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

ChIP analyses with Snf2p-myc and snf2p-R15C-myc. (A) ChIPs of Snf2p-myc were performed in a VHR1 WT (YBC3010), a Δvhr1 mutant (MWY8003) and in a VHR1 control strain (FY3) expressing untagged SNF2. ChIP was also performed in the SNF2-myc, VHR1 strain (YBC3010) with a control antibody (αGFP). Yeast cells were grown on SD medium containing the indicated biotin concentrations. Binding of Snf2p-myc to the VHT1 promoter was assayed relative to its binding at the ACT1 ORF. Input control and immunoprecipitated DNA (IP) were amplified with primers spanning a region containing the VHT1 promoter vitamin H-responsive element (VHRE). Snf2p occupancy was calculated as ratio of the VHT1 signal in the IP-to-input samples and normalized for the corresponding ratio calculated for ACT1. The mean value for Snf2p-myc under inducing conditions was set to 100% (n = 3; ±SE). (B) ChIPs of Snf2p (control), Snf2p-myc and snf2p-R15C-myc were performed in the Δsnf2 mutant strain Y01586 expressing SNF2 (control), SNF2-myc or snf2-R15C-myc from a single copy plasmid under the control of the SNF2 promoter. Binding of Snf2p-myc and snf2p-R15C-myc to the VHT1 promoter was assayed as described in (A). The mean signal obtained with the control strain expressing untagged SNF2 was set to 1 (n = 3; ±SE).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: ChIP analyses with Snf2p-myc and snf2p-R15C-myc. (A) ChIPs of Snf2p-myc were performed in a VHR1 WT (YBC3010), a Δvhr1 mutant (MWY8003) and in a VHR1 control strain (FY3) expressing untagged SNF2. ChIP was also performed in the SNF2-myc, VHR1 strain (YBC3010) with a control antibody (αGFP). Yeast cells were grown on SD medium containing the indicated biotin concentrations. Binding of Snf2p-myc to the VHT1 promoter was assayed relative to its binding at the ACT1 ORF. Input control and immunoprecipitated DNA (IP) were amplified with primers spanning a region containing the VHT1 promoter vitamin H-responsive element (VHRE). Snf2p occupancy was calculated as ratio of the VHT1 signal in the IP-to-input samples and normalized for the corresponding ratio calculated for ACT1. The mean value for Snf2p-myc under inducing conditions was set to 100% (n = 3; ±SE). (B) ChIPs of Snf2p (control), Snf2p-myc and snf2p-R15C-myc were performed in the Δsnf2 mutant strain Y01586 expressing SNF2 (control), SNF2-myc or snf2-R15C-myc from a single copy plasmid under the control of the SNF2 promoter. Binding of Snf2p-myc and snf2p-R15C-myc to the VHT1 promoter was assayed as described in (A). The mean signal obtained with the control strain expressing untagged SNF2 was set to 1 (n = 3; ±SE).

Mentions: We tested this hypothesis in ChIP analyses with strain YBC3010 that contains a myc-tagged version of Snf2p (Snf2p-myc). We assayed binding of Snf2p-myc to the vitamin H-responsive element (VHRE) relative to its binding to the ACT1 ORF. We could confirm that Snf2p-myc is present at the VHRE at low, VHT1-inducing biotin concentrations (Figure 4). The signal obtained at non-inducing, high biotin concentrations was significantly lower and comparable with the signals obtained in two negative controls (Snf2p with no myc-tag or Snf2p-myc with a control antiserum against GFP, αGFP; Figure 4A). We also assayed binding of Snf2p-myc in a Δvhr1 deletion strain. In this strain, Snf2p-myc was not recruited to the VHRE, even when the cells were grown on low biotin. In summary, these data demonstrate (i) that Snf2p occupies the pVHT1, (ii) that this recruitment occurs at or near the VHRE element, (iii) that the presence of Vhr1p is essential to recruit Snf2p to the VHRE and (iv) that Vhr1p targets Snf2p to the VHT1 promoter only at low biotin concentrations, i.e. under conditions, when VHT1 expression is induced.Figure 4.


