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The PTEN induced putative kinase 1 (PINK1) mutation is the second most common cause of autosomal recessive adolescent Parkinson's disease (PD). Furthermore, mitochondrial disorders and oxidative stress are important mechanisms in the pathogenesis of PD. Numerous members of the Wnt family have been found to be associated with neurodegenerative diseases. Therefore, the present study investigated the role of the Wnt2 gene in PINK1B9 transgenic flies, which is a PD model, and its underlying mechanism. It was identified that overexpression of Wnt2 reduced the abnormality rate of PD transgenic Drosophila and improved their flight ability, while other intervention groups had no significant effect. Furthermore, an increase in ATP concentration normalized mitochondrial morphology, and increased the mRNA expression levels of NADH‑ubiquinone oxidoreductase chain 1 (ND1), ND42, ND75, succinate dehydrogenase complex subunits B, Cytochrome b and Cyclooxygenase 1, which are associated with Wnt2 overexpression. Moreover, overexpression of Wnt2 in PD transgenic Drosophila resulted in the downregulation of reactive oxygen species and malondialdehyde production, and increased manganese superoxide dismutase (MnSOD), while glutathione was not significantly affected. It was found that overexpression of Wnt2 did not alter the protein expression of β‑catenin in PINK1B9 transgenic Drosophila, but did increase the expression levels of PPARG coactivator 1α (PGC‑1α) and forkhead box sub‑group O (FOXO). Collectively, the present results indicated that the Wnt2 gene may have a protective effect on PD PINK1B9 transgenic Drosophila. Thus, it was speculated that the reduction of oxidative stress and the restoration of mitochondrial function via Wnt2 overexpression may be related to the PGC‑1α/FOXO/MnSOD signaling pathway in PINK1 mutant transgenic Drosophila.

Summary RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5′ cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex., eLife digest In animals, an enzyme known as RNA polymerase II (Pol II for short) is a key element of the transcription process, whereby the genetic information contained in DNA is turned into messenger RNA molecules in the cells, which can then be translated to proteins. To perform this task, Pol II needs to be activated by a complex of proteins called P-TEFb; however, P-TEFb is usually found in an inactive form held by another group of proteins. Yet, it is unclear how P-TEFb is released and allowed to activate Pol II. Scientists have speculated that another protein called JMJD6 (Jumonji domain-containing 6) is important for P-TEFb to activate Pol II. Various roles for JMJD6 have been proposed, but its exact purpose remains unclear. Recently, two enzymes closely related to JMJD6 were found to be able to make precise cuts in other proteins; Lee, Liu et al. therefore wanted to test whether this is also true of JMJD6. Experiments using purified JMJD6 showed that it could make a cut in an enzyme called MePCE, which belongs to the group of proteins that hold P-TEFb in its inactive form. Lee, Liu et al. then tested the relationships between these proteins in living human and mouse cells. The levels of activated Pol II were lower in cells without JMJD6 and higher in those without MePCE. Together, the results suggest that JMJD6 cuts MePCE to release P-TEFb, which then activates Pol II. JMJD6 appears to know where to cut by following a specific pattern of elements in the structure of MePCE. When MePCE was mutated so that the pattern changed, JMJD6 was unable to cut it. These results suggest that JMJD6 and related enzymes belong to a new family of proteases, the molecular scissors that can cleave other proteins. The molecules that regulate transcription often are major drug targets, for example in the fight against cancer. Ultimately, understanding the role of JMJD6 might help to identify new avenues for cancer drug development.

The positive transcription elongation factor b (P-TEFb) was first identified as a general factor that stimulates transcription elongation by RNA polymerase II (RNAPII), but soon afterwards it turned out to be an essential cellular co-factor of human immunodeficiency virus (HIV) transcription mediated by viral Tat proteins. Studies on the mechanisms of Tat-dependent HIV transcription have led to radical advances in our knowledge regarding the mechanism of eukaryotic transcription, including the discoveries that P-TEFb-mediated elongation control of cellular transcription is a main regulatory step of gene expression in eukaryotes, and deregulation of P-TEFb activity plays critical roles in many human diseases and conditions in addition to HIV/AIDS. P-TEFb is now recognized as an attractive and promising therapeutic target for inflammation/autoimmune diseases, cardiac hypertrophy, cancer, infectious diseases, etc. In this review article, I will summarize our knowledge about basic P-TEFb functions, the regulatory mechanism of P-TEFb-dependent transcription, P-TEFb’s involvement in biological processes and diseases, and current approaches to manipulating P-TEFb functions for the treatment of these diseases.

The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.

Mitochondrial functions are often considered purely from the standpoint of catabolism, but in growing cells they are mainly dedicated to anabolic processes, and can have a profound impact on the rate of growth. The Drosophila larva, which increases in body mass ∼200-fold over the course of ∼3 days at 25°C, provides an excellent model to study the underlying regulatory machinery that connects mitochondrial metabolic capacity to growth. In this review, we will focus on several key aspects of this machinery: nutrient sensing, endocrine control of feeding and nutrient mobilization, metabolic signalling, protein synthesis regulation and pathways of steroid biosynthesis and activity. In all these aspects, mitochondria appear to play a crucial role.

