Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in <i>Saccharomyces cerevisiae</i>

Braglia, Priscilla; Dugas, Sandra L.; Donze, David; Dieci, Giorgio

doi:10.1128/mcb.00773-06

Cited by 41 publications

(27 citation statements)

References 64 publications

(110 reference statements)

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“…7A, lane 1), which was not the case in the presence of the crude fraction BЉ (Andrau and Werner 2001). This fraction contains additional factors, like Nhp6 (Braglia et al 2007), that can restore transcriptional initiation specificity (Kassavetis and Steiner 2006). Western blotting analysis also revealed the presence of TFIIS in the BЉ fraction (data not shown).…”

Section: Tfiis Affects Pol III Transcription In Vitromentioning

confidence: 84%

Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription

Ghavi-Helm¹,

Michaut²,

Acker³

et al. 2008

Genes Dev.

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TFIIS is a transcription elongation factor that stimulates transcript cleavage activity of arrested RNA polymerase II (Pol II). Recent studies revealed that TFIIS has also a role in Pol II transcription initiation. To improve our understanding of TFIIS function in vivo, we performed genome-wide location analysis of this factor. Under normal growth conditions, TFIIS was detected on Pol II-transcribed genes, and TFIIS occupancy was well correlated with that of Pol II, indicating that TFIIS recruitment is not restricted to NTP-depleted cells. Unexpectedly, TFIIS was also detected on almost all Pol III-transcribed genes. TFIIS and Pol III occupancies correlated well genome-wide on this novel class of targets. In vivo, some dst1 mutants were partly defective in tRNA synthesis and showed a reduced Pol III occupancy at the restrictive temperature. In vitro transcription assays suggested that TFIIS may affect Pol III start site selection. These data provide strong in vivo and in vitro evidence in favor of a role of TFIIS as a general Pol III transcription factor. Gene transcription is a complex and highly regulated process that, in eukaryotes, is carried out by three specialized RNA polymerases (Pol I, II, and III), dedicated to the transcription of different sets of genes. Transcription starts by the assembly of large preinitiation complexes (PIC) comprising TBP (the TATA-binding protein common to all three transcription systems) and general transcription factors specific to the RNA polymerase considered. Other transcription factors facilitate RNA elongation by Pol II (Shilatifard et al. 2003). One of these elongation factors, TFIIS, was initially discovered in human (Natori et al. 1973) and yeast cells (Sawadogo et al. 1980b), and is highly conserved in the eukaryotic and archaeal kingdoms (Hausner et al. 2000;Fish and Kane 2002). Structurally unrelated but functionally equivalent factors (GreA and GreB) also operate in bacteria (Borukhov et al. 1993;Opalka et al. 2003). TFIIS forms a binary complex with Pol II and stimulates an intrinsic transcript cleavage activity of RNA polymerase allowing elongating enzymes to resume RNA synthesis after accidental transcription arrest (Fish and Kane 2002;Kettenberger et al. 2003).TFIIS is organized in three domains as determined by limited proteolysis and nuclear magnetic resonance (NMR) (Awrey et al. 1997;Olmsted et al. 1998). The three-dimensional structure of the Pol II-TFIIS complex, without the TFIIS N-terminal domain, has been determined by crystallographic studies (Kettenberger et al. 2003). The Nterminal domain I of TFIIS protrudes from Pol II and is not required for elongation (Nakanishi et al. 1995;Awrey et al. 1998), but binds the yeast Mediator and SAGA coactivator (Wery et al. 2004). The TFIIS central domain II is inserted into the funnel-shaped pore of Pol II and brings the C-terminal conserved RSADE motif very close to the enzyme active site (Kettenberger et al. 2003). Remarkably, the C-terminal domain III of TFIIS shares the RSADE motif with the C-terminal parts o...

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Section: Tfiis Affects Pol III Transcription In Vitromentioning

confidence: 84%

Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription

Ghavi-Helm¹,

Michaut²,

Acker³

et al. 2008

Genes Dev.

