Articoli di riviste: "Spt3"

1

Winston, Fred, Catherine Dollard, Elizabeth A. Malone, Jeffrey Clare, James G. Kapakos, Philip Farabaugh e Patricia L. Minehart. "Three Genes Are Required for trans-Activation of Ty Transcription in Yeast". Genetics 115, n. 4 (1 aprile 1987): 649–56. http://dx.doi.org/10.1093/genetics/115.4.649.

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ABSTRACT Mutations in the SPT3 gene were isolated as one class of suppressors of Ty and solo δ insertion mutations in Saccharomyces cerevisiae. Previous work has shown that null mutations in SPT3 abolish the normal Ty δ-δ transcript; instead, a transcript that initiates 800 bases farther downstream is made, suggesting that SPT3 is required for transcription initiation in δ sequences. We have selected for new spt mutations and have screened for those with the unique suppression pattern of spt3 mutations with respect to two insertion mutations. Our selection and screen has identified two additional genes, SPT7 and SPT8, that are also required for transcription initiation in δ sequences. We show that mutations in SPT7 or SPT8 result in the same alteration of Ty transcription as do mutations in SPT3. In addition, mutations in all three genes cause a sporulation defect. By assay of a Ty-lacZ fusion we have shown that spt3, spt7 and spt8 mutations reduce transcription from a δ sequence by 10-25-fold. Finally, we show that SPT3 mRNA levels are unaffected in either spt7 or spt8 mutants, suggesting that these two genes do not regulate transcription of SPT3.

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2

Happel, A. M., M. S. Swanson e F. Winston. "The SNF2, SNF5 and SNF6 genes are required for Ty transcription in Saccharomyces cerevisiae." Genetics 128, n. 1 (1 maggio 1991): 69–77. http://dx.doi.org/10.1093/genetics/128.1.69.

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Abstract The Saccharomyces cerevisiae SNF2, SNF5 and SNF6 genes were initially identified as genes required for expression of SUC2 and other glucose repressible genes. The Suc- defect in all three of these classes of mutants is suppressed by mutations in the SPT6 gene. Since mutations in SPT6 had also been identified as suppressors of Ty and solo delta insertion mutations at the HIS4 and LYS2 loci, we have examined Ty transcription in snf2, snf5 and snf6 mutants and have found that Ty transcription is abolished or greatly reduced. The snf2, snf5 and snf6 defect for Ty transcription, like the defect for SUC2 transcription, is suppressed by spt6 mutations. In contrast to other mutations that abolish or greatly reduce Ty transcription (in the SPT3, SPT7 and SPT8 genes), mutations in these SNF genes do not cause suppression of insertion mutations. This result suggests that the SNF2, SNF5 and SNF6 gene products act by a distinct mechanism from the SPT3, SPT7 and SPT8 gene products to promote transcription of Ty elements. This result also suggests that a reduction of Ty transcription is not always sufficient for activation of adjacent gene expression.

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3

Laprade, Lisa, Victor L. Boyartchuk, William F. Dietrich e Fred Winston. "Spt3 Plays Opposite Roles in Filamentous Growth in Saccharomyces cerevisiae and Candida albicans and Is Required for C. albicans Virulence". Genetics 161, n. 2 (1 giugno 2002): 509–19. http://dx.doi.org/10.1093/genetics/161.2.509.

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Abstract Spt3 of Saccharomyces cerevisiae is required for the normal transcription of many genes in vivo. Past studies have shown that Spt3 is required for both mating and sporulation, two events that initiate when cells are at G1/START. We now show that Spt3 is needed for two other events that begin at G1/START, diploid filamentous growth and haploid invasive growth. In addition, Spt3 is required for normal expression of FLO11, a gene required for filamentous growth, although this defect is not the sole cause of the spt3Δ/spt3Δ filamentous growth defect. To extend our studies of Spt3's role in filamentous growth to the pathogenic yeast Candida albicans, we have identified the C. albicans SPT3 gene and have studied its role in C. albicans filamentous growth and virulence. Surprisingly, C. albicans spt3Δ/spt3Δ mutants are hyperfilamentous, the opposite phenotype observed for S. cerevisiae spt3Δ/spt3Δ mutants. Furthermore, C. albicans spt3Δ/spt3Δ mutants are avirulent in mice. These experiments demonstrate that Spt3 plays important but opposite roles in filamentous growth in S. cerevisiae and C. albicans.

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4

Happel, A. M., e F. Winston. "A mutant tRNA affects delta-mediated transcription in Saccharomyces cerevisiae." Genetics 132, n. 2 (1 ottobre 1992): 361–74. http://dx.doi.org/10.1093/genetics/132.2.361.

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Abstract Mutations in the SPT3, SPT7, SPT8 and SPT15 genes define one class of trans-acting mutations that are strong suppressors of insertion mutations caused by Ty elements or by the Ty long terminal repeat sequence, delta. These SPT genes are required for normal transcription of Ty elements, and their gene products are believed to be involved in initiation of Ty transcription from delta sequences. We have isolated and analyzed extragenic suppressors of spt3 mutations. These new mutations, named rsp, partially suppress the requirement for SPT3, SPT7, SPT8 and SPT15 functions. In addition, rsp mutations cause changes in transcription of some delta insertions in an SPT+ genetic background. Interactions between mutations in the four identified RSP genes show a number of interesting genetic properties, including the failure of unlinked rsp mutations to complement for recessive phenotypes. Cloning and sequencing of one rsp mutant gene, rsp4-27, showed that it encodes a frameshift suppressor glycine tRNA. Our results indicate that the other three RSP genes also encode frameshift suppressor glycine tRNAs. In addition, other types of frameshift suppressor glycine tRNAs can confer some Rsp- phenotypes.

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5

Roberts, S. M., e F. Winston. "SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae." Molecular and Cellular Biology 16, n. 6 (giugno 1996): 3206–13. http://dx.doi.org/10.1128/mcb.16.6.3206.

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Mutations selected as suppressors of Ty and solo delta insertion mutations is Saccharomyces cerevisiae have identified a number of genes important for transcription initiation. One of these gens, SPT15, encodes the TATA-binding protein, and three others, SPT3, SPT7, and SPT8, encode proteins functionally related to the TATA-binding protein. To identify additional related functions, we have selected for new spt mutations. This work has identified one new gene, SPT20. Null mutations in SPT20 cause poor growth and a set of severe transcriptional defects very similar to those caused by null mutations in SPT3, SPT7, and SPT8 and also very similar to those caused by certain missense mutations in SPT15. Consistent with its having an important function in transcription in vivo, SPT20 was also recently identified as ADA5 and has been shown to be important for transcriptional activation (G.A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996.

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6

Eisenmann, D. M., C. Chapon, S. M. Roberts, C. Dollard e F. Winston. "The Saccharomyces cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein." Genetics 137, n. 3 (1 luglio 1994): 647–57. http://dx.doi.org/10.1093/genetics/137.3.647.

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Abstract Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8 gene and have shown that it encodes a predicted protein of 602 amino acids with an extremely acidic amino terminus. In addition, the predicted SPT8 amino acid sequence contains one copy of a sequence motif found in multiple copies in a number of other eukaryotic proteins, including the beta subunit of heterotrimeric G proteins. To investigate further the relationship between SPT8, SPT3 and TBP, we have analyzed the effect of an spt8 null mutation in combination with different spt3 and spt15 mutations. This genetic analysis has shown that an spt8 deletion mutation is suppressed by particular spt3 alleles. Taken together with previous results, these data suggest that the SPT8 protein is required, directly or indirectly, for TBP function at particular promoters and that the role of SPT8 may be to promote a functional interaction between SPT3 and TBP.

