RNA Polymerase-associated Factor Research Articles

HnRNPA2B1 deacetylation by SIRT6 restrains local transcription and safeguards genome stability.

Repair of double strand breaks (DSBs) by RNA-binding proteins (RBPs) is vital for ensuring genome integrity. DSB repair is accompanied by local transcriptional repression in the vicinity of transcriptionally active genes, but the mechanism by which RBPs regulate transcriptional regulation is unclear. Here, we demonstrated that RBP hnRNPA2B1 functions as a RNA polymerase-associated factor that stabilizes the transcription complex under physiological conditions. Following a DSB, hnRNPA2B1 is released from damaged chromatin, reducing the efficiency of RNAPII complex assembly, leading to local transcriptional repression. Mechanistically, SIRT6 deacetylates hnRNPA2B1 at K113/173 residues, enforcing its rapid detachment from DSBs. This process disrupts the integrity of the RNAPII complex on active chromatin, which is a pre-requisite for transient but complete repression of local transcription. Functionally, the overexpression of an acetylation mimic stabilizes the transcription complex and facilitates the functioning of the transcription machinery. hnRNPA2B1 acetylation status was negatively correlated with SIRT6 expression, and acetylation mimic enhanced radio-sensitivity in vivo. Our findings demonstrate that hnRNPA2B1 is crucial for transcriptional repression. We have uncovered the missing link between DSB repair and transcriptional regulation in genome stability maintenance, highlighting the potential of hnRNPA2B1 as a therapeutic target.

ATX1 and HUB1/2 promote recruitment of the transcription elongation factor VIP2 to modulate the floral transition in Arabidopsis.

Histone 2B ubiquitination (H2Bub) and trimethylation of H3 at lysine 4 (H3K4me3) are associated with transcription activation. However, the function of these modifications in transcription in plants remains largely unknown. Here, we report that coordination of H2Bub and H3K4me3 deposition with the binding of the RNA polymerase-associated factor VERNALIZATION INDEPENDENCE2 (VIP2) to FLOWERING LOCUS C (FLC) modulates flowering time in Arabidopsis. We found that RING domain protein HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 (we refer as HUB1/2), which are responsible for H2Bub, interact with ARABIDOPSIS TRITHORAX1 (ATX1), which is required for H3K4me3 deposition, to promote the transcription of FLC and repress the flowering time. The atx1-2 hub1-10 hub2-2 triple mutant in FRIGIDIA (FRI) background displayed early flowering like FRI hub1-10 hub2-2 and overexpression of ATX1 failed to rescue the early flowering phenotype of hub1-10 hub2-2. Mutations in HUB1 and HUB2 reduced the ATX1 enrichment at FLC, indicating that HUB1 and HUB2 are required for ATX1 recruitment and H3K4me3 deposition at FLC. We also found that the VIP2 directly binds to HUB1, HUB2, and ATX1 and that loss of VIP2 in FRI hub1-10 hub2-2 and FRI atx1-2 plants resulted in early flowering like that observed in FRI vip2-10. Loss of function of HUB2 and ATX1 impaired VIP2 enrichment at FLC, and reduced the transcription initiation and elongation of FLC. In addition, mutations in VIP2 reduced HUB1 and ATX1 enrichment and H2Bub and H3K4me3 levels at FLC. Together, our findings revealed that HUB1/2, ATX1, and VIP2 coordinately modulate H2Bub and H3K4me3 deposition, FLC transcription, and flowering time.

NELF and PAF1C complexes are core transcriptional machineries controlling colon cancer stemness

Mutations in APC, found in 80% of colon caner, enhance β-catenin stabilization, which is the initial step of colonic tumorigenesis. However, the core transcriptional mechanism underlying the induction of colon cancer stemness by stable β-catenin remains unclear. Here, we found that inducible inhibition of β-catenin suppressed elongation of Pol II and RNA polymerase-associated factor 1 complex (PAF1C) around the transcription start site (TSS) of LGR5. Moreover, stable β-catenin enhanced the formation of active Pol II complex cooperatively with CDC73 and CDK9 by facilitating the recruitment of DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) complexes to the Pol II complex. Subsequently, stable β-catenin facilitated the formation of the Pol II–DSIF–PAF1C complex, suggesting that stable β-catenin induces cancer stemness by stimulating active Pol II complex through NELF and PAF1C. Furthermore, NELF or PAF1C inhibition recapitulated the changes in cancer stemness-related gene expression induced by the inhibition of stable β-catenin and suppressed colon cancer stemness. Additionally, the chemical inhibition of CDK12 (a downstream transcription CDK of PAF1C) suppressed colon cancer stemness. These results suggest that NELF and PAF1C are the core transcriptional machineries that control expression of colon cancer stemness-inducing genes and may be therapeutic targets for colon cancer.