Formation of biomolecular condensates by phase separation has recently emerged as a new principle for regulating gene expression in response to extracellular signaling. However, the molecular mechanisms underlying the coupling of signal transduction and gene activation through condensate formation, and how dysregulation of these mechanisms contributes to disease progression, remain elusive. Here, the authors report that CREB-regulated transcription coactivator 2 (CRTC2) translocates to the nucleus and forms phase-separated condensates upon activation of cAMP signaling. They show that intranuclear CRTC2 interacts with positive transcription elongation factor b (P-TEFb) and activates P-TEFb by disrupting the inhibitory 7SK snRNP complex. Aberrantly elevated cAMP signaling plays central roles in the development of autosomal dominant polycystic kidney disease (ADPKD). They find that CRTC2 localizes to the nucleus and forms condensates in cystic epithelial cells of both mouse and human ADPKD kidneys. Genetic depletion of CRTC2 suppresses cyst growth in an orthologous ADPKD mouse model. Using integrative transcriptomic and cistromic analyses, they identify CRTC2-regulated cystogenesis-associated genes, whose activation depends on CRTC2 condensate-facilitated P-TEFb recruitment and the release of paused RNA polymerase II. Together, their findings elucidate a mechanism by which CRTC2 nuclear condensation conveys cAMP signaling to transcription elongation activation and thereby promotes cystogenesis in ADPKD.

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat–TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181–TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

Marié et al. describe a futile cycle between constitutive histone acetyltransferases and deacetylases (HDACs) that regulates interferon-stimulated gene transcription through availability of the epigenetic reader, Brd4. Pharmacologic targeting of the HDAC/Brd4 axis resolves aberrant gene expression associated with genetic susceptibility to autoimmune interferonopathies., In contrast to the common role of histone deacetylases (HDACs) for gene repression, HDAC activity provides a required positive function for IFN-stimulated gene (ISG) expression. Here, we show that HDAC1/2 as components of the Sin3A complex are required for ISG transcriptional elongation but not for recruitment of RNA polymerase or transcriptional initiation. Transcriptional arrest by HDAC inhibition coincides with failure to recruit the epigenetic reader Brd4 and elongation factor P-TEFb due to sequestration of Brd4 on hyperacetylated chromatin. Brd4 availability is regulated by an equilibrium cycle between opposed acetyltransferase and deacetylase activities that maintains a steady-state pool of free Brd4 available for recruitment to inducible promoters. An ISG expression signature is a hallmark of interferonopathies and other autoimmune diseases. Combined inhibition of HDAC1/2 and Brd4 resolved the aberrant ISG expression detected in cells derived from patients with two inherited interferonopathies, ISG15 and USP18 deficiencies, defining a novel therapeutic approach to ISG-associated autoimmune diseases., Graphical Abstract

Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.

The transcriptional co-activator PGC-1α is a key regulator of mitochondrial function and muscle fiber specification in the skeletal muscle. The E3 ubiquitin ligase RNF34 ubiquitinates PGC-1α and negatively regulates mammalian brown fat cell metabolism. However, the functional importance of RNF34 in the skeletal muscle and its impact on energy metabolism remain unknown. The Drosophila PGC-1 homolog dPGC-1 and its mammalian counterparts have conserved functions in mitochondria and insulin signaling. Here, we showed that the Drosophila RNF34 (dRNF34) ubiquitinates the Drosophila PGC-1α (dPGC-1) and promotes its degradation in HEK293T cells by immunoprecipitation and western blot analysis. This allows us to use Drosophila as a powerful model system to study the physiological role of RNF34 in mitochondrial function and metabolism. In the in vivo studies, by separately expressing two independent UAS-dRNF34 RNAi transgenes driven by the muscle-specific 24B-Gal4 driver, we found that knockdown of dRNF34 specifically in muscle promotes mitochondrial biogenesis, improves negative geotaxis, extends climbing time to exhaustion in moderate aged flies and counteracts high-fat-diet-induced high triglyceride content. Furthermore, we showed that knockdown of dPGC-1 reversed the effects of the dRNF34 knockdown phenotypes described above. Our results reveal that dRNF34 plays an important role in regulating mitochondrial biogenesis in muscle and lipid metabolism through dPGC-1. Thus, inhibition of RNF34 activity provides a potential novel therapeutic strategy for the treatment of age-related muscle dysfunction.

ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEClike complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.

Jejunal sodium/glucose co-transporter (Sglt1) displays circadian expression. The jejunum was collected every 4 h from mice, and we examined histone acetylation and binding of bromodomain-containing protein-4 (BRD4) around of the gene. Histone acetylation increased in the transcribed region of Sglt1 prior to induction of the gene. Furthermore, the binding of mRNA elongation factor around the gene showed circadian rhythm.

Autoimmune regulator (AIRE) and nuclear factor-κB (NF-κB) are transcription factors (TFs) that direct the expression of individual genes and gene clusters. Bromodomain-containing protein 4 (BRD4) is an epigenetic regulator that recognizes and binds to acetylated histones. BRD4 also has been reported to promote interactions between the positive transcription elongation factor b (P-TEFb) and AIRE or P-TEFb and NF-κB subunit p65. Here, we report that AIRE and p65 bind to P-TEFb independently of BRD4. JQ1, a compound that disrupts interactions between BRD4 and acetylated proteins, does not decrease transcriptional activities of AIRE or p65. Moreover, siRNA-mediated inactivation of BRD4 alone or in combination with JQ1 had no effects on AIRE- and NF-κB-targeted genes on plasmids and in chromatin and on interactions between P-TEFb and AIRE or NF-κB. Finally, ChIP experiments revealed that recruitment of P-TEFb to AIRE or p65 to transcription complexes was independent of BRD4. We conclude that direct interactions between AIRE, NF-κB, and P-TEFb result in efficient transcription of their target genes.

