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A novel mechanism for target gene-specific SWI/SNF recruitment via the Snf2p N-terminus.

Weider M, Schröder A, Klebl F, Sauer N - Nucleic Acids Res. (2011)

Bottom Line: Chromatin-remodeling complexes regulate the expression of genes in all eukaryotic genomes.Here, we show that the N-terminus of Snf2p, the chromatin remodeling core unit of the SWI/SNF complex, is essential for the expression of VHT1, the gene of the plasma membrane H(+)/biotin symporter, and of BIO5, the gene of a 7-keto-8-aminopelargonic acid transporter, biotin biosynthetic precursor. chromatin immunoprecipitation (ChIP) analyses demonstrate that Vhr1p, the transcriptional regulator of VHT1 and BIO5 expression, is responsible for the targeting of Snf2p to the VHT1 promoter at low biotin.We identified an Snf2p mutant, Snf2p-R(15)C, that specifically abolishes the induction of VHT1 and BIO5 but not of other Snf2p-regulated genes, such as GAL1, SUC2 or INO1.

View Article: PubMed Central - PubMed

Affiliation: Molekulare Pflanzenphysiologie, FAU Erlangen-Nürnberg, Staudtstraße 5, D-91058 Erlangen, Germany.

ABSTRACT
Chromatin-remodeling complexes regulate the expression of genes in all eukaryotic genomes. The SWI/SNF complex of Saccharomyces cerevisiae is recruited to its target promoters via interactions with selected transcription factors. Here, we show that the N-terminus of Snf2p, the chromatin remodeling core unit of the SWI/SNF complex, is essential for the expression of VHT1, the gene of the plasma membrane H(+)/biotin symporter, and of BIO5, the gene of a 7-keto-8-aminopelargonic acid transporter, biotin biosynthetic precursor. chromatin immunoprecipitation (ChIP) analyses demonstrate that Vhr1p, the transcriptional regulator of VHT1 and BIO5 expression, is responsible for the targeting of Snf2p to the VHT1 promoter at low biotin. We identified an Snf2p mutant, Snf2p-R(15)C, that specifically abolishes the induction of VHT1 and BIO5 but not of other Snf2p-regulated genes, such as GAL1, SUC2 or INO1. We present a novel mechanism of target gene-specific SWI/SNF recruitment via Vhr1p and a conserved N-terminal Snf2p domain.

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Identification of the ySWI/SNF complex as an essential component of VHT1 induction. (A) SNF2 complements the non-fluorescence phenotype of the EMS mutant AMYmut153. Images show the GFP fluorescence resulting from a pVHT1::GFP construct in the WT (MWY760gc) or in the AMYmut153 mutant. Strains were complemented with one of the SNF2-containing multicopy (mc) plasmids identified in the complementation screen or with a single copy (sc) plasmid (pMW814) harboring SNF2. Vector controls (A) carried the plasmid YEp24. Numbers show insert lengths relative to the SNF2 start ATG. Plates were photographed in white light and under GFP excitation light. (B) Real-time RT-PCR analysis of VHT1 mRNA levels in the WT and in mutants lacking subunits of the ySWI/SNF complex (Δsnf2 or Δswi3). RT-PCRs were performed with total RNA from yeasts grown on SD medium containing the indicated biotin concentrations. Results were standardized to ACT1 mRNA levels (n = 3; ±SE).
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Figure 1: Identification of the ySWI/SNF complex as an essential component of VHT1 induction. (A) SNF2 complements the non-fluorescence phenotype of the EMS mutant AMYmut153. Images show the GFP fluorescence resulting from a pVHT1::GFP construct in the WT (MWY760gc) or in the AMYmut153 mutant. Strains were complemented with one of the SNF2-containing multicopy (mc) plasmids identified in the complementation screen or with a single copy (sc) plasmid (pMW814) harboring SNF2. Vector controls (A) carried the plasmid YEp24. Numbers show insert lengths relative to the SNF2 start ATG. Plates were photographed in white light and under GFP excitation light. (B) Real-time RT-PCR analysis of VHT1 mRNA levels in the WT and in mutants lacking subunits of the ySWI/SNF complex (Δsnf2 or Δswi3). RT-PCRs were performed with total RNA from yeasts grown on SD medium containing the indicated biotin concentrations. Results were standardized to ACT1 mRNA levels (n = 3; ±SE).

Mentions: To identify additional components of the biotin-dependent signaling cascade, we performed a new EMS mutagenesis on MWY760gc cells and screened additional 600 000 clones for loss of GFP fluorescence on low biotin. Fifteen newly obtained mutants were transformed with an S. cerevisiae genomic library (31) and a minimum of 200 000 transformants of each line was screened for recovery GFP fluorescence on low biotin. With 10 lines the complementation was successful, and for 8 lines the complementing sequences could be determined and assigned to complementation groups. The largest complementation group with 6 lines contained fragments carrying the VHR1 gene (data not shown) confirming our previous results (28). One line was complemented by the ECM1 gene (40) encoding a poorly characterized protein involved in cell wall biosynthesis (data not shown). This gene was not further analyzed. The last line, AMYmut153, was complemented by two different library inserts, each containing SNF2 as the only full-length gene (Figure 1A).Figure 1.


