Journal articles: 'Spt8'

1

Winston, Fred, Catherine Dollard, Elizabeth A. Malone, Jeffrey Clare, James G. Kapakos, Philip Farabaugh, and Patricia L. Minehart. "Three Genes Are Required for trans-Activation of Ty Transcription in Yeast." Genetics 115, no. 4 (April 1, 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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Happel, A. M., M. S. Swanson, and F. Winston. "The SNF2, SNF5 and SNF6 genes are required for Ty transcription in Saccharomyces cerevisiae." Genetics 128, no. 1 (May 1, 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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Belotserkovskaya, Rimma, David E. Sterner, Min Deng, Michael H. Sayre, Paul M. Lieberman, and Shelley L. Berger. "Inhibition of TATA-Binding Protein Function by SAGA Subunits Spt3 and Spt8 at Gcn4-Activated Promoters." Molecular and Cellular Biology 20, no. 2 (January 15, 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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Eisenmann, D. M., C. Chapon, S. M. Roberts, C. Dollard, and F. Winston. "The Saccharomyces cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein." Genetics 137, no. 3 (July 1, 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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Roberts, S. M., and 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, no. 6 (June 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

Happel, A. M., and F. Winston. "A mutant tRNA affects delta-mediated transcription in Saccharomyces cerevisiae." Genetics 132, no. 2 (October 1, 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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Sterner, David E., Patrick A. Grant, Shannon M. Roberts, Laura J. Duggan, Rimma Belotserkovskaya, Lisa A. Pacella, Fred Winston, Jerry L. Workman, and 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, no. 1 (January 1, 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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8

van Oevelen, Chris J. C., Hetty A. A. M. van Teeffelen, and H. T. Marc Timmers. "Differential Requirement of SAGA Subunits for Mot1p and Taf1p Recruitment in Gene Activation." Molecular and Cellular Biology 25, no. 12 (June 15, 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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9

Swanson, M. S., and F. Winston. "SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae." Genetics 132, no. 2 (October 1, 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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10

Shao, Wei, Zhan Ding, Zeng-Zhang Zheng, Ji-Jia Shen, Yu-Xian Shen, Jia Pu, Yu-Jie Fan, Charles C. Query, and Yong-Zhen Xu. "Prp5−Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription." Nucleic Acids Research 48, no. 11 (May 13, 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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11

Lloyd, Amanda, Katie Pratt, Erica Siebrasse, Matthew D. Moran, and 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, no. 2 (December 1, 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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12

Dudley, Aimée M., Lisa J. Gansheroff, and 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, no. 4 (April 1, 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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Wu, Pei-Yun Jenny, and Fred Winston. "Analysis of Spt7 Function in the Saccharomyces cerevisiae SAGA Coactivator Complex." Molecular and Cellular Biology 22, no. 15 (August 1, 2002): 5367–79. http://dx.doi.org/10.1128/mcb.22.15.5367-5379.2002.

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ABSTRACT The Saccharomyces cerevisiae SAGA complex is required for the normal transcription of a large number of genes. Complex integrity depends on three core subunits, Spt7, Spt20, and Ada1. We have investigated the role of Spt7 in the assembly and function of SAGA. Our results show that Spt7 is important in controlling the levels of the other core subunits and therefore of SAGA. In addition, partial SAGA complexes containing Spt7 can be assembled in the absence of both Spt20 and Ada1. Through biochemical and genetic analyses of a series of spt7 deletion mutants, we have identified a region of Spt7 required for interaction with the SAGA component Spt8. An adjacent Spt7 domain was found to be required for a processed form of Spt7 that is present in a previously identified altered form of SAGA called SLIK, SAGAalt, or SALSA. Analysis of an spt7 mutant with greatly reduced levels of SLIK/SAGAalt/SALSA suggests a subtle role for this complex in transcription that may be redundant with a subset of SAGA functions.

