Transcript elongation controlled by RNA polymerase II (RNAP II) represents a key regulatory event in numerous cellular processes. However, the precise mechanisms underlying the regulation of RNAP II distribution and progression in plants remain largely elusive. Here, we positionally cloned the causal mutation in the defective kernel 59 (dek59) maize (Zea mays) mutant and demonstrated that Dek59 encodes Structure-Specific Recognition Protein 1 (ZmSSRP1), a subunit of the FAcilitates Chromatin Transcription (FACT) complex that regulates RNAP II. Using genome-wide mapping assays, we determined that ZmSSRP1-binding sites co-localize with those of RNAP II phosphorylated at its serine 2 residue (Ser2P) and are highly enriched within actively transcribed genes. Mutation of ZmSSRP1 resulted in Ser2P accumulation around the +1 nucleosome of genes, affecting gene expression in a gene length-dependent manner. The reduced amount of RNAP II in the dek59 mutant was rescued to wild-type-like levels by inhibiting the proteasome, indicating that arrested RNAP II degradation is proteasome-dependent. These findings reveal the indispensable role of ZmSSRP1 in regulating RNAP II-mediated transcription, which is critical for the proper expression of thousands of genes during maize seed development.

RNA polymerase II-associated protein 2 (RPAP2) plays a critical role in transcriptional regulation. However, little is known about whether and how RPAP2 regulates hepatocellular carcinoma (HCC) cell growth, and how the RPAP2 stability is precisely maintained. Here it is reported that high RPAP2 levels in HCC tissues correlate with poor patient survival. RPAP2 depletion suppresses the growth and survival of HCC cells. F-box and WD repeat domain-containing 7 (FBXW7) targets RPAP2 for polyubiquitylation and degradation after RPAP2 being pre-phosphorylated at Ser562 and Thr565 by p38 and GSK3, respectively. HSP90 inhibition significantly promotes RPAP2 degradation by CRL5FBXW7 ligase, whereas USP7 deubiquitylates and stabilizes RPAP2. FBXW7 knockdown promotes HCC cell growth via RPAP2 accumulation in vitro and in vivo. In mice, the hepatic-specific deletion of Fbxw7 leads to hepatic cystogenesis with consequential accumulation of RPAP2. Simultaneous deletion of Rpap2 completely reverses the hepatic cystogenesis, indicating a causal role of RPAP2. Taken together, this study demonstrates that the RPAP2 stability is negatively regulated by FBXW7, but positively regulated by HSP90 and USP7. The FBXW7-RPAP2 axis regulates HCC cell growth and modulates the fate of liver cell differentiation. These findings provide proof-of-concept evidence that oncogenic RPAP2 could be a promising therapeutic target for HCC.

Transcriptional control of gene expression is fundamental to all cellular processes. Conversely, transcriptional dysregulation is a hallmark of cancer. While this hallmark is a key driver of all malignancy-related process, it also represents a vulnerability that can be exploited therapeutically. Prostate cancer is a prime example of this phenomenon: it is characterised by aberrant transcription and treated with drugs that influence transcriptional pathways. Indeed, the primary oncogenic driver and therapeutic target of prostate cancer, the androgen receptor (AR), is a transcription factor. Moreover, a plethora of other transcriptional regulators, including transcriptional cyclin-dependent kinases (CDK7, CDK8 and CDK9), MYC and Bromodomain-containing protein 4 (BRD4), play prominent roles in disease progression. In this review, we focus on the roles of transcriptional CDKs in prostate cancer growth, metastasis and therapy resistance and discuss their interplay with AR, MYC and BRD4. Additionally, we explore recent advances in the therapeutic targeting of transcriptional CDKs and propose how these strategies could be effectively harnessed for the treatment of prostate cancer.

