SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits.

RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3' end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3' end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells.

INTAC endonuclease and phosphatase modules differentially regulate transcription by RNA polymerase II.

Gene expression in metazoans is controlled by promoter-proximal pausing of RNA polymerase II, which can undergo productive elongation or promoter-proximal termination. Integrator-PP2A (INTAC) plays a crucial role in determining the fate of paused polymerases, but the underlying mechanisms remain unclear. Here, we establish a rapid degradation system to dissect the functions of INTAC RNA endonuclease and phosphatase modules. We find that both catalytic modules function at most if not all active promoters and enhancers, yet differentially affect polymerase fate. The endonuclease module induces promoter-proximal termination, with its disruption leading to accumulation of elongation-incompetent polymerases and downregulation of highly expressed genes, while elongation-competent polymerases accumulate at lowly expressed genes and non-coding elements, leading to their upregulation. The phosphatase module primarily prevents the release of paused polymerases and limits transcriptional activation, especially for highly paused genes. Thus, both INTAC catalytic modules have unexpectedly general yet distinct roles in dynamic transcriptional control.

N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation.

N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. How m6A regulates RNA polymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regulator of RNA Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight m6A sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the m6A-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcription. Mechanistically, removal of m6A leads to 7SK structural rearrangements that facilitate sequestration of the positive transcription elongation factor b (P-TEFb) complex, which results in reduction of serine 2 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter-proximal region. Taken together, we uncover that m6A modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.

Structural insights into human co-transcriptional capping.

Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is ∼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to ∼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).

Inducible transcriptional condensates drive 3D genome reorganization in the heat shock response.

Mammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, whereas an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become constitutively active without intergenic coalescence, which comes at a fitness cost. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.

Structure of a backtracked hexasomal intermediate of nucleosome transcription.

During gene transcription, RNA polymerase II (RNA Pol II) passes nucleosomes with the help of various elongation factors. Here, we show that RNA Pol II achieves efficient nucleosome passage when the human elongation factors DSIF, PAF1 complex (PAF), RTF1, SPT6, and TFIIS are present. The cryo-EM structure of an intermediate of the nucleosome passage shows a partially unraveled hexasome that lacks the proximal H2A-H2B dimer and interacts with the RNA Pol II jaw, DSIF, and the CTR9trestle helix. RNA Pol II adopts a backtracked state with the RNA 3' end dislodged from the active site and bound in the RNA Pol II pore. Additional structures and biochemical data show that human TFIIS enters the RNA Pol II pore and stimulates the cleavage of the backtracked RNA and nucleosome passage.

Beyond rRNA: nucleolar transcription generates a complex network of RNAs with multiple roles in maintaining cellular homeostasis.

Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes ∼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.

Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements.

Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency. Four intergenic elements form a super-enhancer, and most of the elements contribute to Fgf5 induction at distinct time points. A fifth, poised enhancer located in the first intron contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. Despite low individual enhancer activity, together these elements strongly induce Fgf5 expression in a super-additive fashion that involves strong accumulation of RNA polymerase II at the intronic enhancer. Finally, we observe a strong anti-correlation between RNA polymerase II levels at enhancers and their distance to the closest promoter, and we identify candidate elements with properties similar to the intronic enhancer.

Allosteric transcription stimulation by RNA polymerase II super elongation complex.

The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.

Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination.

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.

Cohesin Removal Reprograms Gene Expression upon Mitotic Entry.

As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.

MeCP2 Represses the Rate of Transcriptional Initiation of Highly Methylated Long Genes.

Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.

RNA Pol II Length and Disorder Enable Cooperative Scaling of Transcriptional Bursting.

RNA polymerase II (RNA Pol II) contains a disordered C-terminal domain (CTD) whose length enigmatically correlates with genome size. The CTD is crucial to eukaryotic transcription, yet the functional and evolutionary relevance of this variation remains unclear. Here, we investigate how CTD length and disorder influence transcription. We find that length modulates the size and frequency of transcriptional bursting. Disorder is highly conserved and facilitates CTD-CTD interactions, an ability we show is separable from protein sequence and necessary for efficient transcription. We build a data-driven quantitative model, simulations of which recapitulate experiments and support that CTD length promotes initial polymerase recruitment to the promoter and slows down its release from it and that CTD-CTD interactions enable recruitment of multiple polymerases. Our results reveal how these parameters provide access to a range of transcriptional activity, offering a new perspective for the mechanistic significance of CTD length and disorder in transcription across eukaryotes.