DnaA and SspA regulation of the iraD gene of Escherichia coli: an alternative DNA damage response independent of LexA/RecA.

The transcription factor RpoS of Escherichia coli controls many genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by azidothymidine. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp and are regulated by regulatory inactivation of DnaA, dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays, we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced regulatory inactivation of DnaA. We also demonstrate that the RNA-polymerase associated factor, stringent starvation protein A, induced by the accumulation of ppGpp, also affects iraD expression, with a positive effect on constitutive expression and a negative effect on azidothymidine-induced expression.

Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence

BackgroundCellular quiescence is a reversible differentiation state during which cells modify their gene expression program to inhibit metabolic functions and adapt to a new cellular environment. The epigenetic changes accompanying these alterations are not well understood. We used fission yeast cells as a model to study the regulation of quiescence. When these cells are starved for nitrogen, the cell cycle is arrested in G1, and the cells enter quiescence (G0). A gene regulatory program is initiated, including downregulation of thousands of genes—for example, those related to cell proliferation—and upregulation of specific genes—for example, autophagy genes—needed to adapt to the physiological challenge. These changes in gene expression are accompanied by a marked alteration of nuclear organization and chromatin structure.ResultsHere, we investigated the role of Leo1, a subunit of the conserved RNA polymerase-associated factor 1 (Paf1) complex, in the quiescence process using fission yeast as the model organism. Heterochromatic regions became very dynamic in fission yeast in G0 during nitrogen starvation. The reduction of heterochromatin in early G0 was correlated with reduced target of rapamycin complex 2 (TORC2) signaling. We demonstrated that cells lacking Leo1 show reduced survival in G0. In these cells, heterochromatic regions, including subtelomeres, were stabilized, and the expression of many genes, including membrane transport genes, was abrogated. TOR inhibition mimics the effect of nitrogen starvation, leading to the expression of subtelomeric genes, and this effect was suppressed by genetic deletion of leo1.ConclusionsWe identified a protein, Leo1, necessary for survival during quiescence. Leo1 is part of a conserved protein complex, Paf1C, linked to RNA polymerase II. We showed that Leo1, acting downstream of TOR, is crucial for the dynamic reorganization of chromosomes and the regulation of gene expression during cellular quiescence. Genes encoding membrane transporters are not expressed in quiescent leo1 mutant cells, and cells die after 2 weeks of nitrogen starvation. Taken together, our results suggest that Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence.

Abstract 4438: Role of post translational modification of PAF1/PD2 in gemcitabine resistance of pancreatic cancer

