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A Col9a1 enhancer element activated by two interdependent SOX9 dimers.

Genzer MA, Bridgewater LC - Nucleic Acids Res. (2007)

Bottom Line: Increasing the spacing between the pairs of sites eliminated enhancer activity in chondrocytic cells, as did the mutation of any one of the four sites.The COL9A1 enhancer is ordinarily inactive in 10T1/2 cells, but cotransfection with a SOX9 expression plasmid was sufficient to activate the enhancer, and mutation of any one of the four sites reduced responsiveness to SOX9 overexpression.These results suggest a novel mechanism for transcriptional activation by SOX9, in which two SOX9 dimers that are bound at the two pairs of sites are required to interact with one another, either directly or indirectly, in order to produce a functional transcriptional activation complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah 84602, USA.

ABSTRACT
The transcription factor SOX9 plays a critical role in chondrogenesis as well as in sex determination. Previous work has suggested that SOX9 functions as a DNA-dependent dimer when it activates genes involved in chondrogenesis, but functions as a monomer to activate genes involved in sex determination. We present evidence herein for a third binding configuration through which SOX9 can activate transcription. We have identified four separate SOX consensus sequences in a COL9A1 collagen gene enhancer. The sites are arranged as two pairs, and each pair is similar to previously discovered dimeric SOX9 binding sites. Increasing the spacing between the pairs of sites eliminated enhancer activity in chondrocytic cells, as did the mutation of any one of the four sites. The COL9A1 enhancer is ordinarily inactive in 10T1/2 cells, but cotransfection with a SOX9 expression plasmid was sufficient to activate the enhancer, and mutation of any one of the four sites reduced responsiveness to SOX9 overexpression. These results suggest a novel mechanism for transcriptional activation by SOX9, in which two SOX9 dimers that are bound at the two pairs of sites are required to interact with one another, either directly or indirectly, in order to produce a functional transcriptional activation complex.

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Dimeric and monomeric SOX9 bind to the COL9A1 D1/D2 and M1/M2 elements. EMSA was performed using SOX9 made in vitro and wild-type or mutant probes as indicated. Anti-SOX9 antibody supershifted both monomeric and dimeric complexes of SOX9 with the wild-type D1/D2 probe (compare lanes 1 and 2). Mutation of the D1 site (D1*/D2) allowed only monomeric SOX9 binding (lanes 3 and 4). Mutation of the D2 site (D1/D2*) completely prevented all SOX9 binding (lanes 5 and 6). Anti-SOX9 antibody also supershifted both monomeric and dimeric complexes of SOX9 with the wild-type M1/M2 probe (compare lanes 7 and 8). Mutation of the M1 site (M1*/M2) prevented dimeric SOX9 binding (lanes 9 and 10). Mutation of the M2 site (M1/M2*) completely prevented all SOX9 binding (lanes 11 and 12). The right and left panels contain lanes from the same EMSA experiment, but the panel on the right was exposed longer because SOX9 binding to the M1/M2 element was very weak compared to D1/D2 binding. * denotes a nonspecific band.
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Figure 2: Dimeric and monomeric SOX9 bind to the COL9A1 D1/D2 and M1/M2 elements. EMSA was performed using SOX9 made in vitro and wild-type or mutant probes as indicated. Anti-SOX9 antibody supershifted both monomeric and dimeric complexes of SOX9 with the wild-type D1/D2 probe (compare lanes 1 and 2). Mutation of the D1 site (D1*/D2) allowed only monomeric SOX9 binding (lanes 3 and 4). Mutation of the D2 site (D1/D2*) completely prevented all SOX9 binding (lanes 5 and 6). Anti-SOX9 antibody also supershifted both monomeric and dimeric complexes of SOX9 with the wild-type M1/M2 probe (compare lanes 7 and 8). Mutation of the M1 site (M1*/M2) prevented dimeric SOX9 binding (lanes 9 and 10). Mutation of the M2 site (M1/M2*) completely prevented all SOX9 binding (lanes 11 and 12). The right and left panels contain lanes from the same EMSA experiment, but the panel on the right was exposed longer because SOX9 binding to the M1/M2 element was very weak compared to D1/D2 binding. * denotes a nonspecific band.

Mentions: After the putative SOX9 binding site D1 was identified adjacent to the known site, D2, EMSAs were performed to determine whether SOX9 binds to D1 in vitro. Probes used included wild-type D1/D2, as well as mutants D1*/D2 and D1/D2* (Figure 1A). Mutant binding sites contained four mismatches with the SOX binding consensus sequence A/TA/TCAAA/TG. The EMSAs showed that SOX9 bound as a dimer and a monomer to the wild-type COL9A1 D1/D2 element (Figure 2, lane 1). Inclusions of anti-SOX9 antibody supershifted both complexes, confirming that they both contain SOX9 (Figure 2, lane 2). Mutation of the D1 site (D1*/D2) allowed for monomeric binding at the D2 site, but prevented dimeric SOX9 binding (Figure 2, lanes 3 and 4). Mutation of the D2 site (D1/D2*), however, prevented both dimeric and monomeric binding, even though the D1 site was still intact (Figure 2, lanes 5 and 6). This result suggested that binding of SOX9‚ÄČat the D1/D2 pair of sites occurs cooperatively and sequentially, with binding at the D2 site required before binding at the D1 site can occur. The D1 site is not capable of binding a SOX9 monomer.Figure 2.