The structure of chromatin at the nucleosome level inside cells is still incompletely understood. Here we present in situ electron cryotomography analyses of chromatin in both G1 and metaphase RPE-1 cells. G1 nucleosomes are concentrated in globular chromatin domains, and metaphase nucleosomes are concentrated in the chromatids. Classification analysis reveals that canonical mononucleosomes, and in some conditions ordered stacked dinucleosomes and mononucleosomes with a disordered gyre-proximal density, are abundant in both cell-cycle states. We do not detect class averages that have more than two stacked nucleosomes or side-by-side dinucleosomes, suggesting that groups of more than two nucleosomes are heterogeneous. Large multi-megadalton structures are abundant in G1 nucleoplasm, but not found in G1 chromatin domains and metaphase chromatin. The macromolecular phenotypes studied here represent a starting point for the comparative analysis of compaction in normal vs. unhealthy human cells, in other cell-cycle states, other organisms, and in vitro chromatin assemblies.

Medulloblastoma is the most common malignant brain tumor of childhood. The highest-risk tumors are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB.

Independent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with CDK9/7 inhibitors to evaluate effect on Myc-driven programs and tumor growth.

Eight CRISPR-Cas9 screens performed across three independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cells are susceptible to CDK9 inhibition, which is synergistic with concurrent inhibition of CDK7. Inhibition of transcriptional CDKs disrupts enhancer-promoter activity in Myc-MB and downregulates Myc-driven transcriptional programs, exerting potent anti-tumor effect.

Our findings identify CDK9 inhibition as a translationally promising strategy for the treatment of Myc-MB.

CDK9 is an intrinsic dependency in Myc-driven medulloblastomaDual CDK9/7 inhibition disrupts Myc-driven transcriptional circuitryCDK9 inhibitors should be developed as pharmaceutical agents for Myc-MB.

Medulloblastoma is the most common malignant brain tumor of childhood, and outcomes for high-risk subgroups remain unsatisfactory despite intensive multimodal therapy. In this study, we pool multiple independent CRISPR-Cas9 screens to identify transcriptional cofactors such as CDK9 as conserved dependencies in Myc-MB. Using Hi-C from primary patient samples, we map Myc enhancer-promoter interactions and show that they can be disrupted using inhibition of transcriptional CDKs. CDK9 inhibitor treatment depletes Myc-driven transcriptional programs, leading to potent anti-tumor effect in vitro and prolongation of xenograft survival in vivo . With a large number of CDK9 inhibitory compounds now in clinical development, this study highlights the opportunity for clinical translation of these for children diagnosed with Myc-MB.

When starved of nitrogen, cells of the fission yeast Schizosaccharomyces pombe enter a quiescent 'G0' state with smaller nuclei and transcriptional repression. The genomics of S. pombe G0 cells has been well studied, but much of its nuclear cell biology remains unknown. Here, we use confocal microscopy, immunoblots and electron cryotomography to investigate the cytological, biochemical and ultrastructural differences between S. pombe proliferating, G1-arrested and G0 cell nuclei, with an emphasis on the histone acetylation, RNA polymerase II fates and macromolecular complex packing. Compared to proliferating cells, G0 cells have lower levels of histone acetylation, nuclear RNA polymerase II and active transcription. The G0 nucleus has similar macromolecular crowding yet fewer chromatin-associated multi-megadalton globular complexes. Induced histone hyperacetylation during nitrogen starvation results in cells that have larger nuclei and therefore chromatin that is less compact. However, these histone-hyperacetylated cells remain transcriptionally repressed with similar nuclear crowding. Canonical nucleosomes - those that resemble the crystal structure - are rare in proliferating, G1-arrested and G0 cells. Our study therefore shows that extreme changes in nucleus physiology are possible without extreme reorganization at the macromolecular level.