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7

Belotserkovskaya, Rimma, David E. Sterner, Min Deng, Michael H. Sayre, Paul M. Lieberman e Shelley L. Berger. "Inhibition of TATA-Binding Protein Function by SAGA Subunits Spt3 and Spt8 at Gcn4-Activated Promoters". Molecular and Cellular Biology 20, n. 2 (15 gennaio 2000): 634–47. http://dx.doi.org/10.1128/mcb.20.2.634-647.2000.

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ABSTRACT SAGA is a 1.8-MDa yeast protein complex that is composed of several distinct classes of transcription-related factors, including the adaptor/acetyltransferase Gcn5, Spt proteins, and a subset of TBP-associated factors. Our results indicate that mutations that completely disrupt SAGA (deletions of SPT7 orSPT20) strongly reduce transcriptional activation at theHIS3 and TRP3 genes and that Gcn5 is required for normal HIS3 transcriptional start site selection. Surprisingly, mutations in Spt proteins involved in the SAGA-TBP interaction (Spt3 and Spt8) cause derepression of HIS3 andTRP3 transcription in the uninduced state. Consistent with this finding, wild-type SAGA inhibits TBP binding to theHIS3 promoter in vitro, while SAGA lacking Spt3 or Spt8 is not inhibitory. We detected two distinct forms of SAGA in cell extracts and, strikingly, one lacks Spt8. Conditions that induceHIS3 and TRP3 transcription result in an altered balance between these complexes strongly in favor of the form without Spt8. These results suggest that the composition of SAGA may be dynamic in vivo and may be regulated through dissociable inhibitory subunits.

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8

Dudley, Aimée M., Lisa J. Gansheroff e Fred Winston. "Specific Components of the SAGA Complex Are Required for Gcn4- and Gcr1-Mediated Activation of the his4-912δ Promoter in Saccharomyces cerevisiae". Genetics 151, n. 4 (1 aprile 1999): 1365–78. http://dx.doi.org/10.1093/genetics/151.4.1365.

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Abstract Mutations selected as suppressors of Ty or solo δ insertion mutations in Saccharomyces cerevisiae have identified several genes, SPT3, SPT7, SPT8, and SPT20, that encode components of the SAGA complex. However, the mechanism by which SAGA activates transcription of specific RNA polymerase II-dependent genes is unknown. We have conducted a fine-structure mutagenesis of one widely used SAGA-dependent promoter, the δ element of his4-912δ, to identify sequence elements important for its promoter activity. Our analysis has characterized three δ regions necessary for full promoter activity and accurate start site selection: an upstream activating sequence, a TATA region, and an initiator region. In addition, we have shown that factors present at the adjacent UASHIS4 (Gcn4, Bas1, and Pho2) also activate the δ promoter in his4-912δ. Our results suggest a model in which the δ promoter in his4-912δ is primarily activated by two factors: Gcr1 acting at the UASδ and Gcn4 acting at the UASHIS4. Finally, we tested whether activation by either of these factors is dependent on components of the SAGA complex. Our results demonstrate that Spt3 and Spt20 are required for full δ promoter activity, but that Gcn5, another member of SAGA, is not required. Spt3 appears to be partially required for activation of his4-912δ by both Gcr1 and Gcn4. Thus, our work suggests that SAGA exerts a large effect on δ promoter activity through a combination of smaller effects on multiple factors.

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9

Boeke, J. D., C. A. Styles e G. R. Fink. "Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements". Molecular and Cellular Biology 6, n. 11 (novembre 1986): 3575–81. http://dx.doi.org/10.1128/mcb.6.11.3575-3581.1986.

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Mutations in the Saccharomyces cerevisiae SPT3 gene have dramatic effects on the expression of Ty elements and genes adjacent to the element. The SPT3 gene is essential for Ty transposition, because transposition of chromosomal Ty elements ceased when the SPT3 gene was replaced with the frameshift mutation spt3-101. Presumably, the elimination of transposition was due to the effect of the SPT3 gene product on Ty transcription; the transcripts of chromosomal Ty elements were largely abolished in the spt3-101 strain (F. Winston, K. J. Durbin, and G. R. Fink, Cell 39:675-682, 1984). Ty transcription in an spt3-101 strain could be reestablished by introduction of the pGTyH3 plasmid, in which transcription of the Ty element TyH3 is under the control of the GAL1 promoter; these plasmid-derived Ty transcripts were SPT3-independent. Ty transposition resumed after galactose induction in spt3-101 strains containing the pGTyH3 plasmid. In spt3 mutants nearly all of the resulting transposition events derived from pGTyH3 plasmids and not from chromosomal elements.

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10

Boeke, J. D., C. A. Styles e G. R. Fink. "Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements." Molecular and Cellular Biology 6, n. 11 (novembre 1986): 3575–81. http://dx.doi.org/10.1128/mcb.6.11.3575.

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Abstract (sommario):

Mutations in the Saccharomyces cerevisiae SPT3 gene have dramatic effects on the expression of Ty elements and genes adjacent to the element. The SPT3 gene is essential for Ty transposition, because transposition of chromosomal Ty elements ceased when the SPT3 gene was replaced with the frameshift mutation spt3-101. Presumably, the elimination of transposition was due to the effect of the SPT3 gene product on Ty transcription; the transcripts of chromosomal Ty elements were largely abolished in the spt3-101 strain (F. Winston, K. J. Durbin, and G. R. Fink, Cell 39:675-682, 1984). Ty transcription in an spt3-101 strain could be reestablished by introduction of the pGTyH3 plasmid, in which transcription of the Ty element TyH3 is under the control of the GAL1 promoter; these plasmid-derived Ty transcripts were SPT3-independent. Ty transposition resumed after galactose induction in spt3-101 strains containing the pGTyH3 plasmid. In spt3 mutants nearly all of the resulting transposition events derived from pGTyH3 plasmids and not from chromosomal elements.

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11

Madison, J. M., e F. Winston. "Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae." Molecular and Cellular Biology 17, n. 1 (gennaio 1997): 287–95. http://dx.doi.org/10.1128/mcb.17.1.287.

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Spt3 of Saccharomyces cerevisiae is a factor required for normal transcription from particular RNA polymerase II-dependent promoters. Previous genetic and biochemical analyses have shown that Spt3 interacts with the yeast TATA-binding protein (TBP). To identify other factors that might interact with Spt3, we have screened for mutations that, in combination with an spt3 null mutation, lead to inviability. In this way, we have identified a mutation in MOT1, which encodes an ATP-dependent inhibitor of TBP binding to TATA boxes: Previous analyses suggested that Mot1 causes repression in vivo. However, our analysis of mot1 mutants shows that, similar to spt3 mutants, they have decreased levels of transcription from certain genes, suggesting that Mot1 may function as an activator in vivo. In addition, mot1 mutants have other phenotypes in common with spt3 delta mutants, including suppression of the insertion mutation his4-912 delta. Motivated by these Spt3-Mot1 genetic interactions, we tested for genetic interactions between Spt3 and the general transcription factor TFIIA. TFIIA has been shown previously to be functionally related to Mot1. We found that overexpression of TFIIA partially suppresses an spt3 delta mutation, that toa1 mutants have Spt-phenotypes, and that spt3 delta toa1 double mutants are inviable. We believe that, taken together, these data suggest that Spt3, Mot1, and TFIIA cooperate to regulate TBP-DNA interactions, perhaps at the level of TATA box selection in vivo.

