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Abstract:

Background: BMPR1A is a cell surface receptor in the bone morphogenetic protein (BMP) pathway. Mutations in BMPR1A predispose to juvenile polyposis (JP). Sp1 and related proteins are widely expressed regulators of gene transcription, including members of the BMP pathway. We set out to identify important transcription factor binding sites (TFBS) in the recently identified BMPR1A promoter and to assess for the role of Sp1 and associated proteins in its regulation.

Materials and Methods: The BMPR1A promoter was cloned into a luciferase reporter vector. Deletion fragments of this promoter insert were then constructed, of varying lengths and opposing directions, and were used to transfect HEK-293 and CRL-1459 cells. In silico analysis was performed to screen for relevant TFBS. Site-directed mutagenesis (SDM) was then employed to individually disrupt these TFBS in the wild-type (WT) vector. SDM constructs were then assessed for activity.

Results: Light activity from the deletion constructs ranged between 3% and 129% of the WT promoter. ModelInspector identified eight potential binding sites for Sp1- and Sp1-associated proteins that mapped to areas of marked loss or gain of activity from the deletion constructs. SDM of these TFBS led to a drop in activity in five mutants, which included 3 Sp1 sites, an ETSF site, and NFκB site.

Conclusions: By combining in silico analysis and experimental data, Sp1 was found to be a candidate factor that likely plays a role in the transcriptional regulation of BMPR1A. This study potentially provides further insight toward the molecular basis of JP, and suggests that Sp1 plays a role in BMP signaling.

In Plain English:

BMPR1A is a receptor on the surface of the cell that receives chemical signals from outside the cell and communicates them to the nucleus, changing the overall level of certain genes and their resulting proteins. These changes control a wide variety of processes, but are especially important in preventing cells from becoming cancer. When the BMPR1A gene is mutated, people tend to develop juvenile polyposis, where they form small tumors within their stomach and/or intestines that are likely to become cancerous. BMPR1A levels are controlled by its promoter, an adjacent sequence with many functional sections that can respond to many different signals. In this study, we examined which functional sections have the greatest impact on BMPR1A levels by deleting various parts of the promoter and measuring resulting BMPR1A levels. Deleted sections that bind Sp1, a cancer-linked DNA-binding protein, decreased the amount of BMPR1A by the greatest degree, and we confirmed this effect using computational modeling and targeted mutations as well. These results indicate Sp1 strongly regulates BMPR1A.