Positioned Nucleosomes Due to Sequential Remodeling of the Yeast U6 Small Nuclear RNA Chromatin Are Essential for Its Transcriptional Activation

Abstract

Transcription from the yeast SNR6 (U6 small nuclear RNA) chromatin, a gene transcribed by the enzyme RNA polymerase III, depends on its transcription factor IIIC (TFIIIC) and the promoter elements (the intragenic box A and box B located downstream to its terminator) to which TFIIIC binds. The genes transcribed by polymerase III generally lack the upstream promoter elements where TFIIIC is known to recruit the transcription initiation factor TFIIIB. The TFIIIC-dependent chromatin remodeling of the gene in vitro that involves translational positioning of a nucleosome between boxes A and B is found to be essential for its transcriptional activation. We show here that the role of TFIIIC is not limited to the recruitment of TFIIIB on chromatin templates. The pre-binding of TFIIIB to the SNR6 TATA box in the upstream gene region does not alleviate TFIIIC requirement for transcriptional activation of the chromatin. Binding of TFIIIC to an array of pre-positioned nucleosomes results in an upward shift of the single nucleosome between boxes A and B. The ϳ40-bp shift of this nucleosome in the 3 to 5 direction leads to increased nuclease sensitivity of the ϳ40-bp DNA 3 to the upstream TATA box. Further chromatin remodeling accompanies the binding of TFIIIB in the next step. This two-step remodeling mechanism using the basal factors of the gene yields high transcription levels and generates a chromatin structure similar to that reported for the gene in vivo.Organization of the eukaryotic genome into chromatin links the process of transcription intimately with the structure of its template. Gene expression in eukaryotes is regulated by several mechanisms that alter the chromatin structure either to allow or disallow an activity using it as the substrate or template. These mechanisms include the covalent histone modifications, exchange with histone variants, and ATP-dependent chromatin remodeling, demonstrated in a large number of studies with the genes transcribed by the enzyme RNA polymerase II (pol II) 3 (1-4). Fewer examples of chromatin transcription by the RNA pol III are available that may have a different relationship with its template structure due to the short gene units and characteristically intragenic promoter elements.Pol III transcribes genes encoding structural RNAs required for translation (tRNA, 5 S rRNA), tRNA processing (RPR1), and splicing (U6 snRNA). The pol III transcription machinery in yeast is made up of the 17-subunit polymerase and its two complex factors (TFIIIB and TFIIIC) that are involved in promoter recognition and/or initiation of all the three classes of the pol III genes (5-8). Transcription factor IIIA, which binds to the intragenic box C, is required only for the 5 S ribosomal DNA transcription (9). TFIIIC binds to the intragenic promoter elements, boxes A and B (9), and the role attributed to it in pol III transcription has been to recruit the true transcription initiation factor TFIIIB in the upstream region of the pol III-transcribed genes (10). The TFIIIB-DNA complex is extremely stab...

Chromatin Structure and Expression of a Gene Transcribed by RNA Polymerase III Are Independent of H2A.Z Deposition

“…Binding of TFIIIC and TFIIIB to SNR6 chromatin in vitro leads to ATP-dependent chromatin remodeling whereby one nucleosome is positioned between boxes A and B and one nucleosome over the TATA box is shifted further upstream (56). This study shows that a similar mechanism operates in vivo and identifies RSC as the remodeler, which shifts the nucleosome from the TATA box region to a position further upstream, resulting in stabilization of the TFIIIB-DNA complex and transcriptional activation.…”

“…This result confirms that the loss in remodeling activity of RSC under the repression conditions is responsible for the increased occupancy of histones over the TATA region under repression. Results also show that loss of RSC from the gene after TFIIIC binding probably helps repression set in, as TFIIIB alone is not enough to transcribe SNR6 chromatin (56).…”

“…This observation reveals the possibility that during activation in the wild-type cells, RSC slides a positioned nucleosome from the TATA box to the upstream region. We had reported earlier a similar chromatin remodeling after TFIIIB binding to the TATA box in vitro that results in positioning of a nucleosome upstream of the TATA box (56). Similarly, recruitment of Isw2p by TFIIIB is also VOL.…”

“…Our previous in vitro studies have shown that binding of TFIIIC and TFIIIB to SNR6 leads to the positioning of a nucleosome between boxes A and B as well as upstream of the TATA box (56,57). As these results strongly advocate for the presence of a nucleosome between boxes A and B in vivo, we used histone depletion and repression conditions to find the chromatin structure of the SNR6 gene region in vivo.…”

CHD8 Associates with Human Staf and Contributes to Efficient U6 RNA Polymerase III Transcription

“…For example, transcription of an Alu element in vitro is profoundly repressed by packaging of the template into nucleosomes (10), and histone acetylation dramatically enhances transcription by Pol III through dinucleosomal templates as well as nucleosomal arrays (68,69). Moreover, nucleosome depletion in vivo activates Pol III transcription at certain normally repressed Pol III promoters in the Saccharomyces cerevisiae (yeast) genome (16), and in the case of the yeast U6 snRNA gene, binding of the transcription factors IIIC and IIIB is accompanied by changes in nucleosome position (56). Such observations are consistent with the proposal that chromatin modification, either histone modification or chromatin remodeling or both, is indeed required.…”