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

Abstract

The genes transcribed by RNA polymerase III (Pol III) generally have intragenic promoter elements. One of them, the yeast U6 snRNA (SNR6) gene is activated in vitro by a positioned nucleosome between its intragenic box A and extragenic, downstream box B separated by ϳ200 bp. We demonstrate here that the in vivo chromatin structure of the gene region is characterized by the presence of an array of positioned nucleosomes, with only one of them in the 5 end of the gene having a regulatory role. A positioned nucleosome present between boxes A and B in vivo does not move when the gene is repressed due to nutritional deprivation. In contrast, the upstream nucleosome which covers the TATA box under repressed conditions is shifted ϳ50 bp further upstream by the ATP-dependent chromatin remodeler RSC upon activation. It is marked with the histone variant H2A.Z and H4K16 acetylation in active state. In the absence of H2A.Z, the chromatin structure of the gene does not change, suggesting that H2A.Z is not required for establishing the active chromatin structure. These results show that the chromatin structure directly participates in regulation of a Pol IIItranscribed gene under different states of its activity in vivo.In the budding yeast, ϳ300 genes transcribed by RNA polymerase III (Pol III) yield the short noncoding RNAs like tRNA, U6 snRNA, and 5S rRNA, for example (25). The basal transcription factor of Pol III, TFIIIC, binds to the two intragenic promoter elements, boxes A and B, and recruits the initiation factor TFIIIB 30 bp upstream of the transcription start site, which recruits Pol III in turn (18, 52). U6 snRNA is conserved from yeast to mammals and is transcribed by RNA Pol III (9). Unlike other Pol III-transcribed genes, box B in the U6 snRNA gene (SNR6) of Saccharomyces cerevisiae is found 120 bases downstream of the terminator (10), positioning it ϳ200 bp away from box A, in contrast to the optimal 50-to 60-bp separation of the two found in most tDNAs. The TATA box at the Ϫ30-bp position can drive the transcription in vitro on naked DNA templates, but box B and TFIIIC are required to relieve the chromatin-mediated repression of SNR6 (10,11,17), indicating that TFIIIC helps alter the chromatin structure in favor of TFIIIB binding.Active participation of chromatin in the transcription by Pol II is well documented (30). In contrast, little is known about the role of chromatin in regulation of Pol III-transcribed genes. This may be partly because of the prevailing notion that Pol III-transcribed genes are devoid of nucleosomes (37, 66). Recently, several genome-wide studies have reported association of chromatin modifying complexes with Pol III-transcribed genes. The chromatin remodeling complex of yeast, Isw2, localizes to a large number of tDNAs (20) while RSC is targeted to virtually all Pol III-transcribed genes (38). Histone chaperone Asf1 also localizes to many Pol III genes including SNR6 (53). Global nucleosome depletion activates transcription of some unusual Pol III genes that are not transcribed unde...

Yeast H2A.Z, FACT complex and RSC regulate transcription of tRNA gene through differential dynamics of flanking nucleosomes

“…This profile of H2A.Z is similar to the general pattern of H2A.Z-containing nucleosomes flanking an NFR around the TSS, reported for active pol II-transcribed genes (10). However, IEL analysis shows that similar to SNR6 (17); the chromatin structure remains unperturbed in the absence of H2A.Z, even under repression (Figure 2D, lanes 7–10), supporting a previous report showing no role of H2A.Z in general nucleosome positioning or maintenance of nucleosomal organization (27). H2A.Z associates with both active and inactive genes (28).…”

A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes

“…Similar NFR is seen on two other pol III-transcribed genes, RNA170 and ZOD1 (Figure 1C). We had earlier reported a similar arrangement on SNR6 with NFR spanning over its TATA box and A box regions [17]. Such a common scheme of arrangement on rest of the non-tRNA genes or any special arrangement with reference to upstream TATA box or introns found in some of tDNAs is not seen.…”

“…Presence of chromatin remodelers RSC and Isw2 on pol III-transcribed genes has also been reported [13,14]. RSC shows different remodeling activity on pol II and pol III-transcribed genes [15] and regulates pol III-transcribed SNR6 and SUP4 genes [16,17]. Recent studies with human pol III-transcribed genes have reported that the presence of pol II and pol II-specific chromatin marks near pol III-transcribed genes generates an active chromatin milieu for them [18-20].…”

Regulation of pol III transcription by nutrient and stress signaling pathways

“…The Nhp6 proteins were originally implicated in pol III transcription through their positive effect on SNR6 gene transcription, which requires a positioned nucleosome for optimal expression from its atypically-spaced promoter elements [96–99]. The Nhp6 proteins also affect pol III transcription from tRNA genes through their effect on TFIIIB positioning, specifically TBP binding, and the fidelity of transcription initiation [98,100–102].…”