An Investigation Into The Role Of Cfia 3\u27 End Processing Complex In The Termination And Initiation/reinitiation Of Transcription

In budding yeast, as in higher eukaryotes, transcription of protein coding genes is executed by a highly specialized, conserved polymerase called RNA polymerase II (RNAPII). The transcription cycle of RNAPII has four major steps: initiation, elongation, termination, and reinitiation. The successful accomplishment of each of these steps requires a number of accessory factors. Many of these factors operate at multiple steps in the transcription cycle. The major focus of this study was to examine the function of Clp1, which is an RNA processing factor operating at the 3′ end of genes, in the transcription cycle. Clp1 is one of the four subunits of the CFIA 3′ end processing complex. It is the least investigated CFIA subunit. The role of the other three subunits of the CFIA complex in 3\u27 end processing and termination of transcription is well documented. Here we investigate the role of Clp1 in the initiation as well as the termination of transcription. We used a temperature-sensitive mutant of Clp1 to assess its function. We demonstrated a direct role for this factor in the termination of transcription of CHA1. We used three different approaches; TRO assay, RNAPII-ChIP assay, and strand specific RT-PCR, to demonstrate the termination function of Clp1. In addition, we showed that Clp1 is also involved in the early steps of the transcription. Our results strongly suggest that Clp1 participates in promoter-associated transcription. We provide multiple lines of evidence in support of a role for Clp1 at the 5′ end of genes. First, the presence of Clp1 in the vicinity of the promoter region implies its involvement early in the transcription cycle. Second, the decrease in RNAPII density near the promoter without a parallel decrease in the level of the GTFs suggested a role for Clp1 in reinitiation of transcription. Third, an increase in 5\u27 initiated antisense divergent transcripts in the Clp1 mutant supports a role for the factor in providing directionality to the promoter-bound polymerase. To assess the generality of the observed functions of Clp1, we investigated the role of Clp1 in the transcription cycle on a genomewide scale using GRO-Seq approach. Our results show that the number of transcriptionally active genes decreased by at least two-fold in the clp1mutant. The GRO-Seq results strongly suggest a genomewide function for Clp1 in the termination of transcription, and indicate that Clp1 is required for the pausing of RNAPII that is a pre-requisite for the termination of transcription. We also observed a dramatic increase in 3\u27 initiated antisense transcription in the absence of a functional Clp1 protein. Using the chromosome conformation capture approach, CCC, we observed a role for Clp1 in gene loop formation. We found a strong correlation between the Clp1 function in gene looping, and its role in promoter-associated transcription which implies gene looping as the means through which this factor is exerting its functions at the 5′ end of genes

A Role for CF1A 3′ End Processing Complex in Promoter-Associated Transcription

<div><p>The Cleavage Factor 1A (CF1A) complex, which is required for the termination of transcription in budding yeast, occupies the 3′ end of transcriptionally active genes. We recently demonstrated that CF1A subunits also crosslink to the 5′ end of genes during transcription. The presence of CF1A complex at the promoter suggested its possible involvement in the initiation/reinitiation of transcription. To check this possibility, we performed transcription run-on assay, RNAP II-density ChIP and strand-specific RT-PCR analysis in a mutant of CF1A subunit Clp1. As expected, RNAP II read through the termination signal in the temperature-sensitive mutant of <i>clp1</i> at elevated temperature. The transcription readthrough phenotype was accompanied by a decrease in the density of RNAP II in the vicinity of the promoter region. With the exception of TFIIB and TFIIF, the recruitment of the general transcription factors onto the promoter, however, remained unaffected in the <i>clp1</i> mutant. These results suggest that the CF1A complex affects the recruitment of RNAP II onto the promoter for reinitiation of transcription. Simultaneously, an increase in synthesis of promoter-initiated divergent antisense transcript was observed in the <i>clp1</i> mutant, thereby implicating CF1A complex in providing directionality to the promoter-bound polymerase. Chromosome Conformation Capture (3C) analysis revealed a physical interaction of the promoter and terminator regions of a gene in the presence of a functional CF1A complex. Gene looping was completely abolished in the <i>clp1</i> mutant. On the basis of these results, we propose that the CF1A-dependent recruitment of RNAP II onto the promoter for reinitiation and the regulation of directionality of promoter-associated transcription are accomplished through gene looping.</p></div

The promoter occupancy of the general transcription factors is not appreciably affected in the <i>clp1^ts</i> mutant.

<p>(A, C) Schematic depictions of <i>INO1</i> and <i>CHA1</i> indicating the position of ChIP primer pairs. (B, D) Quantification of ChIP analysis showing crosslinking of the general transcription factors TFIID, TFIIB, TFIIF, TFIIE and TFIIH to different regions of <i>INO1</i> and <i>CHA1</i> in the <i>clp1</i> mutant at the permissive (25°C, black bars) and non-permissive (37°C, grey bars) temperatures. The results shown are an average of at least six independent PCRs from four separate immunoprecipitates from two independently grown cultures. Error bars indicate one unit of standard deviation.</p

TRO analysis showing transcription readthrough and alteration in promoter-associated transcription in the <i>clp1^ts</i> mutant.

<p>(A) Schematic depiction of the <i>CHA1</i> gene indicating the positions of the probes (A–I) used in the TRO assay. (B) TRO analysis of <i>CHA1</i> in the wild type strain at 25°C and 37°C under induced transcription of the gene. (C) TRO analysis of <i>CHA1</i> in the temperature-sensitive mutant of Clp1 at the permissive (25°C) and non-permissive (37°C) temperatures. (D) and (E) Quantification of the data shown in B and C respectively. Error bars indicate one unit of standard deviation. The results shown are an average of at least four independent replicates. ST = Serine/Threonine, WT = Wild type, TRO = Transcription run-on.</p

RNAP II density in the promoter region exhibits a decline in the <i>clp1^ts</i> mutant.

<p>(A, D) Schematic depictions of <i>INO1</i> and <i>CHA1</i> showing the positions of ChIP primer pairs. (B, E) ChIP analysis showing polymerase density in different regions of <i>INO1</i> and <i>CHA1</i> in the <i>clp1</i> mutant at the permissive (25°C) and non-permissive (37°C) temperatures. (C and F) Quantification of data shown in B and E respectively. The input signals represent DNA prior to immunoprecipitation. The results shown are an average of at least eight independent PCRs from four separate immunoprecipitates from two independently grown cultures. Error bars indicate one unit of standard deviation. IP = immunoprecipitate.</p

Transcript levels of <i>INO1</i> and <i>CHA1</i> are adversely affected in the <i>clp1^ts</i> mutant.

<p>(A, D) Schematic representations of <i>INO1</i> and <i>CHA1</i> showing the positions of the primer pairs used in RT-PCR analysis. (B) and (E) RT-PCR analysis of <i>INO1</i> and <i>CHA1</i> in <i>clp1^ts</i> and isogenic wild type strains during induced and non-induced transcription at 25°C and 37°C. (C) and (F) Quantification of the data shown in B and E respectively. The transcript level of 18S was used as the control. The results shown are an average of four PCRs from two cDNA preparations from two different RNA extractions. Error bars indicate one unit of standard deviation. AS = Ammonium sulfate, ST = Serine/Threonine, WT = Wild type.</p