Joseph A. Martens

Intergenic transcription is required to repress the Saccharomyces cerevisiae SER3 gene

Transcription by RNA polymerase II in Saccharomyces cerevisiae and in humans is widespread, even in genomic regions that do not encode proteins. The purpose of such intergenic transcription is largely unknown, although it can be regulatory. We have discovered a role for one case of intergenic transcription by studying the S. cerevisiae SER3 gene. Our previous results demonstrated that transcription of SER3 is tightly repressed during growth in rich medium. We now show that the regulatory region of this gene is highly transcribed under these conditions and produces a non-protein-coding RNA (SRG1). Expression of the SRG1 RNA is required for repression of SER3. Additional experiments have demonstrated that repression occurs by a transcription-interference mechanism in which SRG1 transcription across the SER3 promoter interferes with the binding of activators. This work identifies a previously unknown class of transcriptional regulatory genes.

Recent advances in understanding chromatin remodeling by Swi/Snf complexes.

Members of the Swi/Snf family of chromatin-remodeling complexes play critical roles in transcriptional control. Recent studies have made significant advances in our understanding of the fundamental aspects of Swi/Snf complexes, including the roles of specific subunits, the repression of transcription, and the mechanism of remodeling. In addition, new findings also indicate an important role for the Swi/Snf-related complex, RSC, in controlling gene expression.

Intergenic transcription causes repression by directing nucleosome assembly

Transcription of non-protein-coding DNA (ncDNA) and its noncoding RNA (ncRNA) products are beginning to emerge as key regulators of gene expression. We previously identified a regulatory system in Saccharomyces cerevisiae whereby transcription of intergenic ncDNA (SRG1) represses transcription of an adjacent protein-coding gene (SER3) through transcription interference. We now provide evidence that SRG1 transcription causes repression of SER3 by directing a high level of nucleosomes over SRG1, which overlaps the SER3 promoter. Repression by SRG1 transcription is dependent on the Spt6 and Spt16 transcription elongation factors. Significantly, spt6 and spt16 mutations reduce nucleosome levels over the SER3 promoter without reducing intergenic SRG1 transcription, strongly suggesting that nucleosome levels, not transcription levels, cause SER3 repression. Finally, we show that spt6 and spt16 mutations allow transcription factor access to the SER3 promoter. Our results raise the possibility that transcription of ncDNA may contribute to nucleosome positioning on a genome-wide scale where, in some cases, it negatively impacts protein-DNA interactions.

Transcription regulation by the noncoding RNA SRG1 requires Spt2-dependent chromatin deposition in the wake of RNA polymerase II.

ABSTRACT Spt2 is a chromatin component with roles in transcription and posttranscriptional regulation. Recently, we found that Spt2 travels with RNA polymerase II (RNAP II), is involved in elongation, and plays important roles in chromatin modulations associated with this process. In this work, we dissect the function of Spt2 in the repression of SER3. This gene is repressed by a transcription interference mechanism involving the transcription of an adjacent intergenic region, SRG1, that leads to the production of a noncoding RNA (ncRNA). We find that Spt2 and Spt6 are required for the repression of SER3 by SRG1 transcription. Intriguingly, we demonstrate that these effects are not mediated through modulations of the SRG1 transcription rate. Instead, we show that the SRG1 region overlapping the SER3 promoter is occluded by randomly positioned nucleosomes that are deposited behind RNAP II transcribing SRG1 and that their deposition is dependent on the presence of Spt2. Our data indicate that Spt2 is required for the major chromatin deposition pathway that uses old histones to refold nucleosomes in the wake of RNAP II at the SRG1-SER3 locus. Altogether, these observations suggest a new mechanism of repression by ncRNA transcription involving a repressive nucleosomal structure produced by an Spt2-dependent pathway following RNAP II passage.

