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Transcriptional regulation of the promoter of the rat frizzled related protein gene by CREB

机译：CREB对大鼠卷曲相关蛋白基因启动子的转录调控

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Frizzled related proteins (Frps) are secreted proteins structurally similar to frizzled receptors; they bind Wnt via the cysteine-rich domain and antagonize the Wnt signaling pathway. In this study, we have investigated the mechanisms regulating the transcriptional regulation of rat Frp (rFrp) promoter. From previous findings, we know that the transcriptional activation domain of rFrp resides in the region -202 to -144 relative to the transcription start site, and that it is essential for efficient promoter activity. The study presented here was designed to identify trans-acting factors that bind to this critical domain of the rFrp promoter and to elucidate the pathway involved in the regulation of rFrp expression. Electrophoretic mobility shift assay (EMSA) demonstrated that specific DNA–protein binding activities fall into two adjacent core sequences with (CTTTGGGGG) at -197 to -189 and (AGATGATGTAA) at -151 to -141 of the rFrp promoter. Reporter assay showed that these core sequences are both required for the activation of rFrp promoter. Mutation within either one or both core sequence drastically reduced the promoter activity. Southwestern blotting showed that the estimated molecular mass of the distinct binding protein to the (AGATGATGTAA) domain is about 43kDa. Further EMSA suggested CREB as the trans-acting factor in the DNA–protein complex, which was out competed by CREB consensus oligonucleotides and supershifted by anti-CREB antibody. Overexpression of PKA and CREB also transactivated rFrp promoter, and dominant-negative CREB inhibited the promoter activity in transient reporter assays. More importantly, CREB, phosphorylated CREB and the adaptor protein CBP were found binding to the endogenous rFrp promoter using chromatin immunoprecipitation assay. Collectively, our results demonstrate the induction of rFrp promoter activity by PKA and CREB in vitro, and the binding of CREB and CBP to the rFrp promoter core motif in vivo.
机译：毛躁相关蛋白（Frps）是结构类似于毛躁受体的分泌蛋白。它们通过富含半胱氨酸的结构域结合Wnt，并拮抗Wnt信号通路。在这项研究中，我们研究了调控大鼠Frp（rFrp）启动子转录调控的机制。从以前的发现，我们知道rFrp的转录激活域位于相对于转录起始位点的-202至-144区域，这对于有效的启动子活性至关重要。此处进行的研究旨在鉴定与rFrp启动子的关键域结合的反式作用因子，并阐明涉及rFrp表达调控的途径。电泳迁移率迁移分析（EMSA）表明，特定的DNA-蛋白质结合活性分为两个相邻的核心序列，其中rFrp启动子在-197至-189处具有（CTTTGGGGG），在-151至-141处具有（AGATGATGTAA）。记者分析表明，这些核心序列都是激活rFrp启动子所必需的。一个或两个核心序列中的突变大大降低了启动子活性。西南印迹显示，与（AGATGATGTAA）结构域的独特结合蛋白的估计分子量约为43kDa。进一步的EMSA认为CREB是DNA-蛋白质复合物中的反式作用因子，它被CREB共有寡核苷酸所竞争，而被抗CREB抗体所取代。 PKA和CREB的过表达也可激活rFrp启动子，而显性阴性CREB在瞬时报告基因检测中可抑制启动子活性。更重要的是，使用染色质免疫沉淀测定法发现CREB，磷酸化CREB和衔接蛋白CBP与内源性rFrp启动子结合。总的来说，我们的结果证明了体外由PKA和CREB诱导rFrp启动子活性，以及​​在体内CREB和CBP与rFrp启动子核心基序的结合。

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