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Stebbins, John L., and Steven J. Triezenberg. "Identification, Mutational Analysis, and Coactivator Requirements of Two Distinct Transcriptional Activation Domains of the Saccharomyces cerevisiae Hap4 Protein." Eukaryotic Cell 3, no. 2 (April 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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Lindstrom, D. L., S. L. Squazzo, N. Muster, T. A. Burckin, K. C. Wachter, C. A. Emigh, J. A. McCleery, J. R. Yates, and 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, no. 4 (February 15, 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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16

Schilling, Silke, and Christine Oesterhelt. "Structurally reduced monosaccharide transporters in an evolutionarily conserved red alga." Biochemical Journal 406, no. 2 (August 13, 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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Lee, Pam, Hong Liu, and Scott Filler. "2599. Studying the Effects of Altering Histone Modification on Aspergillus fumigatus Virulence." Open Forum Infectious Diseases 6, Supplement_2 (October 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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Krogan, Nevan J., Minkyu Kim, Seong Hoon Ahn, Guoqing Zhong, Michael S. Kobor, Gerard Cagney, Andrew Emili, Ali Shilatifard, Stephen Buratowski, and Jack F. Greenblatt. "RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach." Molecular and Cellular Biology 22, no. 20 (October 15, 2002): 6979–92. http://dx.doi.org/10.1128/mcb.22.20.6979-6992.2002.

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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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Badarinarayana, Vasudeo, Yueh-Chin Chiang, and Clyde L. Denis. "Functional Interaction of CCR4-NOT Proteins With TATAA-Binding Protein (TBP) and Its Associated Factors in Yeast." Genetics 155, no. 3 (July 1, 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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Gao, Tao, Zhitian Zheng, Yiping Hou, and Mingguo Zhou. "Transcription factors spt3 and spt8 are associated with conidiation, mycelium growth, and pathogenicity inFusarium graminearum." FEMS Microbiology Letters 351, no. 1 (December 19, 2013): 42–50. http://dx.doi.org/10.1111/1574-6968.12350.

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Prather, Donald, Nevan J. Krogan, Andrew Emili, Jack F. Greenblatt, and Fred Winston. "Identification and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae." Molecular and Cellular Biology 25, no. 22 (November 15, 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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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, no. 8 (April 15, 2004): 3324–36. http://dx.doi.org/10.1128/mcb.24.8.3324-3336.2004.

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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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Sutton, P. R., and S. W. Liebman. "Rearrangements occurring adjacent to a single Ty1 yeast retrotransposon in the presence and absence of full-length Ty1 transcription." Genetics 131, no. 4 (August 1, 1992): 833–50. http://dx.doi.org/10.1093/genetics/131.4.833.

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Abstract The structures of two unusual deletions from the yeast Saccharomyces cerevisiae are described. These deletions extend from a single Ty1 retrotransposon to an endpoint near a repetitive tRNA(Gly) gene. The deletions suggest that unique sequences flanked by two nonidentical repetitive sequences, or bordered on only one side by a transposable element, have the potential to be mobilized in the yeast genome. Models for the formation of these two unusual deletions were tested by isolating and analyzing 32 additional unusual deletions of the CYC1 region that extend from a single Ty1 retrotransposon. Unlike the most common class of deletions recovered in this region, these deletions are not attributable solely to homologous recombination among repetitive Ty1 or delta elements. They arose by two distinct mechanisms. In an SPT8 genetic background, most unusual deletions arose by transposition of a Ty1 element to a position adjacent to a tRNA(Gly) gene followed by Ty1-Ty1 recombination. In an spt8 strain, where full-length Ty1 transcription and, therefore, transposition are reduced, most deletions were due to gene conversion of a 7-kb chromosomal interval flanked by a Ty1 element and a tRNA(Gly) gene.

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Laprade, Lisa, David Rose, and 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, no. 4 (December 2007): 2007–17. http://dx.doi.org/10.1534/genetics.107.081976.

