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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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Alignment of N-terminal Snf2 protein sequences and comparative expression analyses. (A) The first 70 amino acids of S. cerevisiae Snf2p and the corresponding residues of Snf2 proteins from other members of the order Saccharomycetales. Amino acids identical in all proteins are highlighted. The arginine at position 15 in Snf2p is highlighted in black, predicted α-helices are underlined. (B) Model of the predicted secondary structure for Snf2p EVA domains. The position of the conserved arginine/lysine is indicated. (C) Western blot analysis of the relative amount of Snf2p-myc and snf2p-R15C-myc in extracts of cells expressing the corresponding genes. An extract of cells expressing SNF2 without a C-terminal fusion is shown to demonstrate the specificity of the anti-myc antiserum (anti-myc). Each lane was loaded with 5 µg of protein extract. Signals obtained from the same blot with an anti-Adh-antiserum (anti-Adh) are shown as loading controls.
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Figure 2: Alignment of N-terminal Snf2 protein sequences and comparative expression analyses. (A) The first 70 amino acids of S. cerevisiae Snf2p and the corresponding residues of Snf2 proteins from other members of the order Saccharomycetales. Amino acids identical in all proteins are highlighted. The arginine at position 15 in Snf2p is highlighted in black, predicted α-helices are underlined. (B) Model of the predicted secondary structure for Snf2p EVA domains. The position of the conserved arginine/lysine is indicated. (C) Western blot analysis of the relative amount of Snf2p-myc and snf2p-R15C-myc in extracts of cells expressing the corresponding genes. An extract of cells expressing SNF2 without a C-terminal fusion is shown to demonstrate the specificity of the anti-myc antiserum (anti-myc). Each lane was loaded with 5 µg of protein extract. Signals obtained from the same blot with an anti-Adh-antiserum (anti-Adh) are shown as loading controls.

Mentions: When we sequenced the snf2 mutant allele of the EMS-mutant strain, AMYmut153 from −830 bp upstream from the start-ATG to 52 bp downstream of the stop-codon, we found a single point mutation in the codon for the arginine residue at position 15 in Snf2p. This mutation changed CGC into TGC, which replaced the arginine at position 15 by a cysteine yielding snf2p-R15C. A comparison of the Snf2p sequences from different fungi demonstrated that this amino acid is highly conserved in Snf2 proteins from other species of the order Saccharomycetales (Figure 2A). Occasionally, this arginine can be replaced by a lysine residue (see the sequence of the Candida dubliniensis Snf2 protein in Figure 2A).


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)

Alignment of N-terminal Snf2 protein sequences and comparative expression analyses. (A) The first 70 amino acids of S. cerevisiae Snf2p and the corresponding residues of Snf2 proteins from other members of the order Saccharomycetales. Amino acids identical in all proteins are highlighted. The arginine at position 15 in Snf2p is highlighted in black, predicted α-helices are underlined. (B) Model of the predicted secondary structure for Snf2p EVA domains. The position of the conserved arginine/lysine is indicated. (C) Western blot analysis of the relative amount of Snf2p-myc and snf2p-R15C-myc in extracts of cells expressing the corresponding genes. An extract of cells expressing SNF2 without a C-terminal fusion is shown to demonstrate the specificity of the anti-myc antiserum (anti-myc). Each lane was loaded with 5 µg of protein extract. Signals obtained from the same blot with an anti-Adh-antiserum (anti-Adh) are shown as loading controls.
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Related In: Results  -  Collection

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Figure 2: Alignment of N-terminal Snf2 protein sequences and comparative expression analyses. (A) The first 70 amino acids of S. cerevisiae Snf2p and the corresponding residues of Snf2 proteins from other members of the order Saccharomycetales. Amino acids identical in all proteins are highlighted. The arginine at position 15 in Snf2p is highlighted in black, predicted α-helices are underlined. (B) Model of the predicted secondary structure for Snf2p EVA domains. The position of the conserved arginine/lysine is indicated. (C) Western blot analysis of the relative amount of Snf2p-myc and snf2p-R15C-myc in extracts of cells expressing the corresponding genes. An extract of cells expressing SNF2 without a C-terminal fusion is shown to demonstrate the specificity of the anti-myc antiserum (anti-myc). Each lane was loaded with 5 µg of protein extract. Signals obtained from the same blot with an anti-Adh-antiserum (anti-Adh) are shown as loading controls.
Mentions: When we sequenced the snf2 mutant allele of the EMS-mutant strain, AMYmut153 from −830 bp upstream from the start-ATG to 52 bp downstream of the stop-codon, we found a single point mutation in the codon for the arginine residue at position 15 in Snf2p. This mutation changed CGC into TGC, which replaced the arginine at position 15 by a cysteine yielding snf2p-R15C. A comparison of the Snf2p sequences from different fungi demonstrated that this amino acid is highly conserved in Snf2 proteins from other species of the order Saccharomycetales (Figure 2A). Occasionally, this arginine can be replaced by a lysine residue (see the sequence of the Candida dubliniensis Snf2 protein in Figure 2A).

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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