Abstract Background: RNA polymerase associated factor 1 (PAF1)/Pancreatic differentiation 2 (PD2) is one of the core subunit of the human PAF1 complex (PAF1C), which regulates various cellular functions such as transcriptional elongation and histone modification. We have previously demonstrated its unique role in oncogenesis and stem cell maintenance. Studies have demonstrated that PAF1/PD2 gene yields a protein of 59.9 Kda (531 amino acids), however it has been found that it always gives a band at 80 Kda. Further, previous studies suggests that the 60 Kda protein represents the precursor, which rapidly process into an 80 Kda mature protein. SUMOylation is a process of reversible posttranslational modification that adds a small ubiquitin-related modifier (SUMO)-1 protein to the target protein. SUMOylation plays various molecular biology function such as transcriptional regulation, protein-protein interaction, and DNA damage repair and in cell cycle. DNA damage happen due to several physiological processes, but can also be caused by genotoxic agents. Promyelocytic Leukemia (PML) is protein that forms nuclear bodies and may be modified by SUMO1 and act as a DNA-damage sensor. Hypothesis: PAF1/PD2 is interacting with SUMO-1 and PML and, thus, plays an important function in providing gemcitabine resistance to pancreatic cancer cells Methods: SW1990, F9 (mouse embryonic cells) and CD18/HPAF cells were treated with 2-D08, a potent SUMOylation inhibitor for 24 hours and with siRNA against SUMO1 for 48 hours followed by protein isolation and western blotting. Immunoprecipitation and immunofluorescence were done to show the interaction between PAF1/PD2 and SUMO1 and with PML. To study the effect of PAF1/PD2 on gemcitabine resistance, SW1990 and Capan-1 cells were treated with different concentration of gemcitabine and then the expression of PAF1/PD2 along with SUMO1 was checked through immunoblotting and confocal imaging. Results: Results shows that inhibiting SUMOylation with both 2D08 and siRNA resulted in a decrease expression of PAF1/PD2 80 Kda protein. Immunoprecipitation and immunofluorescence analysis showed that endogenous PAF1/PD2 interacts with SUMO1. This finding was further verified using ectopically overexpressed Flag- tagged PAF1/PD2 and HA-tagged SUMO1, which showed a physical interaction between PAF1/PD2 and SUMO1. Interestingly, we observed that gemcitabine treatment significantly increased the SUMOylated status of PAF1/PD2 in pancreatic cancer cells. Further our results proved that SUMOylated PAF1/PD2 form nuclear bodies along with PML in pancreatic cancer cells. Conclusions: Our observation suggest that PAF1/PD2 undergoes SUMOylation and covalently interacts with SUMO1. Treatment with gemcitabine results in enhanced expression of PAF1/PD2 and increased co-localization with SUMO1 and PML, indicates a role of SUMOylated PAF1/PD2 in gemcitabine resistance. Citation Format: Sanchita Rauth, Saswati Karmakar, Ashu Shah, Rama Krishna Nimmakayala, Rakesh Bhatia, Sakthivel Muniyan, Sushil Kumar, Samikshan Dutta, Kaustubh Datta, Surinder K. Batra, Moorthy Palanimuthu Ponnusamy. Role of post translational modification of PAF1/PD2 in gemcitabine resistance of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4438.

Inheritance of a Phenotypically Neutral Epimutation Evokes Gene Silencing in Later Generations.

SummarySmall RNAs trigger the formation of epialleles that are silenced across generations. Consequently, RNA-directed epimutagenesis is associated with persistent gene repression. Here, we demonstrate that small interfering RNA-induced epimutations in fission yeast are still inherited even when the silenced gene is reactivated, and descendants can reinstate the silencing phenotype that only occurred in their ancestors. This process is mediated by the deposition of a phenotypically neutral molecular mark composed of tri-methylated histone H3 lysine 9 (H3K9me3). Its stable propagation is coupled to RNAi and requires maximal binding affinity of the Clr4/Suvar39 chromodomain to H3K9me3. In wild-type cells, this mark has no visible impact on transcription but causes gene silencing if RNA polymerase-associated factor 1 complex (Paf1C) activity is impaired. In sum, our results reveal a distinct form of epigenetic memory in which cells acquire heritable, transcriptionally active epialleles that confer gene silencing upon modulation of Paf1C.

The RNA Polymerase-Associated Factor 1 Complex Is Required for Plant Touch Responses.

Thigmomorphogenesis is a stereotypical developmental alteration in the plant body plan that can be induced by repeatedly touching plant organs. To unravel how plants sense and record multiple touch stimuli we performed a novel forward genetic screen based on the development of a shorter stem in response to repetitive touch. The touch insensitive (ths1) mutant identified in this screen is defective in some aspects of shoot and root thigmomorphogenesis. The ths1 mutant is an intermediate loss-of-function allele of VERNALIZATION INDEPENDENCE 3 (VIP3), a previously characterized gene whose product is part of the RNA polymerase II-associated factor 1 (Paf1) complex. The Paf1 complex is found in yeast, plants and animals, and has been implicated in histone modification and RNA processing. Several components of the Paf1 complex are required for reduced stem height in response to touch and normal root slanting and coiling responses. Global levels of histone H3K36 trimethylation are reduced in VIP3 mutants. In addition, THS1/VIP3 is required for wild type histone H3K36 trimethylation at the TOUCH3 (TCH3) and TOUCH4 (TCH4) loci and for rapid touch-induced upregulation of TCH3 and TCH4 transcripts. Thus, an evolutionarily conserved chromatin-modifying complex is required for both short- and long-term responses to mechanical stimulation, providing insight into how plants record mechanical signals for thigmomorphogenesis.

Cln 3-requiring 9 is a negative regulator of Th17 pathway-driven inflammation in anti-glomerular basement membrane glomerulonephritis.