The Paf1 Complex Represses SER3 Transcription in Saccharomyces cerevisiae by Facilitating Intergenic Transcription-Dependent Nucleosome Occupancy of the SER3 Promoter

ABSTRACT Previous studies have shown that repression of the Saccharomyces cerevisiae SER3 gene is dependent on transcription of SRG1 from noncoding DNA initiating within the intergenic region 5′ of SER3 and extending across the SER3 promoter region. By a mechanism dependent on the activities of the Swi/Snf chromatin remodeling factor, the HMG-like factor Spt2, and the Spt6 and Spt16 histone chaperones, SRG1 transcription deposits nucleosomes over the SER3 promoter to prevent transcription factors from binding and activating SER3. In this study, we uncover a role for the Paf1 transcription elongation complex in SER3 repression. We find that SER3 repression is primarily dependent on the Paf1 and Ctr9 subunits of this complex, with minor contributions by the Rtf1, Cdc73, and Leo1 subunits. We show that the Paf1 complex localizes to the SRG1 transcribed region under conditions that repress SER3, consistent with it having a direct role in mediating SRG1 transcription-dependent SER3 repression. Importantly, we show that the defect in SER3 repression in strains lacking Paf1 subunits is not a result of reduced SRG1 transcription or reduced levels of known Paf1 complex-dependent histone modifications. Rather, we find that strains lacking subunits of the Paf1 complex exhibit reduced nucleosome occupancy and reduced recruitment of Spt16 and, to a lesser extent, Spt6 at the SER3 promoter. Taken together, our results suggest that Paf1 and Ctr9 repress SER3 by maintaining SRG1 transcription-dependent nucleosome occupancy.

Identification of Histone Mutants That Are Defective for Transcription-Coupled Nucleosome Occupancy

ABSTRACT Our previous studies of Saccharomyces cerevisiae described a gene repression mechanism where the transcription of intergenic noncoding DNA (ncDNA) (SRG1) assembles nucleosomes across the promoter of the adjacent SER3 gene that interfere with the binding of transcription factors. To investigate the role of histones in this mechanism, we screened a comprehensive library of histone H3 and H4 mutants for those that derepress SER3. We identified mutations altering eight histone residues (H3 residues V46, R49, V117, Q120, and K122 and H4 residues R36, I46, and S47) that strongly increase SER3 expression without reducing the transcription of the intergenic SRG1 ncDNA. We detected reduced nucleosome occupancy across SRG1 in these mutants to degrees that correlate well with the level of SER3 derepression. The histone chromatin immunoprecipitation experiments on several other genes suggest that the loss of nucleosomes in these mutants is specific to highly transcribed regions. Interestingly, two of these histone mutants, H3 R49A and H3 V46A, reduce Set2-dependent methylation of lysine 36 of histone H3 and allow transcription initiation from cryptic intragenic promoters. Taken together, our data identify a new class of histone mutants that is defective for transcription-dependent nucleosome occupancy.

Identification of Mutant Versions of the Spt16 Histone Chaperone That Are Defective for Transcription-Coupled Nucleosome Occupancy in Saccharomyces cerevisiae

The highly conserved FACT (Facilitates Chromatin Transactions) complex performs essential functions in eukaryotic cells through the reorganization of nucleosomes. During transcription, FACT reorganizes nucleosomes to allow passage of RNA Polymerase II and then assists in restoring these nucleosomes after RNA Polymerase II has passed. We have previously shown, consistent with this function, that Spt16 facilitates repression of the Saccharomyces cerevisiae SER3 gene by maintaining nucleosome occupancy over the promoter of this gene as a consequence of intergenic transcription of SRG1 noncoding DNA. In this study, we report the results of a genetic screen to identify mutations in SPT16 that derepress SER3. Twenty-five spt16 mutant alleles were found to derepress SER3 without causing significant reductions in either SRG1 RNA levels or Spt16 protein levels. Additional phenotypic assays indicate that these mutants have general transcription defects related to altered chromatin structure. Our analyses of a subset of these spt16 mutants reveal defects in SRG1 transcription-coupled nucleosome occupancy over the SER3 promoter. We provide evidence that these mutants broadly impair transcription-coupled nucleosome occupancy at highly transcribed genes but not at lowly transcribed genes. Finally, we show that one consequence shared by these mutations is the reduced binding of mutant Spt16 proteins across SRG1 and other highly transcribed genes. Taken together, our results highlight an important role for Spt16 in orchestrating transcription-coupled nucleosome assembly at highly transcribed regions of the genome, possibly by facilitating the association of Spt16 during this process.