The interplay between circadian clocks, the cell cycle, and DNA repair has been extensively documented, yet the epigenetic control of circadian clocks by DNA damage responses remains relatively unexplored. Here, we showed that checkpoint kinases CHK1/2 regulate chromatin structure during DNA damage in Neurospora crassa to maintain robust circadian rhythms. Under DNA damage stress, deletion of chk1/2 disrupted the rhythmic transcription of the clock gene frq by suppressing the rhythmic binding of the transcription activator White Collar complex (WCC) at the frq promoter, as the chromatin structure remained condensed. Mechanistically, CHK1/2 interacted with WC-2 and were recruited by WCC to bind at the frq promoter to phosphorylate H3T11, promoting H3 acetylation, especially H3K56 acetylation, to counteract the histone variant H2A.Z deposition, thereby establishing a suitable chromatin state to maintain robust circadian rhythms despite DNA damage. Additionally, a genome-wide correlation was discovered between H3T11 phosphorylation and H3K56 acetylation, showing a specific function at the frq promoter that is dependent on CHK1/2. Furthermore, transcriptome analysis revealed that CHK1/2 are responsible for robust rhythmic transcription of metabolic and DNA repair genes during DNA damage. These findings highlight the essential role of checkpoint kinases in maintaining robust circadian rhythms under DNA damage stress.

Nucleotide excision repair (NER) is a universal cut-and-paste DNA repair mechanism that corrects bulky DNA lesions such as those caused by UV radiation, environmental mutagens, and some chemotherapy drugs. In this review, we focus on the human transcription/DNA repair factor TFIIH, a key player of the NER pathway in eukaryotes. This 10-subunit multiprotein complex notably verifies the presence of a lesion and opens the DNA around the damage via its XPB and XPD subunits, two proteins identified in patients suffering from Xeroderma Pigmentosum syndrome. Isolated as a class II gene transcription factor in the late 1980s, TFIIH is a prototypic molecular machine that plays an essential role in both DNA repair and transcription initiation and harbors a DNA helicase, a DNA translocase, and kinase activity. More recently, TFIIH subunits have been identified as participating in other cellular processes, including chromosome segregation during mitosis, maintenance of mitochondrial DNA integrity, and telomere replication.

Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used a small interfering RNA (siRNA)-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1 (SR-related C-terminal domain-associated factor 1), using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery is synthetically lethal.

The 5'-3' exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by Rai1, which is recruited to the termination site by Rtt103, an adaptor protein binding to the RNAPII C-terminal domain (CTD). The overall architecture of the Xrn2/Rat1-Rai1-Rtt103 complex remains unknown. We combined structural biology methods to characterize the torpedo complex from Saccharomyces cerevisiae and Chaetomium thermophilum. Comparison of the structures from these organisms revealed a conserved protein core fold of the subunits, but significant variability in their interaction interfaces. We found that in the mesophile, Rtt103 utilizes an unstructured region to augment a Rai1 β-sheet, while in the thermophile Rtt103 binds to a C-terminal helix of Rai1 via its CTD-interacting domain with an α-helical fold. These different torpedo complex assemblies reflect adaptations to the environment and impact complex recruitment to RNAPII.

Anillin (ANLN), a mitotic protein that regulates contractile ring assembly, has been reported as an oncoprotein. However, the function of ANLN in cancer cells, especially in the nucleus, has not been fully understood. Here, we report a role of nuclear ANLN in gene transcriptional regulation. We find that nuclear ANLN directly interacts with the RNA polymerase II (Pol II) large subunit to form transcriptional condensates. ANLN enhances initiated Pol II clustering and promotes Pol II CTD phase separation. Short-term depletion of ANLN alters the chromatin binding and enhancer-mediated transcriptional activity of Pol II. The target genes of ANLN-Pol II axis are involved in oxidoreductase activity, Wnt signaling and cell differentiation. THZ1, a super-enhancer inhibitor, specifically inhibits ANLN-Pol II clustering, target gene expression and esophageal squamous cell carcinoma (ESCC) cell proliferation. Our results reveal the function of nuclear ANLN in transcriptional regulation, providing a theoretical basis for ESCC treatment.

