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Sp1, but not Sp3, functions to mediate promoter activation by TGF-beta through canonical Sp1 binding sites
1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27708, USA.
Abstract:
Transforming growth factor beta (TGF-beta) causes growth arrest at the G1 phase of the cell cycle in most cell types. Both the cyclin dependent kinase inhibitor p15(INK4B) and p21(Cip1/WAF1) genes have been found to be induced by TGF-beta in human keratinocyte HaCaT cells. Analyses of the human p15 and p21 promoters have led to the identification of GC-rich sequences capable of binding to Sp1 transcription factors as necessary elements for the TGF-beta induction of both promoters. We report here that canonical Sp1 binding sites derived from the SV40 21 bp repeat could also support promoter induction by TGF-beta when placed upstream of a minimal luciferase reporter construct containing only the TATA and Inr elements. Gel retardation assays identified Sp1, Sp3 and DeltaSp3 as major factors binding to the canonical Sp1 sites in HaCaT cells and that TGF-beta treatment did not change their binding activities over a 24 h period. More importantly, GAL4-Sp1, but not GAL4-Sp3, chimeric protein supported TGF-beta mediated gene induction from a luciferase reporter construct driven by five GAL4 DNA binding sites. Our results suggest that Sp1 binding site can function as a distinct TGF-beta responsive element for TGF-beta mediated promoter expression and Sp1 per se can mediate this response.
Insights
Transforming growth factor beta (TGF-beta) induces cell cycle arrest by activating specific gene promoters. The transcription factor Sp1 is identified as a key mediator of this TGF-beta response.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Transforming growth factor beta (TGF-beta) is a crucial regulator of cell growth and differentiation, often inducing G1 phase cell cycle arrest.
- Previous studies identified p15(INK4B) and p21(Cip1/WAF1) gene induction by TGF-beta in HaCaT cells, with GC-rich Sp1 binding sites implicated in promoter regulation.
- The precise mechanism by which TGF-beta mediates gene induction through these elements requires further elucidation.
Purpose of the Study:
- To investigate the role of Sp1 transcription factors in mediating TGF-beta-induced gene expression.
- To determine if canonical Sp1 binding sites can confer TGF-beta responsiveness to minimal promoters.
- To identify the specific Sp1 family members involved in this signaling pathway.
Main Methods:
- Analysis of human p15 and p21 gene promoters to identify Sp1 binding sites.
- Reporter gene assays using luciferase constructs with canonical Sp1 binding sites and minimal promoters.
- Gel retardation assays to assess Sp1, Sp3, and DeltaSp3 binding to Sp1 sites in HaCaT cells.
- Chimeric protein experiments (GAL4-Sp1, GAL4-Sp3) to evaluate the role of specific Sp1 family members in TGF-beta-mediated induction.
Main Results:
- Canonical Sp1 binding sites, when upstream of TATA and Inr elements, supported TGF-beta-mediated promoter induction.
- Gel retardation assays revealed Sp1, Sp3, and DeltaSp3 binding to Sp1 sites, with binding activity unaffected by TGF-beta treatment over 24 hours.
- GAL4-Sp1, but not GAL4-Sp3, chimeric proteins mediated TGF-beta-induced gene expression from a reporter construct.
Conclusions:
- Sp1 binding sites function as distinct TGF-beta responsive elements, mediating promoter expression.
- The transcription factor Sp1, independently, can mediate the TGF-beta-induced gene expression response.
- These findings provide critical insights into the molecular mechanisms of TGF-beta signaling in cell cycle regulation.