Summary: RNAP II switching from the paused to the productive transcription elongation state is a pivotal regulatory step that requires specific phosphorylations catalyzed by the P-TEFb kinase. Nucleosolic P-TEFb activity is inhibited by its interaction with the ribonuclear protein complex built around the 7SK small nuclear RNA (7SK snRNP). MePCE is the RNA methyltransferase that methylates and stabilizes 7SK in the nucleosol. Here, we report that MePCE also binds chromatin through the histone H4 tail to serve as a P-TEFb activator at specific genes important for cellular identity. Notably, this histone binding abolishes MePCE’s RNA methyltransferase activity toward 7SK, which explains why MePCE-bound P-TEFb on chromatin may not be associated with the full 7SK snRNP and is competent for RNAP II activation. Overall, our results suggest that crosstalk between the histone-binding and RNA methylation activities of MePCE regulates P-TEFb activation on chromatin in a 7SK- and Brd4-independent manner.

Although the definition of a noncoding RNA (ncRNA) is an RNA molecule that does not encode a protein, recent evidence has revealed that some ncRNAs are indeed translated to give rise to small polypeptides (usually containing fewer than 100 amino acids). Despite their small size, however, these peptides are often biologically relevant in that they are required for a variety of cellular processes. In this review, we summarize the production and functions of peptides that have been recently identified as translation products of putative ncRNAs.Key words: long noncoding RNA (lncRNA), circular RNA (circRNA), primary miRNA (pri-miRNA), translation, peptide.

Lipid storage in fat tissue is important for energy homeostasis and cellular functions. Through RNAi screening in Drosophila fat body, we found that knockdown of a Drosophila NAD kinase (NADK), which phosphorylates NAD to synthesize NADP de novo, causes lipid storage defects. NADK sustains lipogenesis by maintaining the pool of NADPH. Promoting NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals. Furthermore, NADK and fatty acid synthase 1 (FASN1) regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids. Reducing the level of acetyl-CoA or increasing the synthesis of cardiolipin (CL), a mitochondrion-specific phospholipid, partially rescues the mitochondrial defects of NADK RNAi. Therefore, NADK- and FASN1-mediated fatty acid synthesis coordinates lipid storage and mitochondrial function.

Cyclin-Dependent Kinase 9 (CDK9) is part of a functional diverse group of enzymes responsible for cell cycle control and progression. It associates mainly with Cyclin T1 and forms the Positive Transcription Elongation Factor b (p-TEFb) complex responsible for regulation of transcription elongation and mRNA maturation. Recent studies have highlighted the importance of CDK9 in many relevant pathologic processes, like cancer, cardiovascular diseases, and viral replication. Herein we provide an overview of the different pathways in which CDK9 is directly and indirectly involved.

The SERPINB2 gene is strongly upregulated in inflammatory states. In monocytes, it can constitute up to 1% of total cellular protein. It functions in protection from proteotoxic stress and plays a role in angioedema. The purpose of this study was to define the roles of enhancer RNAs embedded in the SERPIN gene complex. We found that the upstream enhancer RNAs upregulated SERPINB2 and the enhancer RNAs were expressed prior to those of SERPINB2 mRNA. Studies of the SERPINB2 promoter demonstrated the presence of an RNA polymerase II pause-inducing protein, NELF. Stimulation with LPS led to recruitment of the pause-releasing kinase P-TEFb and departure of the pause-inducing protein NELF. RNA immunoprecipitation revealed that NELF and the CDK9 component of P-TEFb bound to the enhancer RNAs after stimulation with distinct kinetics. Knock-down of the enhancer RNAs compromised stimulus induction of promoter and enhancer chromatin changes. Conversely, over-expression was associated with enhanced recruitment of c-JUN and increased expression of SERPINB2 mRNA expression. This study is the first to associate enhancer RNAs with SERPINB2 and is the first demonstration of acquisition of NELF binding by enhancer RNAs on chromatin.

SIRT7 is an NAD+-dependent protein deacetylase that regulates cell growth and proliferation. Previous studies have shown that SIRT7 is required for RNA polymerase I (Pol I) transcription and pre-rRNA processing. Here, we took a proteomic approach to identify novel molecular targets and characterize the role of SIRT7 in non-nucleolar processes. We show that SIRT7 interacts with numerous proteins involved in transcriptional regulation and RNA metabolism, the majority of interactions requiring ongoing transcription. In addition to its role in Pol I transcription, we found that SIRT7 also regulates transcription of snoRNAs and mRNAs. Mechanistically, SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex and deacetylates CDK9, a subunit of the elongation factor P-TEFb, which activates transcription by phosphorylating serine 2 within the C-terminal domain (CTD) of Pol II. SIRT7 counteracts GCN5-directed acetylation of lysine 48 within the catalytic domain of CDK9, deacetylation promoting CTD phosphorylation and transcription elongation.

