Rsc4 Connects the Chromatin Remodeler RSC to RNA Polymerases

Abstract

RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling.Transcription occurs in the crowded context of the nucleus in which genes are wrapped in chromatin. The first step in gene expression involves the modification and/or the remodeling of repressive chromatin by specialized complexes. For polymerase II (Pol II)-transcribed genes, these steps are followed by the recruitment of Mediator, the general transcription factors (GTFs) and the Pol II itself, although in an order that can vary from one promoter to another (9, 34). The pathway leading from silent chromatin to transcription by Pol I and Pol III has not been studied as thoroughly but is globally similar, with an additional contribution of cognate GTFs. In yeast and human cells, the Pol III-specific transcription factor TFIIIC has been found to be required for the proper nucleosomal organization of Pol III genes (4, 23, 32). In the case of Pol I transcription, the mammalian termination factor TTF-I is able to activate transcription by promoting chromatin remodeling in synergy with ATP-dependent cofactors in vitro (24). Transcription initiation is not the only step at which chromatin might interfere with transcription. Nucleosomes residing in the transcribed region can inhibit the movement of RNA polymerases during elongation. To contend with this, the FACT complex helps human Pol II transcribe through nucleosome-induced blocks (28, 38). These observations suggest that factors that relieve the repressive effect of nucleosomes might act in conjunction with the transcription machinery at the successive stages of the transcription cycle.The repressive effect of nucleosomes is overcome by two cooperative mechanisms. The first involves the covalent modification of the histones, including the acetylation of specific histone tail lysines by acetyl transferases (...

TFIIS elongation factor and Mediator act in conjunction during transcription initiation in vivo

“…Gene expression was monitored with DNA microarrays manufactured by Service de Génomique Fonctionnelle (Commissariat à l'Energie Atomique/Evry, France), as described (13), except that an indirect cDNA-labeling protocol of the targets was used (35). The microarrays were scanned with a GENEPIX 4000B scanner.…”

Nucleosome Remodeling and Epigenetics

“…Yeast Swi/Snf has been implicated in histone eviction during polymerase elongation in vivo (Schwabish and Struhl 2007). The yeast RSC complex can associate with RNA polymerase II (Soutourina et al 2006) in vitro and stimulate transcription through a nucleosome in a manner enhanced by histone acetylation (Li et al 2007). Yeast Chd1 colocalizes with RNA polymerase II and interacts with a number of factors involved in elongation, such as the PAF complex, the Spt4/5 elongation factors, and the FACT histone chaperone complex (Simic et al 2003).…”

Asymmetric nucleosomes flank promoters in the budding yeast genome

“…Remodeler action at the +1 nucleosome position is associated with RNA polymerase II (RNAPII) transit across nucleosomal DNA, both in facilitating RNAPII elongation (Soutourina et al 2006;Schwabish and Struhl 2007) and in repositioning nucleosomes in the wake of RNAPII transit (Smolle et al 2012). If RSC is acting at the asymmetric +1 position to facilitate transcription, its depletion should result in expression changes at genes with asymmetric +1 nucleosome positions.…”