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25

Ingvarsdottir, Kristin, Nevan J. Krogan, N. C. Tolga Emre, Anastasia Wyce, Natalie J. Thompson, Andrew Emili, Timothy R. Hughes, Jack F. Greenblatt, and 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, no. 3 (February 1, 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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Ivanov, Dmitri, Youn Tae Kwak, Jun Guo, and Richard B. Gaynor. "Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties." Molecular and Cellular Biology 20, no. 9 (May 1, 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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Trosok, Steve P., John H. T. Luong, David F. Juck, and Brian T. Driscoll. "Characterization of two novel yeast strains used in mediated biosensors for wastewater." Canadian Journal of Microbiology 48, no. 5 (May 1, 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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Compagnone-Post, Patricia A., and Mary Ann Osley. "Mutations in the SPT4, SPT5, and SPT6 Genes Alter Transcription of a Subset of Histone Genes in Saccharomyces cerevisiae." Genetics 143, no. 4 (August 1, 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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Batheja, Ameesha D., David J. Uhlinger, Jill M. Carton, George Ho, and Michael R. D'Andrea. "Characterization of Serine Palmitoyltransferase in Normal Human Tissues." Journal of Histochemistry & Cytochemistry 51, no. 5 (May 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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Meyer, Peter A., Sheng Li, Mincheng Zhang, Kentaro Yamada, Yuichiro Takagi, Grant A. Hartzog, and Jianhua Fu. "Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5." Molecular and Cellular Biology 35, no. 19 (July 27, 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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Keegan, Brian R., Jessica L. Feldman, Diana H. Lee, David S. Koos, Robert K. Ho, Didier Y. R. Stainier, and Deborah Yelon. "The elongation factors Pandora/Spt6 and Foggy/Spt5 promote transcription in the zebrafish embryo." Development 129, no. 7 (April 1, 2002): 1623–32. http://dx.doi.org/10.1242/dev.129.7.1623.

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Precise temporal and spatial control of transcription is a fundamental component of embryonic development. Regulation of transcription elongation can act as a rate-limiting step during mRNA synthesis. The mechanisms of stimulation and repression of transcription elongation during development are not yet understood. We have identified a class of zebrafish mutations (pandora, sk8 and s30) that cause multiple developmental defects, including discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. We demonstrate that the pandora gene encodes a protein similar to Spt6, a proposed transcription elongation factor. Additionally, the sk8 and s30 mutations are null alleles of the foggy/spt5 locus, which encodes another transcription elongation factor. Through real-time RT-PCR analysis, we demonstrate that Spt6 and Spt5 are both required for efficient kinetics of hsp70 transcription in vivo. Altogether, our results suggest that Spt6 and Spt5 play essential roles of comparable importance for promoting transcription during embryogenesis. This study provides the first genetic evidence for parallel functions of Spt6 and Spt5 in metazoans and establishes a system for the future analysis of transcription elongation during development. Supplemental figure available on-line

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Swanson, M. S., E. A. Malone, and F. Winston. "SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat." Molecular and Cellular Biology 11, no. 6 (June 1991): 3009–19. http://dx.doi.org/10.1128/mcb.11.6.3009-3019.1991.

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Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae.

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Swanson, M. S., E. A. Malone, and F. Winston. "SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat." Molecular and Cellular Biology 11, no. 6 (June 1991): 3009–19. http://dx.doi.org/10.1128/mcb.11.6.3009.

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Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae.

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Owsian, Dawid, Julita Gruchota, Olivier Arnaiz, and Jacek K. Nowak. "The transient Spt4-Spt5 complex as an upstream regulator of non-coding RNAs during development." Nucleic Acids Research 50, no. 5 (February 21, 2022): 2603–20. http://dx.doi.org/10.1093/nar/gkac106.

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Abstract The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.

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Soegiarto, Gatot, David Kurnia, Chairul Effendi, and Putu Gedhe Konthen. "CETIRIZINE SUPPRESSION TO SKIN PRICK TEST RESULTS IN ATOPIC ALLERGY PATIENTS." Folia Medica Indonesiana 53, no. 2 (November 3, 2017): 152. http://dx.doi.org/10.20473/fmi.v53i2.6432.

