scholarly journals Roles for the Saccharomyces cerevisiae SDS3, CBK1 and HYM1 Genes in Transcriptional Repression by SIN3

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 573-586 ◽  
Author(s):  
Scott Dorland ◽  
Michelle L Deegenaars ◽  
David J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Fusion Protein ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Cell Separation ◽  
Multiprotein Complex ◽  
Genetic Pathway ◽  
Lexa Binding ◽  
Sin3 Complex

Abstract The Saccharomyces cerevisiae Sin3 transcriptional repressor is part of a large multiprotein complex that includes the Rpd3 histone deacetylase. A LexA-Sin3 fusion protein represses transcription of promoters with LexA binding sites. To identify genes involved in repression by Sin3, we conducted a screen for mutations that reduce repression by LexA-Sin3. One of the mutations identified that reduces LexA-Sin3 repression is in the RPD3 gene, consistent with the known roles of Rpd3 in transcriptional repression. Mutations in CBK1 and HYM1 reduce repression by LexA-Sin3 and also cause defects in cell separation and altered colony morphology. cbk1 and hym1 mutations affect some but not all genes regulated by SIN3 and RPD3, but the effect on transcription is much weaker. Genetic analysis suggests that CBK1 and HYM1 function in the same pathway, but this genetic pathway is separable from that of SIN3 and RPD3. The remaining gene from this screen described in this report is SDS3, previously identified in a screen for mutations that increase silencing at HML, HMR, and telomere-linked genes, a phenotype also seen in sin3 and rpd3 mutants. Genetic analysis demonstrates that SDS3 functions in the same genetic pathway as SIN3 and RPD3, and coimmunoprecipitation experiments show that Sds3 is physically present in the Sin3 complex.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

scholarly journals ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences.

1990 ◽  
Vol 10 (1) ◽  
pp. 426-429 ◽  
Author(s):  
C F Evans ◽  
D R Engelke ◽  
D J Thiele
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Fusion Protein ◽  
Binding Sites ◽  
Recognition Sequence ◽  
Multiple Regions

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5'-TC(T)4-6GCTG-3', which we propose constitutes an important part of the ACE1 consensus recognition sequence.

scholarly journals Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (6) ◽  
pp. 2690-2700 ◽  
Author(s):  
M A Huie ◽  
E W Scott ◽  
C M Drazinic ◽  
M C Lopez ◽  
I K Hornstra ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Binding Site ◽  
Gene Function ◽  
Binding Sites ◽  
Sequence Motif ◽  
Wild Type ◽  
Upstream Activating Sequence ◽  
Sequence Elements

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

scholarly journals Both TEL and AML-1 Contribute Repression Domains to the t(12;21) Fusion Protein

1999 ◽  
Vol 19 (10) ◽  
pp. 6566-6574 ◽  
Author(s):  
Randy Fenrick ◽  
Joseph M. Amann ◽  
Bart Lutterbach ◽  
Lilin Wang ◽  
Jennifer J. Westendorf ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Wild Type ◽  
Deletion Mutagenesis ◽  
Binding Domains ◽  
Active Mechanism ◽  
Dna Binding Sites ◽  
Interaction Domains

ABSTRACT t(12;21) is the most frequent translocation found in pediatric B-cell acute lymphoblastic leukemias. This translocation fuses a putative repressor domain from the TEL DNA-binding protein to nearly all of the AML-1B transcription factor. Here, we demonstrate that fusion of the TEL pointed domain to the GAL4 DNA-binding domain resulted in sequence-specific transcriptional repression, indicating that the pointed domain is a portable repression motif. The TEL pointed domain functioned equally well when the GAL4 DNA-binding sites were moved 600 bp from the promoter, suggesting an active mechanism of repression. This lead us to demonstrate that wild-type TEL and the t(12;21) fusion protein bind the mSin3A corepressor. In the fusion protein, both TEL and AML-1B contribute mSin3 interaction domains. Deletion mutagenesis indicated that both the TEL and AML-1B mSin3-binding domains contribute to repression by the fusion protein. While both TEL and AML-1B associate with mSin3A, TEL/AML-1B appears to bind this corepressor much more stably than either wild-type protein, suggesting a mode of action for the t(12;21) fusion protein.

scholarly journals The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

1991 ◽  
Vol 11 (5) ◽  
pp. 2641-2646 ◽  
Author(s):  
S Jones ◽  
M L Vignais ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cyclic Amp ◽  
Genetic Analysis ◽  
Fusion Protein ◽  
Guanine Nucleotides ◽  
Biochemical Activity ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction. This biochemical activity is consistent with previous genetic analysis of CDC25 function.

scholarly journals Saccharomyces cerevisiae BUF protein binds to sequences participating in DNA replication in addition to those mediating transcriptional repression (URS1) and activation.

1993 ◽  
Vol 13 (9) ◽  
pp. 5749-5761 ◽  
Author(s):  
R M Luche ◽  
W C Smart ◽  
T Marion ◽  
M Tillman ◽  
R A Sumrada ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Negative Control ◽  
Multiple Roles ◽  
Protein Binding Sites ◽  
Mating Type Switching ◽  
Transcription And Replication

The heteromeric BUF protein was originally shown to bind to URS1 elements which are situated upstream of many genes in Saccharomyces cerevisiae and mediate negative control of their transcription. Among the genes regulated through the URS1 site and the proteins interacting with it are those participating in carbon, nitrogen, and inositol metabolism; electron transport; meiosis; sporulation; and mating-type switching. We show here that pure BUF protein, in addition to binding to the negatively acting URS1 site, also binds to CAR1 sequences supporting transcriptional activation (upstream activation sequences). To determine the BUF protein structure, we cloned and sequenced the BUF1 and BUF2 genes and found them to be identical to the RF-A (RP-A) gene whose products participate in yeast DNA replication as single-stranded DNA binding proteins. These data argue that BUF protein-binding sites serve multiple roles in transcription and replication.

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae

1992 ◽  
Vol 12 (6) ◽  
pp. 2690-2700
Author(s):  
M A Huie ◽  
E W Scott ◽  
C M Drazinic ◽  
M C Lopez ◽  
I K Hornstra ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Binding Site ◽  
Gene Function ◽  
Binding Sites ◽  
Sequence Motif ◽  
Wild Type ◽  
Upstream Activating Sequence ◽  
Sequence Elements

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

Saccharomyces cerevisiae BUF protein binds to sequences participating in DNA replication in addition to those mediating transcriptional repression (URS1) and activation

1993 ◽  
Vol 13 (9) ◽  
pp. 5749-5761
Author(s):  
R M Luche ◽  
W C Smart ◽  
T Marion ◽  
M Tillman ◽  
R A Sumrada ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Negative Control ◽  
Multiple Roles ◽  
Protein Binding Sites ◽  
Mating Type Switching ◽  
Transcription And Replication

The heteromeric BUF protein was originally shown to bind to URS1 elements which are situated upstream of many genes in Saccharomyces cerevisiae and mediate negative control of their transcription. Among the genes regulated through the URS1 site and the proteins interacting with it are those participating in carbon, nitrogen, and inositol metabolism; electron transport; meiosis; sporulation; and mating-type switching. We show here that pure BUF protein, in addition to binding to the negatively acting URS1 site, also binds to CAR1 sequences supporting transcriptional activation (upstream activation sequences). To determine the BUF protein structure, we cloned and sequenced the BUF1 and BUF2 genes and found them to be identical to the RF-A (RP-A) gene whose products participate in yeast DNA replication as single-stranded DNA binding proteins. These data argue that BUF protein-binding sites serve multiple roles in transcription and replication.

scholarly journals A large protein complex containing the yeast Sin3p and Rpd3p transcriptional regulators.

1997 ◽  
Vol 17 (8) ◽  
pp. 4852-4858 ◽  
Author(s):  
M M Kasten ◽  
S Dorland ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Gel Filtration ◽  
Transcriptional Regulators ◽  
Dna Binding Proteins ◽  
Multiprotein Complex ◽  
Large Protein ◽  
Genetic Studies ◽  
Large Protein Complex

The SIN3 gene is required for the transcriptional repression of diverse genes in Saccharomyces cerevisiae. Sin3p does not bind directly to DNA but is thought to be targeted to promoters by interacting with sequence-specific DNA-binding proteins. We show here that Sin3p is present in a large multiprotein complex with an apparent molecular mass, estimated by gel filtration chromatography, of greater than 2 million Da. Genetic studies have shown that the yeast RPD3 gene has a function similar to that of SIN3 in transcriptional regulation, as SIN3 and RPD3 negatively regulate the same set of genes. The SIN3 and RPD3 genes are conserved from yeasts to mammals, and recent work suggests that RPD3 may encode a histone deacetylase. We show that Rpd3p is present in the Sin3p complex and that an rpd3 mutation eliminates SIN3-dependent repression. Thus, Sin3p may function as a bridge to recruit the Rpd3p histone deacetylase to specific promoters.

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