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“…A non-histone protein like Nhp6 and not a nucleosome was suggested to occupy the DNA between boxes A and B of SNR6 (21,29). However, Nhp6 deletion does not show any change in the MNase digestion pattern between boxes A and B (34), and recent reports suggest that the major role for Nhp6 in Pol III transcription may be in stabilization of the TFIIIB-DNA complex (7). Evidence provided by our studies for the presence of a nucleosome between the boxes A and B of yeast SNR6 in vivo has revealed the possibility that Pol III transcribes the DNA wound over a nucleosome in SNR6.…”

Section: Discussionmentioning

confidence: 99%

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

Arimbasseri

Bhargava

2008

Molecular and Cellular Biology

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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...

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“…For example, tRNAs represent approximately 15% of the total RNA of exponentially growing S. cerevisiae cells (57), implying that there are on the order of 3 ϫ 10 6 tRNA molecules per yeast cell. Thus, each of the 274 nucleus-encoded (24) and additional mitochondrially encoded tDNAs of this organism must be (on average) transcribed on the order of 10 4 times per generation (or approaching about twice per second, given a 90-min generation time), a value that is considerably higher than the maximal transcription initiation frequency of one initiation every 6 to 8 s estimated for RNA Pol II-transcribed genes in yeast (31).…”

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confidence: 99%

TFIIIC Binding Sites Function as both Heterochromatin Barriers and Chromatin Insulators in Saccharomyces cerevisiae

et al. 2008

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Chromosomal sites of RNA polymerase III (Pol III) transcription have been demonstrated to have "extratranscriptional" functions, as the assembled Pol III complex can act as chromatin boundaries or pause sites for replication forks, can alter nucleosome positioning or affect transcription of neighboring genes, and can play a role in sister chromatid cohesion. Several studies have demonstrated that assembled Pol III complexes block the propagation of heterochromatin-mediated gene repression. Here we show that in Saccharomyces cerevisiae tRNA genes (tDNAs) and even partially assembled Pol III complexes containing only the transcription factor TFIIIC can exhibit chromatin boundary functions both as heterochromatin barriers and as insulators to gene activation. Both the TRT2 tDNA and the ETC4 site which binds only the TFIIIC complex prevented an upstream activation sequence from activating the GAL promoters in our assay system, effectively acting as chromatin insulators. Additionally, when placed downstream from the heterochromatic HMR locus, ETC4 blocked the ectopic spread of Sir protein-mediated silencing, thus functioning as a barrier to repression. Finally, we show that TRT2 and the ETC6 site upstream of TFC6 in their natural contexts display potential insulator-like functions, and ETC6 may represent a novel case of a Pol III factor directly regulating a Pol II promoter. The results are discussed in the context of how the TFIIIC transcription factor complex may function to demarcate chromosomal domains in yeast and possibly in other eukaryotes.Eukaryotic genomes are organized into structurally and functionally distinct domains as one layer of transcriptional regulation to allow the expression of particular sets of genes when required and to restrict their expression when necessary. Mechanisms of activation usually involve DNA-bound transcription factors that recruit RNA polymerase or general transcription factors or recruit proteins that promote the formation of chromatin structures compatible with RNA polymerase preinitiation complex formation and transcriptional elongation. Repressive chromatin domains can inhibit gene expression at either of these stages. Chromatin boundary elements function to separate chromosomal domains so that regulatory regions of one domain do not inappropriately influence adjacent domains, either by insulating promoters from activation or by acting as a barrier to propagating repressive heterochromatin (55, 58).Evidence has accumulated over the past several years that RNA polymerase III (Pol III) promoter sequences, mainly studied using tRNA genes (tDNAs), can possess an intrinsic chromatin boundary activity. This was first demonstrated at the heterochromatic HMR locus in Saccharomyces cerevisiae, as the downstream tDNA is a critical component of the barrier that prevents the inappropriate spreading of silencing from HMR (16), and the characterization of this activity was the first demonstration of a natural chromatin boundary in yeast. Another yeast tDNA, TRT2, was shown to prevent the...

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Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae

Cited by 41 publications

References 64 publications

Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription

Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription

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

TFIIIC Binding Sites Function as both Heterochromatin Barriers and Chromatin Insulators in Saccharomyces cerevisiae

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