Explore the vast range of titles available.

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6–HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6–HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

Transcriptional pause-release critically regulates cellular RNA biogenesis, yet how dysregulation of this process impacts embryonic development is not fully understood. Rtf1 is a multifunctional transcription regulatory protein involved in modulating promoter-proximal pausing of RNA Polymerase II (RNA Pol II). Using zebrafish and mouse as model systems, we show that Rtf1 activity is essential for the differentiation of the myocardial lineage from mesoderm. Ablation of rtf1 impairs the formation of nkx2.5+ / tbx5a+ cardiac progenitor cells, resulting in the development of embryos without cardiomyocytes. Structure-function analysis demonstrates that Rtf1’s cardiogenic activity requires its Plus3 domain, which confers interaction with the pausing/elongation factor Spt5. In Rtf1-deficient embryos, the occupancy of RNA Pol II at transcription start sites was reduced relative to downstream occupancy, suggesting a reduction in transcriptional pausing. Intriguingly, attenuating pause release by pharmacological inhibition or morpholino targeting of CDK9 improved RNA Pol II occupancy at the transcription start sites of key cardiac genes and restored cardiomyocytes in Rtf1-deficient embryos. Thus, our findings demonstrate the crucial role that Rtf1-mediated transcriptional pausing plays in controlling the precise spatiotemporal transcription programs that govern early heart development.

The multicomponent polymerase associated factor 1 (Paf1) complex (PAF1C) is an important transcription elongation factor that upregulates RNA polymerase II-mediated genome-wide transcription. PAF1C can regulate transcription through direct association with the polymerase or by impacting the chromatin structure epigenetically. In recent years, significant progress has been made in understanding the molecular mechanisms of PAF1C. However, high-resolution structures that can clarify the interaction details among the components of the complex are still needed. In this study, we evaluated the structural core of the yeast PAF1C containing the four components Ctr9, Paf1, Cdc73 and Rtf1 at high resolution. We observed the interaction details among these components. In particular, we identified a new binding surface of Rtf1 on PAF1C and found that the C-terminal sequence of Rtf1 dramatically changed during evolution, which may account for its different binding affinities to PAF1C among species. Our work presents a precise model of PAF1C, which will facilitate our understanding of the molecular mechanism and the in vivo function of the yeast PAF1C.

Rtf1 is generally considered to be a subunit of the Paf1 complex (Paf1C), which is a multifunctional protein complex involved in histone modification and RNA biosynthesis at multiple stages. Rtf1 is stably associated with the Paf1C in Saccharomyces cerevisiae , but not in other species including humans. Little is known about its function in human fungal pathogens. Here, we show that Rtf1 is required for facilitating H2B monoubiquitination (H2Bub1), and regulates fungal morphogenesis and pathogenicity in the meningitis-causing fungal pathogen Cryptococcus neoformans . Rtf1 is not tightly associated with the Paf1C, and its histone modification domain (HMD) is sufficient to promote H2Bub1 and the expression of genes related to fungal mating and filamentation. Moreover, Rtf1 HMD fully restores fungal morphogenesis and pathogenicity; however, it fails to restore defects of thermal tolerance and melanin production in the rtf1 Δ strain background. The present study establishes a role for cryptococcal Rtf1 as a Paf1C-independent regulator in regulating fungal morphogenesis and pathogenicity, and highlights the function of HMD in facilitating global H2Bub1 in C. neoformans .

We are using biochemical and genetic approaches to study Rtf1 and the Spt4–Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II‐associated Paf1 complex. Further analysis revealed allele‐specific genetic interactions between Paf1 complex members, Spt4–Spt5, and Spt16–Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold‐sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4–Spt5 and Spt16–Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6‐azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4–Spt5 and Spt16–Pob3.

Maintenance of chromatin structure under the disruptive force of transcription requires cooperation among numerous regulatory factors. Histone post-translational modifications can regulate nucleosome stability and influence the disassembly and reassembly of nucleosomes during transcription elongation. The Paf1 transcription elongation complex, Paf1C, is required for several transcription-coupled histone modifications, including the mono-ubiquitylation of H2B. In Saccharomyces cerevisiae, amino acid substitutions in the Rtf1 subunit of Paf1C greatly diminish H2B ubiquitylation and cause transcription to initiate at a cryptic promoter within the coding region of the FLO8 gene, an indicator of chromatin disruption. In a genetic screen to identify factors that functionally interact with Paf1C, we identified mutations in HDA3, a gene encoding a subunit of the Hda1C histone deacetylase (HDAC), as suppressors of an rtf1 mutation. Absence of Hda1C also suppresses the cryptic initiation phenotype of other mutants defective in H2B ubiquitylation. The genetic interactions between Hda1C and the H2B ubiquitylation pathway appear specific: loss of Hda1C does not suppress the cryptic initiation phenotypes of other chromatin mutants and absence of other HDACs does not suppress the absence of H2B ubiquitylation. Providing further support for an appropriate balance of histone acetylation in regulating cryptic initiation, absence of the Sas3 histone acetyltransferase elevates cryptic initiation in rtf1 mutants. Our data suggest that the H2B ubiquitylation pathway and Hda1C coordinately regulate chromatin structure during transcription elongation and point to a potential role for a HDAC in supporting chromatin accessibility.

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1-Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

The PAF1 complex component Rtf1 is an RNA Polymerase II-interacting transcription regulatory protein that promotes transcription elongation and the co-transcriptional monoubiquitination of histone 2B. Rtf1 plays an essential role in the specification of cardiac progenitors from the lateral plate mesoderm during early embryogenesis, but its requirement in mature cardiac cells is unknown. Here, we investigate the importance of Rtf1 in neonatal and adult cardiomyocytes using knockdown and knockout approaches. We demonstrate that loss of Rtf1 activity in neonatal cardiomyocytes disrupts cell morphology and results in a breakdown of sarcomeres. Similarly, Rtf1 ablation in mature cardiomyocytes of the adult mouse heart leads to myofibril disorganization, disrupted cell–cell junctions, fibrosis, and systolic dysfunction. Rtf1 knockout hearts eventually fail and exhibit structural and gene expression defects resembling dilated cardiomyopathy. Intriguingly, we observed that loss of Rtf1 activity causes a rapid change in the expression of key cardiac structural and functional genes in both neonatal and adult cardiomyocytes, suggesting that Rtf1 is continuously required to support expression of the cardiac gene program.

Histone modification is a critical determinant of the frequency and location of meiotic double-strand breaks (DSBs), and thus recombination. Set1-dependent histone H3K4 methylation and Dot1-dependent H3K79 methylation play important roles in this process in budding yeast. Given that the RNA polymerase II associated factor 1 complex, Paf1C, promotes both types of methylation, we addressed the role of the Paf1C component, Rtf1, in the regulation of meiotic DSB formation. Similar to a set1 mutation, disruption of RTF1 decreased the occurrence of DSBs in the genome. However, the rtf1  set1 double mutant exhibited a larger reduction in the levels of DSBs than either of the single mutants, indicating independent contributions of Rtf1 and Set1 to DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a manner that was different from the distributions observed in both set1 and set1  dot1 mutants, including enhanced DSB formation at some DSB-cold regions that are occupied by nucleosomes in wild-type cells. These observations suggest that Rtf1, and by extension the Paf1C, modulate the genomic DSB landscape independently of H3K4 methylation.

Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of \"slow-moving\" Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin.