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Eukaryotic genomes are pervasively transcribed by RNA polymerase II. Yet, the molecular and biological implications of such a phenomenon are still largely puzzling. Here, we describe noncoding RNA transcription upstream of the Arabidopsis thaliana DOG1 gene, which governs salt stress responses and is a key regulator of seed dormancy. We find that expression of the DOG1 gene is induced by salt stress, thereby causing a delay in seed germination. We uncover extensive transcriptional activity on the promoter of the DOG1 gene, which produces a variety of lncRNAs. These lncRNAs, named PUPPIES, are co‐directionally transcribed and extend into the DOG1 coding region. We show that PUPPIES RNAs respond to salt stress and boost DOG1 expression, resulting in delayed germination. This positive role of pervasive PUPPIES transcription on DOG1 gene expression is associated with augmented pausing of RNA polymerase II, slower transcription and higher transcriptional burst size. These findings highlight the positive role of upstream co‐directional transcription in controlling transcriptional dynamics of downstream genes. Synopsis: DOG1 is a key regulator of plant seed dormancy, whose expression is regulated by multiple mechanisms. Here, salt stress is found to induce pervasive lncRNA transcription within the DOG1 promoter region, resulting in augmented RNA Polymerase II pausing, slower transcription and increased DOG1 expression. The DOG1 gene restricts seed germination under salt stress independently of its function in primary dormancy.The DOG1 promoter region gives rise to salt stress‐responsive PUPPIES lncRNAs, which regulate DOG1 expression in cis to delay germination.Co‐directional pervasive transcription of PUPPIES activates DOG1 expression through augmented RNA polymerase II pausing and enhanced transcriptional bursting.The identified regulatory mechanism involves an increase of RNA molecules produced per DOG1 transcriptional burst, augmented pausing and slower transcription. [ABSTRACT FROM AUTHOR]

Proper timing of flowering, a phase transition from vegetative to reproductive development, is crucial for plant fitness. The floral repressor FLOWERING LOCUS C (FLC) is the major determinant of flowering in Arabidopsis thaliana. In rapid-cycling A. thaliana accessions, which bloom rapidly, FLC is constitutively repressed by autonomous pathway (AP) genes, regardless of photoperiod. Diverse AP genes have been identified over the past two decades, and most of them repress FLC through histone modifications. However, the detailed mechanism underlying such modifications remains unclear. Several recent studies have revealed novel mechanisms to control FLC repression in concert with histone modifications. This review summarizes the latest advances in understanding the novel mechanisms by which AP proteins regulate FLC repression, including changes in chromatin architecture, RNA polymerase pausing, and liquid-liquid phase separation- and ncRNA-mediated gene silencing. Furthermore, we discuss how each mechanism is coupled with histone modifications in FLC chromatin. [ABSTRACT FROM AUTHOR]

RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing.

Abstract: TFIIH is a 10‐subunit complex that regulates RNA polymerase II (pol II) transcription but also serves other important biological roles. Although much remains unknown about TFIIH function in eukaryotic cells, much progress has been made even in just the past few years, due in part to technological advances (e.g. cryoEM and single molecule methods) and the development of chemical inhibitors of TFIIH enzymes. This review focuses on the major cellular roles for TFIIH, with an emphasis on TFIIH function as a regulator of pol II transcription. We describe the structure of TFIIH and its roles in pol II initiation, promoter‐proximal pausing, elongation, and termination. We also discuss cellular roles for TFIIH beyond transcription (e.g. DNA repair, cell cycle regulation) and summarize small molecule inhibitors of TFIIH and diseases associated with defects in TFIIH structure and function. [ABSTRACT FROM AUTHOR]

The 7SK small nuclear RNA is a multifunctional transcriptional regulatory RNA that controls the nuclear activity of the positive transcription elongation factor b (P-TEFb), specifically targets P-TEFb to the promoter regions of selected protein-coding genes and promotes transcription of RNA polymerase II-specific spliceosomal small nuclear RNA genes. [ABSTRACT FROM AUTHOR]

We recently identified PHF13 as an H3K4me2/3 chromatin reader and transcriptional co-regulator. We found that PHF13 interacts with RNAPIIS5P and PRC2 stabilizing their association with active and bivalent promoters. Furthermore, mass spectrometry analysis identified ∼50 spliceosomal proteins in PHF13s interactome. Here, we will discuss the potential role of PHF13 in RNAPII pausing and co-transcriptional splicing. [ABSTRACT FROM PUBLISHER]