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This study was done to determine the suppression index of Cetirizine to the skin prick test results to obtain a correction constant or factor that can be used to assess the results of the skin prick test in patients who cannot stop the use of antihistamines (Cetirizine). This pre and post test study design clinical trial involved 22 atopic allergy patients who seek medical treatment at the Allergy and Immunology Outpatient Clinic Dr. Soetomo Hospital. Skin prick tests were done twice (SPT1 and SPT2) using house dust mite allergen extract to all study subjecs. The first (SPT1) were done after washout of all antihistamine for 1 week prior the test. All study subjects were then given Cetirizine 10 mg once daily for 5 days and on day 6 we performed the second test (SPT2). Cetirizine suppresion index and correction factor were calculated by comparing the wheal area of SPT1 and SPT2. All 22 study subjects (6 males and 16 females) were sensitized to house dust mite allergen. Mean serum total IgE levels were 176.42 + 352.5 IU/dL. Mean wheal area generated by the positive control (histamine 1 mg/mL) in SPT1 was 7.53 + 7.31 mm2, and in SPT2 was 1.08 + 1.46 mm2. Mean wheal area generated by house dust mite allergen in SPT1 was 43.57 + 36 mm2, and in SPT2 was 10.28 + 8.47 mm2. Cetirizine suppression index for positive controls (histamine 10 mg/mL) was 94.63 + 7.90% (p=0.000), while the Cetirizine suppression index for house dust mite allergen is 72.31 + 13.96% (p=0.000). There was no significant influence of serum total IgE levels to Cetirizine suppression index (p=0.381). The correction constant based on the calculation was 1.9. In conclusion, Cetirizine suppression index to the mean wheals area generated by house dust mite allergen was 72.31% and the correction constant was 1.9. In allergic patients who cannot stop their antihistamine drugs, Cetirizine 10 mg once daily can be used as a replacement and they still be able to undergo skin prick tests. The actual wheal diameter (or area) of the skin prick test results can be calculated by multiplying the measured wheal diameter (or area) under the Cetirizine administration with the correction constant.

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Malagón, Francisco, and Andrés Aguilera. "Yeastspt6-140Mutation, Affecting Chromatin and Transcription, Preferentially Increases Recombination in Which Rad51p-Mediated Strand Exchange Is Dispensable." Genetics 158, no. 2 (June 1, 2001): 597–611. http://dx.doi.org/10.1093/genetics/158.2.597.

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AbstractWe have shown that the spt6-140 and spt4-3 mutations, affecting chromatin structure and transcription, stimulate recombination between inverted repeats by a RAD52-dependent mechanism that is very efficient in the absence of RAD51, RAD54, RAD55, and RAD57. Such a mechanism of recombination is RAD1-RAD59-dependent and yields gene conversions highly associated with the inversion of the repeat. The spt6-140 mutation alters transcription and chromatin in our inverted repeats, as determined by Northern and micrococcal nuclease sensitivity analyses, respectively. Hyper-recombination levels are diminished in the absence of transcription. We believe that the chromatin alteration, together with transcription impairment caused by spt6-140, increases the incidence of spontaneous recombination regardless of whether or not it is mediated by Rad51p-dependent strand exchange. Our results suggest that spt6, as well as spt4, primarily stimulates a mechanism of break-induced replication. We discuss the possibility that the chromatin alteration caused by spt6-140 facilitates a Rad52p-mediated one-ended strand invasion event, possibly inefficient in wild-type chromatin. Our results are consistent with the idea that the major mechanism leading to inversions might not be crossing over but break-induced replication followed by single-strand annealing.

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DeSilva, Heshani, Kenneth Lee, and Mary Ann Osley. "Functional Dissection of Yeast Hir1p, a WD Repeat–Containing Transcriptional Corepressor." Genetics 148, no. 2 (February 1, 1998): 657–67. http://dx.doi.org/10.1093/genetics/148.2.657.