ncRNA transcription makes its mark

EMBO J 28, 1697–1707 (2009); published online 17 June 2009 [PMC free article] [PubMed] A recently recognized strategy for gene regulation involves transcription of a non-coding RNA (ncRNA) transcript that overlaps the gene targeted for regulation. In many cases, it seems that it is the act of transcription itself rather than the ncRNA transcript that mediates regulation. A paper in this issue of the EMBO Journal shows one mechanism by which these transcription events regulate transcription; elongating RNA polymerases direct a set of regulatory histone modifications that modulate expression of an overlapping gene. A major surprise of the past few years has been the discovery of significant transcription activity across entire eukaryotic genomes, showing a large class of ncRNAs that are often rapidly degraded (Yazgan and Krebs, 2007). In a number of cases, these ncRNAs have been found to regulate gene expression. Most of these regulatory ncRNAs function through RNAi-mediated pathways of gene repression. However, some ncRNAs regulate gene expression in cis. In these cases, the act of transcription itself, rather than the RNA product of transcription, mediates regulation of an overlapping gene. The proposed mechanisms for regulation in cis include promoter occlusion or transcriptional interference by RNA polymerases transcribing ncRNAs (Yazgan and Krebs, 2007). Other genes show regulated transcription start-site choice from a single promoter, giving rise to either a coding transcript or an ncRNA (Jenks et al, 2008; Kuehner and Brow, 2008). A cryptic promoter that lies at the 3′ end of the PHO5 gene and drives an antisense transcript is required for the normal kinetics of PHO5 activation (Uhler et al, 2007). Transcription of a series of ncRNAs upstream of the Schizosaccharomyces pombe fbp1+ promoter is required for its induction when cells are shifted to inducing conditions (Hirota et al, 2008). Passage of RNA polymerase II through the fbp1+ promoter during transcription of these ncRNAs promotes the formation of open chromatin, allowing the transcription factor access to the fbp1+ promoter during induction. At present, reports by Houseley et al (2008) and by Pinskaya et al (2009) provide compelling evidence that transcription of ncRNAs influences post-translational modifications of histones that facilitate the repression of overlapping genes. Chromatin immunoprecipitation (ChIP) experiments carried out by Houseley et al showed a surprising pattern of Set1-dependent histone H3K4 trimethylation across the well-characterized GAL1–10 gene locus (Figure 1). A significant peak of this histone methylation mark, normally associated with the 5′ end of transcribed genes, was found within the 3′ end of GAL10 when cells were grown in glucose medium (GAL1-10 repressing conditions). These observations led Houseley et al to identify and characterize a set of ncRNAs that are transcribed from the 3′ end of GAL10 across the promoter region shared by the divergent GAL1 and GAL10 genes, which they named GAL10 ncRNAs. Figure 1 Model for ncRNA-based regulation of GAL1–GAL10 expression. Cells grown in glucose (repressing conditions) transcribe an ncRNA, GAL1ucut, from the 3′ end of GAL10. This directs H3K4 methylation at the 5′ end of the GAL1ucut and ... Pinskaya et al observed that cells lacking Set1 induced GAL1–10 expression more rapidly than wild-type cells when cells were shifted to galactose medium, although the final, fully induced levels of GAL mRNA were unchanged. The increased expression of GAL1–10 in set1 cells correlated with TBP occupancy at the GAL1–10 promoter, suggesting that Set1 regulates transcription initiation at GAL1–10. Furthermore, an H3K4A mutant showed a similar induction phenotype, indicating a role for H3K4 methylation in GAL1–10 induction. Subsequent experiments identified a set of ncRNA transcripts similar to those reported by Houseley et al, which they named GAL1ucut (GAL1 upstream cryptic unstable transcripts). Both groups mapped the GAL1ucut promoter to a location in the 3′ end of GAL10 near a pair of binding sites for the Reb1 transcription factor. Mutation of REB1, or