A novel mechanism for target gene-specific SWI/SNF recruitment via the Snf2p N-terminus.

Weider M, Schröder A, Klebl F, Sauer N - Nucleic Acids Res. (2011)

Identification of the ySWI/SNF complex as an essential component of VHT1 induction. (A) SNF2 complements the non-fluorescence phenotype of the EMS mutant AMYmut153. Images show the GFP fluorescence resulting from a pVHT1::GFP construct in the WT (MWY760gc) or in the AMYmut153 mutant. Strains were complemented with one of the SNF2-containing multicopy (mc) plasmids identified in the complementation screen or with a single copy (sc) plasmid (pMW814) harboring SNF2. Vector controls (A) carried the plasmid YEp24. Numbers show insert lengths relative to the SNF2 start ATG. Plates were photographed in white light and under GFP excitation light. (B) Real-time RT-PCR analysis of VHT1 mRNA levels in the WT and in mutants lacking subunits of the ySWI/SNF complex (Δsnf2 or Δswi3). RT-PCRs were performed with total RNA from yeasts grown on SD medium containing the indicated biotin concentrations. Results were standardized to ACT1 mRNA levels (n = 3; ±SE).
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Related In: Results  -  Collection

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Figure 1: Identification of the ySWI/SNF complex as an essential component of VHT1 induction. (A) SNF2 complements the non-fluorescence phenotype of the EMS mutant AMYmut153. Images show the GFP fluorescence resulting from a pVHT1::GFP construct in the WT (MWY760gc) or in the AMYmut153 mutant. Strains were complemented with one of the SNF2-containing multicopy (mc) plasmids identified in the complementation screen or with a single copy (sc) plasmid (pMW814) harboring SNF2. Vector controls (A) carried the plasmid YEp24. Numbers show insert lengths relative to the SNF2 start ATG. Plates were photographed in white light and under GFP excitation light. (B) Real-time RT-PCR analysis of VHT1 mRNA levels in the WT and in mutants lacking subunits of the ySWI/SNF complex (Δsnf2 or Δswi3). RT-PCRs were performed with total RNA from yeasts grown on SD medium containing the indicated biotin concentrations. Results were standardized to ACT1 mRNA levels (n = 3; ±SE).
Mentions: To identify additional components of the biotin-dependent signaling cascade, we performed a new EMS mutagenesis on MWY760gc cells and screened additional 600 000 clones for loss of GFP fluorescence on low biotin. Fifteen newly obtained mutants were transformed with an S. cerevisiae genomic library (31) and a minimum of 200 000 transformants of each line was screened for recovery GFP fluorescence on low biotin. With 10 lines the complementation was successful, and for 8 lines the complementing sequences could be determined and assigned to complementation groups. The largest complementation group with 6 lines contained fragments carrying the VHR1 gene (data not shown) confirming our previous results (28). One line was complemented by the ECM1 gene (40) encoding a poorly characterized protein involved in cell wall biosynthesis (data not shown). This gene was not further analyzed. The last line, AMYmut153, was complemented by two different library inserts, each containing SNF2 as the only full-length gene (Figure 1A).Figure 1.

Bottom Line: Chromatin-remodeling complexes regulate the expression of genes in all eukaryotic genomes.Here, we show that the N-terminus of Snf2p, the chromatin remodeling core unit of the SWI/SNF complex, is essential for the expression of VHT1, the gene of the plasma membrane H(+)/biotin symporter, and of BIO5, the gene of a 7-keto-8-aminopelargonic acid transporter, biotin biosynthetic precursor. chromatin immunoprecipitation (ChIP) analyses demonstrate that Vhr1p, the transcriptional regulator of VHT1 and BIO5 expression, is responsible for the targeting of Snf2p to the VHT1 promoter at low biotin.We identified an Snf2p mutant, Snf2p-R(15)C, that specifically abolishes the induction of VHT1 and BIO5 but not of other Snf2p-regulated genes, such as GAL1, SUC2 or INO1.

View Article: PubMed Central - PubMed

Affiliation: Molekulare Pflanzenphysiologie, FAU Erlangen-Nürnberg, Staudtstraße 5, D-91058 Erlangen, Germany.

ABSTRACT
Chromatin-remodeling complexes regulate the expression of genes in all eukaryotic genomes. The SWI/SNF complex of Saccharomyces cerevisiae is recruited to its target promoters via interactions with selected transcription factors. Here, we show that the N-terminus of Snf2p, the chromatin remodeling core unit of the SWI/SNF complex, is essential for the expression of VHT1, the gene of the plasma membrane H(+)/biotin symporter, and of BIO5, the gene of a 7-keto-8-aminopelargonic acid transporter, biotin biosynthetic precursor. chromatin immunoprecipitation (ChIP) analyses demonstrate that Vhr1p, the transcriptional regulator of VHT1 and BIO5 expression, is responsible for the targeting of Snf2p to the VHT1 promoter at low biotin. We identified an Snf2p mutant, Snf2p-R(15)C, that specifically abolishes the induction of VHT1 and BIO5 but not of other Snf2p-regulated genes, such as GAL1, SUC2 or INO1. We present a novel mechanism of target gene-specific SWI/SNF recruitment via Vhr1p and a conserved N-terminal Snf2p domain.

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