scholarly journals The Saccharomyces cerevisiae Srb8-Srb11 Complex Functions with the SAGA Complex during Gal4-Activated Transcription

2005 ◽  
Vol 25 (1) ◽  
pp. 114-123 ◽  
Author(s):  
Erica Larschan ◽  
Fred Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Immunoprecipitation ◽  
Kinase Activity ◽  
Saga Complex ◽  
Functional Interaction ◽  
Complex Functions ◽  
Complementation Groups ◽  
Stable Association ◽  
Activation Sequence

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.

scholarly journals The ketogenic diet increases Neuregulin 1 expression via elevating histone acetylation and its anti-seizure effect requires ErbB4 kinase activity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jin Wang ◽  
Jie Huang ◽  
Shan Yao ◽  
Jia-Hui Wu ◽  
Hui-Bin Li ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Ketogenic Diet ◽  
Chromatin Immunoprecipitation ◽  
Kinase Activity ◽  
Control Diet ◽  
Synaptic Activity ◽  
Therapeutic Interventions ◽  
Type I ◽  
Neuregulin 1

Abstract Background The ketogenic diet (KD)has been considered an effective treatment for epilepsy, whereas its underlying mechanisms remain obscure. We have previously reported that the KD feeding increased Neuregulin 1 (NRG1) expression in the hippocampus; disruption of NRG1 signaling by genetically deleting its receptor-ErbB4 abolished KD’s effects on inhibitory synaptic activity and seizures. However, it is still unclear about the mechanisms underlying the effect of KD on NRG1 expression and whether the effects of KD require ErbB4 kinase activity. Methods The effects of the KD on NRG1 expression were assessed via western blotting and real-time PCR. Acetylation level at the Nrg1 promoter locus was examined using the chromatin immunoprecipitation technique. Kainic acid (KA)-induced acute seizure model was utilized to examine the effects of KD and histone deacetylase inhibitor-TSA on seizures. Synaptic activities in the hippocampus were recorded with the technique of electrophysiology. The obligatory role of ErbB4 kinase activity in KD’s effects on seizures and inhibitory synaptic activity was evaluated by using ErbB kinase antagonist and transgenic mouse-T796G. Results We report that KD specifically increases Type I NRG1 expression in the hippocampus. Using the chromatin immunoprecipitation technique, we observe increased acetylated-histone occupancy at the Nrg1 promoter locus of KD-fed mice. Treatment of TSA dramatically elevates NRG1 expression and diminishes the difference between the effects of the control diet (CD) and KD. These data indicate that KD increases NRG1 expression via up-regulating histone acetylation. Moreover, both pharmacological and genetic inhibitions of ErbB4 kinase activity significantly block the KD’s effects on inhibitory synaptic activity and seizure, suggesting an essential role of ErbB4 kinase activity. Conclusion These results strengthen our understanding of the role of NRG1/ErbB4 signaling in KD and shed light on novel therapeutic interventions for epilepsy.

Identification of SAS4 and SAS5, Two Genes That Regulate Silencing in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Eugenia Y Xu ◽  
Susan Kim ◽  
Kirstin Replogle ◽  
Jasper Rine ◽  
David H Rivier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acute Myeloid Leukemia ◽  
Binding Site ◽  
Myeloid Leukemia ◽  
Null Alleles ◽  
Human Genes ◽  
Complementation Groups ◽  
A Site ◽  
Acute Myeloid

Abstract In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the α-mating phenotype to MATα HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia.

scholarly journals Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

1999 ◽  
Vol 19 (7) ◽  
pp. 4888-4896 ◽  
Author(s):  
Guy Oshiro ◽  
Julia C. Owens ◽  
Yiqun Shellman ◽  
Robert A. Sclafani ◽  
Joachim J. Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
S Phase ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Initiation Of Dna Replication

ABSTRACT In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

scholarly journals Mitotic Cyclins Regulate Telomeric Recombination in Telomerase-Deficient Yeast Cells

2003 ◽  
Vol 23 (24) ◽  
pp. 9162-9177 ◽  
Author(s):  
Nathalie Grandin ◽  
Michel Charbonneau
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Mitotic Cell ◽  
Nuclease Activity ◽  
Yeast Cells ◽  
Growth Defect ◽  
Mitotic Cell Cycle ◽  
Functional Interaction ◽  
Synthetic Growth