Transcription elongation, especially RNA polymerase II (Pol II) pause-release, is less studied than transcription initiation in regulating gene expression during meiosis. It is also unclear how transcription elongation interplays with transcription initiation. Here, we show that depletion of NKAPL, a testis-specific protein distantly related to RNA splicing factors, causes male infertility in mice by blocking the meiotic exit and downregulating haploid genes. NKAPL binds to promoter-associated nascent transcripts and co-localizes with DNA-RNA hybrid R-loop structures at GAA-rich loci to enhance R-loop formation and facilitate Pol II pause-release. NKAPL depletion prolongs Pol II pauses and stalls the SOX30/HDAC3 transcription initiation complex on the chromatin. Genetic variants in NKAPL are associated with azoospermia in humans, while mice carrying an NKAPL frameshift mutation (M349fs) show defective meiotic exit and transcriptomic changes similar to NKAPL depletion. These findings identify NKAPL as an R-loop-recognizing factor that regulates transcription elongation, which coordinates the meiotic-to-postmeiotic transcriptome switch in alliance with the SOX30/HDAC3-mediated transcription initiation.

Defining the beginning of a eukaryotic protein-coding gene is relatively simple. It corresponds to the first ribonucleotide incorporated by RNA polymerase II (Pol II) into the nascent RNA molecule. This nucleotide is protected by capping and maintained in the mature messenger RNA (mRNA). However, in higher eukaryotes, the end of mRNA is separated from the sites of transcription termination by hundreds to thousands of base pairs. Currently used genomic annotations only take account of the end of the mature transcript - the sites where pre-mRNA cleavage occurs, while the regions in which transcription terminates are unannotated. Here, we describe the evidence for a marker of transcription termination, which could be widely applicable in genomic studies. Pol II termination regions can be determined genome-wide by detecting Pol II phosphorylated on threonine 4 of its C-terminal domain (Pol II CTD-T4ph). Pol II in this state pauses before leaving the DNA template. Up to date this potent mark has been underused because the evidence for its place and role in termination is scattered across multiple publications. We summarize the observations regarding Pol II CTD-T4ph in termination regions and present bioinformatic analyses that further support Pol II CTD-T4ph as a global termination mark in animals.

CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- and TFIID-dependent. Similarly in human cells, CDK7 inhibition reduced transcription by suppressing RNAPII activity at promoters, consistent with reduced initiation and/or re-initiation. Moreover, widespread 3'-end readthrough transcription was observed in CDK7-inhibited cells; mechanistically, this occurred through rapid nuclear depletion of RNAPII elongation and termination factors, including high-confidence CDK7 targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5'-ends and 3'-ends, and reveal that nuclear abundance of elongation and termination factors is kinase-dependent. Because 3'-readthrough transcription is commonly induced during stress, our results further suggest regulated suppression of CDK7 activity may enable this RNAPII transcriptional response.

A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.

Understanding the mechanisms behind MYC-driven oncogenic transformation could pave the way for identifying novel drug targets. This study explored the role of CREPT in MYC-induced malignancy by generating MYC-transformed mouse embryonic fibroblasts (MEFs) with conditional CREPT deletion. Our results demonstrated that the loss of CREPT significantly impaired MYC-induced colony formation and cell proliferation, indicating that CREPT is essential for the malignant transformation of MEFs. Reintroducing CREPT in CREPT-deficient cells restored malignant properties. Furthermore, CREPT overexpression alone enhanced colony formation upon MYC induction but was insufficient to induce transformation without MYC, suggesting a cooperative interaction between CREPT and MYC in malignant transformation. CREPT deletion resulted in delayed cell cycle progression during the G2/M and S phases. CREPT enhanced the expression of MYC target genes by directly interacting with MYC through the CID domain of CREPT and the PEST domain of MYC. Arginine 34 of CREPT was identified as a critical residue for the interaction with MYC, and its mutation lost the ability of CREPT to promote MYC-driven colony formation and tumor growth in colorectal cancer models. Additionally, CREPT facilitated the recruitment of RNA Polymerase II to MYC-binding promoters, promoting transcriptional initiation of MYC-targeted genes. Our study also revealed a strong correlation between CREPT and MYC expression in various human cancers, particularly in colorectal cancer, where their interaction appears to play a significant role in tumorigenesis. These findings suggest that the CREPT-MYC interaction is crucial for the progression of MYC-driven cancers and presents a potential target for therapeutic intervention.