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12

Hirschhorn, J. N., e F. Winston. "SPT3 is required for normal levels of a-factor and alpha-factor expression in Saccharomyces cerevisiae". Molecular and Cellular Biology 8, n. 2 (febbraio 1988): 822–27. http://dx.doi.org/10.1128/mcb.8.2.822-827.1988.

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Mutations in the Saccharomyces cerevisiae SPT3 gene were previously found to cause suppression of Ty and delta insertion mutations in 5'-noncoding regions of genes. This suppression likely results from the fact that SPT3 is required for transcription initiation in delta sequences. Other additional phenotypes of spt3 mutants, including a mating defect, suggest that SPT3 is required for normal levels of expression of other genes. We analyzed the mating defect in spt3 mutants and showed that the levels of transcripts of the three major mating pheromone genes, MF alpha 1, MFa1, MFa2, were all reduced. The reduction in expression of these genes in spt3 mutants was not due to expression of a silent mating type cassette. Furthermore, we showed that the spt3 mating defect was manifest at the levels of both cellular fusion and nuclear fusion.

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13

Hirschhorn, J. N., e F. Winston. "SPT3 is required for normal levels of a-factor and alpha-factor expression in Saccharomyces cerevisiae." Molecular and Cellular Biology 8, n. 2 (febbraio 1988): 822–27. http://dx.doi.org/10.1128/mcb.8.2.822.

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Abstract (sommario):

Mutations in the Saccharomyces cerevisiae SPT3 gene were previously found to cause suppression of Ty and delta insertion mutations in 5'-noncoding regions of genes. This suppression likely results from the fact that SPT3 is required for transcription initiation in delta sequences. Other additional phenotypes of spt3 mutants, including a mating defect, suggest that SPT3 is required for normal levels of expression of other genes. We analyzed the mating defect in spt3 mutants and showed that the levels of transcripts of the three major mating pheromone genes, MF alpha 1, MFa1, MFa2, were all reduced. The reduction in expression of these genes in spt3 mutants was not due to expression of a silent mating type cassette. Furthermore, we showed that the spt3 mating defect was manifest at the levels of both cellular fusion and nuclear fusion.

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14

Swanson, M. S., e F. Winston. "SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae." Genetics 132, n. 2 (1 ottobre 1992): 325–36. http://dx.doi.org/10.1093/genetics/132.2.325.

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Abstract The SPT4, SPT5 and SPT6 genes of Saccharomyces cerevisiae were identified originally by mutations that suppress delta insertion mutations at HIS4 and LYS2. Subsequent analysis has demonstrated that spt4, spt5 and spt6 mutations confer similar pleiotropic phenotypes. They suppress delta insertion mutations by altering transcription and are believed to be required for normal transcription of several other loci. We have now analyzed interactions between SPT4, SPT5 and SPT6. First, the combination of mutations in any two of these three genes causes lethality in haploids. Second, some recessive mutations in different members of this set fail to complement each other. Third, mutations in all three genes alter transcription in similar ways. Finally, the results of coimmunoprecipitation experiments demonstrate that at least the SPT5 and SPT6 proteins interact physically. Taken together, these genetic and biochemical results indicate that SPT4, SPT5 and SPT6 function together in a transcriptional process that is essential for viability in yeast.

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15

Collart, M. A. "The NOT, SPT3, and MOT1 genes functionally interact to regulate transcription at core promoters." Molecular and Cellular Biology 16, n. 12 (dicembre 1996): 6668–76. http://dx.doi.org/10.1128/mcb.16.12.6668.

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Previous studies demonstrated that mutations in the Saccharomyces cerevisiae NOT genes increase transcription from TATA-less promoters. In this report, I show that in contrast, mutations in the yeast MOT1 gene decrease transcription from TATA-less promoters. I also demonstrate specific genetic interactions between the Not complex, Mot1p, and another global regulator of transcription in S. cerevisiae, Spt3p. Five distinct genetic interactions have been established. First, a null allele of SPT3, or a mutation in SPT15 that disrupts the interaction between Spt3p and TATA-binding protein (TBP), allele specifically suppressed the not1-2 mutation. Second, in contrast to not mutations, mutations in MOT1 decreased HIS3 and HIS4 TATA-less transcription. Third, not mutations suppressed toxicity due to overexpression of TBP in mot1-1 mutants. Finally, overexpression of SPT3 caused a weak Not- mutant phenotype in mot1-1 mutants. Collectively, these results suggest a novel type of transcriptional regulation whereby the distribution of limiting TBP (TFIID) on weak and strong TBP-binding core promoters is regulated: Mot1p releases stably bound TBP to allow its redistribution to low-affinity sites, and the Not proteins negatively regulate the activity of factors such as Spt3p that favor distribution of TBP to these low-affinity sites.

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16

Badarinarayana, Vasudeo, Yueh-Chin Chiang e Clyde L. Denis. "Functional Interaction of CCR4-NOT Proteins With TATAA-Binding Protein (TBP) and Its Associated Factors in Yeast". Genetics 155, n. 3 (1 luglio 2000): 1045–54. http://dx.doi.org/10.1093/genetics/155.3.1045.

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Abstract The CCR4-NOT transcriptional regulatory complex affects expression of a number of genes both positively and negatively. We report here that components of the CCR4-NOT complex functionally and physically interact with TBP and TBP-associated factors. First, mutations in CCR4-NOT components suppressed the his4-912δ insertion in a manner similar to that observed for the defective TBP allele spt15-122. Second, using modified HIS3 promoter derivatives containing specific mutations within the TATA sequence, we found that the NOT proteins were general repressors that disrupt TBP function irrespective of the DNA sequence. Third, increasing the dosage of NOT1 specifically inhibited the ability of spt15-122 to suppress the his4-912δ insertion but did not affect the Spt− phenotype of spt3 or spt10 at this locus. Fourth, spt3, spt8, and spt15-21 alleles (all involved in affecting interaction of SPT3 with TBP) suppressed ccr4 and caf1 defects. Finally, we show that NOT2 and NOT5 can be immunoprecipitated by TBP. NOT5 was subsequently shown to associate with TBP and TAFs and this association was dependent on the integrity of TFIID. These genetic and physical interactions indicate that one role of the CCR4-NOT proteins is to inhibit functional TBP-DNA interactions, perhaps by interacting with and modulating the function of TFIID.

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17

Schwertz, A., C. Villaume, L. Mejean, B. Decaris e G. Percebois. "New identification of the strain Rhizopus microsporus var. oligosporus spT3 as Rhizopus microsporus var. chinensis". Canadian Journal of Microbiology 43, n. 10 (1 ottobre 1997): 971–76. http://dx.doi.org/10.1139/m97-139.