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency., A leading pharmacological strategy toward HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

Antiretroviral therapy (ART) suppresses HIV-1 replication but fails to cure the infection. The presence of an extremely stable viral latent reservoir, primarily in resting memory CD4(+) T cells, remains a major obstacle to viral eradication. The “shock and kill” strategy targets these latently infected cells and boosts immune recognition and clearance, and thus, it is a promising approach for an HIV-1 functional cure. Although some latency-reversing agents (LRAs) have been reported, no apparent clinical progress has been made, so it is still vital to seek novel and effective LRAs. Here, we report that thiostrepton (TSR), a proteasome inhibitor, reactivates latent HIV-1 effectively in cellular models and in primary CD4(+) T cells from ART-suppressed individuals ex vivo. TSR does not induce global T cell activation, severe cytotoxicity, or CD8(+) T cell dysfunction, making it a prospective LRA candidate. We also observed a significant synergistic effect of reactivation when TSR was combined with JQ1, prostratin, or bryostatin-1. Interestingly, six TSR analogues also show reactivation abilities that are similar to or more effective than that of TSR. We further verified that TSR upregulated expression of heat shock proteins (HSPs) in CD4(+) T cells, which subsequently activated positive transcriptional elongation factor b (p-TEFb) and NF-κB signals, leading to viral reactivation. In summary, we identify TSR as a novel LRA which could have important significance for applications to an HIV-1 functional cure in the future.

7SK RNA, as part of the 7SK ribonucleoprotein complex, is crucial to the regulation of transcription by RNA-polymerase II, via its interaction with the positive transcription elongation factor P-TEFb. The interaction is induced by binding of the protein HEXIM to the 5′ hairpin (HP1) of 7SK RNA. Four distinct structural models have been obtained experimentally for HP1. Here, we employ computational methods to investigate the relative stability of these structures, transitions between them, and the effects of mutations on the observed structural ensembles. We further analyse the results with respect to mutational binding assays, and hypothesize a mechanism for HEXIM binding. Our results indicate that the dominant structure in the wild type exhibits a triplet involving the unpaired nucleotide U40 and the base pair A43-U66 in the GAUC/GAUC repeat. This conformation leads to an open major groove with enough potential binding sites for peptide recognition. Sequence mutations of the RNA change the relative stability of the different structural ensembles. Binding affinity is consequently lost if these changes alter the dominant structure.

International audience; How nuclear proteins diffuse and find their targets remains a key question in the transcription field. Dynamic proteins in the nucleus are classically subdiffusive and undergo anomalous diffusion, yet the underlying physical mechanisms are still debated. In this study, we explore the contribution of interactions to the generation of anomalous diffusion by the means of fluorescence spectroscopy and simulation. Using interaction-deficient mutants, our study indicates that HEXIM1 interactions with both 7SK RNA and positive transcription elongation factor b are critical for HEXIM1 subdiffusion and thus provides evidence of the effects of protein-RNA interaction on molecular diffusion. Numerical simulations allowed us to establish that the proportions of distinct oligomeric HEXIM1 subpopulations define the apparent anomaly parameter of the whole population. Slight changes in the proportions of these oligomers can lead to significant shifts in the diffusive features and recapitulate the modifications observed in cells with the various interaction-deficient mutants. By combining simulations and experiments, our work opens new prospects in which the anomaly a coefficient in diffusion becomes a helpful tool to infer alterations in molecular interactions.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway has cell-specific functions. Suppressors of cytokine signaling (SOCS) proteins are negative-feedback regulators of JAK-STAT signaling. STAT5 plays a significant role in adipocyte development and function, and bromodomain and extraterminal (BET) proteins may be involved in STAT5 transcriptional activity. We treated 3T3-L1 adipocytes with the BET inhibitor JQ1 and observed that growth hormone (GH)-induced expression of 2 STAT5 target genes from the SOCS family, Socs3 and Cish, were inversely regulated (increased and decreased, respectively) by BET inhibition. Chromatin immunoprecipitation analyses revealed that changes in STAT5 binding did not correlate with gene expression changes. GH promoted the recruitment of the BET protein BRD2 to the Cish, but not Socs3, promoter. JQ1 treatment ablated this effect as well as the GH-induced binding of ribonucleic acid polymerase II (RNA Pol II) to the Cish transcription start site. BRD2 knockdown also suppressed GH induction of Cish, further supporting the role of BRD2 in Cish transcriptional activation. In contrast, JQ1 increased the binding of activated Pol II to the Socs3 coding region, suggesting enhanced messenger RNA (mRNA) elongation. Our finding that JQ1 transiently reduced the interaction between the positive transcription elongation factor (P-TEFb) and its inhibitor hexamethylene bis-acetamide inducible 1 (HEXIM1) is consistent with a previously described off-target effect of JQ1, whereby P-TEFb becomes more available to be recruited by genes that do not depend on BET proteins for activating transcription. These results demonstrate substantially different transcriptional regulation of Socs3 and Cish and suggest distinct roles in adipocytes for these 2 closely related proteins.