RNA polymerase II (RNAPII) controls expression of all protein-coding genes and most noncoding loci in higher eukaryotes. Calibrating RNAPII activity requires an assortment of polymerase-associated factors that are recruited at sites of active transcription. The Integrator complex is one of the most elusive transcriptional regulators in metazoans, deemed to be recruited after initiation to help establish and modulate paused RNAPII. Integrator is known to be composed of 14 subunits that assemble and operate in a modular fashion. We employed proteomics and machine-learning structure prediction (AlphaFold2) to identify an additional Integrator subunit, INTS15. We report that INTS15 assembles primarily with the INTS13/14/10 module and interfaces with the Int-PP2A module. Functional genomics analysis further reveals a role for INTS15 in modulating RNAPII pausing at a subset of genes. Our study shows that omics approaches combined with AlphaFold2-based predictions provide additional insights into the molecular architecture of large and dynamic multiprotein complexes. [Display omitted] • Proteomics, machine learning, and functional genomics characterize C7ORF26 as INTS15 • INTS15 forms the tetrameric Arm module with INTS13/INTS14/INTS10 • INTS15 bridges Integrator's Arm module to INTS5 and the core integrator complex • INTS15 functions in promoting RNA polymerase II pause-release Integrated multiomic approaches, paired with AlphaFold2 modeling, can provide insight into the structure/function of large, multisubunit complexes. Offley et al. characterize C7ORF26 as INTS15, an additional subunit of the Integrator complex. INTS15 bridges the peripheral INTS13/14/10 subunits to the core complex and supports productive elongation by RNA polymerase II. [ABSTRACT FROM AUTHOR]

Background: The transcriptional corepressor Groucho (Gro) is required for the function of many developmentally regulated DNA binding repressors, thus helping to define the gene expression profile of each cell during development. The ability of Gro to repress transcription at a distance together with its ability to oligomerize and bind to histones has led to the suggestion that Gro may spread along chromatin. However, much is unknown about the mechanism of Gro-mediated repression and about the dynamics of Gro targeting. Results: Our chromatin immunoprecipitation sequencing analysis of temporally staged Drosophila embryos shows that Gro binds in a highly dynamic manner primarily to clusters of discrete (<1 kb) segments. Consistent with the idea that Gro may facilitate communication between silencers and promoters, Gro binding is enriched at both cis-regulatory modules, as well as within the promotors of potential target genes. While this Gro-recruitment is required for repression, our data show that it is not sufficient for repression. Integration of Gro binding data with transcriptomic analysis suggests that, contrary to what has been observed for another Gro family member, Drosophila Gro is probably a dedicated repressor. This analysis also allows us to define a set of high confidence Gro repression targets. Using publically available data regarding the physical and genetic interactions between these targets, we are able to place them in the regulatory network controlling development. Through analysis of chromatin associated pre-mRNA levels at these targets, we find that genes regulated by Gro in the embryo are enriched for characteristics of promoter proximal paused RNA polymerase II. Conclusions: Our findings are inconsistent with a one-dimensional spreading model for long-range repression and suggest that Gro-mediated repression must be regulated at a post-recruitment step. They also show that Gro is likely a dedicated repressor that sits at a prominent highly interconnected regulatory hub in the developmental network. Furthermore, our findings suggest a role for RNA polymerase II pausing in Gro-mediated repression. [ABSTRACT FROM AUTHOR]

Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency. [ABSTRACT FROM AUTHOR]

CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early elongation checkpoint ofc-mycby affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate elongation of transcription ofc-mycand recognition of the snRNA gene-specific 3’ box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the elongation checkpoint of the protein-codingc-mycand the termination site of the non-codingU2, by regulating the recruitment and/or activity of key players in these processes. [ABSTRACT FROM AUTHOR]

Abstract: Activation induced deaminase (AID) is globally targeted to immunoglobulin loci, preferentially focused to switch (S) regions and variable (V) regions, and prone to attack hotspot motifs. Nevertheless, AID deamination is not exclusive to Ig loci and the rules regulating AID targeting remain unclear. Transcription is critically required for class switch recombination and somatic hypermutation. Here, I consider the unique features associated with S region transcription leading to RNA polymerase II pausing, that in turn promote the introduction of activating chromatin remodeling, histone modifications and recruitment of AID to targeted S regions. These findings allow for a better understanding of the interplay between transcription, AID targeting and mistargeting to Ig and non-Ig loci. [Copyright &y& Elsevier]

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation. [Display omitted] • m6A MTC recruitment to the chromatin regulates pausing of RNAP II • Mettl3 induces pause release, and this effect depends on its catalytic domain • m6A MTC recruitment can be predicted by RNAP II and pause factor occupancies • m6A MTC interacts with Spt6, and its recruitment is partially dependent on Spt6 Akhtar et al. show that the m6A RNA methyl transferase complex (MTC) releases pausing of RNAP II through its recruitment to the chromatin, predictable by RNAP II and associated pause factor binding. This regulation depends on the catalytic activity of Mettl3 and partially works through its interaction with elongation factor Spt6. [ABSTRACT FROM AUTHOR]