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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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The D1/D2 element has no enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing four tandem copies of the short D1/D2 element or a single copy of the longer M1/M2/D1/D2 enhancer. Neither wild-type D1/D2 nor corresponding mutants activated gene expression. The longer M1/M2/D1/D2 enhancer, however, was transcriptionally active in RCS cells, as previously reported (16). Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the D1/D2 element.
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Figure 3: The D1/D2 element has no enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing four tandem copies of the short D1/D2 element or a single copy of the longer M1/M2/D1/D2 enhancer. Neither wild-type D1/D2 nor corresponding mutants activated gene expression. The longer M1/M2/D1/D2 enhancer, however, was transcriptionally active in RCS cells, as previously reported (16). Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the D1/D2 element.

Mentions: To determine whether the binding of SOX9 to the wild-type and mutant COL9A1 D1/D2 elements in vitro correlated with transcriptional activity in vivo, transient transfections were performed using enhancer/reporter plasmids in RCS cells where SOX9 is endogenously expressed. To our surprise, neither the wild-type nor mutant D1/D2 elements displayed any enhancer activity in RCS cells (Figure 3).Figure 3.


A Col9a1 enhancer element activated by two interdependent SOX9 dimers.

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

The D1/D2 element has no enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing four tandem copies of the short D1/D2 element or a single copy of the longer M1/M2/D1/D2 enhancer. Neither wild-type D1/D2 nor corresponding mutants activated gene expression. The longer M1/M2/D1/D2 enhancer, however, was transcriptionally active in RCS cells, as previously reported (16). Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the D1/D2 element.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851653&req=5

Figure 3: The D1/D2 element has no enhancer activity. Transient transfections were performed in RCS cells using reporter plasmids containing four tandem copies of the short D1/D2 element or a single copy of the longer M1/M2/D1/D2 enhancer. Neither wild-type D1/D2 nor corresponding mutants activated gene expression. The longer M1/M2/D1/D2 enhancer, however, was transcriptionally active in RCS cells, as previously reported (16). Graph includes data from three independent experiments, each performed in triplicate, and data was normalized to the activity of the D1/D2 element.
Mentions: To determine whether the binding of SOX9 to the wild-type and mutant COL9A1 D1/D2 elements in vitro correlated with transcriptional activity in vivo, transient transfections were performed using enhancer/reporter plasmids in RCS cells where SOX9 is endogenously expressed. To our surprise, neither the wild-type nor mutant D1/D2 elements displayed any enhancer activity in RCS cells (Figure 3).Figure 3.

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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