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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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Sequences from the COL9A1 enhancer region. SOX9 binding consensus sequences are marked with grey background, and arrows indicate site orientation. Mismatches with the SOX9 consensus sequence are underlined, and introduced mutations are bold. (A) The D1/D2 element contains the SOX9 binding site (D2) that was previously shown by Zhang et al. (16) to be critical for COL9A1 enhancer activity. Mutations were introduced into the D1 site (D1*/D2) or the D2 site (D1/D2*) to produce a total of four mismatches with the SOX binding consensus sequence A/TA/TCAAA/TG. (B) The M1/M2 element includes the newly identified binding sites upstream of the D1/D2 element. M1*/M2 and M1/M2* mutants contain four mismatches with the SOX binding consensus sequence. (C) Sequence of the full-length M1/M2/D1/D2 enhancer. Brackets indicate the boundaries of the probes used for EMSA. (D) Mutants of the M1/M2/D1/D2 enhancer.
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Figure 1: Sequences from the COL9A1 enhancer region. SOX9 binding consensus sequences are marked with grey background, and arrows indicate site orientation. Mismatches with the SOX9 consensus sequence are underlined, and introduced mutations are bold. (A) The D1/D2 element contains the SOX9 binding site (D2) that was previously shown by Zhang et al. (16) to be critical for COL9A1 enhancer activity. Mutations were introduced into the D1 site (D1*/D2) or the D2 site (D1/D2*) to produce a total of four mismatches with the SOX binding consensus sequence A/TA/TCAAA/TG. (B) The M1/M2 element includes the newly identified binding sites upstream of the D1/D2 element. M1*/M2 and M1/M2* mutants contain four mismatches with the SOX binding consensus sequence. (C) Sequence of the full-length M1/M2/D1/D2 enhancer. Brackets indicate the boundaries of the probes used for EMSA. (D) Mutants of the M1/M2/D1/D2 enhancer.

Mentions: Complementary oligonucleotides were designed for a 50-bp portion of the COL9A1 D enhancer region described by Zhang et al. (16), containing either wild-type paired SOX9 binding sites (D1/D2) or substitution mutations in either the upstream or downstream site within each pair to generate D1*/D2 and D1/D2*, respectively (Figure 1A). Mutant sites contained a total of four mismatches with the consensus SOX binding sequence, A/TA/TCAAA/TG. Complementary single-strand DNA oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis, annealed and cloned into the BamHI-BglII site of the p89Col2a1Bs plasmid. The enhancer elements were multimerized to four tandem copies as previously described and transferred with the minimal promoter to the luciferase reporter plasmid p95Luc, resulting in four repeats of the enhancer upstream of the Col2a1 minimal promoter and luciferase reporter gene (7). Constructs were verified by sequencing in the BYU DNA Sequencing Center (BYU DNASC).Figure 1.


A Col9a1 enhancer element activated by two interdependent SOX9 dimers.

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

Sequences from the COL9A1 enhancer region. SOX9 binding consensus sequences are marked with grey background, and arrows indicate site orientation. Mismatches with the SOX9 consensus sequence are underlined, and introduced mutations are bold. (A) The D1/D2 element contains the SOX9 binding site (D2) that was previously shown by Zhang et al. (16) to be critical for COL9A1 enhancer activity. Mutations were introduced into the D1 site (D1*/D2) or the D2 site (D1/D2*) to produce a total of four mismatches with the SOX binding consensus sequence A/TA/TCAAA/TG. (B) The M1/M2 element includes the newly identified binding sites upstream of the D1/D2 element. M1*/M2 and M1/M2* mutants contain four mismatches with the SOX binding consensus sequence. (C) Sequence of the full-length M1/M2/D1/D2 enhancer. Brackets indicate the boundaries of the probes used for EMSA. (D) Mutants of the M1/M2/D1/D2 enhancer.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 1: Sequences from the COL9A1 enhancer region. SOX9 binding consensus sequences are marked with grey background, and arrows indicate site orientation. Mismatches with the SOX9 consensus sequence are underlined, and introduced mutations are bold. (A) The D1/D2 element contains the SOX9 binding site (D2) that was previously shown by Zhang et al. (16) to be critical for COL9A1 enhancer activity. Mutations were introduced into the D1 site (D1*/D2) or the D2 site (D1/D2*) to produce a total of four mismatches with the SOX binding consensus sequence A/TA/TCAAA/TG. (B) The M1/M2 element includes the newly identified binding sites upstream of the D1/D2 element. M1*/M2 and M1/M2* mutants contain four mismatches with the SOX binding consensus sequence. (C) Sequence of the full-length M1/M2/D1/D2 enhancer. Brackets indicate the boundaries of the probes used for EMSA. (D) Mutants of the M1/M2/D1/D2 enhancer.
Mentions: Complementary oligonucleotides were designed for a 50-bp portion of the COL9A1 D enhancer region described by Zhang et al. (16), containing either wild-type paired SOX9 binding sites (D1/D2) or substitution mutations in either the upstream or downstream site within each pair to generate D1*/D2 and D1/D2*, respectively (Figure 1A). Mutant sites contained a total of four mismatches with the consensus SOX binding sequence, A/TA/TCAAA/TG. Complementary single-strand DNA oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis, annealed and cloned into the BamHI-BglII site of the p89Col2a1Bs plasmid. The enhancer elements were multimerized to four tandem copies as previously described and transferred with the minimal promoter to the luciferase reporter plasmid p95Luc, resulting in four repeats of the enhancer upstream of the Col2a1 minimal promoter and luciferase reporter gene (7). Constructs were verified by sequencing in the BYU DNA Sequencing Center (BYU DNASC).Figure 1.

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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