Control of RNA polymerase II (RNA Pol II) through ubiquitylation is essential for the DNA-damage response. Here, we reveal a distinct ubiquitylation pathway in human cells, mediated by CRL3ARMC5, that targets excessive and defective RNA Pol II molecules at the initial stages of the transcription cycle. Upon ARMC5 loss, RNA Pol II accumulates in the free pool and in the promoter-proximal zone but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess RNA Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has detrimental effects on cell growth and results in the uncontrolled release of excessive RNA Pol II complexes into early elongation, many of which are transcriptionally incompetent and fail to reach the ends of genes. These findings uncover CRL3ARMC5 and Integrator as two distinct pathways acting in parallel to monitor the quantity and quality of transcription complexes before they are licensed into elongation.

Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass spectrometry and other proteomics profiling. Here, we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light-dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates.

Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here, we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

The transcription and transportation of mRNA are coupled processes; however, the mechanisms linking these processes remain unclear. Additionally, the significance of this connection in cancer drug development is poorly understood. To address these issues, we investigated the role of CDK12 kinase, which regulates RNA transcription through the phosphorylation of RNA polymerase II (Pol II) and has a repeated serine-arginine dipeptide (RS domain) involved in mRNA transport. Despite the anticipated uniqueness of CDK12 function, the mechanism by which CDK12 bridges and manages mRNA transcription and transport has not been fully analyzed. Our study revealed that CDK12 interacts with NXF1, a key molecule involved in the export of mRNA from the nucleus to the cytosol. Although CDK12 does not phosphorylate NXF1, we found that NXF1 unexpectedly stabilized the CDK12 protein, suggesting that NXF1 mRNA export activity indirectly affects mRNA transcriptional activity by modifying the protein level of CDK12. Furthermore, CDK12 recruited other essential RNA transporters, specifically the exon junction complex (EJC) and THO complexes, into the CDK12-NXF1 axis through its kinase activity. These observations provide insights into the mechanisms linking mRNA transcription and transport through the formation of a novel CDK12-NXF1 complex that involves EJC and THO. Importantly, the expression level of NXF1 influences sensitivity to CDK12 inhibitors, which are emerging as novel anti-cancer drug candidates. This highlights the importance of considering the relationship between mRNA transcription and transport when targeting RNA transcription in cancer therapy.

Cryptic transcription refers to the unintended expression of non-canonical sites within the genome, producing aberrant RNA and proteins that may disrupt cellular functions. In this opinion piece, I will explore the role of histone modifications in modulating cryptic transcription and its implications for gene expression and cellular integrity, particularly with a focus on H3K36 and H3K4 methylation marks. H3K36 tri-methylation plays a crucial role in maintaining chromatin integrity by facilitating the recruitment of the Rpd3S histone deacetylase (HDAC) complex, which helps restore closed chromatin states following transcription and prevents cryptic initiation within gene bodies. In parallel, crosstalk between H3K4 di-methylation and histone ubiquitylation and sumoylation is critical for recruiting the Set3 HDAC complex, which maintains low histone acetylation levels in gene bodies and further suppresses cryptic transcription. Therefore, by elucidating these regulatory mechanisms, this opinion highlights the intricate interplay of histone modifications in preserving transcriptional fidelity and suggests potential pathways for future research to develop novel therapies for age-related disorders and other diseases associated with dysregulated gene expression.

The intrinsically disordered carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, drastically accelerating phosphorylation and promoting the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation.

RNA polymerase II (Pol II) C-terminal domain (CTD) is known to have crucial roles in regulating transcription. CTD has also been highly recognized for undergoing phase separation, which is further associated with its regulatory functions. However, the molecular interactions that the CTD forms to induce clustering to drive phase separations and how the phosphorylation of the CTD affects clustering are not entirely known. In this work, we studied the concentrated solutions of two heptapeptide repeat (2CTD) models at different phosphorylation patterns and protein and ion concentrations using all-atom molecular dynamics simulations to investigate clustering behavior and molecular interactions driving the cluster formation. Our results show that salt concentration and phosphorylation patterns play an important role in determining the clustering pattern, specifically at low protein concentrations. The balance between inter- and intrapeptide interactions and counterion coordination together impact the clustering behavior upon phosphorylation.

