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Cloning and characterization of the human SH3BP2 promoter
Chun Fan1, Robert J Gaivin, Thomas A Marth
1Department of Biomedical Engineering, The Cleveland Clinic, Cleveland, OH 44195, USA. chunf@ccf.org
Abstract:
SH3BP2 activating mutations lead to an unique clinical condition in which patients develop symmetrical bone resorptive lesions of the jaw, a condition termed cherubism. Due to this specific temporal sequence and location of bone resorption, we investigated the transcriptional regulation of SH3BP2 expression. Analyses of 5'- and 3'-serial promoter deletions defined the core promoter/regulatory elements, including two repressor sites (from -1,200 to -1,000 and from +86 to +115, respectively) and two activator sites (a PARP1 binding site from -44 to -21 and a second activator site from +57 to +86). We identified that PARP1 binds to DNA from -44 to -21 by Streptavidin-biotin purification and confirmed this binding by electrophoretic mobility shift assay (EMSA). Mutagenesis of the PARP1 binding site on the SH3BP2 promoter showed that this binding site is essential for SH3BP2 expression. EMSA and chromatin immunoprecipitation (ChIP) assays confirmed that PARP1 was able to bind to the SH3BP2 promoter in vitro and in vivo. Indeed, knockout of Parp1 in mice BMMs reduced expression of SH3BP2. These results demonstrate that PARP1 regulates expression of SH3BP2.
Insights
Poly(ADP-ribose) polymerase 1 (PARP1) regulates SH3BP2 expression, a gene linked to cherubism. This study identifies PARP1 binding to the SH3BP2 promoter, crucial for its gene expression.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Activating mutations in SH3BP2 cause cherubism, characterized by jaw bone resorption.
- The specific pattern of bone resorption suggests a need to understand SH3BP2 transcriptional regulation.
Purpose of the Study:
- To investigate the transcriptional regulation of SH3BP2 expression.
- To identify regulatory elements and binding factors controlling SH3BP2 gene expression.
Main Methods:
- 5' and 3' serial promoter deletions to define regulatory regions.
- Streptavidin-biotin purification and electrophoretic mobility shift assay (EMSA) to identify protein-DNA interactions.
- Mutagenesis, EMSA, and chromatin immunoprecipitation (ChIP) assays to confirm binding and functional significance.
- PARP1 knockout in mouse bone marrow-derived macrophages (BMMs) to assess in vivo regulation.
Main Results:
- Core promoter elements of SH3BP2 were identified, including repressor and activator sites.
- A specific Poly(ADP-ribose) polymerase 1 (PARP1) binding site (-44 to -21) essential for SH3BP2 expression was discovered.
- PARP1 binding to the SH3BP2 promoter was confirmed both in vitro and in vivo.
- PARP1 knockout significantly reduced SH3BP2 expression in mouse BMMs.
Conclusions:
- PARP1 directly binds to the SH3BP2 promoter and is a key regulator of its expression.
- Understanding PARP1's role in SH3BP2 regulation provides insights into cherubism pathogenesis.
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