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The wild Rhizopus strain spT3 extracted from tempeh of Bali, Indonesia (Institute of Microbial Resources, Taichung, Taiwan) had first been identified as Rhizopus microspores var. oligosporus spT3. This strain is used to ferment soybeans, to detoxify them and improve their nutritional value. Analysis of fermented seeds showed that this strain strongly degrades numerous noxious antinutritional factors, which is unusual for R. microsporus var. oligosporus. The activity of strain spT3 on carbohydrates, compared with that of R. microsporus var. oligosporus NRRL 2710 (= CBS 338.62 = ATCC 22959 = IMI 174457 = IFO 8631 = Scholer's M140), presented differences suggesting that strain spT3 was not a R. microsporus var. oligosporus. As a matter of fact, strain spT3 hydrolyzed sucrose and raffinose, whereas R. microsporus var. oligosporus NRRL 2710 did not. A taxonomic study including morphology, growth temperature, and mating showed that the strain spT3 was similar to R. microsporus var. chinensis CBS 261.28 (= Scholer's M213).Key words: Rhizopus microsporus var. oligosporus, Rhizopus microsporus var. chinensis, Rhizopus microsporus var. rhizopodiformis, mating, carbohydrate utilization, taxonomy, scanning electron microscopy.

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18

Lloyd, Amanda, Katie Pratt, Erica Siebrasse, Matthew D. Moran e Andrea A. Duina. "Uncoupling of the Patterns of Chromatin Association of Different Transcription Elongation Factors by a Histone H3 Mutant in Saccharomyces cerevisiae". Eukaryotic Cell 8, n. 2 (1 dicembre 2008): 257–60. http://dx.doi.org/10.1128/ec.00348-08.

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ABSTRACT The transcription elongation complexes yFACT, Spt4/Spt5, and Spt6/Iws1 were previously shown to follow similar patterns of association across transcribed genes in Saccharomyces cerevisiae. Using a histone H3 mutant, we now provide evidence that the mechanism of association of yFACT across genes is separable from that adopted by Spt4/Spt5 and Spt6/Iws1.

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19

Laprade, Lisa, David Rose e Fred Winston. "Characterization of New Spt3 and TATA-Binding Protein Mutants of Saccharomyces cerevisiae: Spt3–TBP Allele-Specific Interactions and Bypass of Spt8". Genetics 177, n. 4 (dicembre 2007): 2007–17. http://dx.doi.org/10.1534/genetics.107.081976.

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20

Yu, Yaxin, Peter Eriksson, Leena T. Bhoite e David J. Stillman. "Regulation of TATA-Binding Protein Binding by the SAGA Complex and the Nhp6 High-Mobility Group Protein". Molecular and Cellular Biology 23, n. 6 (15 marzo 2003): 1910–21. http://dx.doi.org/10.1128/mcb.23.6.1910-1921.2003.

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ABSTRACT Transcriptional activation of the yeast HO gene involves the sequential action of DNA-binding and chromatin-modifying factors. Here we examine the role of the SAGA complex and the Nhp6 architectural transcription factor in HO regulation. Our data suggest that these factors regulate binding of the TATA-binding protein (TBP) to the promoter. A gcn5 mutation, eliminating the histone acetyltransferase present in SAGA, reduces the transcription of HO, but expression is restored in a gcn5 spt3 double mutant. We conclude that the major role of Gcn5 in HO activation is to overcome repression by Spt3. Spt3 is also part of SAGA, and thus two proteins in the same regulatory complex can have opposing roles in transcriptional regulation. Chromatin immunoprecipitation experiments show that TBP binding to HO is very weak in wild-type cells but markedly increased in an spt3 mutant, indicating that Spt3 reduces HO expression by inhibiting TBP binding. In contrast, it has been shown previously that Spt3 stimulates TBP binding to the GAL1 promoter as well as GAL1 expression, and thus, Spt3 regulates these promoters differently. We also find genetic interactions between TBP and either Gcn5 or the high-mobility-group protein Nhp6, including multicopy suppression and synthetic lethality. These results suggest that, while Spt3 acts to inhibit TBP interaction with the HO promoter, Gcn5 and Nhp6 act to promote TBP binding. The result of these interactions is to limit TBP binding and HO expression to a short period within the cell cycle. Furthermore, the synthetic lethality resulting from combining a gcn5 mutation with specific TBP point mutations can be suppressed by the overexpression of transcription factor IIA (TFIIA), suggesting that histone acetylation by Gcn5 can stimulate transcription by promoting the formation of a TBP/TFIIA complex.

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21

van Oevelen, Chris J. C., Hetty A. A. M. van Teeffelen e H. T. Marc Timmers. "Differential Requirement of SAGA Subunits for Mot1p and Taf1p Recruitment in Gene Activation". Molecular and Cellular Biology 25, n. 12 (15 giugno 2005): 4863–72. http://dx.doi.org/10.1128/mcb.25.12.4863-4872.2005.

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ABSTRACT Transcription activation in yeast (Saccharomyces cerevisiae) involves ordered recruitment of transcription factor complexes, such as TFIID, SAGA, and Mot1p. Previously, we showed that both Mot1p and Taf1p are recruited to the HXT2 and HXT4 genes, which encode hexose transporter proteins. Here, we show that SAGA also binds to the HXT2 and HXT4 promoters and plays a pivotal role in the recruitment of Mot1p and Taf1p. The deletion of either SPT3 or SPT8 reduces Mot1p binding to HXT2 and HXT4. Surprisingly, the deletion of GCN5 reduces Taf1p binding to both promoters. When GCN5 is deleted in spt3Δ or spt8Δ strains, neither Mot1p nor Taf1p binds, and this results in a diminished recruitment of TATA binding protein and polymerase II to the HXT4 but not the HXT2 promoter. This is reflected by the SAGA-dependent expression of HXT4. In contrast, SAGA-independent induction of HXT2 suggests a functional redundancy with other factors. A functional interplay of different SAGA subunits with Mot1p and Taf1p was supported by phenotypic analysis of MOT1 SAGA or TAF1/SAGA double mutant strains, which revealed novel genetic interactions between MOT1 and SPT8 and between TAF1 and GCN5. In conclusion, our data demonstrate functional links between SAGA, Mot1p, and TFIID in HXT gene regulation.

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22

Barbaric, Slobodan, Hans Reinke e Wolfram Hörz. "Multiple Mechanistically Distinct Functions of SAGA at the PHO5 Promoter". Molecular and Cellular Biology 23, n. 10 (15 maggio 2003): 3468–76. http://dx.doi.org/10.1128/mcb.23.10.3468-3476.2003.

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ABSTRACT Our previous studies have shown that the rate of chromatin remodeling and consequently the rate of PHO5 activation are strongly decreased in the absence of Gcn5 histone acetyltransferase activity. Using chromatin immunoprecipitation, we demonstrate that SAGA is physically recruited to the PHO5 promoter. Recruitment is dependent on the specific activator Pho4 and occurs only under inducing conditions. Spt3, another subunit of SAGA, also plays a role in PHO5 activation but has a function that is completely different from that of Gcn5. An SPT3 deletion severely compromises the PHO5 promoter and reduces the extent of transcriptional activation by diminishing the binding of the TATA binding protein to the promoter without, however, affecting the rate or the extent of chromatin remodeling. A gcn5 spt3 double mutant shows a synthetic phenotype almost as severe as that observed for an spt7 or spt20 mutant. The latter two mutations are known to prevent the assembly of the complex and consequently lead to the loss of all SAGA functions. The absence of the Ada2 subunit causes a strong delay in chromatin remodeling and promoter activation that closely resembles the delay observed in the absence of Gcn5. A deletion of only the Ada2 SANT domain has exactly the same effect, strongly suggesting that Ada2 controls Gcn5 activity by virtue of its SANT domain. Finally, the Gcn5 bromodomain also contributes to but is not essential for Gcn5 function at the PHO5 promoter. Taken together, the results provide a detailed and differentiated description of the role of SAGA as a coactivator at the PHO5 promoter.