The recognition of core promoter sequences by TFIID is the first step in RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is a trilobular complex, composed of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs). Why and how TAFs are necessary for the formation of TFIID domains and how they contribute to transcription initiation remain unclear. Inducible TAF7 or TAF10 depletion, followed by comprehensive analysis of TFIID subcomplex formation, chromatin binding, and nascent transcription in mouse embryonic stem cells, result in the formation of a TAF7-lacking TFIID or a minimal core-TFIID complex, respectively. These partial complexes support TBP recruitment at promoters and nascent Pol II transcription at most genes early after depletion, but importantly, TAF10 is necessary for efficient Pol II pausing. We show that partially assembled TFIID complexes can sustain Pol II transcription initiation but cannot replace holo-TFIID over several cell divisions and/or development.

Phosphatidylinositol phosphates are powerful signaling molecules that orchestrate signaling and direct membrane trafficking in the cytosol. Interestingly, phosphatidylinositol phosphates also localize within the membrane-less compartments of the cell nucleus, where they participate in the regulation of gene expression. Nevertheless, current models of gene expression, which include condensates of proteins and nucleic acids, do not include nuclear phosphatidylinositol phosphates. This gap is partly a result of the missing detailed analysis of the subnuclear distribution of phosphatidylinositol phosphates and their relationships with gene expression. Here, we used quantitative dual-color direct stochastic optical reconstruction microscopy to analyze the nanoscale co-patterning between RNA polymerase II transcription initiation and elongation markers with respect to phosphatidylinositol 4,5- or 3,4-bisphosphate in the nucleoplasm and nuclear speckles and compared it with randomized data and cells with inhibited transcription. We found specific co-patterning of the transcription initiation marker P-S5 with phosphatidylinositol 4,5-bisphosphate in the nucleoplasm and with phosphatidylinositol 3,4-bisphosphate at the periphery of nuclear speckles. We showed the specific accumulation of the transcription elongation marker PS-2 and of nascent RNA in the proximity of phosphatidylinositol 3,4-bisphosphate associated with nuclear speckles. Taken together, this shows that the distinct spatial associations between the consecutive stages of RNA polymerase II transcription and nuclear phosphatidylinositol phosphates exhibit specificity within the gene expression compartments. Thus, in analogy to the cellular membranes, where phospholipid composition orchestrates signaling pathways and directs membrane trafficking, we propose a model in which the phospholipid identity of gene expression compartments orchestrates RNA polymerase II transcription.

The polymerase-associated factor 1 (Paf1) complex (Paf1C) is a conserved protein complex with critical functions during eukaryotic transcription. Previous studies showed that Paf1C is multi-functional, controlling specific aspects of transcription ranging from RNA polymerase II (RNAPII) processivity to histone modifications. However, it is unclear how specific Paf1C subunits directly impact transcription and coupled processes. We have compared conditional depletion to steady-state deletion for each Paf1C subunit to determine the direct and indirect contributions to gene expression in Saccharomyces cerevisiae. Using nascent transcript sequencing, RNAPII profiling, and modeling of transcription elongation dynamics, we have demonstrated direct effects of Paf1C subunits on RNAPII processivity and elongation rate and indirect effects on transcript splicing and repression of antisense transcripts. Further, our results suggest that the direct transcriptional effects of Paf1C cannot be readily assigned to any particular histone modification. This work comprehensively analyzes both the immediate and the extended roles of each Paf1C subunit in transcription elongation and transcript regulation.