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)

ChIP analyses with Snf2p-myc and snf2p-R15C-myc. (A) ChIPs of Snf2p-myc were performed in a VHR1 WT (YBC3010), a Δvhr1 mutant (MWY8003) and in a VHR1 control strain (FY3) expressing untagged SNF2. ChIP was also performed in the SNF2-myc, VHR1 strain (YBC3010) with a control antibody (αGFP). Yeast cells were grown on SD medium containing the indicated biotin concentrations. Binding of Snf2p-myc to the VHT1 promoter was assayed relative to its binding at the ACT1 ORF. Input control and immunoprecipitated DNA (IP) were amplified with primers spanning a region containing the VHT1 promoter vitamin H-responsive element (VHRE). Snf2p occupancy was calculated as ratio of the VHT1 signal in the IP-to-input samples and normalized for the corresponding ratio calculated for ACT1. The mean value for Snf2p-myc under inducing conditions was set to 100% (n = 3; ±SE). (B) ChIPs of Snf2p (control), Snf2p-myc and snf2p-R15C-myc were performed in the Δsnf2 mutant strain Y01586 expressing SNF2 (control), SNF2-myc or snf2-R15C-myc from a single copy plasmid under the control of the SNF2 promoter. Binding of Snf2p-myc and snf2p-R15C-myc to the VHT1 promoter was assayed as described in (A). The mean signal obtained with the control strain expressing untagged SNF2 was set to 1 (n = 3; ±SE).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: ChIP analyses with Snf2p-myc and snf2p-R15C-myc. (A) ChIPs of Snf2p-myc were performed in a VHR1 WT (YBC3010), a Δvhr1 mutant (MWY8003) and in a VHR1 control strain (FY3) expressing untagged SNF2. ChIP was also performed in the SNF2-myc, VHR1 strain (YBC3010) with a control antibody (αGFP). Yeast cells were grown on SD medium containing the indicated biotin concentrations. Binding of Snf2p-myc to the VHT1 promoter was assayed relative to its binding at the ACT1 ORF. Input control and immunoprecipitated DNA (IP) were amplified with primers spanning a region containing the VHT1 promoter vitamin H-responsive element (VHRE). Snf2p occupancy was calculated as ratio of the VHT1 signal in the IP-to-input samples and normalized for the corresponding ratio calculated for ACT1. The mean value for Snf2p-myc under inducing conditions was set to 100% (n = 3; ±SE). (B) ChIPs of Snf2p (control), Snf2p-myc and snf2p-R15C-myc were performed in the Δsnf2 mutant strain Y01586 expressing SNF2 (control), SNF2-myc or snf2-R15C-myc from a single copy plasmid under the control of the SNF2 promoter. Binding of Snf2p-myc and snf2p-R15C-myc to the VHT1 promoter was assayed as described in (A). The mean signal obtained with the control strain expressing untagged SNF2 was set to 1 (n = 3; ±SE).
Mentions: We tested this hypothesis in ChIP analyses with strain YBC3010 that contains a myc-tagged version of Snf2p (Snf2p-myc). We assayed binding of Snf2p-myc to the vitamin H-responsive element (VHRE) relative to its binding to the ACT1 ORF. We could confirm that Snf2p-myc is present at the VHRE at low, VHT1-inducing biotin concentrations (Figure 4). The signal obtained at non-inducing, high biotin concentrations was significantly lower and comparable with the signals obtained in two negative controls (Snf2p with no myc-tag or Snf2p-myc with a control antiserum against GFP, αGFP; Figure 4A). We also assayed binding of Snf2p-myc in a Δvhr1 deletion strain. In this strain, Snf2p-myc was not recruited to the VHRE, even when the cells were grown on low biotin. In summary, these data demonstrate (i) that Snf2p occupies the pVHT1, (ii) that this recruitment occurs at or near the VHRE element, (iii) that the presence of Vhr1p is essential to recruit Snf2p to the VHRE and (iv) that Vhr1p targets Snf2p to the VHT1 promoter only at low biotin concentrations, i.e. under conditions, when VHT1 expression is induced.Figure 4.

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