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

2007 ◽  
Vol 27 (24) ◽  
pp. 8729-8738 ◽  
Author(s):  
Chih-Chi Yuan ◽  
Xinyang Zhao ◽  
Laurence Florens ◽  
Selene K. Swanson ◽  
Michael P. Washburn ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Chromatin Remodeling ◽  
Rna Polymerase Iii ◽  
Chromatin Modification ◽  
Small Nuclear Rna ◽  
Polymerase Iii ◽  
Chromatin Template ◽  
Pol Iii

ABSTRACT Chromatin remodeling and histone modification are essential for eukaryotic transcription regulation, but little is known about chromatin-modifying activities acting on RNA polymerase III (Pol III)-transcribed genes. The human U6 small nuclear RNA promoter, located 5′ of the transcription start site, consists of a core region directing basal transcription and an activating region that recruits the transcription factors Oct-1 and Staf (ZNF143). Oct-1 activates transcription in part by helping recruit core binding factors, but nothing is known about the mechanisms of transcription activation by Staf. We show that Staf activates U6 transcription from a preassembled chromatin template in vitro and associates with several proteins linked to chromatin modification, among them chromodomain-helicase-DNA binding protein 8 (CHD8). CHD8 binds to histone H3 di- and trimethylated on lysine 4. It resides on the human U6 promoter as well as the mRNA IRF3 promoter in vivo and contributes to efficient transcription from both these promoters. Thus, Pol III transcription from type 3 promoters uses some of the same factors used for chromatin remodeling at Pol II promoters.

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

2008 ◽  
Vol 28 (8) ◽  
pp. 2598-2607 ◽  
Author(s):  
Aneeshkumar Gopalakrishnan Arimbasseri ◽  
Purnima Bhargava
Keyword(s):  
Rna Polymerase ◽  
Chromatin Structure ◽  
Rna Polymerase Iii ◽  
Nutritional Deprivation ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Downstream Box ◽  
Pol Iii

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 ∼200 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 ∼50 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 III-transcribed gene under different states of its activity in vivo.

scholarly journals RNA Polymerase III Transcription Factor IIIB Is a Target for Repression by Pocket Proteins p107 and p130

1999 ◽  
Vol 19 (6) ◽  
pp. 4255-4261 ◽  
Author(s):  
Josephine E. Sutcliffe ◽  
Carol A. Cairns ◽  
Angela McLees ◽  
Simon J. Allison ◽  
Kerrie Tosh ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Direct Interaction ◽  
Double Knockout ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Pol Iii ◽  
Related Proteins

ABSTRACT RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88–90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061–2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755–14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.

scholarly journals Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (15) ◽  
pp. 5031-5040 ◽  
Author(s):  
Krzysztof Pluta ◽  
Olivier Lefebvre ◽  
Nancy C. Martin ◽  
Wieslaw J. Smagowicz ◽  
David R. Stanford ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Growth Defect ◽  
Class Iii ◽  
Gene Encoding ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.

1995 ◽  
Vol 15 (9) ◽  
pp. 4873-4883 ◽  
Author(s):  
K S Ullman ◽  
D J Forbes
Keyword(s):  
Rna Polymerase ◽  
Nuclear Structure ◽  
De Novo ◽  
Rna Polymerase Iii ◽  
Transcriptional Analysis ◽  
Dna Encoding ◽  
Nuclear Assembly ◽  
Polymerase Iii ◽  
Pol Iii

Although much is known of the basic control of transcription, little is understood of the way in which the structural organization of the nucleus affects transcription. Synthetic nuclei, assembled de novo in extracts of Xenopus eggs, would be predicted to have a large potential for approaching the role of nuclear structure in RNA biogenesis. Synthetic nuclei provide a system in which the genetic content of the nuclei, as well as the structural and enzymatic proteins within the nuclei, can be manipulated. In this study, we have begun to examine transcription in such nuclei by using the most simple of templates, RNA polymerase III (pol III)-transcribed genes. DNA encoding tRNA or 5S genes was added to an assembly extract, and nuclei were formed entirely from the pol III templates. Conditions which allowed nuclear assembly and pol III transcription to take place efficiently and simultaneously in the assembly extract were found. To examine whether pol III transcription could initiate within synthetic nuclei, or instead was inhibited in nuclei and initiated only on rare unincorporated templates, we identified transcriptional inhibitors that were excluded from nuclei. We found that these inhibitors, heparin and dextran sulfate, blocked pol III transcription in the absence of assembly but did not do so following nuclear assembly. At the concentrations used, the inhibitors had no deleterious effect on nuclear structure itself or on nuclear import. We conclude that pol III transcription is active in synthetic nuclei, and this conclusion is further strengthened by the finding that pol III transcripts could be coisolated with synthetic nuclei. The rapid and direct transcriptional analysis possible with pol III templates, coupled with the simple experimental criteria developed in this study for distinguishing between nuclear and non-nuclear transcription, should now allow a molecular analysis of the effect of nuclear structure on transcriptional and posttranscriptional control.

scholarly journals Mechanism of RNA polymerase III termination-associated reinitiation-recycling conferred by the essential function of the N terminal-and-linker domain of the C11 subunit

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saurabh Mishra ◽  
Shaina H. Hasan ◽  
Rima M. Sakhawala ◽  
Shereen Chaudhry ◽  
Richard J. Maraia
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Cleavage Activity ◽  
Terminal Domain ◽  
Polymerase Iii ◽  
Essential Function ◽  
High Level ◽  
Pol Iii

AbstractRNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3′-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3′-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3′-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3′-cleavage activity increases growth rate but is nonessential.

Induction of specific transcription by RNA polymerase III in transformed cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3068-3076
Author(s):  
M F Carey ◽  
K Singh ◽  
M Botchan ◽  
N R Cozzarelli
Keyword(s):  
Rna Polymerase ◽  
Gene Family ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Intrinsic Signals ◽  
Polymerase Iii ◽  
Pol Iii

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.

scholarly journals A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

2001 ◽  
Vol 21 (19) ◽  
pp. 6429-6439 ◽  
Author(s):  
Michael P. Martin ◽  
Valerie L. Gerlach ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Tata Box ◽  
Initiation Factor ◽  
Base Pairs ◽  
Polymerase Iii ◽  
U6 Rna

ABSTRACT The Saccharomyces cerevisiae U6 RNA gene,SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription ofSNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position −30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele ofSNR6 with decreased spacing between the A and B blocks,snr6-Δ42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Δ42 is much more sensitive to mutations in a (dT-dA)7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Δ42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription ofSNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

scholarly journals Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila

2016 ◽  
Author(s):  
Shrivani Sriskanthadevan-Pirahas ◽  
Rujuta Deshpande ◽  
Byoungchun Lee ◽  
Savraj S. Grewal
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Tissue Growth ◽  
Polymerase Iii ◽  
Kinase Cascade ◽  
Important Challenge ◽  
Pol Iii ◽  
And Function ◽  
Stimulation Of

ABSTRACTThe small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that the main way that Ras promotes Pol III function and tRNA synthesis is by inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.

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