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23

Imhof, M. O., e D. P. McDonnell. "Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors." Molecular and Cellular Biology 16, n. 6 (giugno 1996): 2594–605. http://dx.doi.org/10.1128/mcb.16.6.2594.

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We have developed a system in Saccharomyces cerevisiae in which agonist-dependent transcriptional activity of the human progesterone receptor (hPR) is elevated to the point that it compromises cell growth. Screens for suppressors of this phenotype led to the demonstration that RSP5 is involved in hPR transactivation. Expression of RSP5 in yeast cells potentiated hPR and human glucocorticoid receptor (hGR) transcriptional activity and increased the efficacy of weak agonists of these receptors. Remarkably, expression of this yeast protein in mammalian cells had a similar effect on PR and GR transcriptional activity. Importantly, a human homolog of RSP5, hRPF1, functioned identically in mammalian cells. Previously, it has been demonstrated that RSP5 overexpression in yeast cells suppressed mutations within SPT3, a protein which interacts with the TATA-box-binding protein (TBP), suggesting that RSP5 and SPT3 operate in the same regulatory pathway. In support of this observation, we have shown that SPT3 enhances the activity of RSP5 on GR and PR when tested in yeast or mammalian cells. We conclude from these experiments that the regulatory pathways in which RSP5 and SPT3 operate in yeast cells are conserved in higher eukaryotes. Additionally, since SPT3 has been shown to contact yeast TBP directly and is the likely homolog of human TBP-associated factor TAFII18, we propose that RSP5/hRPF1 and SPT3 establish a functional link between activated PR and GR and the general transcription apparatus.

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24

Shao, Wei, Zhan Ding, Zeng-Zhang Zheng, Ji-Jia Shen, Yu-Xian Shen, Jia Pu, Yu-Jie Fan, Charles C. Query e Yong-Zhen Xu. "Prp5−Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription". Nucleic Acids Research 48, n. 11 (13 maggio 2020): 5799–813. http://dx.doi.org/10.1093/nar/gkaa311.

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Abstract Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt–Ada–Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

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25

Sterner, David E., Patrick A. Grant, Shannon M. Roberts, Laura J. Duggan, Rimma Belotserkovskaya, Lisa A. Pacella, Fred Winston, Jerry L. Workman e Shelley L. Berger. "Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction". Molecular and Cellular Biology 19, n. 1 (1 gennaio 1999): 86–98. http://dx.doi.org/10.1128/mcb.19.1.86.

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ABSTRACT SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δand gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar tospt7Δ, spt20Δ, or ada1Δmutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.

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26

Madison, Jon M., Aimée M. Dudley e Fred Winston. "Identification and Analysis of Mot3, a Zinc Finger Protein That Binds to the Retrotransposon Ty Long Terminal Repeat (δ) in Saccharomyces cerevisiae". Molecular and Cellular Biology 18, n. 4 (1 aprile 1998): 1879–90. http://dx.doi.org/10.1128/mcb.18.4.1879.

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Abstract (sommario):

ABSTRACT Spt3 and Mot1 are two transcription factors of Saccharomyces cerevisiae that are thought to act in a related fashion to control the function of TATA-binding protein (TBP). Current models suggest that while Spt3 and Mot1 do not directly interact, they do function in a related fashion to stabilize the TBP-TATA interaction at particular promoters. Consistent with this model, certain combinations of spt3 and mot1 mutations are inviable. To identify additional proteins related to Spt3 and Mot1 functions, we screened for high-copy-number suppressors of the mot1 spt3inviability. This screen identified a previously unstudied gene,MOT3, that encodes a zinc finger protein. We show that Mot3 binds in vitro to three sites within the retrotransposon Ty long terminal repeat (δ) sequence. One of these sites is immediately 5′ of the δ TATA region. Although a mot3 null mutation causes no strong phenotypes, it does cause some mild phenotypes, including a very modest increase in Ty mRNA levels, partial suppression of transcriptional defects caused by a mot1 mutation, and partial suppression of an spt3 mutation. These results, in conjunction with those of an independent study of Mot3 (A. Grishin, M. Rothenberg, M. A. Downs, and K. J. Blumer, Genetics, in press), suggest that this protein plays a varied role in gene expression that may be largely redundant with other factors.

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27

Lindstrom, D. L., S. L. Squazzo, N. Muster, T. A. Burckin, K. C. Wachter, C. A. Emigh, J. A. McCleery, J. R. Yates e G. A. Hartzog. "Dual Roles for Spt5 in Pre-mRNA Processing and Transcription Elongation Revealed by Identification of Spt5-Associated Proteins". Molecular and Cellular Biology 23, n. 4 (15 febbraio 2003): 1368–78. http://dx.doi.org/10.1128/mcb.23.4.1368-1378.2003.

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ABSTRACT During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.

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28

Liu, Xiaohui, Marina Vorontchikhina, Yuan-Liang Wang, Francesco Faiola e Ernest Martinez. "STAGA Recruits Mediator to the MYC Oncoprotein To Stimulate Transcription and Cell Proliferation". Molecular and Cellular Biology 28, n. 1 (27 ottobre 2007): 108–21. http://dx.doi.org/10.1128/mcb.01402-07.

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ABSTRACT Activation of eukaryotic gene transcription involves the recruitment by DNA-binding activators of multiprotein histone acetyltransferase (HAT) and Mediator complexes. How these coactivator complexes functionally cooperate and the roles of the different subunits/modules remain unclear. Here we report physical interactions between the human HAT complex STAGA (SPT3-TAF9-GCN5-acetylase) and a “core” form of the Mediator complex during transcription activation by the MYC oncoprotein. Knockdown of the STAF65γ component of STAGA in human cells prevents the stable association of TRRAP and GCN5 with the SPT3 and TAF9 subunits; impairs transcription of MYC-dependent genes, including MYC transactivation of the telomerase reverse transcriptase (TERT) promoter; and inhibits proliferation of MYC-dependent cells. STAF65γ is required for SPT3/STAGA interaction with core Mediator and for MYC recruitment of SPT3, TAF9, and core Mediator components to the TERT promoter but is dispensable for MYC recruitment of TRRAP, GCN5, and p300 and for acetylation of nucleosomes and loading of TFIID and RNA polymerase II on the promoter. These results suggest a novel STAF65γ-dependent function of STAGA-type complexes in cell proliferation and transcription activation by MYC postloading of TFIID and RNA polymerase II that involves direct recruitment of core Mediator.

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29

Stebbins, John L., e Steven J. Triezenberg. "Identification, Mutational Analysis, and Coactivator Requirements of Two Distinct Transcriptional Activation Domains of the Saccharomyces cerevisiae Hap4 Protein". Eukaryotic Cell 3, n. 2 (aprile 2004): 339–47. http://dx.doi.org/10.1128/ec.3.2.339-347.2004.