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Abstract The HIR1 gene product is required to repress transcription of three of the four histone gene loci in Saccharomyces cerevisiae, and like its counterpart, the HIR2 protein, it functions as a transcriptional corepressor. Although Hir1p and Hir2p are physically associated in yeast, Hir1p is able to function independently of Hir2p when it is artificially recruited to the histone HTA1 promoter. A deletion analysis of HIR1 has revealed two separate repression domains: one in its N terminus, where seven copies of the β-transducin or WD40 motif reside, and the second in the remaining C-terminal amino acids. Overexpression of the WD repeats in a hir1Δ strain complemented its Hir− phenotype, while overexpression of the C terminus in a wild-type strain caused both Hir− and Spt− phenotypes. The Hir1p C terminus physically interacted in vivo with Hir2p, and both Hir1p repression domains interacted with full-length Hir1p. It was additionally found that the Hir1p WD repeats functionally interacted with the SPT4, SPT5, and SPT6 gene products, suggesting that these repeats may direct Hir1p to different protein complexes.

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Batta, Gyula, Zsolt Szilagyi, Miklos Laczik, and Matthias Sipiczki. "The involvement of âtheSchizosaccharomyces pombe sep9/spt8+gene in the regulation of septum cleavage." FEMS Yeast Research 9, no. 5 (August 2009): 757–67. http://dx.doi.org/10.1111/j.1567-1364.2009.00522.x.

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Sermwittayawong, Decha, and Song Tan. "SAGA binds TBP via its Spt8 subunit in competition with DNA: implications for TBP recruitment." EMBO Journal 25, no. 16 (August 3, 2006): 3791–800. http://dx.doi.org/10.1038/sj.emboj.7601265.

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40

Lindstrom, Derek L., and Grant A. Hartzog. "Genetic Interactions of Spt4-Spt5 and TFIIS With the RNA Polymerase II CTD and CTD Modifying Enzymes in Saccharomyces cerevisiae." Genetics 159, no. 2 (October 1, 2001): 487–97. http://dx.doi.org/10.1093/genetics/159.2.487.

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Abstract Genetic and biochemical studies have identified many factors thought to be important for transcription elongation. We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD. Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an spt16 mutation did not show these interactions. These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function.

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Engel, Krysta L., Sarah L. French, Olga V. Viktorovskaya, Ann L. Beyer, and David A. Schneider. "Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I." Molecular and Cellular Biology 35, no. 13 (April 27, 2015): 2321–31. http://dx.doi.org/10.1128/mcb.01499-14.

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Spt6 (suppressor ofTy6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele,spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression ofRRN3rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA.

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Duan, Mingrui, Kathiresan Selvam, John J. Wyrick, and Peng Mao. "Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin." Proceedings of the National Academy of Sciences 117, no. 31 (July 20, 2020): 18608–16. http://dx.doi.org/10.1073/pnas.2003868117.

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Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion ofSPT4significantly restores TC-NER across the genome in arad26∆mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.

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Farnung, Lucas, Moritz Ochmann, Maik Engeholm, and Patrick Cramer. "Structural basis of nucleosome transcription mediated by Chd1 and FACT." Nature Structural & Molecular Biology 28, no. 4 (April 2021): 382–87. http://dx.doi.org/10.1038/s41594-021-00578-6.

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AbstractEfficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II−Spt4/5−nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A−H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA.

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Huffines, Abigail K., Yvonne J. K. Edwards, and David A. Schneider. "Spt4 Promotes Pol I Processivity and Transcription Elongation." Genes 12, no. 3 (March 12, 2021): 413. http://dx.doi.org/10.3390/genes12030413.

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RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo.

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Hartzog, G. A., T. Wada, H. Handa, and F. Winston. "Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae." Genes & Development 12, no. 3 (February 1, 1998): 357–69. http://dx.doi.org/10.1101/gad.12.3.357.

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Mavreli, Danai, Mariana Theodora, Margaritis Avgeris, Nikolas Papantoniou, Panagiotis Antsaklis, George Daskalakis, and Aggeliki Kolialexi. "First Trimester Maternal Plasma Aberrant miRNA Expression Associated with Spontaneous Preterm Birth." International Journal of Molecular Sciences 23, no. 23 (November 29, 2022): 14972. http://dx.doi.org/10.3390/ijms232314972.