In eukaryotes, the elongation phase of transcription by RNA polymerase II (RNAP II) is regulated by the transcription elongation factor b (P-TEFb), composed of Cyclin-T1 and cyclin-dependent kinase 9. The release of RNAP II is mediated by phosphorylation through P-TEFb that in turn is under control by the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex. The 7SK snRNP consists of the 7SK non-coding RNA and the proteins MEPCE, LARP7, and HEXIM1/2. Biallelic LARP7 loss-of-function variants underlie Alazami syndrome characterized by growth retardation and intellectual disability. We report a boy with global developmental delay and seizures carrying the de novo MEPCE nonsense variant c.1552 C > T/p.(Arg518*). mRNA and protein analyses identified nonsense-mediated mRNA decay to underlie the decreased amount of MEPCE in patient fibroblasts followed by LARP7 and 7SK snRNA downregulation and HEXIM1 upregulation. Reduced binding of HEXIM1 to Cyclin-T1, hyperphosphorylation of the RNAP II C-terminal domain, and upregulated expression of ID2, ID3, MRPL11 and snRNAs U1, U2 and U4 in patient cells are suggestive of enhanced activation of P-TEFb. Flavopiridol treatment and ectopic MEPCE protein expression in patient fibroblasts rescued increased expression of six RNAP II-sensitive genes and suggested a possible repressive effect of MEPCE on P-TEFb-dependent transcription of specific genes.

Background: The transcriptional co-activators of the PGC-1 family have been proposed as master regulators of mitochondrial biogenesis. Drosophila has a single member of the family, Spargel (srl). Functional studies of srl allow PGC-1 role(s) in development and physiology to be elucidated without the confounding effects of tissue-specificity and redundancy. Spargel was previously shown to govern the expression of genes for mitochondrial functions in the adult fat body. Results: Here we analysed the role of srl in early development. srl RNA was abundant in ovaries and early embryos but, upon cellularization, fell to low levels where it remained for the rest of development. Knockdown of srl in the female germline resulted in embryonic semilethality, with only ~10% of embryos able to complete development. Embryos from germline srl-knockdown mothers were predominantly small, with the majority suffering a catastrophic derangement of development marked by failure to form a uniform blatstoderm, irregular cell size and chaotic cell movements instead of orderly gastrulation. However, genes whose expression in early embryos depends on localized determinants were expressed normally. The abundance of representative mitochondrial proteins was not decreased in knockdown ovaries or embryos, and their mRNAs were generally increased rather than decreased. Conclusions: We infer that srl has a more general role in early development, distinct from specifically promoting mitochondrial biogenesis.

P-TEFb activation by cAMP-PKA signaling promotes cystogenic gene transcription elongation and disease progression in ADPKD., Positive transcription elongation factor b (P-TEFb) functions as a central regulator of transcription elongation. Activation of P-TEFb occurs through its dissociation from the transcriptionally inactive P-TEFb/HEXIM1/7SK snRNP complex. However, the mechanisms of signal-regulated P-TEFb activation and its roles in human diseases remain largely unknown. Here, we demonstrate that cAMP-PKA signaling disrupts the inactive P-TEFb/HEXIM1/7SK snRNP complex by PKA-mediated phosphorylation of HEXIM1 at serine-158. The cAMP pathway plays central roles in the development of autosomal dominant polycystic kidney disease (ADPKD), and we show that P-TEFb is hyperactivated in mouse and human ADPKD kidneys. Genetic activation of P-TEFb promotes cyst formation in a zebrafish ADPKD model, while pharmacological inhibition of P-TEFb attenuates cyst development by suppressing the pathological gene expression program in ADPKD mice. Our study therefore elucidates a mechanism by which P-TEFb activation by cAMP-PKA signaling promotes cystogenesis in ADPKD.

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes., eLife digest DNA contains the instructions for making proteins, which build and maintain our cells. So that the information encoded in DNA can be used, a molecular machine called RNA polymerase II makes copies of specific genes. These copies, in the form of a molecule called RNA, convey the instructions for making proteins to the rest of the cell. To ensure that RNA polymerase II copies the correct genes at the correct time, a group of regulatory proteins are needed to control its activity. Many of these proteins interact with RNA polymerase II at a region known as the C-terminal domain, or CTD for short. For example, before RNA polymerase can make a full copy of a gene, a small molecule called a phosphate group must first be added to CTD at specific units known as Ser2. The regulatory protein P-TEFb was thought to be responsible for phosphorylating Ser2. However, it was previously not known how P-TEFb added this phosphate group, and why it did not also add phosphate groups to other positions in the CTD domain that are structurally similar to Ser2. To investigate this, Mayfield, Irani et al. mixed the CTD domain with different regulatory proteins, and used various biochemical approaches to examine which specific positions of the domain had phosphate groups attached. These experiments revealed a previously unknown aspect of P-TEFb activity: its specificity for Ser2 increased dramatically if a different regulatory protein first added a phosphate group to a nearby location in CTD. This additional phosphate group directed P-TEFb to then add its phosphate specifically at Ser2. To confirm the activity of this mechanism in living human cells, Mayfield, Irani et al. used a drug that prevented the first phosphate from being added. In the drug treated cells, RNA polymerase II was found more frequently ‘stalled’ at positions on the DNA just before a gene starts. This suggests that living cells needs this two-phosphate code system in order for RNA polymerase II to progress and make copies of specific genes. These results are a step forward in understanding the complex control mechanisms cells use to make proteins from their DNA. Moreover, the model presented here – one phosphate addition priming a second specific phosphate addition – provides a template that may underlie similar regulatory processes.