Gene-Specific Control of tRNA Expression by RNA Polymerase II.

Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

Pre-mRNA splicing and its cotranscriptional connections.

Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.

The role of centromeric repeats and transcripts in kinetochore assembly and function.

Centromeres are the chromosomal domains, where the kinetochore protein complex is formed, mediating proper segregation of chromosomes during cell division. Although the function of centromeres has remained conserved during evolution, centromeric DNA is highly variable, even in closely related species. In addition, the composition of the kinetochore complexes varies among organisms. Therefore, it is assumed that the centromeric position is determined epigenetically, and the centromeric histone H3 (CENH3) serves as an epigenetic marker. The loading of CENH3 onto centromeres depends on centromere-licensing factors, chaperones, and transcription of centromeric repeats. Several proteins that regulate CENH3 loading and kinetochore assembly interact with the centromeric transcripts and DNA in a sequence-independent manner. However, the functional aspects of these interactions are not fully understood. This review discusses the variability of centromeric sequences in different organisms and the regulation of their transcription through the RNA Pol II and RNAi machinery. The data suggest that the interaction of proteins involved in CENH3 loading and kinetochore assembly with centromeric DNA and transcripts plays a role in centromere, and possibly neocentromere, formation in a sequence-independent manner.

Transcriptional repression across mitosis: mechanisms and functions.

Transcription represents a central aspect of gene expression with RNA polymerase machineries (RNA Pol) driving the synthesis of RNA from DNA template molecules. In eukaryotes, a total of three RNA Pol enzymes generate the plethora of RNA species and RNA Pol II is the one transcribing all protein-coding genes. A high number of cis- and trans-acting factors orchestrates RNA Pol II-mediated transcription by influencing the chromatin recruitment, activation, elongation, and/or termination steps. The levels of DNA accessibility, defining open-euchromatin versus close-heterochromatin, delimits RNA Pol II activity as well as the encounter with other factors acting on chromatin such as the DNA replication or DNA repair machineries. The stage of the cell cycle highly influences RNA Pol II activity with mitosis representing the major challenge. In fact, there is a massive inhibition of transcription during the mitotic entry coupled with chromatin dissociation of most of the components of the transcriptional machinery. Mitosis, as a consequence, highly compromises the transcriptional memory and the perpetuation of cellular identity. Once mitosis ends, transcription levels immediately recover to define the cell fate and to safeguard the proper progression of daughter cells through the cell cycle. In this review, we evaluate our current understanding of the transcriptional repression associated with mitosis with a special focus on the molecular mechanisms involved, on the potential function behind the general repression, and on the transmission of the transcriptional machinery into the daughter cells. We finally discuss the contribution that errors in the inheritance of the transcriptional machinery across mitosis might play in stem cell aging.

The Myc-associated zinc finger protein (MAZ) works together with CTCF to control cohesin positioning and genome organization.

The Myc-associated zinc finger protein (MAZ) is often found at genomic binding sites adjacent to CTCF, a protein which affects large-scale genome organization through its interaction with cohesin. We show here that, like CTCF, MAZ physically interacts with a cohesin subunit and can arrest cohesin sliding independently of CTCF. It also shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and consequently affects RNA alternative splicing. CTCF/MAZ double sites are more effective at sequestering cohesin than sites occupied only by CTCF. Furthermore, depletion of CTCF results in preferential loss of CTCF from sites not occupied by MAZ. In an assay for insulation activity like that used for CTCF, binding of MAZ to sites between an enhancer and promoter results in down-regulation of reporter gene expression, supporting a role for MAZ as an insulator protein. Hi-C analysis of the effect of MAZ depletion on genome organization shows that local interactions within topologically associated domains (TADs) are disrupted, as well as contacts that establish the boundaries of individual TADs. We conclude that MAZ augments the action of CTCF in organizing the genome, but also shares properties with CTCF that allow it to act independently.

Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.

Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription.