Biomedical subjects

Nataly Kacherovsky

Publications and source records attributed to Nataly Kacherovsky.

5 recordsLinked to original sources

PubMed2006

Snf1-dependent and Snf1-independent pathways of constitutive ADH2 expression in Saccharomyces cerevisiae.

The transcription factor Adr1 directly activates the expression of genes encoding enzymes in numerous pathways that are upregulated after the exhaustion of glucose in the yeast Saccharomyces cerevisiae. ADH2, encoding the alcohol dehydrogenase isozyme required for ethanol oxidation, is a highly glucose-repressed, Adr1-dependent gene. Using a genetic screen we isolated >100 mutants in 12 complementation groups that exhibit ADR1-dependent constitutive ADH2 expression on glucose. Temperature-sensitive alleles are present among the new constitutive mutants, indicating that essential genes play a role in ADH2 repression. Among the genes we cloned is MOT1, encoding a repressor that inhibits TBP binding to the promoter, thus linking glucose repression with TBP access to chromatin. Two genes encoding proteins involved in vacuolar function, FAB1 and VPS35, and CDC10, encoding a nonessential septin, were also uncovered in the search, suggesting that vacuolar function and the cytoskeleton have previously unknown roles in regulating gene expression. Constitutive activation of ADH2 expression by Adr1 is SNF1-dependent in a strain with a defective MOT1 gene, whereas deletion of SNF1 did not affect constitutive ADH2 expression in the mutants affecting vacuolar or septin function. Thus, the mutant search revealed previously unknown Snf1-dependent and -independent pathways of ADH2 expression.

Alcohol Dehydrogenase↗

PubMed2005

Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8.

In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.

Chromatin Immunoprecipitation↗

PubMed2004

The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.

14-3-3 Proteins↗

PubMed2002

Snf1 protein kinase regulates Adr1 binding to chromatin but not transcription activation.

The yeast transcriptional activator Adr1 controls the expression of genes required for ethanol, glycerol, and fatty acid utilization. We show that Adr1 acts directly on the promoters of ADH2, ACS1, GUT1, CTA1, and POT1 using chromatin immunoprecipitation assays. The yeast homolog of the AMP-activated protein kinase, Snf1, promotes Adr1 chromatin binding in the absence of glucose, and the protein phosphatase complex, Glc7.Reg1, represses its binding in the presence of glucose. A post-translational process is implicated in the regulation of Adr1 binding activity. Chromatin binding by Adr1 is not the only step in ADH2 transcription that is regulated by glucose repression. Adr1 can bind to chromatin in repressed conditions in the presence of hyperacetylated histones. To study steps subsequent to promoter binding we utilized miniAdr1 transcription factors to characterize Adr1-dependent transcription in vitro. Yeast nuclear extracts prepared from glucose-repressed and glucose-derepressed cells are equally capable of supporting miniAdr1-dependent transcription and pre-initiation complex formation. Nuclear extracts prepared from a snf1 mutant support miniAdr1-dependent transcription but are partially defective in the formation of pre-initiation complexes with Mediator components being particularly depleted. We conclude that Snf1 regulates Adr1-dependent transcription primarily at the level of chromatin binding.

Chromatin↗

PubMed2002

Hyperacetylation of chromatin at the ADH2 promoter allows Adr1 to bind in repressed conditions.

We report that in vivo increased acetylation of the repressed Saccharomyces cerevisiae ADH2 promoter chromatin, as obtained by disrupting the genes for the two deacetylases HDA1 and RPD3, destabilizes the structure of the TATA box-containing nucleosome. This acetylation-dependent chromatin remodeling is not sufficient to allow the binding of the TATA box-binding protein, but facilitates the recruitment of the transcriptional activator Adr1 and induces faster kinetics of mRNA accumulation when the cells are shifted to derepressing conditions.

Acetylation↗