRT protein CHMP5 promotes T cell leukemia by controlling BRD4-p300-dependent transcription.

Oncogene activity rewires cellular transcription, creating new transcription networks to which cancer cells become addicted, by mechanisms that are still poorly understood. Using human and mouse models of T cell acute lymphoblastic leukemia (T-ALL), we identify an essential nuclear role for CHMP5, a cytoplasmic endosomal sorting complex required for transport (ESCRT) protein, in establishing and maintaining the T-ALL transcriptional program. Nuclear CHMP5 promoted the T-ALL gene program by augmenting recruitment of the co-activator BRD4 by the histone acetyl transferase p300 selectively at enhancers and super-enhancers, an interaction that potentiated H3K27 acetylation at these regulatory enhancers. Consequently, loss of CHMP5 diminished BRD4 occupancy at enhancers and super-enhancers and impaired RNA polymerase II pause release, which resulted in downregulation of key T-ALL genes, notably MYC. Reinforcing its importance in T-ALL pathogenesis, CHMP5 deficiency mitigated chemoresistance in human T-ALL cells and abrogated T-ALL induction by oncogenic NOTCH1 in vivo. Thus, the ESCRT protein CHMP5 is an essential positive regulator of the transcriptional machinery promoting T-ALL disease.

Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation.

The RNA polymerase II (Pol II) C-terminal domain (CTD) serves as a docking site for numerous proteins, bridging various nuclear processes to transcription. The recruitment of these proteins is mediated by CTD phospho-epitopes generated during transcription. The mechanisms regulating the kinases that establish these phosphorylation patterns on the CTD are not known. We report that three CTD kinases, CDK7, CDK9, and BRD4, engage in cross-talk, modulating their subsequent CTD phosphorylation. BRD4 phosphorylates PTEFb/CDK9 at either Thr-29 or Thr-186, depending on its relative abundance, which represses or activates CDK9 CTD kinase activity, respectively. Conversely, CDK9 phosphorylates BRD4 enhancing its CTD kinase activity. The CTD Ser-5 kinase CDK7 also interacts with and phosphorylates BRD4, potently inhibiting BRD4 kinase activity. Additionally, the three kinases regulate each other indirectly through the general transcription factor TAF7. An inhibitor of CDK9 and CDK7 CTD kinase activities, TAF7 also binds to BRD4 and inhibits its kinase activity. Each of these kinases phosphorylates TAF7, affecting its subsequent ability to inhibit the other two. Thus, a complex regulatory network governs Pol II CTD kinases.

BRD4 and MYC: power couple in transcription and disease.

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.

Novel functions for TAF7, a regulator of TAF1-independent transcription.

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TAF7 in regulating TAF1-independent transcription has not been defined. The IFNγ-induced transcriptional co-activator CIITA activates MHC class I and II genes, which are vital for immune responses, in a TAF1-independent manner. Activation by CIITA depends on its intrinsic AT activity. We now show that TAF7 binds to CIITA and inhibits its AT activity, thereby repressing activated transcription. Consistent with this TAF7 function, siRNA-mediated depletion of TAF7 resulted in increased CIITA-dependent transcription. A more global role for TAF7 as a regulator of transcription was revealed by expression profiling analysis: expression of 30-40% of genes affected by TAF7 depletion was independent of either TAF1 or CIITA. Surprisingly, although TAF1-dependent transcripts were largely down-regulated by TAF7 depletion, TAF1-independent transcripts were predominantly up-regulated. We conclude that TAF7, until now considered only a TFIID component and regulator of TAF1-dependent transcription, also regulates TAF1-independent transcription.

The nuclear transcription factor, TAF7, is a cytoplasmic regulator of protein synthesis.

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 1­dependent 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 4: a cellular Swiss army knife.

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.

BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin.

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. Although chromatin decompaction and transcriptional activation of target genes are associated with BRD4 binding, the mechanisms involved are unknown. We report that BRD4 is a histone acetyltransferase (HAT) that acetylates histones H3 and H4 with a pattern distinct from those of other HATs. Both mouse and human BRD4 have intrinsic HAT activity. Importantly, BRD4 acetylates H3 K122, a residue critical for nucleosome stability, thus resulting in nucleosome eviction and chromatin decompaction. Nucleosome clearance by BRD4 occurs genome wide, including at its targets MYC, FOS and AURKB (Aurora B kinase), resulting in increased transcription. These findings suggest a model wherein BRD4 actively links chromatin structure and transcription: it mediates chromatin decompaction by acetylating and evicting nucleosomes at target genes, thereby activating transcription.

Two faces of brd4: mitotic bookmark and transcriptional lynchpin.

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.

CIITA and Its Dual Roles in MHC Gene Transcription.

Class II transactivator (CIITA) is a transcriptional coactivator that regulates γ-interferon-activated transcription of Major Histocompatibility Complex (MHC) class I and 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, cyclic AMP response element binding protein, 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.

TAF7: traffic controller in transcription initiation.

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.

Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis.

The limited availability of phosphate (Pi) in most soils results in the manifestation of Pi starvation responses in plants. To dissect the transcriptional regulation of Pi stress-response mechanisms, we have characterized the biological role of MYB62, an R2R3-type MYB transcription factor that is induced in response to Pi deficiency. The induction of MYB62 is a specific response in the leaves during Pi deprivation. The MYB62 protein localizes to the nucleus. The overexpression of MYB62 resulted in altered root architecture, Pi uptake, and acid phosphatase activity, leading to decreased total Pi content in the shoots. The expression of several Pi starvation-induced (PSI) genes was also suppressed in the MYB62 overexpressing plants. Overexpression of MYB62 resulted in a characteristic gibberellic acid (GA)-deficient phenotype that could be partially reversed by exogenous application of GA. In addition, the expression of SOC1 and SUPERMAN, molecular regulators of flowering, was suppressed in the MYB62 overexpressing plants. Interestingly, the expression of these genes was also reduced during Pi deprivation in wild-type plants, suggesting a role for GA biosynthetic and floral regulatory genes in Pi starvation responses. Thus, this study highlights the role of MYB62 in the regulation of phosphate starvation responses via changes in GA metabolism and signaling. Such cross-talk between Pi homeostasis and GA might have broader implications on flowering, root development and adaptive mechanisms during nutrient stress.

Transcriptional Regulation of Pi Starvation Responses by WRKY75.

Adaptive responses during phosphate (Pi) starvation are regulated by complex molecular mechanisms in plants. Transcription factors are believed to be the key determinants of Pi starvation responses. We have recently identified the plant-specific WRKY75 transcription factor as an important component of the Pi stress responses. WRKY75 is a positive regulator of several phosphate starvation induced (PSI) genes including phosphatases, Mt4/TPS1-like genes and high affinity Pi transporters. It also acts as a negative regulator of some components of root development, independent of Pi stress response. WRKY75 has considerable effect on anthocyanin accumulation, Pi uptake and Pi content in the plant. Here we present a hypothetical model of transcriptional regulation during phosphate starvation induced processes in plants which help in the maintenance of Pi homeostasis.