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ABSTRACT The Hap4 protein of the budding yeast Saccharomyces cerevisiae activates the transcription of genes that are required for growth on nonfermentable carbon sources. Previous reports suggested the presence of a transcriptional activation domain within the carboxyl-terminal half of Hap4 that can function in the absence of Gcn5, a transcriptional coactivator protein and histone acetyltransferase. The boundaries of this activation domain were further defined to a region encompassing amino acids 359 to 476. Within this region, several clusters of hydrophobic amino acids are critical for transcriptional activity. This activity does not require GCN5 or two other components of the SAGA coactivator complex, SPT3 and SPT8, but it does require SPT7 and SPT20. Contrary to previous reports, a Hap4 fragment comprising amino acids 1 to 330 can support the growth of yeast on lactate medium, and when tethered to lexA, can activate a reporter gene with upstream lexA binding sites, demonstrating the presence of a second transcriptional activation domain. In contrast to the C-terminal activation domain, the transcriptional activity of this N-terminal region depends on GCN5. We conclude that the yeast Hap4 protein has at least two transcriptional activation domains with strikingly different levels of dependence on specific transcriptional coactivator proteins.

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30

Schilling, Silke, e Christine Oesterhelt. "Structurally reduced monosaccharide transporters in an evolutionarily conserved red alga". Biochemical Journal 406, n. 2 (13 agosto 2007): 325–31. http://dx.doi.org/10.1042/bj20070448.

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The unicellular red alga Galdieria sulphuraria is a facultative heterotrophic member of the Cyanidiaceae, a group of evolutionary highly conserved extremophilic red algae. Uptake of various sugars and polyols is accomplished by a large number of distinct plasma membrane transporters. We have cloned three transporters [GsSPT1 (G. sulphuraria sugar and polyol transporter 1), GsSPT2 and GsSPT4], followed their transcriptional regulation and assayed their transport capacities in the heterologous yeast system. SPT1 is a conserved type of sugar/H+ symporter with 12 predicted transmembrane-spanning domains, whereas SPT2 and SPT4 represent monosaccharide transporters, characterized by only nine hydrophobic domains. Surprisingly, all three proteins are functional plasma membrane transporters, as demonstrated by genetic complementation of a sugar uptake-deficient yeast mutant. Substrate specificities were broad and largely redundant, except for glucose, which was only taken up by SPT1. Comparison of SPT1 and truncated SPT1(Δ1–3) indicated that the N-terminus of the protein is not required for sugar transport or substrate recognition. However, its deletion affected substrate affinity as well as maximal transport velocity and released the pH dependency of sugar uptake. In line with these results, uptake by SPT2 and SPT4 was active but not pH-dependent, making a H+ symport mechanism unlikely for the truncated proteins. We postulate SPT2 and SPT4 as functional plasma membrane transporters in G. sulphuraria. Most likely, they originated from genes encoding active monosaccharide/H+ symporters with 12 transmembrane-spanning domains.

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Biswas, Debabrata, Yaxin Yu, Matthew Prall, Tim Formosa e David J. Stillman. "The Yeast FACT Complex Has a Role in Transcriptional Initiation". Molecular and Cellular Biology 25, n. 14 (luglio 2005): 5812–22. http://dx.doi.org/10.1128/mcb.25.14.5812-5822.2005.

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Abstract (sommario):

ABSTRACT A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.

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32

Gao, Tao, Zhitian Zheng, Yiping Hou e Mingguo Zhou. "Transcription factors spt3 and spt8 are associated with conidiation, mycelium growth, and pathogenicity inFusarium graminearum". FEMS Microbiology Letters 351, n. 1 (19 dicembre 2013): 42–50. http://dx.doi.org/10.1111/1574-6968.12350.

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33

Krogan, Nevan J., Minkyu Kim, Seong Hoon Ahn, Guoqing Zhong, Michael S. Kobor, Gerard Cagney, Andrew Emili, Ali Shilatifard, Stephen Buratowski e Jack F. Greenblatt. "RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach". Molecular and Cellular Biology 22, n. 20 (15 ottobre 2002): 6979–92. http://dx.doi.org/10.1128/mcb.22.20.6979-6992.2002.

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Abstract (sommario):

ABSTRACT To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3′ end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

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Lee, Pam, Hong Liu e Scott Filler. "2599. Studying the Effects of Altering Histone Modification on Aspergillus fumigatus Virulence". Open Forum Infectious Diseases 6, Supplement_2 (ottobre 2019): S903. http://dx.doi.org/10.1093/ofid/ofz360.2277.

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Abstract Background As there are few drugs for treating invasive aspergillosis, there is an urgent need for new antifungal agents. Enzymes involved in histone modification are possible antifungal drug targets. We set out to investigate whether genes whose products are involved in histone modifications influence the virulence of Aspergillus fumigatus (Af). Methods Genes whose products were likely involved in histone modification were deleted in strain Af293 using CRISPR-Cas9. Virulence was assessed in a triamcinolone-treated mouse model of invasive pulmonary aspergillosis. The extent of Af-induced damage to the A549 pulmonary epithelial cell line was determined by Cr51 release assay. Results Af genes were selected for investigation based on their homology to genes encoding known histone modifying proteins and their high expression level in vivo. The genes were predicted to encode members of the COMPASS histone methyltransferase complex (cclA/bre2, set2/Afu5g06000), the SAGA histone acetyltransferase complex (spt3, spt8), and the RPDL histone deacetylase complex (hosA). The ΔcclA and Δset2 mutants had significant growth defects on rich media and were not tested further. The Δspt3 and Δspt8 mutants grew normally and had mild conidiation defects. The ΔhosA mutant had wild-type (WT) growth and conidiation in vitro. Mice infected with the WT strain had 100% mortality within 9 days whereas mice infected the Δspt3, Δspt8, and ΔhosA mutants had only 40% mortality by 21 days. The ΔhosA mutant also had impaired capacity to damage pulmonary epithelial cells in vitro. Conclusion Ccla and Set2, components of the COMPASS complex, are required for normal growth in vitro. Spt3 and Spt8, members of the SAGA complex, are required for normal conidiation and virulence. HosA, part of the RPD3L complex, is necessary for maximal virulence and induction of host cell damage. Our results suggest that the HosA histone deacetylase may be a promising drug target for treating invasive aspergillosis. Disclosures All authors: No reported disclosures.

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35

Giesbert, S., J. Schumacher, V. Kupas, J. Espino, N. Segmüller, I. Haeuser-Hahn, P. H. Schreier e P. Tudzynski. "Identification of Pathogenesis-Associated Genes by T-DNA–Mediated Insertional Mutagenesis in Botrytis cinerea: A Type 2A Phosphoprotein Phosphatase and an SPT3 Transcription Factor Have Significant Impact on Virulence". Molecular Plant-Microbe Interactions® 25, n. 4 (aprile 2012): 481–95. http://dx.doi.org/10.1094/mpmi-07-11-0199.

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Abstract (sommario):

Agrobacterium tumefaciens–mediated transformation (ATMT) was used to generate an insertional mutant library of the gray mold fungus Botrytis cinerea. From a total of 2,367 transformants, 68 mutants showing significant reduction in virulence on tomato and bean plants were analyzed in detail. As reported for other fungal ATMT libraries, integrations were mostly single copy, occurred preferentially in noncoding (regulatory) regions, and were frequently accompanied by small deletions of the target sequences and loss of parts of the border sequence. Two T-DNA integration events that were found to be linked to virulence were characterized in more detail: a catalytic subunit of a PP2A serine/threonine protein phosphatase (BcPP2Ac) and the SPT3 subunit of a Spt-Ada-Gcn5-acetyltransferase (SAGA-like) transcriptional regulator complex. Gene replacement and silencing approaches revealed that both Bcpp2Ac and SPT3 are crucial for virulence, growth, and differentiation as well as for resistance to H2O2 in B. cinerea.