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Spontaneous Preterm Delivery (sPTD) is one of the leading causes of perinatal mortality and morbidity worldwide. The present case–control study aims to detect miRNAs differentially expressed in the first trimester maternal plasma with the view to identify predictive biomarkers for sPTD, between 320/7 and 366/7 weeks, that will allow for timely interventions for this serious pregnancy complication. Small RNA sequencing (small RNA-seq) of five samples from women with a subsequent sPTD and their matched controls revealed significant down-regulation of miR-23b-5p and miR-125a-3p in sPTD cases compared to controls, whereas miR-4732-5p was significantly overexpressed. Results were confirmed by qRT-PCR in an independent cohort of 29 sPTD cases and 29 controls. Statistical analysis demonstrated that miR-125a is a promising early predictor for sPTL (AUC: 0.895; 95% CI: 0.814-0.972; p < 0.001), independent of the confounding factors tested, providing a useful basis for the development of a novel non-invasive predictive test to assist clinicians in estimating patient-specific risk.

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Carton, Jill M., David J. Uhlinger, Ameesha D. Batheja, Claudia Derian, George Ho, Dennis Argenteri, and Michael R. D'Andrea. "Enhanced Serine Palmitoyltransferase Expression in Proliferating Fibroblasts, Transformed Cell Lines, and Human Tumors." Journal of Histochemistry & Cytochemistry 51, no. 6 (June 2003): 715–26. http://dx.doi.org/10.1177/002215540305100603.

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Metastatic processes, including cell invasion, extracellular matrix degradation, and tissue remodeling, require cellular reorganization and proliferation. The cell signaling molecules required and the proteins involved in cell restructuring have not been completely elucidated. We have been studying the role of sphingolipids in normal cell activity and in several pathophysiological states. In this study we used immunohistochemistry to observe the presence of the two known subunits of serine palmitoyltransferase (SPT) in proliferating cells, in an in vitro model of wound repair, and in human malignant tissue. We report increased expression of the two subunits, SPT1 and SPT2, in the proliferating cells in these models. We also demonstrate a change in subcellular localization of the SPT subunits from predominantly cytosolic in quiescent cells to nuclear in proliferating cells. In addition, we observed SPT1 and SPT2 immunoreactivity in reactive stromal fibroblasts surrounding the carcinoma cells of some of the tumors. This enhanced SPT expression was absent in the stromal fibroblasts surrounding normal epithelial cells. Our results suggest a potential role for overexpression of SPT in the processes of cell metastasis.

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Basrai, M. A., J. Kingsbury, D. Koshland, F. Spencer, and P. Hieter. "Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae." Molecular and Cellular Biology 16, no. 6 (June 1996): 2838–47. http://dx.doi.org/10.1128/mcb.16.6.2838.

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A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and kinetochore mutation ndc10-42 or ctf13-30 show a synthetic conditional phenotype. Both spt4-138 and spt4 delta strains exhibit synergistic chromosome instability in combination with CEN DNA mutations and show in vitro defects in microtubule binding to minichromosomes. These results indicate that Spt4p plays a role in chromosome segregation. The results of in vivo genetic interactions with mutations in kinetochore proteins and CEN DNA and of in vitro biochemical assays suggest that Spt4p is important for kinetochore function.

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Xue, Ting, Duo Chen, Qiuqiong Su, Xue Yuan, Kui Liu, Luqiang Huang, Jingping Fang, Jiebo Chen, Wenjin He, and Youqiang Chen. "Improved ethanol tolerance and production of Saccharomyces cerevisiae by global transcription machinery engineering via directed evolution of the SPT8 gene." Food Biotechnology 33, no. 2 (March 22, 2019): 155–73. http://dx.doi.org/10.1080/08905436.2019.1572517.

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Zhou, Karen, Wei Hung William Kuo, Jeffrey Fillingham, and Jack F. Greenblatt. "Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5." Proceedings of the National Academy of Sciences 106, no. 17 (April 13, 2009): 6956–61. http://dx.doi.org/10.1073/pnas.0806302106.

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Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

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Journal articles on the topic 'Spt8'

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