T helper 17 (Th17) lymphocytes promote renal inflammation in anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), and signal transducer and activator of transcription 3 (STAT3) mediates activation of Th17 lymphocytes by IL-6 and transforming growth factor-β (TGF-β). Cln 3-requiring 9 (Ctr9), a subunit of RNA polymerase-associated factor complex (PAFc), regulates the transcription of IL-6/STAT3-dependent genes. Here, we investigated the role of Ctr9 in regulating Th17-driven inflammation in anti-GBM GN. In mice, STAT3β or IL-17 knockout ameliorated anti-GBM autoantibody-induced renal injury. This phenomenon was associated with decreases in retinoic acid receptor-related orphan receptor γt (RORγt), IL-17, phosphorylated STAT3, and proinflammatory cytokines. Compared with wild-type mice, Ctr9 increased in both STAT3β(-/-) and IL-17(-/-) mice injected with anti-GBM IgG, showing a negative correlation with Th17-related transcripts. Small interfering RNA (siRNA)-mediated knockdown of Ctr9 in intrarenal lymphocytes further upregulated Th17-related transcripts, consistent with repression of Th17 differentiation by Ctr9. Interestingly, Ctr9 was also expressed in human and mouse mesangial cells and downregulated in response to anti-GBM IgG or to TGF-β plus IL-17. Ctr9 in mesangial cells was even more repressed in the presence of both anti-GBM IgG and Th17-activating cytokines. Consistent with these findings, renal biopsies obtained from patients with anti-GBM GN showed consistent downregulation of Ctr9 and upregulation of phosphorylated STAT3 and IL-17 in the glomerulus. We conclude that Ctr9 is a negative regulator of Th17 differentiation in anti-GBM GN and repressed by anti-GBM IgG and IL-17 in mesangial cells.

Regulating infidelity: RNA-mediated recruitment of AID to DNA during class switch recombination.

The mechanism by which the DNA deaminase activation-induced cytidine deaminase (AID) is specifically recruited to repetitive switch region DNA during class switch recombination is still poorly understood. Work over the past decade has revealed a strong link between transcription and RNA polymerase-associated factors in AID recruitment, yet none of these processes satisfactorily explain how AID specificity is affected. Here, we review a recent finding wherein AID is guided to switch regions not by a protein factor but by an RNA moiety, and especially one associated with a noncoding RNA that has been long thought of as being inert. This work explains the long-standing requirement of splicing of noncoding transcripts during class switching, and has implications in both B cell-mediated immunity as well as the underlying pathological syndromes associated with the recombination reaction.

Structural and biochemical insights into the DNA-binding mode of MjSpt4p:Spt5 complex at the exit tunnel of RNAPII.

Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription. In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length MjSpt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region was found on the Spt4:Spt5 surface in both crystal forms, and identified as double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, the Spt4:Spt5-DNA binding model was built for the first time.

The Paf1 complex represses small-RNA-mediated epigenetic gene silencing.

RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA (dsRNA) to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the dsRNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically post-transcriptional gene silencing (PTGS) and chromatin-dependent gene silencing1. Although endogenous small RNAs play critical roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model where Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing (RITS) complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of bi-stable heterochromatin by trans-acting siRNAs, impairment of both transcription termination and nascent transcript release is imperative to confer stability to the repressed state. Our work uncovers a novel mechanism for small RNA- mediated epigenome regulation and highlights fundamental roles for Paf1C and the RNAi machinery in building epigenetic memory.

Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein

RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of σ-factors (σ70, σS, σ54, σ32, σE) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σE-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the σE-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency.

A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

Histone H3R17me2a mark recruits human RNA Polymerase-Associated Factor 1 Complex to activate transcription

The histone coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator for a number of transcription factors, including nuclear receptors. Although CARM1 and its asymmetrically deposited dimethylation at histone H3 arginine 17 (H3R17me2a) are associated with transcription activation, the mechanism by which CARM1 activates transcription remains unclear. Using an unbiased biochemical approach, we discovered that the transcription elongation-associated PAF1 complex (PAF1c) directly interacts with H3R17me2a. PAF1c binds to histone H3 tails harboring dimethylation at R17 in CARM1-methylated histone octamers. Knockdown of either PAF1c subunits or CARM1 affected transcription of CARM1-regulated, estrogen-responsive genes. Furthermore, either CARM1 knockdown or CARM1 enzyme-deficient mutant knockin resulted in decreased H3R17me2a accompanied by the reduction of PAF1c occupancy at the proximal promoter estrogen-responsive elements. In contrast, PAF1c knockdown elicited no effects on H3R17me2a but reduced the H3K4me3 level at estrogen-responsive elements. These observations suggest that, apart from PAF1c's established roles in directing histone modifications, PAF1c acts as an arginine methyl histone effector that is recruited to promoters and activates a subset of genes, including targets of estrogen signaling.