The 5´-3´ exoribonuclease Rat1/Xrn2 is responsible for the termination of eukaryotic mRNA transcription by RNAPII. Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a "torpedo" to bind transcribing RNAPII and dissociate DNA/RNA from it. Here we report the cryo-electron microscopy structures of the Rat1-Rai1-Rtt103 complex and three Rat1-Rai1-associated RNAPII complexes (type-1, type-1b, and type-2) from the yeast, Komagataella phaffii. The Rat1-Rai1-Rtt103 structure revealed that Rat1 and Rai1 form a heterotetramer with a single Rtt103 bound between two Rai1 molecules. In the type-1 complex, Rat1-Rai1 forms a heterodimer and binds to the RNA exit site of RNAPII to extract RNA into the Rat1 exonuclease active site. This interaction changes the RNA path in favor of termination (the "pre-termination" state). The type-1b and type-2 complexes have no bound DNA/RNA, likely representing the "post-termination" states. These structures illustrate the termination mechanism of eukaryotic mRNA transcription.

Transcription-blocking lesions (TBLs) stall elongating RNA polymerase II (Pol II), which then initiates transcription-coupled repair (TCR) to remove TBLs and allow transcription recovery. In the absence of TCR, eviction of lesion-stalled Pol II is required for alternative pathways to address the damage, but the mechanism is unclear. Using Protein-Associated DNA Damage Sequencing (PADD-seq), this study reveals that the p97-proteasome pathway can evict lesion-stalled Pol II independently of repair. Both TCR and repair-independent eviction require CSA and ubiquitination. However, p97 is dispensable for TCR and Pol II eviction in TCR-proficient cells, highlighting repair's prioritization over repair-independent eviction. Moreover, ubiquitination of RPB1-K1268 is important for both pathways, with USP7's deubiquitinase activity promoting TCR without abolishing repair-independent Pol II release. In summary, this study elucidates the fate of lesion-stalled Pol II, and may shed light on the molecular basis of genetic diseases caused by the defects of TCR genes.

How gene activation works in heterochromatin, and how the mechanism might differ from the one used in euchromatin, has been largely unexplored. Previous work has shown that in SIR-regulated heterochromatin of Saccharomyces cerevisiae, gene activation occurs in the absence of covalent histone modifications and other alterations of chromatin commonly associated with transcription.Here we demonstrate that such activation occurs in a substantial fraction of cells, consistent with frequent transcriptional bursting, and this raises the possibility that an alternative activation pathway might be used. We address one such possibility, Pol II CTD phosphorylation, and explore this idea using a natural telomere-linked gene, YFR057w, as a model. Unlike covalent histone modifications, we find that Ser2, Ser5 and Ser7 CTD phosphorylated Pol II is prevalent at the drug-induced heterochromatic gene. Particularly enriched relative to the euchromatic state is Ser2 phosphorylation. Consistent with a functional role for Ser2P, YFR057w is negligibly activated in cells deficient in the Ser2 CTD kinases Ctk1 and Bur1 even though the gene is strongly stimulated when it is placed in a euchromatic context. Collectively, our results are consistent with a critical role for Ser2 CTD phosphorylation in driving Pol II recruitment and transcription of a natural heterochromatic gene - an activity that may supplant the need for histone epigenetic modifications.

The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3' end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

RNA polymerase II (Pol II) has a C-terminal domain (CTD) that is unstructured, consisting of a large number of heptad repeats, and whose precise function remains unclear. Here, we investigate how altering the CTD's length and fusing it with protein tags affects transcriptional output on a genome-wide scale in mammalian cells at single-cell resolution. While transcription generally appears to occur in burst-like fashion, where RNA is predominantly made during short bursts of activity that are interspersed with periods of transcriptional silence, the CTD's role in shaping these dynamics seems gene-dependent; global patterns of bursting appear mostly robust to CTD alterations. Introducing protein tags with defined structures to the N terminus cause transcriptome-wide effects, however. We find the type of tag to dominate characteristics of the resulting transcriptomes. This is possibly due to Pol II-interacting factors, including non-coding RNAs, whose expression correlates with the tags. Proteins involved in liquid-liquid phase separation appear prominently.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.