Cloning and characterization of the human SH3BP2 promoter

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.

Published by Elsevier Inc.

Figures

Figure 1. Mapping of the transcription start site of the SH3BP2 gene

(A) The nucleotide sequence of the human SH3BP2 promoter/regulatory region is shown. (B) Representation of the two longer transcript transcription start sites of the SH3BP2 gene. (C) Identification of the putative TSS of SH3BP2 by RT-PCR analysis. The top panel shows results from RT-PCR, and the bottom panel shows positive control PCR primers (regular PCR with genomic DNA). (D) 5′ – RACE followed by nested PCR product from circularized cDNA (left). A water control with no DNA served as a negative control (right).

Figure 2

Deletion analysis of the human SH3BP2 promoter and fine mapping of the SH3BP2 transcription factor binding site. (A) Schematic representation of the SH3BP2 promoter luciferase reporter. (B) Serial 3′-deletion was generated based on −2,000 bp to +115 bp-SH3BP2 p-LUC. The locations of activator or repressor sites (olive-shaped) are indicated. (C) Further 5′-deletions were created based on −200 bp to + 86 bp-SH3BP2 p-LUC. The relative luciferase activity of each deletion mutant in RAW264.7 cells is shown on the right. Data shown represent three independent experiments. (D) Probes used for EMSA and mutagenesis are shown. (E) DNA-protein complexes from incubation of oligonucleotides with RAW264.7 lysates were detected with the EMSA probe, particularly in the GC-box II and III regions. (F) The functional identification of binding proteins to the SH3BP2 promoter region is shown. The relative luciferase activity of each mutant in RAW264.7 cells is shown on the right. Data represent three independent experiments. GC-II: GC-box II site; GC-III: GC-box III site.

Figure 3

The PARP1 binds to the SH3BP2 core promoter and PARP1 is required for maximal SH3BP2 promoter activity. (A) EMSA data is shown, with 32-p labeled GC-box III probe incubated with (lanes 2–4) or without (lane 1) nuclear extract from RAW264.7 cells. In lanes 3 and 4, there was preincubation of a 100x excess of cold competitor WT or mutant GC-box III probe, respectively. (B) Supershift EMSA is shown with (lanes 2–4) or without (lane 1) nuclear extract from RAW264.7 lysates. (C) Streptavidin-biotin purification of the PARP1 protein from RAW264.7 cells after binding to the SH3BP2 promoter in vivo is shown. (D) Confirmation of PARP1 as a DNA binding protein is demonstrated by immunoblot. Single stranded sense or antisense, or double stranded GC-box III probes with (lane 1, 3, 5) or without (lane 2, 4, 6) Exo I (hydrolysis of single stranded DNA) were used for this experiment. FL indicates full length. (E) Immunoblotting of in vitro translated human PARP1 protein is shown. (F) EMSA assay of unlabeled GC—box III probe binding to full length PARP1 protein. Sense (EMSA4) and double stranded (EMSA6) probes bound PARP1 in a dose-dependent manner, while anti-sense (EMSA5) probe binding is not detected in these experiments. (G) Quantification of PARP1 binding is shown based on three independent experiments (n = 3). DNA was stained with ethidium bromide and the ratio of band densities of shift/free probe were plotted. DS, double stranded probe. (H) Supershift EMSA is shown with (lanes 2–4) or without (lane 1) RAW264.7 nuclear extract (RAW NE). (I) Overexpression of PARP1 increased expression of SH3BP2 and PARP1 inhibitor 3-AB inhibited the transcriptional activation of the SH3BP2 promoter. Transcriptional activity for wild-type and GC-box III mutant −108/+86-SH3BP2p-LUC reporter genes (left panel) are shown. RAW264.7 cells with or without RANKL treatment were co-transfected with a reporter gene and pcDNA3.1-PARP1 mammalian expression plasmid. (J) ChIP analysis detected binding of the PARP1 protein to the 2 kb SH3BP2 promoter in vivo in RAW264.7 cells. Primers amplifying the SH3BP2 promoter were used for the PCR analysis. For the 1 kb analysis, ChIP with PCR primers covering the SH3BP2 promoter fragment (with the PARP1 binding site located within the 1 kb region upstream from the transcription start site) were used; and for the 2 kb analysis, ChIPs with PCR primers covering other SH3BP2 promoter fragments without the PARP1 binding site were used.

Figure 4

Knockout of PARP1 reduced the activity of core SH3BP2 promoter and SH3BP2 protein synthesis in vivo. (A) Luciferase reporter assay. BMMs were electroporated with the core SH3BP2 promoter/Luc. reporter or empty vector pGL4.17. Data shown represent two independent experiments with the luciferase activity in triplicate. (B) Immunoblot demonstrates the expression levels of PARP1, SH3BP2, PARP2 and β-ACTIN in WT and Parp1 (−/−) BMMs. (C) Quantification was based on two independent experiments (n = 2). Immunoblot analysis shows that Parp1 (−/−) reduced expression of SH3BP2. β-ACTIN or PARP2 served as a loading control.