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Compromised RNA polymerase III complex assembly leads to local alterations of intergenic RNA polymerase II transcription in Saccharomyces cerevisiae.

Wang Q, Nowak CM, Korde A, Oh DH, Dassanayake M, Donze D - BMC Biol. (2014)

Bottom Line: Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast.The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Assembled RNA polymerase III (Pol III) complexes exert local effects on chromatin processes, including influencing transcription of neighboring RNA polymerase II (Pol II) transcribed genes. These properties have been designated as 'extra-transcriptional' effects of the Pol III complex. Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.

Results: Given our recent results demonstrating that tDNAs block progression of intergenic Pol II transcription, we hypothesized that extra-transcriptional effects within intergenic regions were not identified in the microarray study. To reconsider global impacts of Pol III complex binding, we used RNA sequencing to compare transcriptomes of wild type versus Pol III transcription factor TFIIIC depleted mutants. The results reveal altered intergenic Pol II transcription near TFIIIC binding sites in the mutant strains, where we observe readthrough of upstream transcripts that normally terminate near these sites, 5'- and 3'-extended transcripts, and de-repression of adjacent genes and intergenic regions.

Conclusions: The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast. The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

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Model for the appearance of 5′-extended and de-repressed Pol II transcripts in Pol III complex mutants. In wild type cells, the bidirectional activity of transcription factor binding sites at some promoters is inhibited by the presence of a nearby fully or partially assembled Pol III complex (for example, ETC site). Compromised Pol III complex formation allows Pol II transcription factors to bidirectionally load Pol II preinitiation complexes (PIC). These events lead to the creation of aberrant transcription start sites (TSS), resulting in the extension of the 5′-UTR of the divergent gene. Upstream initiating Pol II may also inhibit normal PIC formation by transcriptional interference. This scenario may also be involved in the de-repression of SPO74 (and other intergenic regions adjacent to tDNAs) observed when Pol III assembly at an upstream tDNA is compromised. In this case the aberrant transcript reads through chromatin-bound factors responsible for repression of SPO74 in haploid cells. Pol II, polymerase II; Pol III, polymerase III.
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Fig8: Model for the appearance of 5′-extended and de-repressed Pol II transcripts in Pol III complex mutants. In wild type cells, the bidirectional activity of transcription factor binding sites at some promoters is inhibited by the presence of a nearby fully or partially assembled Pol III complex (for example, ETC site). Compromised Pol III complex formation allows Pol II transcription factors to bidirectionally load Pol II preinitiation complexes (PIC). These events lead to the creation of aberrant transcription start sites (TSS), resulting in the extension of the 5′-UTR of the divergent gene. Upstream initiating Pol II may also inhibit normal PIC formation by transcriptional interference. This scenario may also be involved in the de-repression of SPO74 (and other intergenic regions adjacent to tDNAs) observed when Pol III assembly at an upstream tDNA is compromised. In this case the aberrant transcript reads through chromatin-bound factors responsible for repression of SPO74 in haploid cells. Pol II, polymerase II; Pol III, polymerase III.

Mentions: Manual inspection and computational analysis of our RNA-Seq data at these loci reveal that the extended 5′-ends observed in tfc6 mutants may normally be constrained by assembled Pol III complexes that prevent an upstream promoter from acting in a bidirectional manner. With the advent of tiling array and RNA-Seq technologies, the presence of pervasive and intergenic transcription in both prokaryotes and eukaryotes has been revealed. A significant fraction of such transcripts appears to arise from intrinsic bidirectional preinitiation complex formation directed by transcription factors bound upstream of active Pol II promoters [34,44]. Of the list of 5′-UTR extended loci in tfc6 mutants given in Additional file 1: Table S6, the origin of many of the extensions is consistent with a model shown in Figure 8, which is based on the bidirectional model proposed by the Steinmetz lab. In these instances, it appears that one of a divergently transcribed gene pair is expressed at a high level, but in wild type cells, intrinsic bidirectional initiation is inhibited by the presence of adjacent chromatin bound Pol III complexes (Figure 8, upper panel). When Pol III complex formation is compromised, the bidirectional capacity of DNA bound transcription factors is enabled, allowing formation of a new intergenic Pol II TSS. Progression of Pol II from the new upstream TSS then leads to transcriptional interference of the normal divergent promoter, resulting in the extended 5′-UTRs (Figure 8, lower panel). Given that Pol III complexes exhibit both chromatin insulator and heterochromatin barriers [5], this bidirectional blocking activity could be considered as another type of ‘boundary’ element. Boundaries are defined as sequences that prevent regulatory elements from inappropriately affecting adjacent chromosomal regions; therefore, the blocking of bidirectional transcription by Pol III complexes can be viewed as insulating the divergent gene from the interfering effects of cryptic bidirectional transcription.Figure 8


Compromised RNA polymerase III complex assembly leads to local alterations of intergenic RNA polymerase II transcription in Saccharomyces cerevisiae.