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. Here, we show that Nos represses somatic gene expression by inhibiting translation of maternal importin-α2 (impα2) mRNA. Mis-expression of Impα2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, we propose that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a ‘double-lock’ mechanism to inhibit somatic gene expression in germline progenitors., Author summary Identification of the molecular mechanism underlying germline segregation from the soma is a fundamental goal of reproductive, cellular, and developmental biology. In many animal species, repression of somatic gene expression in germline progenitors is critical for the germ/soma segregation. In Drosophila, germ plasm, a specialized ooplasm partitioned into germline progenitors, contains maternal factors sufficient to repress somatic differentiation. Here, we show that a subset of somatic genes is derepressed when two maternal factors, Nanos (Nos) and Polar granule component (Pgc) are concomitantly suppressed. While Pgc is known to suppress RNA polymerase II (Pol II) activity, how Nos achieves this effect remains obscure. We find that Nos represses production of Importin-α2 that is essential for nuclear import of transcriptional activators for somatic gene expression in germline progenitors. Thus, we propose that Nos-dependent inhibition of nuclear import of transcriptional activators and Pgc-dependent suppression of Pol II activity acts as a ‘double-lock’ mechanism to ensure tight inhibition of somatic gene expression in germline progenitors. Since Nos is evolutionarily conserved, and a transient suppression of Pol II is a trait of germline progenitors of diverse animal species, the ‘double-lock’ mechanism may play a widespread role in germ/soma segregation.

There is increasing interest in the potential role of epigallocatechin gallate (EGCG) in changing body composition to lower body fat with increased lean mass. In this study, we examined the sex-dependent effect of EGCG on body composition, locomotion, feeding behaviour, sugar levels, and transcription levels of key regulators in lipid, carbohydrate, and energy metabolisms in

We recently reported that the cyclin T1 histidine-rich domain creates a phase-separated environment to promote hyperphosphorylation of RNA polymerase II C-terminal domain and robust transcriptional elongation by P-TEFb. Here, we discuss this and several other recent discoveries to demonstrate that phase separation is important for controlling various aspects of transcription.

The nuclear factor kappa B (NF-κB) family of transcription factors plays a central role in coordinating the expression of genes that control inflammation, immune responses, cell proliferation, and a variety of other biological processes. In an attempt to identify novel regulators of this pathway, we performed whole-genome RNA interference (RNAi) screens in physiologically relevant human macrophages in response to lipopolysaccharide and tumor necrosis factor alpha (TNF-α). The top hit was SNW1, a splicing factor and transcriptional coactivator. SNW1 does not regulate the cytoplasmic components of the NF-κB pathway but complexes with the NF-κB heterodimer in the nucleus for transcriptional activation. We show that SNW1 detaches from its splicing complex (formed with SNRNP200 and SNRNP220) upon NF-κB activation and binds to NF-κB's transcriptional elongation partner p-TEFb. We also show that SNW1 is indispensable for the transcriptional elongation of NF-κB target genes such as the interleukin 8 (IL-8) and TNF genes. SNW1 is a unique protein previously shown to be involved in both splicing and transcription, and in this case, its role involves binding to the NF-κB-p-TEFb complex to facilitate transcriptional elongation of some NF-κB target genes.

Multipotent progenitor populations are necessary for generating diverse tissue types during embryogenesis. We show the Polymerase Associated Factor 1 Complex (Paf1C) is required to maintain multipotent progenitors of the neural crest (NC) lineage in zebrafish. Mutations affecting each Paf1C component result in near-identical NC phenotypes; alyron mutant embryos carrying a null mutation in paf1 were analyzed in detail. In the absence of zygotic paf1 function, definitive premigratory NC progenitors arise but fail to maintain expression of the sox10 specification gene. The mutant NC progenitors migrate aberrantly and fail to differentiate appropriately. Blood and germ cell progenitor development is affected similarly. Development of mutant NC could be rescued by additional loss of Positive Transcription Elongation Factor b (P-TEFb) activity, a key factor in promoting transcription elongation. Consistent with the interpretation that inhibiting/delaying expression of some genes is essential for maintaining progenitors, mutant embryos lacking the CDK9 kinase component of P-TEFb exhibit a surfeit of NC progenitors and their derivatives. We propose Paf1C and P-TEFb act antagonistically to regulate the timing of the expression of genes needed for NC development.

DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.

Eukaryotic gene transcription is often controlled at the level of RNA polymerase II (Pol II) pausing in the promoter-proximal region. Pausing Pol II limits the frequency of transcription initiation (‘pause-initiation limit’), predicting that the pause duration must be decreased for transcriptional activation. To test this prediction, we conduct a genome-wide kinetic analysis of the heat shock response in human cells. We show that the pause-initiation limit restricts transcriptional activation at most genes. Gene activation generally requires the activity of the P-TEFb kinase CDK9, which decreases the duration of Pol II pausing and thereby enables an increase in the productive initiation frequency. The transcription of enhancer elements is generally not pause limited and can be activated without CDK9 activity. Our results define the kinetics of Pol II transcriptional regulation in human cells at all gene classes during a natural transcription response., Gene activation requires an increase of successful initiation events. Here, by employing a genome-wide kinetic analysis of transcription, the authors showed that gene activation generally requires a decrease in RNA Polymerase II (Pol II) promoter-proximal pausing while transcription of enhancer elements is not limited by Pol II pausing.