A Col9a1 enhancer element activated by two interdependent SOX9 dimers.

Genzer MA, Bridgewater LC - Nucleic Acids Res. (2007)

Dimeric and monomeric SOX9 bind to the COL9A1 D1/D2 and M1/M2 elements. EMSA was performed using SOX9 made in vitro and wild-type or mutant probes as indicated. Anti-SOX9 antibody supershifted both monomeric and dimeric complexes of SOX9 with the wild-type D1/D2 probe (compare lanes 1 and 2). Mutation of the D1 site (D1*/D2) allowed only monomeric SOX9 binding (lanes 3 and 4). Mutation of the D2 site (D1/D2*) completely prevented all SOX9 binding (lanes 5 and 6). Anti-SOX9 antibody also supershifted both monomeric and dimeric complexes of SOX9 with the wild-type M1/M2 probe (compare lanes 7 and 8). Mutation of the M1 site (M1*/M2) prevented dimeric SOX9 binding (lanes 9 and 10). Mutation of the M2 site (M1/M2*) completely prevented all SOX9 binding (lanes 11 and 12). The right and left panels contain lanes from the same EMSA experiment, but the panel on the right was exposed longer because SOX9 binding to the M1/M2 element was very weak compared to D1/D2 binding. * denotes a nonspecific band.
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Figure 2: Dimeric and monomeric SOX9 bind to the COL9A1 D1/D2 and M1/M2 elements. EMSA was performed using SOX9 made in vitro and wild-type or mutant probes as indicated. Anti-SOX9 antibody supershifted both monomeric and dimeric complexes of SOX9 with the wild-type D1/D2 probe (compare lanes 1 and 2). Mutation of the D1 site (D1*/D2) allowed only monomeric SOX9 binding (lanes 3 and 4). Mutation of the D2 site (D1/D2*) completely prevented all SOX9 binding (lanes 5 and 6). Anti-SOX9 antibody also supershifted both monomeric and dimeric complexes of SOX9 with the wild-type M1/M2 probe (compare lanes 7 and 8). Mutation of the M1 site (M1*/M2) prevented dimeric SOX9 binding (lanes 9 and 10). Mutation of the M2 site (M1/M2*) completely prevented all SOX9 binding (lanes 11 and 12). The right and left panels contain lanes from the same EMSA experiment, but the panel on the right was exposed longer because SOX9 binding to the M1/M2 element was very weak compared to D1/D2 binding. * denotes a nonspecific band.
Mentions: After the putative SOX9 binding site D1 was identified adjacent to the known site, D2, EMSAs were performed to determine whether SOX9 binds to D1 in vitro. Probes used included wild-type D1/D2, as well as mutants D1*/D2 and D1/D2* (Figure 1A). Mutant binding sites contained four mismatches with the SOX binding consensus sequence A/TA/TCAAA/TG. The EMSAs showed that SOX9 bound as a dimer and a monomer to the wild-type COL9A1 D1/D2 element (Figure 2, lane 1). Inclusions of anti-SOX9 antibody supershifted both complexes, confirming that they both contain SOX9 (Figure 2, lane 2). Mutation of the D1 site (D1*/D2) allowed for monomeric binding at the D2 site, but prevented dimeric SOX9 binding (Figure 2, lanes 3 and 4). Mutation of the D2 site (D1/D2*), however, prevented both dimeric and monomeric binding, even though the D1 site was still intact (Figure 2, lanes 5 and 6). This result suggested that binding of SOX9‚ÄČat the D1/D2 pair of sites occurs cooperatively and sequentially, with binding at the D2 site required before binding at the D1 site can occur. The D1 site is not capable of binding a SOX9 monomer.Figure 2.

Bottom Line: Increasing the spacing between the pairs of sites eliminated enhancer activity in chondrocytic cells, as did the mutation of any one of the four sites.The COL9A1 enhancer is ordinarily inactive in 10T1/2 cells, but cotransfection with a SOX9 expression plasmid was sufficient to activate the enhancer, and mutation of any one of the four sites reduced responsiveness to SOX9 overexpression.These results suggest a novel mechanism for transcriptional activation by SOX9, in which two SOX9 dimers that are bound at the two pairs of sites are required to interact with one another, either directly or indirectly, in order to produce a functional transcriptional activation complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah 84602, USA.

ABSTRACT
The transcription factor SOX9 plays a critical role in chondrogenesis as well as in sex determination. Previous work has suggested that SOX9 functions as a DNA-dependent dimer when it activates genes involved in chondrogenesis, but functions as a monomer to activate genes involved in sex determination. We present evidence herein for a third binding configuration through which SOX9 can activate transcription. We have identified four separate SOX consensus sequences in a COL9A1 collagen gene enhancer. The sites are arranged as two pairs, and each pair is similar to previously discovered dimeric SOX9 binding sites. Increasing the spacing between the pairs of sites eliminated enhancer activity in chondrocytic cells, as did the mutation of any one of the four sites. The COL9A1 enhancer is ordinarily inactive in 10T1/2 cells, but cotransfection with a SOX9 expression plasmid was sufficient to activate the enhancer, and mutation of any one of the four sites reduced responsiveness to SOX9 overexpression. These results suggest a novel mechanism for transcriptional activation by SOX9, in which two SOX9 dimers that are bound at the two pairs of sites are required to interact with one another, either directly or indirectly, in order to produce a functional transcriptional activation complex.

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