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Mitra, Doyel, Emily J. Parnell, Jack W. Landon, Yaxin Yu e David J. Stillman. "SWI/SNF Binding to the HO Promoter Requires Histone Acetylation and Stimulates TATA-Binding Protein Recruitment". Molecular and Cellular Biology 26, n. 11 (1 giugno 2006): 4095–110. http://dx.doi.org/10.1128/mcb.01849-05.

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Abstract (sommario):

ABSTRACT We use chromatin immunoprecipitation assays to show that the Gcn5 histone acetyltransferase in SAGA is required for SWI/SNF association with the HO promoter and that binding of SWI/SNF and SAGA are interdependent. Previous results showed that SWI/SNF binding to HO was Gcn5 independent, but that work used a strain with a mutation in the Ash1 daughter-specific repressor of HO expression. Here, we show that Ash1 functions as a repressor that inhibits SWI/SNF binding and that Gcn5 is required to overcome Ash1 repression in mother cells to allow HO transcription. Thus, Gcn5 facilitates SWI/SNF binding by antagonizing Ash1. Similarly, a mutation in SIN3, like an ash1 mutation, allows both HO expression and SWI/SNF binding in the absence of Gcn5. Although Ash1 has recently been identified in a Sin3-Rpd3 complex, our genetic analysis shows that Ash1 and Sin3 have distinct functions in regulating HO. Analysis of mutant strains shows that SWI/SNF binding and HO expression are correlated and regulated by histone acetylation. The defect in HO expression caused by a mutant SWI/SNF with a Swi2(E834K) substitution can be partially suppressed by ash1 or spt3 mutation or by a gain-of-function V71E substitution in the TATA-binding protein (TBP). Spt3 inhibits TBP binding at HO, and genetic analysis suggests that Spt3 and TBP(V71E) act in the same pathway, distinct from that of Ash1. We have detected SWI/SNF binding at the HO TATA region, and our results suggest that SWI/SNF, either directly or indirectly, facilitates TBP binding at HO.

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Prather, Donald, Nevan J. Krogan, Andrew Emili, Jack F. Greenblatt e Fred Winston. "Identification and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae". Molecular and Cellular Biology 25, n. 22 (15 novembre 2005): 10122–35. http://dx.doi.org/10.1128/mcb.25.22.10122-10135.2005.

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ABSTRACT In order to identify previously unknown transcription elongation factors, a genetic screen was carried out to identify mutations that cause lethality when combined with mutations in the genes encoding the elongation factors TFIIS and Spt6. This screen identified a mutation in YKL160W, hereafter named ELF1 (elongation factor 1). Further analysis identified synthetic lethality between an elf1Δ mutation and mutations in genes encoding several known elongation factors, including Spt4, Spt5, Spt6, and members of the Paf1 complex. Genome-wide synthetic lethality studies confirmed that elf1Δ specifically interacts with mutations in genes affecting transcription elongation. Chromatin immunoprecipitation experiments show that Elf1 is cotranscriptionally recruited over actively transcribed regions and that this association is partially dependent on Spt4 and Spt6. Analysis of elf1Δ mutants suggests a role for this factor in maintaining proper chromatin structure in regions of active transcription. Finally, purification of Elf1 suggests an association with casein kinase II, previously implicated in roles in transcription. Together, these results suggest an important role for Elf1 in the regulation of transcription elongation.

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38

Endoh, Masaki, Wenyan Zhu, Jun Hasegawa, Hajime Watanabe, Dong-Ki Kim, Masatoshi Aida, Naoto Inukai et al. "Human Spt6 Stimulates Transcription Elongation by RNA Polymerase II In Vitro". Molecular and Cellular Biology 24, n. 8 (15 aprile 2004): 3324–36. http://dx.doi.org/10.1128/mcb.24.8.3324-3336.2004.

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Abstract (sommario):

ABSTRACT Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

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39

Périer, F., e J. Carbon. "A colony color assay for Saccharomyces cerevisiae mutants defective in kinetochore structure and function." Genetics 132, n. 1 (1 settembre 1992): 39–51. http://dx.doi.org/10.1093/genetics/132.1.39.

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Abstract We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae). The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter. Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct. A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the "pseudo-operator" led to the identification of mutations (blu) in three complementation groups. The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro. The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes. Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins.

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40

Eisenmann, D. M., K. M. Arndt, S. L. Ricupero, J. W. Rooney e F. Winston. "SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae." Genes & Development 6, n. 7 (1 luglio 1992): 1319–31. http://dx.doi.org/10.1101/gad.6.7.1319.

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Topalidou, Irini, Manolis Papamichos-Chronakis, George Thireos e Dimitris Tzamarias. "Spt3 and Mot1 cooperate in nucleosome remodeling independently of TBP recruitment". EMBO Journal 23, n. 9 (1 aprile 2004): 1943–48. http://dx.doi.org/10.1038/sj.emboj.7600199.

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42

Yu, Jianming, Jon M. Madison, Stephan Mundlos, Fred Winston e Bjorn R. Olsen. "Characterization of a Human Homologue of theSaccharomyces cerevisiaeTranscription Factor Spt3 (SUPT3H)". Genomics 53, n. 1 (ottobre 1998): 90–96. http://dx.doi.org/10.1006/geno.1998.5500.

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43

Larschan, Erica, e Fred Winston. "The Saccharomyces cerevisiae Srb8-Srb11 Complex Functions with the SAGA Complex during Gal4-Activated Transcription". Molecular and Cellular Biology 25, n. 1 (1 gennaio 2005): 114–23. http://dx.doi.org/10.1128/mcb.25.1.114-123.2005.

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ABSTRACT The Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex functions as a coactivator during Gal4-activated transcription. A functional interaction between the SAGA component Spt3 and TATA-binding protein (TBP) is important for TBP binding at Gal4-activated promoters. To better understand the role of SAGA and other factors in Gal4-activated transcription, we selected for suppressors that bypass the requirement for SAGA. We obtained eight complementation groups and identified the genes corresponding to three of the groups as NHP10, HDA1, and SRB9. In contrast to the srb9 suppressor mutation that we identified, an srb9Δ mutation causes a strong defect in Gal4-activated transcription. Our studies have focused on this requirement for Srb9. Srb9 is part of the Srb8-Srb11 complex, associated with the Mediator coactivator. Srb8-Srb11 contains the Srb10 kinase, whose activity is important for GAL1 transcription. Our data suggest that Srb8-Srb11, including Srb10 kinase activity, is directly involved in Gal4 activation. By chromatin immunoprecipitation studies, Srb9 is present at the GAL1 promoter upon induction and facilitates the recruitment or stable association of TBP. Furthermore, the association of Srb9 with the GAL1 upstream activation sequence requires SAGA and specifically Spt3. Finally, Srb9 association also requires TBP. These results suggest that Srb8-Srb11 associates with the GAL1 promoter subsequent to SAGA binding, and that the binding of TBP and Srb8-Srb11 is interdependent.

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44

Madison, Jon M., e Fred Winston. "Identification and analysis of homologues ofSaccharomyces cerevisiae Spt3 suggest conserved functional domains". Yeast 14, n. 5 (30 marzo 1998): 409–17. http://dx.doi.org/10.1002/(sici)1097-0061(19980330)14:5<409::aid-yea237>3.0.co;2-x.

