Karen M. Arndt

Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes.

Transcription in eukaryotes is influenced by the chromatin state of the template, and chromatin remodeling factors have well‐documented roles in regulating transcription initiation by RNA polymerase (pol) II. Chromatin also influences transcription elongation; however, little is known about the role of chromatin remodeling factors in this process. Here, we present evidence that the Saccharomyces cerevisiae chromatin remodeling factor Chd1 functions during transcription elongation. First, we identified Chd1 in a two‐hybrid screen for proteins that interact with Rtf1, a member of the Paf1 complex that associates with RNA pol II and regulates transcription elongation. Secondly, we show through co‐immunoprecipitation studies that Chd1 also interacts with components of two essential elongation factors, Spt4–Spt5 and Spt16–Pob3. Thirdly, we demonstrate that deletion of CHD1 suppresses a cold‐sensitive spt5 mutation that is also suppressed by defects in the Paf1 complex and RNA pol II. Finally, we demonstrate that Chd1, Rtf1 and Spt5 associate with actively transcribed regions of chromatin. Collectively, these findings suggest an important role for Chd1 and chromatin remodeling in the control of transcription elongation.

The Paf1 complex physically and functionally associates with transcription elongation factors in vivo.

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.

Regulation of histone modification and cryptic transcription by the Bur1 and Paf1 complexes

The Bur1–Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1–Bur2 and the Paf1 complex. We find that histone H3 K36 tri‐methylation requires specific components of the Paf1 complex and that K36 tri‐methylation is more strongly affected at the 5′ ends of genes in paf1Δ and bur2Δ strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5′ increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation‐driven deacetylation that occurs at the 3′ ends of genes. Finally, Bur1–Bur2 and the Paf1 complex have a second methylation‐independent function, since bur2Δ set2Δ and paf1Δ set2Δ double mutants display enhanced histone acetylation at the 3′ ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1–Bur2 complexes, provide evidence that histone modifications at the 5′ and 3′ ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1–Bur2 and Paf1 complexes repress cryptic transcription through a Set2‐independent pathway.

Access Denied: Snf1 Activation Loop Phosphorylation Is Controlled by Availability of the Phosphorylated Threonine 210 to the PP1 Phosphatase

Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose-regulated, since immune complex kinase assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon the addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7-Reg1 phosphatase may not itself be directly regulated by glucose, since the Glc7-Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7.

The many roles of the conserved eukaryotic Paf1 complex in regulating transcription, histone modifications, and disease states ☆

The Paf1 complex was originally identified over fifteen years ago in budding yeast through its physical association with RNA polymerase II. The Paf1 complex is now known to be conserved throughout eukaryotes and is well studied for promoting RNA polymerase II transcription elongation and transcription-coupled histone modifications. Through these critical regulatory functions, the Paf1 complex participates in numerous cellular processes such as gene expression and silencing, RNA maturation, DNA repair, cell cycle progression and prevention of disease states in higher eukaryotes. In this review, we describe the historic and current research involving the eukaryotic Paf1 complex to explain the cellular roles that underlie its conservation and functional importance. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

TBP mutants defective in activated transcription in vivo.

The TATA box binding protein (TBP) plays a central and essential role in transcription initiation. At TATA box‐containing genes transcribed by RNA polymerase II, TBP binds to the promoter and initiates the assembly of a multiprotein preinitiation complex. Several studies have suggested that binding of TBP to the TATA box is an important regulatory step in transcription initiation in vitro. To determine whether TBP is a target of regulatory factors in vivo, we performed a genetic screen in yeast for TBP mutants defective in activated transcription. One class of TBP mutants identified in this screen comprises inositol auxotrophs that are also defective in using galactose as a carbon source. These phenotypes are due to promoter‐specific defects in transcription initiation that are governed by the upstream activating sequence (UAS) and apparently not by the sequence of the TATA element. The finding that these TBP mutants are severely impaired in DNA binding in vitro suggests that transcription initiation at certain genes is regulated at the level of TATA box binding by TBP in vivo.

Inhibition of Acetyl Coenzyme A Carboxylase Activity Restores Expression of the INO1 Gene in a snf1 Mutant Strain of Saccharomyces cerevisiae

ABSTRACT Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy ofsnf1Δ strains. We identified mutations inACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the β subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show thatsnf1Δ strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying theACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression ofACC1, suppresses the inositol auxotrophy ofsnf1Δ strains. Together, these findings indicate that the inositol auxotrophy of snf1Δ strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important forINO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

Rtf1 is a multifunctional component of the Paf1 complex that regulates gene expression by directing cotranscriptional histone modification.