The RNA Polymerase-associated Factor 1 Complex (Paf1C) Directly Increases the Elongation Rate of RNA Polymerase I and Is Required for Efficient Regulation of rRNA Synthesis

The rate of ribosome synthesis is proportional to the rate of cell proliferation; thus, transcription of rRNA by RNA polymerase I (Pol I) is an important target for the regulation of this process. Most previous investigations into mechanisms that regulate the rate of ribosome synthesis have focused on the initiation step of transcription by Pol I; however, recent studies in yeast and mammals have identified factors that influence transcription elongation by Pol I. The RNA polymerase-associated factor 1 complex (Paf1C) is a transcription elongation factor with known roles in Pol II transcription. We previously identified a role for Paf1C in transcription elongation by Pol I. In this study, genetic interactions between genes for Paf1C and Pol I subunits confirm this conclusion. In vitro studies demonstrate that purified Paf1C directly increases the rate of transcription elongation by Pol I. Finally, we show that Paf1C function is required for efficient control of Pol I transcription in response to target of rapamycin (TOR) signaling or amino acid limitation. These studies demonstrate that Paf1C plays an important direct role in cellular control of rRNA expression.

Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5—the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

Cell cycle progression in G1 and S phases is CCR4 dependent following ionizing radiation or replication stress in Saccharomyces cerevisiae.

To identify new nonessential genes that affect genome integrity, we completed a screening for diploid mutant Saccharomyces cerevisiae strains that are sensitive to ionizing radiation (IR) and found 62 new genes that confer resistance. Along with those previously reported (Bennett et al., Nat. Genet. 29:426-434, 2001), these genes bring to 169 the total number of new IR resistance genes identified. Through the use of existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4-NOT and RNA polymerase-associated factor 1 (PAF1)-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G1 cell population is radiosensitive. Consistent with a role in G1, diploid ccr4Delta cells irradiated in G1 show enhanced lethality compared to cells exposed as a synchronous G2 population. In addition, a prolonged RAD9-dependent G1 arrest occurred following IR of ccr4Delta cells and CCR4 is a member of the RAD9 epistasis group, thus confirming a role for CCR4 in checkpoint control. Moreover, ccr4Delta cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G2 followed by cellular lysis. This S-phase replication defect is separate from that seen for rad52 mutants, since rad52Delta ccr4Delta cells show increased sensitivity to HU compared to rad52Delta or ccr4Delta mutants alone. These results indicate that cell cycle transition through G1 and S phases is CCR4 dependent following radiation or replication stress.

Paf1p, an RNA Polymerase II-Associated Factor inSaccharomyces cerevisiae, May Have both Positive and Negative Roles in Transcription

Regulated transcription initiation requires, in addition to RNA polymerase II and the general transcription factors, accessory factors termed mediators or adapters. We have used affinity chromatography to identify a collection of factors that associate with Saccharomyces cerevisiae RNA polymerase II (P. A. Wade, W. Werel, R. C. Fentzke, N. E. Thompson, J. F. Leykam, R. R. Burgess, J. A. Jaehning, and Z. F. Burton, submitted for publication). Here we report identification and characterization of a gene encoding one of these factors, PAF1 (for RNA polymerase-associated factor 1). PAF1 encodes a novel, highly charged protein of 445 amino acids. Disruption of PAF1 in S. cerevisiae leads to pleiotropic phenotypic traits, including slow growth, temperature sensitivity, and abnormal cell morphology. Consistent with a possible role in transcription, Paf1p is localized to the nucleus. By comparing the abundances of many yeast transcripts in isogenic wild-type and paf1 mutant strains, we have identified genes whose expression is affected by PAF1. In particular, disruption of PAF1 decreases the induction of the galactose-regulated genes three- to fivefold. In contrast, the transcript level of MAK16, an essential gene involved in cell cycle regulation, is greatly increased in the paf1 mutant strain. Paf1p may therefore be required for both positive and negative regulation of subsets of yeast genes. Like Paf1p, the GAL11 gene product is found associated with RNA polymerase II and is required for regulated expression of many yeast genes including those controlled by galactose. We have found that a gal11 paf1 double mutant has a much more severe growth defect than either of the single mutants, indicating that these two proteins may function in parallel pathways to communicate signals from regulatory factors to RNA polymerase II.