Limits...
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.

Show MeSH

Related in: MedlinePlus

qRT-PCR analysis of mRNA levels of VHT1, BIO5 and GAL1 under inducing conditions and of SER3 under repressing conditions. The Δsnf2 deletion mutant was transformed with the single copy plasmids harboring the SNF2 gene, with the snf2-R15C mutant allele, or with the YEp24 vector (maintaining the Δsnf2 genotype). After growth in CAA liquid medium, cultures were split and incubated in the following media: (A) and (B) in low biotin medium, (C) in CAA medium supplemented with 2% galactose, (D) CAA medium with 2% glucose. Results were standardized to ACT1 mRNA levels; expression levels in SNF2 complemented cells were set to 100% (n = 3; ±SE).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105400&req=5

Figure 8: qRT-PCR analysis of mRNA levels of VHT1, BIO5 and GAL1 under inducing conditions and of SER3 under repressing conditions. The Δsnf2 deletion mutant was transformed with the single copy plasmids harboring the SNF2 gene, with the snf2-R15C mutant allele, or with the YEp24 vector (maintaining the Δsnf2 genotype). After growth in CAA liquid medium, cultures were split and incubated in the following media: (A) and (B) in low biotin medium, (C) in CAA medium supplemented with 2% galactose, (D) CAA medium with 2% glucose. Results were standardized to ACT1 mRNA levels; expression levels in SNF2 complemented cells were set to 100% (n = 3; ±SE).

Mentions: We could not include analyses of VHT1 expression in Figure 7, as the snf2-R15C mutant used in these analyses (AMYmut153; see Figure 1) expressed VHT1 from the constitutive ADH1 promoter. For a direct comparison of the snf2-R15C-dependent induction of VHT1 with the induction/repression of other ySWI/SNF target genes in WT and Δsnf2 cells, we introduced the snf2-R15C mutation into the single copy plasmid pMW814 used in Figure 1 by site-directed mutagenesis. We next transformed the Δsnf2 strain Y01586 either with this plasmid to obtain the snf2-R15C genotype, with the SNF2 WT plasmid to regain the WT gene, or with the empty vector to maintain the Δsnf2 genotype. To study the expression of VHT1, GAL1 and SER3 by quantitative real-time RT-PCR, these three strains were grown either on SD medium with low biotin (0.2 µg l−1; Figure 8A), on galactose medium (Figure 8C), or on CAA medium (Figure 8D). Moreover, we included analyses of BIO5 expression, another gene induced by the Vhr1p transcription factor on low biotin [Figure 8B and ref. (28)].Figure 8.


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)

qRT-PCR analysis of mRNA levels of VHT1, BIO5 and GAL1 under inducing conditions and of SER3 under repressing conditions. The Δsnf2 deletion mutant was transformed with the single copy plasmids harboring the SNF2 gene, with the snf2-R15C mutant allele, or with the YEp24 vector (maintaining the Δsnf2 genotype). After growth in CAA liquid medium, cultures were split and incubated in the following media: (A) and (B) in low biotin medium, (C) in CAA medium supplemented with 2% galactose, (D) CAA medium with 2% glucose. Results were standardized to ACT1 mRNA levels; expression levels in SNF2 complemented cells were set to 100% (n = 3; ±SE).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105400&req=5

Figure 8: qRT-PCR analysis of mRNA levels of VHT1, BIO5 and GAL1 under inducing conditions and of SER3 under repressing conditions. The Δsnf2 deletion mutant was transformed with the single copy plasmids harboring the SNF2 gene, with the snf2-R15C mutant allele, or with the YEp24 vector (maintaining the Δsnf2 genotype). After growth in CAA liquid medium, cultures were split and incubated in the following media: (A) and (B) in low biotin medium, (C) in CAA medium supplemented with 2% galactose, (D) CAA medium with 2% glucose. Results were standardized to ACT1 mRNA levels; expression levels in SNF2 complemented cells were set to 100% (n = 3; ±SE).
Mentions: We could not include analyses of VHT1 expression in Figure 7, as the snf2-R15C mutant used in these analyses (AMYmut153; see Figure 1) expressed VHT1 from the constitutive ADH1 promoter. For a direct comparison of the snf2-R15C-dependent induction of VHT1 with the induction/repression of other ySWI/SNF target genes in WT and Δsnf2 cells, we introduced the snf2-R15C mutation into the single copy plasmid pMW814 used in Figure 1 by site-directed mutagenesis. We next transformed the Δsnf2 strain Y01586 either with this plasmid to obtain the snf2-R15C genotype, with the SNF2 WT plasmid to regain the WT gene, or with the empty vector to maintain the Δsnf2 genotype. To study the expression of VHT1, GAL1 and SER3 by quantitative real-time RT-PCR, these three strains were grown either on SD medium with low biotin (0.2 µg l−1; Figure 8A), on galactose medium (Figure 8C), or on CAA medium (Figure 8D). Moreover, we included analyses of BIO5 expression, another gene induced by the Vhr1p transcription factor on low biotin [Figure 8B and ref. (28)].Figure 8.

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.

Show MeSH
Related in: MedlinePlus