The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity.

HIV-1 infection leads to a gradual loss of T helper cells, chronic immune activation, and eventual immune system breakdown. HIV-1 causes deregulation of the expression of IL-2, a cytokine important for T helper cell growth and survival, which is downregulated in HIV-1 patients. The present study addresses the regulation of IL2 expression via HIV-1 Tat transcriptional activator. We used J-LAT cells, a T cell line that serves as a latency model for studies of HIV-1 expression in T cells, and as controls a T cell line lacking HIV-1 elements and a T cell line with a stably integrated copy of the HIV-1-LTR promoter. We show that endogenously expressed Tat inhibits IL2 transcription in J-Lat cells via its presence in the ARRE-1/2 elements of the IL2 promoter and that the inhibition of IL2 expression is mediated by Tat inhibiting Pol II activity at the IL2 promoter, which is mediated by preventing the presence of Pol II at the ARRE-1/2 elements. Overall, Tat is present at the IL2 promoter, apart from its cognate HIV-1 LTR target. This supports our current knowledge of how HIV-1 affects the host transcriptional machinery and reflects the potential of Tat to disrupt transcriptional regulation of host genes to manipulate cell responses.

Viroids are single-stranded circular noncoding RNAs that infect plants. Research in the past five decades has deciphered the viroid genome structures, viroid replication cycles, numerous host factors for viroid infection, viroid motifs for intracellular and intercellular trafficking, interactions with host defense machinery, etc. In this review, we mainly focus on some significant questions that remain to be tackled, centered around (1) how the RNA polymerase II machinery performs transcription on RNA templates of nuclear-replicating viroids, (2) how viroid RNAs coordinate multiple structural elements for diverse functions, and (3) how viroid RNAs activate plant immunity. Research on viroids has led to seminal discoveries in biology, and we expect the research directions outlined in this review to continue providing key knowledge inspiring other areas of biology.

Genome-wide association studies (GWAS) have identified multiple gastric cancer risk loci and several protein-coding susceptibility genes. However, the role of long-noncoding RNAs (lncRNAs) transcribed from these risk loci in gastric cancer development and progression remains to be explored. Here, we functionally characterize a lncRNA, lncPSCA, as a novel tumor suppressor whose expression is fine-regulated by a gastric cancer risk-associated genetic variant. The rs2978980 T > G change in an intronic enhancer of lncPSCA interrupts binding of transcription factor RORA, which down-regulates lncPSCA expression in an allele-specific manner. LncPSCA interacts with DDX5 and promotes DDX5 degradation through ubiquitination. Increased expression of lncPSCA results in low levels of DDX5, less RNA polymerase II (Pol II) binding with DDX5 in the nucleus, thus activating transcription of multiple p53 signaling genes by Pol II. These findings highlight the importance of functionally annotating lncRNAs in GWAS risk loci and the great potential of modulating lncRNAs as innovative cancer therapy.

The HIV-1 latent reservoir is the major barrier to an HIV cure. Due to low levels or lack of transcriptional activity, HIV-1 latent proviruses in vivo are not easily detectable and cannot be targeted by either natural immune mechanisms or molecular therapies based on protein expression. To target the latent reservoir, further understanding of HIV-1 proviral transcription is required. In this study, we demonstrate a novel role for cleavage and polyadenylation specificity factor 6 (CPSF6) in HIV-1 transcription. We show that knockout of CPSF6 hinders reactivation of latent HIV-1 proviruses by PMA in primary CD4+ cells. CPSF6 knockout reduced HIV-1 transcription, concomitant with a drastic reduction in the phosphorylation levels of Pol II and CDK9. Knockout of CPSF6 led to abnormal stabilization of protein phosphatase 2A (PP2A) subunit A, which then acted to dephosphorylate CDK9, downmodulating CDK9\'s ability to phosphorylate the Pol II carboxy-terminal domain. In agreement with this mechanism, incubation with the PP2A inhibitor, LB100, restored HIV-1 transcription in the CPSF6 knockout cells. Destabilization of PP2A subunit A occurs in the ubiquitin proteasome pathway, wherein CPSF6 acts as a substrate adaptor for the ITCH ubiquitin ligase. Our observations reveal a novel role of CPSF6 in HIV-1 transcription, which appears to be independent of its known roles in cleavage and polyadenylation and the targeting of preintegration complexes to the chromatin for viral DNA integration. IMPORTANCE CPSF6 is a cellular factor that regulates cleavage and polyadenylation of mRNAs and participates in HIV-1 infection by facilitating targeting of preintegration complexes to the chromatin. Our observations reveal a second role of CPSF6 in the HIV-1 life cycle that involves regulation of viral transcription through controlling the stability of protein phosphatase 2A, which in turn regulates the phosphorylation/dephosphorylation status of critical residues in CDK9 and Pol II.

Alternative polyadenylation (APA) is a widespread post-transcriptional modification method that changes the 3\' ends of transcripts by altering poly(A) site usage. However, the longitudinal transcriptomic 3\' end profile and its mechanism of action are poorly understood. We applied diurnal time-course poly(A) tag sequencing (PAT-seq) for Arabidopsis and identified 3284 genes that generated both rhythmic and arrhythmic transcripts. These two classes of transcripts appear to exhibit dramatic differences in expression and translation activisty. The asynchronized transcripts derived by APA are embedded with different poly(A) signals, especially for rhythmic transcripts, which contain higher AAUAAA and UGUA signal proportions. The Pol II occupancy maximum is reached upstream of rhythmic poly(A) sites, while it is present directly at arrhythmic poly(A) sites. Integrating H3K9ac and H3K4me3 time-course data analyses revealed that transcriptional activation of histone markers may be involved in the differentiation of rhythmic and arrhythmic APA transcripts. These results implicate an interplay between histone modification and RNA 3\'-end processing, shedding light on the mechanism of transcription rhythm and alternative polyadenylation.

Regulation of transcription is a tightly choreographed process. The establishment of RNA polymerase II promoter proximal pausing soon after transcription initiation and the release of Pol II into productive elongation are key regulatory processes that occur in early elongation. We describe the techniques and tools that have become available for the study of promoter proximal pausing and their utility for future experiments. We then provide an overview of the factors and interactions that govern a multipartite pausing process and address emerging questions surrounding the mechanism of RNA polymerase II\'s subsequent advancement into the gene body. Finally, we address remaining controversies and future areas of study.

Mediator is a universal adaptor for transcription control. It serves as an interface between gene-specific activator or repressor proteins and the general RNA polymerase II (pol II) transcription machinery. Previous structural studies revealed a relatively small part of Mediator and none of the gene activator-binding regions. We have determined the cryo-EM structure of the Mediator at near-atomic resolution. The structure reveals almost all amino acid residues in ordered regions, including the major targets of activator proteins, the Tail module, and the Med1 subunit of the Middle module. Comparison of Mediator structures with and without pol II reveals conformational changes that propagate across the entire Mediator, from Head to Tail, coupling activator- and pol II-interacting regions.

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).

CCCTC-binding factor (CTCF) and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution stimulated emission depletion (STED) microscopy, we demonstrate that in living cells, CTCF forms clusters typically containing 2-8 molecules. A fraction of CTCF clusters, enriched for those with ≥3 molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition (TI) result in increased CTCF clustering. Furthermore, the effect of TI on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.