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45

Ivanov, Dmitri, Youn Tae Kwak, Jun Guo e Richard B. Gaynor. "Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties". Molecular and Cellular Biology 20, n. 9 (1 maggio 2000): 2970–83. http://dx.doi.org/10.1128/mcb.20.9.2970-2983.2000.

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ABSTRACT SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.

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46

Trosok, Steve P., John H. T. Luong, David F. Juck e Brian T. Driscoll. "Characterization of two novel yeast strains used in mediated biosensors for wastewater". Canadian Journal of Microbiology 48, n. 5 (1 maggio 2002): 418–26. http://dx.doi.org/10.1139/w02-035.

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After isolation from a pulp mill wastewater treatment facility, two yeast strains, designated SPT1 and SPT2, were characterized and used in the development of mediated biochemical oxygen demand (BOD) biosensors for wastewater. 18S rRNA gene sequence analysis revealed a one nucleotide difference between the sequence of SPT1 and those of Candida sojae and Candida viswanthii. While SPT2 had the highest overall homology to Pichia norvegensis, at only 73.5%, it is clearly an ascomycete, based on BLAST comparisons and phylogenetic analyses. Neighbor-joining dendrograms indicated that SPT1 clustered with several Candida spp., and that SPT2 clustered with Starmera spp., albeit as a very deep branch. Physiological tests, microscopic observations, and fatty acid analysis confirmed that SPT1 and SPT2 are novel yeast strains. Physiological tests also indicated that both strains had potential for use in mediated biosensors for estimation of BOD in wastewater. The lower detection limits of SPT1- and SPT2-based K3Fe(CN)6-mediated biosensors for a pulp-mill effluent were 2 and 1 mg BOD/L, respectively. Biosensor-response times for effluents from eight different pulp mills were in the range of 5 min. Reliability and sensitivity of the SPT1- and SPT2-based biosensors were good, but varied with the wastewater.Key words: yeast characterization, 18S rRNA gene sequence, pulp-mill wastewater, BOD5, mediated BOD biosensor.

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47

Compagnone-Post, Patricia A., e Mary Ann Osley. "Mutations in the SPT4, SPT5, and SPT6 Genes Alter Transcription of a Subset of Histone Genes in Saccharomyces cerevisiae". Genetics 143, n. 4 (1 agosto 1996): 1543–54. http://dx.doi.org/10.1093/genetics/143.4.1543.

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Abstract The SPT4, SPT5, and SPT6 gene products define a class of transcriptional repressors in Saccharomyces cermisiae that are thought to function through their effects on chromatin assembly or stability. Mutations in these genes confer a similar range of phenotypes to mutations in HIR genes, which encode transcriptional repressors that regulate expression of many of the core histone genes. Here we show that mutations in the three SPTgenes also affect transcription of the histone genes that reside at the HTA1-HTB1 locus. HTA1-lacZ transcription was reduced in each spt mutant background, an effect that required a negative site in the HTA1 promoter. The transcriptional effect could be reversed by the overproduction of histones H2A and H2B in an spt4 mutant and histones H3 and H4 in all three spt mutants. Suppression of the spt4 transcriptional defect was dependent on the overproduction of both histones H2A and H2B, and required the presence of N-terminal amino acids in both histones. The results are consistent with the idea that the effects of the spt mutations on nucleosome assembly and/or stability activate repressors of HTA1 transcription.

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Ingvarsdottir, Kristin, Nevan J. Krogan, N. C. Tolga Emre, Anastasia Wyce, Natalie J. Thompson, Andrew Emili, Timothy R. Hughes, Jack F. Greenblatt e Shelley L. Berger. "H2B Ubiquitin Protease Ubp8 and Sgf11 Constitute a Discrete Functional Module within the Saccharomyces cerevisiae SAGA Complex". Molecular and Cellular Biology 25, n. 3 (1 febbraio 2005): 1162–72. http://dx.doi.org/10.1128/mcb.25.3.1162-1172.2005.

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ABSTRACT The SAGA complex is a multisubunit protein complex involved in transcriptional regulation in Saccharomyces cerevisiae. SAGA combines proteins involved in interactions with DNA-bound activators and TATA-binding protein (TBP), as well as enzymes for histone acetylation (Gcn5) and histone deubiquitylation (Ubp8). We recently showed that H2B ubiquitylation and Ubp8-mediated deubiquitylation are both required for transcriptional activation. For this study, we investigated the interaction of Ubp8 with SAGA. Using mutagenesis, we identified a putative zinc (Zn) binding domain within Ubp8 as being critical for the association with SAGA. The Zn binding domain is required for H2B deubiquitylation and for growth on media requiring Ubp8's function in gene activation. Furthermore, we identified an 11-kDa subunit of SAGA, Sgf11, and showed that it is required for the Ubp8 association with SAGA and for H2B deubiquitylation. Different approaches indicated that the functions of Ubp8 and Sgf11 are related and separable from those of other components of SAGA. In particular, the profiles of Ubp8 and Sgf11 deletions were remarkably similar in microarray analyses and synthetic genetic interactions and were distinct from those of the Spt3 and Spt8 subunits of SAGA, which are involved in TBP regulation. These data indicate that Ubp8 and Sgf11 likely represent a new functional module within SAGA that is involved in gene regulation through H2B deubiquitylation.

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49

Batheja, Ameesha D., David J. Uhlinger, Jill M. Carton, George Ho e Michael R. D'Andrea. "Characterization of Serine Palmitoyltransferase in Normal Human Tissues". Journal of Histochemistry & Cytochemistry 51, n. 5 (maggio 2003): 687–96. http://dx.doi.org/10.1177/002215540305100514.

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Sphingolipids serve as structural elements of cells and as lipid second messengers. They regulate cellular homeostasis, mitogenesis, and apoptosis. Sphingolipid signaling may also be important in various pathophysiologies such as vascular injury, inflammation, and cancer. Serine palmitoyltransferase (SPT) catalyzes the condensation of serine with palmitoyl-CoA, the first, rate-limiting step in de novo sphingolipid biosynthesis. This integral microsomal membrane protein consists of at least two subunits, SPT1 and SPT2. In this study we analyzed the expression of SPT1 and SPT2 in normal human tissues. Strong SPT1 and SPT2 expression was observed in pyramidal neurons in the brain, in colon epithelium, and in mucosal macrophages. However, SPT2 expression was more prominent than SPT1 in the colon mucosal macrophages, the adrenomedullary chromaffin cells and endothelium, and in the uterine endothelium. SPT2 was localized in both nuclei and cytoplasm of the adrenomedullary chromaffin cells, whereas SPT1 was primarily cytoplasmic. These observations link enhanced SPT expression to proliferating cells, such as the lung, stomach, intestinal epithelium, and renal proximal tubular epithelium, and to potentially activated cells such as neurons, chromaffin cells, and mucosal macrophages. A baseline expression of SPT, established by this study, may serve as a measure for aberrant expression in various disease states.

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50

Meyer, Peter A., Sheng Li, Mincheng Zhang, Kentaro Yamada, Yuichiro Takagi, Grant A. Hartzog e Jianhua Fu. "Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5". Molecular and Cellular Biology 35, n. 19 (27 luglio 2015): 3354–69. http://dx.doi.org/10.1128/mcb.00520-15.

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The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) ofSaccharomyces cerevisiaeSpt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acidsin vitro, as shown in biochemical assays, and is important forin vivofunction, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation.

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