of the Reb1 sites in GAL10, abolished GAL1ucut expression. Furthermore, both groups found an inverse relationship between GAL1ucut and GAL1–10 expression. GAL1ucut is expressed under conditions that repress GAL1–10, and as GAL1–10 is induced GAL1ucut declines. Curiously, Houseley et al did not observe an effect of GAL1ucut on GAL1–10 expression when cells were shifted to a medium with high levels of galactose. Rather, they observed that GAL1ucut antagonized the induction kinetics and final levels of GAL1–10 in a medium with low levels of both glucose and galactose. The basis for the difference in observations between the groups is not obvious, but both agree that GAL1ucut is used to attenuate GAL1–10 expression. Both groups argue that GAL1ucut acts in cis. First, Houseley et al formed a heterozygous diploid yeast strain in which one of the two GAL1–10 loci lacked the GAL1ucut promoter. They observed no attenuation of GAL1–10 expression in this strain. Second, both groups found that GAL1ucut RNA was stabilized by mutations affecting RNA degradation pathways used to target ncRNA, and Pinskaya et al showed that this stabilization had no effect on GAL1–10 induction. Earlier work has shown that Rpd3S histone-deacetylase complex is recruited to the body of protein-coding genes by H3K36-methylated nucleosomes (Lee and Shilatifard, 2007). This serves to inhibit intragenic transcription from cryptic promoters that might otherwise be activated by the passage of transcription elongation complexes. Houseley et al observed that histone modifications, which are the hallmarks of this Rpd3S-mediated intragenic repression mechanism, methylation of histone H3K36 and subsequent histone deacetylation, were found across the repressed GAL1–10 locus. Furthermore, these marks were dependent on GAL1ucut transcription, and deletion of the Eaf3 subunit of the Rpd3S complex relieved glucose repression to a level similar to that observed when the GAL1ucut promoter was deleted. Pinskaya et al also found a role for Rpd3S in GAL1ucut function. As H3K4-methylated histones can be recognized by proteins with the PHD domain (Mellor, 2006), Pinskaya et al systematically tested yeast strains lacking different PHD proteins for an effect on GAL1–10 induction. They found that loss of Rco1, a component of the Rpd3S complex, mimicked the effects of set1 mutations on GAL1–10 expression. In addition, they used ChIP to show that Rpd3 is recruited to the repressed GAL1–10 locus and that this is abolished by H3K4A and set1 mutations. Interestingly, they did not observe any effect of a mutation deleting SET2, which encodes the H3K36 methyltransferase (Lee and Shilatifard, 2007), on GAL1–10 induction kinetics, suggesting that the effects of GAL1ucut transcription might be mediated primarily through H3K4 methylation. Although the different observations regarding the effects of GAL1ucut on induction kinetics and expression in low levels of glucose still need to be resolved, these papers indicate that cryptic transcription events might be used to set the chromatin-modification state of overlapping sequences. This regulatory strategy might be used more widely; both groups present preliminary observations, suggesting that ncRNA might regulate expression of other yeast genes. Furthermore, in higher eukaryotes, ncRNA are implicated in genomic imprinting (Edwards and Ferguson-Smith, 2007) and the function of some enhancers (Drewell et al, 2002). Perhaps these transcription events serve to establish epigenetic marks that influence the function of the overlapping regulatory elements.

Transcription of ncDNA: Many roads lead to local gene regulation

Transcription of ncDNA occurs throughout eukaryotic genomes, generating a wide array of ncRNAs. One large class of ncRNAs include those transcribed over the promoter regions of nearby protein coding genes. Recent studies primarily focusing on individual genes have uncovered multiple mechanisms by which promoter-associated transcriptional activity locally alters gene expression.

Transcription of intergenic DNA deposits nucleosomes on promoter to silence gene expression.

Comment on: Hainer SJ, et al. Genes Dev. 2011; 25:29-40