Wang Q, Nowak CM, Korde A, Oh DH, Dassanayake M, Donze D - BMC Biol. (2014)

Model for the appearance of 5′-extended and de-repressed Pol II transcripts in Pol III complex mutants. In wild type cells, the bidirectional activity of transcription factor binding sites at some promoters is inhibited by the presence of a nearby fully or partially assembled Pol III complex (for example, ETC site). Compromised Pol III complex formation allows Pol II transcription factors to bidirectionally load Pol II preinitiation complexes (PIC). These events lead to the creation of aberrant transcription start sites (TSS), resulting in the extension of the 5′-UTR of the divergent gene. Upstream initiating Pol II may also inhibit normal PIC formation by transcriptional interference. This scenario may also be involved in the de-repression of SPO74 (and other intergenic regions adjacent to tDNAs) observed when Pol III assembly at an upstream tDNA is compromised. In this case the aberrant transcript reads through chromatin-bound factors responsible for repression of SPO74 in haploid cells. Pol II, polymerase II; Pol III, polymerase III.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4228148&req=5

Fig8: Model for the appearance of 5′-extended and de-repressed Pol II transcripts in Pol III complex mutants. In wild type cells, the bidirectional activity of transcription factor binding sites at some promoters is inhibited by the presence of a nearby fully or partially assembled Pol III complex (for example, ETC site). Compromised Pol III complex formation allows Pol II transcription factors to bidirectionally load Pol II preinitiation complexes (PIC). These events lead to the creation of aberrant transcription start sites (TSS), resulting in the extension of the 5′-UTR of the divergent gene. Upstream initiating Pol II may also inhibit normal PIC formation by transcriptional interference. This scenario may also be involved in the de-repression of SPO74 (and other intergenic regions adjacent to tDNAs) observed when Pol III assembly at an upstream tDNA is compromised. In this case the aberrant transcript reads through chromatin-bound factors responsible for repression of SPO74 in haploid cells. Pol II, polymerase II; Pol III, polymerase III.
Mentions: Manual inspection and computational analysis of our RNA-Seq data at these loci reveal that the extended 5′-ends observed in tfc6 mutants may normally be constrained by assembled Pol III complexes that prevent an upstream promoter from acting in a bidirectional manner. With the advent of tiling array and RNA-Seq technologies, the presence of pervasive and intergenic transcription in both prokaryotes and eukaryotes has been revealed. A significant fraction of such transcripts appears to arise from intrinsic bidirectional preinitiation complex formation directed by transcription factors bound upstream of active Pol II promoters [34,44]. Of the list of 5′-UTR extended loci in tfc6 mutants given in Additional file 1: Table S6, the origin of many of the extensions is consistent with a model shown in Figure 8, which is based on the bidirectional model proposed by the Steinmetz lab. In these instances, it appears that one of a divergently transcribed gene pair is expressed at a high level, but in wild type cells, intrinsic bidirectional initiation is inhibited by the presence of adjacent chromatin bound Pol III complexes (Figure 8, upper panel). When Pol III complex formation is compromised, the bidirectional capacity of DNA bound transcription factors is enabled, allowing formation of a new intergenic Pol II TSS. Progression of Pol II from the new upstream TSS then leads to transcriptional interference of the normal divergent promoter, resulting in the extended 5′-UTRs (Figure 8, lower panel). Given that Pol III complexes exhibit both chromatin insulator and heterochromatin barriers [5], this bidirectional blocking activity could be considered as another type of ‘boundary’ element. Boundaries are defined as sequences that prevent regulatory elements from inappropriately affecting adjacent chromosomal regions; therefore, the blocking of bidirectional transcription by Pol III complexes can be viewed as insulating the divergent gene from the interfering effects of cryptic bidirectional transcription.Figure 8

Bottom Line: Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast.The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Assembled RNA polymerase III (Pol III) complexes exert local effects on chromatin processes, including influencing transcription of neighboring RNA polymerase II (Pol II) transcribed genes. These properties have been designated as 'extra-transcriptional' effects of the Pol III complex. Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.

Results: Given our recent results demonstrating that tDNAs block progression of intergenic Pol II transcription, we hypothesized that extra-transcriptional effects within intergenic regions were not identified in the microarray study. To reconsider global impacts of Pol III complex binding, we used RNA sequencing to compare transcriptomes of wild type versus Pol III transcription factor TFIIIC depleted mutants. The results reveal altered intergenic Pol II transcription near TFIIIC binding sites in the mutant strains, where we observe readthrough of upstream transcripts that normally terminate near these sites, 5'- and 3'-extended transcripts, and de-repression of adjacent genes and intergenic regions.

Conclusions: The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast. The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

Show MeSH
Related in: MedlinePlus