When chill-susceptible insects are exposed to low temperatures they enter a temporary state of paralysis referred to as a chill coma. The most well-studied physiological mechanism of chill coma onset and recovery involves regulation of ion homeostasis. Previous studies show that changes in metabolism may also underlie the ability to recovery quickly, but the roles of genes that regulate metabolic homeostasis in chill coma recovery time (CCRT) are not well understood. Here, we investigate the roles of Sestrin and Spargel (Drosophila homolog of PGC-1α), which are involved in metabolic homeostasis and substrate oxidation, on CCRT in Drosophila melanogaster. We find that sestrin and spargel mutants have impaired CCRT. sestrin is required in the muscle and nervous system tissue for normal CCRT and spargel is required in muscle and adipose. On the basis that exercise induces sestrin and spargel, we also test the interaction of cold and exercise. We find that pre-treatment with one of these stressors does not consistently confer acute protection against the other. We conclude that Sestrin and Spargel are important in the chill coma response, independent of their role in exercise.

The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P-TEFb. Release of P-TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P-TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly important for neuron development and maintenance and it can thus be hypothesized that 7SK is at the center of many signaling pathways contributing to neuron function. 7SK activates neuronal gene expression programs that are key for terminal differentiation of neurons. Proteomics studies revealed a complex protein interactome of 7SK that includes several RNA-binding proteins. Some of these novel 7SK subcomplexes exert non-canonical cytosolic functions in neurons by regulating axonal mRNA transport and fine-tuning spliceosome production in response to transcription alterations. Thus, a picture emerges according to which 7SK acts as a multi-functional RNA scaffold that is integral for neuron homeostasis.

Positive transcription elongation factor b (P-TEFb) is an RNA polymerase II kinase that phosphorylates Ser2 of the carboxyl-terminal domain and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in early developmental events. In this study, using immunocytochemical analyses, we find that the P-TEFb components, Cyclin T1, CDK9, and its T-loop phosphorylated form, are localized to nuclear speckles, as well as in nucleoli in mouse germinal vesicle oocytes. Moreover, using fluorescence in situ hybridization, we show that in absence of CDK9 activity, nucleolar integration, as well as production of 28S rRNA is impaired in oocytes and embryos. We also present evidence indicating that P-TEFb kinase activity is essential for completion of mouse oocyte maturation and embryo development. Treatment with CDK9 inhibitor, flavopiridol resulted in metaphase I arrest in maturing oocytes. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when zygotes or 2-cell embryos were treated with flavopiridol only in their G2 phase of the cell cycle, development to the blastocyst stage was impaired. Inhibition of the CDK9 activity after embryonic genome activation resulted in failure to form normal blastocysts and aberrant phosphorylation of RNA polymerase II CTD. In all stages analyzed, treatment with flavopiridol abrogated global transcriptional activity. Collectively, our data suggest that P-TEFb kinase activity is crucial for oocyte maturation, embryo development, and regulation of global RNA transcription in mouse early development.

Poly[adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) onto substrate proteins. Here we report a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.

Release of RNA polymerase II (Pol II) from promoter-proximal pausing has emerged as a critical step regulating gene expression in multicellular organisms. The transition of Pol II into productive elongation requires the kinase activity of positive transcription elongation factor b (P-TEFb), which is itself under a stringent control by the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. Here, we provide an overview on stimulating Pol II pause release by P-TEFb and on sequestering P-TEFb into 7SK snRNP. Furthermore, we highlight mechanisms that govern anchoring of 7SK snRNP to chromatin as well as means that release P-TEFb from the inhibitory complex, and propose a unifying model of P-TEFb activation on chromatin. Collectively, these studies shine a spotlight on the central role of RNA binding proteins (RBPs) in directing the inhibition and activation of P-TEFb, providing a compelling paradigm for controlling Pol II transcription with a non-coding RNA.

The association of DSIF and NELF with initiated RNA Polymerase II (Pol II) is the general mechanism for inducing promoter-proximal pausing of Pol II. However, it remains largely unclear how the paused Pol II is released in response to stimulation. Here, we show that the release of the paused Pol II is cooperatively regulated by multiple P-TEFbs which are recruited by bromodomain-containing protein Brd4 and super elongation complex (SEC) via different recruitment mechanisms. Upon stimulation, Brd4 recruits P-TEFb to Spt5/DSIF via a recruitment pathway consisting of Med1, Med23 and Tat-SF1, whereas SEC recruits P-TEFb to NELF-A and NELF-E via Paf1c and Med26, respectively. P-TEFb-mediated phosphorylation of Spt5, NELF-A and NELF-E results in the dissociation of NELF from Pol II, thereby transiting transcription from pausing to elongation. Additionally, we demonstrate that P-TEFb-mediated Ser2 phosphorylation of Pol II is dispensable for pause release. Therefore, our studies reveal a co-regulatory mechanism of Brd4 and SEC in modulating the transcriptional pause release by recruiting multiple P-TEFbs via a Mediator- and Paf1c-coordinated recruitment network.

RNA polymerase II (Pol II) pausing at promoter-proximal regions is a highly regulated step in the transcription cycle. Pause release is facilitated by the P-TEFb kinase, which phosphorylates Pol II and negative elongation factors. Recent studies suggest that P-TEFb (as part of the inhibitory 7SK snRNP) is recruited to promoter-proximal regions through interaction with KAP1/TRIM28/TIF1β to facilitate ‘on-site’ kinase activation and transcription elongation. Here, I discuss features of this model and future challenges to further hone our understanding of transcriptional regulation including Pol II pausing and pause release.

