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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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Insertion of 4 bp between the two SOX9 binding site pairs eliminates enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing the wild-type enhancer (M1/M2/D1/D2) or enhancers containing one of two different 4-bp insertions between the two SOX9 binding site pairs (M1/M2/A*/D1/D2 and M1/M2/B*/D1/D2). Both insertions eliminated enhancer activity. Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the wild-type enhancer.
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Figure 7: Insertion of 4 bp between the two SOX9 binding site pairs eliminates enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing the wild-type enhancer (M1/M2/D1/D2) or enhancers containing one of two different 4-bp insertions between the two SOX9 binding site pairs (M1/M2/A*/D1/D2 and M1/M2/B*/D1/D2). Both insertions eliminated enhancer activity. Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the wild-type enhancer.

Mentions: To determine whether the spacing between the two pairs of SOX9 binding sites was a significant factor in enhancer activation, RCS cells were transfected with mutant enhancer/reporter plasmids containing two different 4-bp insertion mutations between the two paired SOX9 binding sites (M1/M2/A*/D1/D2 and M1/M2/B*/D1/D2). Two different insertions were used in order to verify that the specific sequence of the inserted DNA had no inherent activity. These insertions increased the space between the two dimeric binding sites from 14 to 18 bp and thus offset the two pairs of binding sites by nearly half a turn of the DNA helix. The presence of either insert resulted in complete inactivation of the enhancer, suggesting that the SOX9 dimers bound at the two pairs of sites must interact, either directly or indirectly, to form a complex that results in transcriptional activation (Figure 7).Figure 7.


A Col9a1 enhancer element activated by two interdependent SOX9 dimers.

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

Insertion of 4 bp between the two SOX9 binding site pairs eliminates enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing the wild-type enhancer (M1/M2/D1/D2) or enhancers containing one of two different 4-bp insertions between the two SOX9 binding site pairs (M1/M2/A*/D1/D2 and M1/M2/B*/D1/D2). Both insertions eliminated enhancer activity. Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the wild-type enhancer.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC1851653&req=5

Figure 7: Insertion of 4 bp between the two SOX9 binding site pairs eliminates enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing the wild-type enhancer (M1/M2/D1/D2) or enhancers containing one of two different 4-bp insertions between the two SOX9 binding site pairs (M1/M2/A*/D1/D2 and M1/M2/B*/D1/D2). Both insertions eliminated enhancer activity. Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the wild-type enhancer.
Mentions: To determine whether the spacing between the two pairs of SOX9 binding sites was a significant factor in enhancer activation, RCS cells were transfected with mutant enhancer/reporter plasmids containing two different 4-bp insertion mutations between the two paired SOX9 binding sites (M1/M2/A*/D1/D2 and M1/M2/B*/D1/D2). Two different insertions were used in order to verify that the specific sequence of the inserted DNA had no inherent activity. These insertions increased the space between the two dimeric binding sites from 14 to 18 bp and thus offset the two pairs of binding sites by nearly half a turn of the DNA helix. The presence of either insert resulted in complete inactivation of the enhancer, suggesting that the SOX9 dimers bound at the two pairs of sites must interact, either directly or indirectly, to form a complex that results in transcriptional activation (Figure 7).Figure 7.

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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