ABSTRACT Telomerase-deficient mutants of Saccharomyces cerevisiae can survive death by senescence by using one of two homologous recombination pathways. The Rad51 pathway amplifies the subtelomeric Y′ sequences, while the Rad50 pathway amplifies the telomeric TG1-3 repeats. Here we show that telomerase-negative cells require Clb2 (the major B-type cyclin in this organism), in association with Cdc28 (Cdk1), to generate postsenescence survivors at a normal rate. The Rad50 pathway was more sensitive to the absence of Clb2 than the Rad51 pathway. We also report that telomerase RAD50 RAD51 triple mutants still generated postsenescence survivors. This novel Rad50- and Rad51-independent pathway of telomeric recombination also appeared to be controlled by Clb2. In telomerase-positive cells, a synthetic growth defect between mutations in CLB2 and RAD50 or in its partners in the conserved MRX complex, MRE11 and XRS2, was observed. This genetic interaction was independent of Mre11 nuclease activity but was dependent on a DNA repair function. The present data reveal an unexpected role of Cdc28/Clb2 in telomeric recombination during telomerase-independent maintenance of telomeres. They also uncover a functional interaction between Cdc28/Clb2 and MRX during the control of the mitotic cell cycle.

scholarly journals Fission yeast Opy1 is an endogenous PI(4,5)P2 sensor that binds to the phosphatidylinositol 4-phosphate 5-kinase Its3

2020 ◽  
Vol 133 (23) ◽  
pp. jcs247973
Author(s):  
Chloe E. Snider ◽  
Alaina H. Willet ◽  
HannahSofia T. Brown ◽  
Jun-Song Chen ◽  
Joshua M. Evers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Kinase Activity ◽  
Striking Difference ◽  
Evolutionary Divergence ◽  
Ph Domain ◽  
Phosphatidylinositol 4 ◽  
Related Proteins

ABSTRACTPhosphoinositides (PIPs) are a dynamic family of lipids that execute diverse roles in cell biology. PIP levels are regulated by numerous enzymes, but our understanding of how these enzymes are controlled in space and time is incomplete. One role of the PIP phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is to anchor the cytokinetic ring (CR) to the plasma membrane (PM) in Schizosaccharomyces pombe. While examining potential PI(4,5)P2-binding proteins for roles in CR anchoring, we identified the dual pleckstrin homology (PH) domain-containing protein Opy1. Although related proteins are implicated in PIP regulation, we found no role for S. pombe Opy1 in CR anchoring, which would be expected if it modulated PM PI(4,5)P2 levels. Our data indicate that although Opy1 senses PM PI(4,5)P2 levels and binds to the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, Opy1 does not regulate Its3 kinase activity or PM PI(4,5)P2 levels, a striking difference from its Saccharomyces cerevisiae homolog. However, overexpression of Opy1 resulted in cytokinesis defects, as might be expected if it sequestered PI(4,5)P2. Our results highlight the evolutionary divergence of dual PH domain-containing proteins and the need for caution when interpreting results based on their overexpression.This article has an associated First Person interview with the first author of the paper.

scholarly journals TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

2016 ◽  
Vol 27 (2) ◽  
pp. 397-409 ◽  
Author(s):  
Seda Yerlikaya ◽  
Madeleine Meusburger ◽  
Romika Kumari ◽  
Alexandre Huber ◽  
Dorothea Anrather ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Activity ◽  
Ribosome Profiling ◽  
Signaling Cascades ◽  
Ribosomal Protein S6 ◽  
Polysome Profiling ◽  
Functional Consequences ◽  
Rps6 Phosphorylation ◽  
Global Translation

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.

scholarly journals Acetylation of GATA-1 is required for chromatin occupancy

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3736-3738 ◽  
Author(s):  
Janine M. Lamonica ◽  
Christopher R. Vakoc ◽  
Gerd A. Blobel
Keyword(s):  
Protein Interactions ◽  
Chromatin Immunoprecipitation ◽  
Protein Levels ◽  
Defective Mutant ◽  
Cellular Target ◽  
Gata Transcription Factors ◽  
Stable Association

