SP1 regulates the transcription of BMPR1A.
SP1 regulates the transcription of BMPR1A.
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DOI：
10.1016/j.jss.2011.06.056
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发表时间：
2011-11
期刊：
影响因子：
--
通讯作者：
Howe JR
中科院分区：
文献类型：
--
作者：
Dahdaleh FS;Carr JC;Calva D;Howe JR;Howe JR
BMPR1A是骨形态发生蛋白（BMP）通路中的细胞表面受体。BMPR1A突变易导致青少年性息肉病（JP）。Sp1和相关蛋白是广泛表达的基因转录调控因子，包括BMP通路的成员。我们开始在最近发现的BMPR1A启动子中确定重要的转录因子结合位点（TFBS），并评估Sp1和相关蛋白在其调控中的作用。将BMPR1A启动子克隆到荧光素酶报告载体中。然后构建该启动子插入的不同长度和相反方向的缺失片段，并用于转染HEK-293和CRL-1459细胞。计算机分析筛选相关的TFBS。然后采用定点突变（SDM）在野生型（WT）载体中单独破坏这些TFBS。然后评估SDM结构的活性。缺失结构的轻活性范围在WT启动子的3%到129%之间。ModelInspector确定了Sp1和Sp1相关蛋白的8个潜在结合位点，这些位点映射到从缺失结构中显着丧失或获得活性的区域。这些TFBS的SDM导致5个突变体的活性下降，其中包括3个Sp1位点，一个ETSF位点和NFKB位点。通过结合硅分析和实验数据，Sp1被发现是可能在BMPR1A转录调控中发挥作用的候选因子。这项研究为进一步了解JP的分子基础提供了可能，并表明Sp1在BMP信号传导中起作用。
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. 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. Light activity from the deletion constructs ranged between 3 and 129% of the WT promoter. ModelInspector identified 8 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 NFKB site. 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.
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