The RNA polymerase II (Pol II) transcribes all mRNA genes in eukaryotes and is among the most highly regulated enzymes in the cell. The classic model of mRNA gene regulation involves recruitment of the RNA polymerase to gene promoters in response to environmental signals. Higher eukaryotes have an additional ability to generate multiple cell types. This extra level of regulation enables each cell to interpret the same genome by committing to one of the many possible transcription programs and executing it in a precise and robust manner. Whereas multiple mechanisms are implicated in cell type-specific transcriptional regulation, how one genome can give rise to distinct transcriptional programs and what mechanisms activate and maintain the appropriate program in each cell remains unclear. This review focuses on the process of promoter-proximal Pol II pausing during early transcription elongation as a key step in context-dependent interpretation of the metazoan genome. We highlight aspects of promoter-proximal Pol II pausing, including its interplay with epigenetic mechanisms, that may enable cell type-specific regulation, and emphasize some of the pertinent questions that remain unanswered and open for investigation.

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a ‘gate-keeper’ for the correct temporal and spatial development of the neural crest.

MYC is an unstable protein, and its turnover is controlled by the ubiquitin system. Ubiquitination enhances MYC-dependent transactivation, but the underlying mechanism remains unresolved. Here we show that MYC proteasomal turnover is dispensable for loading of RNA polymerase II (RNAPII). In contrast, MYC turnover is essential for recruitment of TRRAP, histone acetylation, and binding of BRD4 and P-TEFb to target promoters, leading to phosphorylation of RNAPII and transcriptional elongation. In the absence of histone acetylation and P-TEFb recruitment, MYC associates with the PAF1 complex (PAF1C) through a conserved domain in the MYC amino terminus ("MYC box I"). Depletion of the PAF1C subunit CDC73 enhances expression of MYC target genes, suggesting that the MYC/PAF1C complex can inhibit transcription. Because several ubiquitin ligases bind to MYC via the same domain ("MYC box II") that interacts with TRRAP, we propose that degradation of MYC limits the accumulation of MYC/PAF1C complexes during transcriptional activation.

The positive transcription elongation factor b (P-TEFb) promotes transcription elongation through phosphorylation of the RNA polymerase II C-terminal domain. This process is not well understood, partly due to difficulties in determining the specificity of P-TEFb toward the various heptad repeat motifs within the C-terminal domain. A simple assay using mass spectrometry was developed to identify the substrate specificity of the Drosophila melanogaster P-TEFb (DmP-TEFb) in vitro. This assay demonstrated that DmP-TEFb preferentially phosphorylates Ser5 and, surprisingly, that pre-phosphorylation or conserved amino acid variation at the 7-position in the heptad can alter DmP-TEFb specificity, leading to the creation of distinct double-phosphorylation marks.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.

Comprehensively elucidating the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) latency is a priority to achieve a functional cure. As current 'shock' agents failed to efficiently reactivate the latent reservoir, it is important to discover new targets for developing more efficient latency-reversing agents (LRAs). Here, we found that TRIM28 potently suppresses HIV-1 expression by utilizing both SUMO E3 ligase activity and epigenetic adaptor function. Through global site-specific SUMO-MS study and serial SUMOylation assays, we identified that P-TEFb catalytic subunit CDK9 is significantly SUMOylated by TRIM28 with SUMO4. The Lys44, Lys56 and Lys68 residues on CDK9 are SUMOylated by TRIM28, which inhibits CDK9 kinase activity or prevents P-TEFb assembly by directly blocking the interaction between CDK9 and Cyclin T1, subsequently inhibits viral transcription and contributes to HIV-1 latency. The manipulation of TRIM28 and its consequent SUMOylation pathway could be the target for developing LRAs., eLife digest The human immunodeficiency virus-1, or HIV-1, infects certain human cells, including white blood cells. One reason the infection is incurable is because the virus can integrate its genetic information into its host, and essentially ‘sleep’ within the host cell, a process called latency. This helps to hide HIV-1 from the immune system and stops it getting destroyed. Latency represents a critical challenge in treating and curing HIV-1. One proposed cure for HIV-1 involves ‘shocking’ the viruses out of latency so that they can be eliminated. Applying this so-called shock and kill approach means scientists need to understand more about how latency is maintained. Previous evidence shows that latency requires proteins known as histone deacetylases and histone methyltransferases. Certain gene-silencing proteins called transcription suppressors are also involved. Ma et al. have now examined latent HIV-1 in several kinds of human cells grown in the laboratory. The cells were modified to make certain proteins at much lower levels than normal. The experiments showed that the loss of a protein called TRIM28 ‘wakes up’ latent HIV-1. TRIM28 attaches chemical marks called SUMOylations to gene regulators in the cell. These SUMOylations restrict the activity of HIV-1’s genes, which is important to maintain latency. Specifically, TRIM28 adds SUMOylations to a protein named CDK9 at three key positions. Reducing the levels of TRIM28 made it easier to shock many HIV-1 in infected cells out of latency. With further investigation, targeting TRIM28 may one day be used to treat HIV-1 infection through a shock and kill method.