ABSTRACT Numerous transcription accessory proteins cause alterations in chromatin structure that promote the progression of RNA polymerase II (Pol II) along open reading frames (ORFs). The Saccharomyces cerevisiae Paf1 complex colocalizes with actively transcribing Pol II and orchestrates modifications to the chromatin template during transcription elongation. To better understand the function of the Rtf1 subunit of the Paf1 complex, we created a series of sequential deletions along the length of the protein. Genetic and biochemical assays were performed on these mutants to identify residues required for the various activities of Rtf1. Our results establish that discrete nonoverlapping segments of Rtf1 are necessary for interaction with the ATP-dependent chromatin-remodeling protein Chd1, promoting covalent modification of histones H2B and H3, recruitment to active ORFs, and association with other Paf1 complex subunits. We observed transcription-related defects when regions of Rtf1 that mediate histone modification or association with active genes were deleted, but disruption of the physical association between Rtf1 and other Paf1 complex subunits caused only subtle mutant phenotypes. Together, our results indicate that Rtf1 influences transcription and chromatin structure through several independent functional domains and that Rtf1 may function independently of its association with other members of the Paf1 complex.

Identification of RTF1, a novel gene important for TATA site selection by TATA box-binding protein in Saccharomyces cerevisiae.

Interaction of the TATA box-binding protein (TBP) with promoters of RNA polymerase II-transcribed genes is an early and essential step in mRNA synthesis. Previous studies have demonstrated that the rate-limiting binding of TBP to a TATA element can be influenced by transcriptional regulatory proteins. To identify additional factors that may regulate DNA binding by TBP in vivo, we performed a genetic selection for extragenic suppressors of a yeast TBP mutant that exhibits altered and relaxed DNA binding specificity. This analysis has led to the discovery of a previously unidentified gene, RTF1. The original rtf1 suppressor mutation, which encodes a single amino acid change in Rtf1, and an rtf1 null allele suppress the effects of the TBP specificity mutant by altering transcription initiation. Differences in the patterns of transcription initiation in these strains strongly suggest that the rtf1 missense mutation is distinct from a simple loss-of-function allele. The results of genetic crosses indicate that suppression of TBP mutants by mutations in RTF1 occurs in an allele-specific fashion. In a strain containing wild-type TBP, the rtf1 null mutation suppresses the transcriptional effects of a Ty delta insertion mutation in the promoter of the HIS4 gene, a phenotype also conferred by the TBP altered-specificity mutant. Finally, as shown by indirect immunofluorescence experiments, Rtf1 is a nuclear protein. Taken together, our findings suggest that Rtf1 either directly or indirectly regulates the DNA binding properties of TBP and, consequently, the relative activities of different TATA elements in vivo.

Structural basis for Spt5-mediated recruitment of the Paf1 complex to chromatin

Significance The polymerase associated factor 1 complex (Paf1C) is an RNA polymerase (pol) II accessory factor that broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription. This study shows how phosphorylation of a repeated motif within an additional factor, Spt5, is recognized and used by the Plus3 domain within the Paf1C subunit Rtf1 (restores TBP function 1) to promote recruitment of Paf1C to the transcription machinery. Deletions of both the Rtf1 Plus3 domain and the C domain of Cdc73 (Cell Division Cycle 73) are required to abolish Paf1C-mediated histone modifications and chromatin occupancy suggesting that dual attachment points facilitate the association of Paf1C with RNA pol II. Polymerase associated factor 1 complex (Paf1C) broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription elongation. The Plus3 domain of the Rtf1 subunit mediates Paf1C recruitment to genes by binding a repeating domain within the elongation factor Spt5 (suppressor of Ty). Here we provide a molecular description of this interaction by reporting the structure of human Rtf1 Plus3 in complex with a phosphorylated Spt5 repeat. We find that Spt5 binding is mediated by an extended surface containing phosphothreonine recognition and hydrophobic interfaces that interact with residues outside the Spt5 motif. Changes within these interfaces diminish binding of Spt5 in vitro and chromatin localization of Rtf1 in vivo. The structure reveals the basis for recognition of the repeat motif of Spt5, a key player in the recruitment of gene regulatory factors to RNA polymerase II.