Abstract All 3 hematopoietic GATA transcription factors, GATA-1, GATA-2, and GATA-3, are acetylated, although the in vivo role of this modification remains unclear. We examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 does not impair its nuclear localization, steady-state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to all examined cellular target sites in vivo, including genes that are normally activated and repressed by GATA-1. Together, these results suggest that acetylation regulates chromatin occupancy of GATA-1. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo.

scholarly journals Combinatorial Repression of the Hypoxic Genes of Saccharomyces cerevisiae by DNA Binding Proteins Rox1 and Mot3

2005 ◽  
Vol 4 (4) ◽  
pp. 649-660 ◽  
Author(s):  
Lee G. Klinkenberg ◽  
Thomas A. Mennella ◽  
Katharina Luetkenhaus ◽  
Richard S. Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Aerobic Growth ◽  
Repression Complex ◽  
Combinatorial Interactions

ABSTRACT The hypoxic genes of Saccharomyces cerevisiae are transcriptionally repressed during aerobic growth through recruitment of the Ssn6/Tup1 general repression complex by the DNA binding protein Rox1. A second DNA binding protein Mot3 enhances repression of some hypoxic genes. Previous studies characterized the role of Mot3 at the hypoxic ANB1 gene as promoting synergy among one Mot3 site and two Rox1 sites comprising operator A of that gene. Here we studied the role of Mot3 in enhancing repression by Rox1 at another hypoxic gene, HEM13, which is less strongly regulated than ANB1 and has a very different arrangement of Rox1 and Mot3 binding sites. By assessing the effects of deleting Rox1 and Mot3 sites individually and in combination, we found that the major repression of HEM13 occurred through three Mot3 sites closely spaced with a single Rox1 site. While the Mot3 sites functioned additively, they enhanced repression by the single Rox1 site, and the presence of Rox1 enhanced the additive effects of the Mot3 sites. In addition, using a Rox1-Ssn6 fusion protein, we demonstrated that Mot3 enhances Rox1 repression through helping recruit the Ssn6/Tup1 complex. Chromatin immunoprecipitation assays indicated that Rox1 stabilized Mot3 binding to DNA. Integrating these results, we were able to devise a set of rules that govern the combinatorial interactions between Rox1 and Mot3 to achieve differential repression.

scholarly journals Evolution and Functional Trajectory of Sir1 in Gene Silencing

2016 ◽  
Vol 36 (7) ◽  
pp. 1164-1179 ◽  
Author(s):  
Aisha Ellahi ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Interference ◽  
Deep Sequencing ◽  
Chromatin Immunoprecipitation ◽  
Feedback Regulation ◽  
Content Type ◽  
Torulaspora Delbrueckii ◽  
Heterochromatin Formation ◽  
Sir Proteins

We used the budding yeastsSaccharomyces cerevisiaeandTorulaspora delbrueckiito examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles ofSIRand RNA interference (RNAi) genes inT. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins inT. delbrueckiirevealed a different topography of chromatin at theHMLandHMRloci than was observed inS. cerevisiae. S. cerevisiaeSir1, enriched at the silencers ofHMLα andHMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast toS. cerevisiaeSIR1's partially dispensable role in silencing, theT. delbrueckiiSIR1paralogKOS3was essential for silencing.KOS3was also found at telomeres withT. delbrueckiiSir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping inT. delbrueckiirevealed single silencers atHMLandHMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. TheKOS3gene mapped nearHMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing,T. delbrueckiiRNAi genesAGO1andDCR1did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.

scholarly journals Analysis of Spt7 Function in the Saccharomyces cerevisiae SAGA Coactivator Complex

2002 ◽  
Vol 22 (15) ◽  
pp. 5367-5379 ◽  
Author(s):  
Pei-Yun Jenny Wu ◽  
Fred Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
The Other ◽  
Deletion Mutants ◽  
Saga Complex ◽  
Number Of Genes ◽  
Processed Form ◽  
Altered Form ◽  
And Function ◽  
Coactivator Complex

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.

Sign in / Sign up

Export Citation Format

Share Document