Bien-Willner Gabriel A, Stankiewicz Pawel, Lupski James R
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Hum Mol Genet. 2007 May 15;16(10):1143-56. doi: 10.1093/hmg/ddm061. Epub 2007 Apr 4.
SOX9 is a temporal and tissue-specific transcription factor involved in male sexual development and bone formation. Haploinsufficiency of SOX9 is known to cause campomelic dysplasia (CD). CD cases without SOX9 coding region mutations have been described in association with translocations that have breakpoints mapping as far as 932 kb upstream from the gene. These rearrangements suggest that position effects acting from a great distance regulate SOX9 gene expression. Studies of one such case (900 kb upstream to SOX9) have led to the delineation of a potential 2.1 kb cis-acting regulatory element 1.1 Mb upstream of SOX9, termed SOX9cre1. We investigated the role of this putative regulator in SOX9 expression. SOX9cre1 increases the activity of a minimal SOX9 promoter in reporter constructs in a dose-dependent and tissue-specific manner, consistent with an enhancer role. In silico studies identify a putative binding site within SOX9cre1 for GLI1, a downstream mediator of sonic hedgehog (SHH). Furthermore, the stimulation of primary human chondrocyte cells in culture with SHH increases endogenous SOX9 expression 3-fold. Electrophoresis mobility shift assay (EMSA) studies that demonstrate physical interactions between the GLI1 transcription factor and a putative binding site within SOX9cre1, as well as experiments in which reporter constructs are co-transfected with GLI1, suggest a direct interaction between GLI1 and SOX9cre1. GLI1-SOX9cre1 interactions are verified in chromatin immunoprecipitation experiments. These data support a direct molecular link between the Hh signaling pathway and SOX9 regulation, wherein SHH stimulates SOX9 through its mediator GLI1, and are consistent with a mechanism of SOX9 regulation through distal chromatin interactions.
SOX9是一种参与男性性发育和骨骼形成的时空及组织特异性转录因子。已知SOX9单倍体不足会导致弯肢侏儒症（CD）。已报道了一些没有SOX9编码区突变的CD病例，这些病例与易位有关，其断点位于该基因上游932 kb处。这些重排表明远距离的位置效应调节SOX9基因表达。对其中一个病例（SOX9上游900 kb）的研究导致在SOX9上游1.1 Mb处确定了一个潜在的2.1 kb顺式作用调节元件，称为SOX9cre1。我们研究了这个假定调节因子在SOX9表达中的作用。SOX9cre1以剂量依赖性和组织特异性方式增加报告基因构建体中最小SOX9启动子的活性，这与增强子作用一致。计算机模拟研究在SOX9cre1内鉴定出一个假定的GLI1结合位点，GLI1是音猬因子（SHH）的下游介质。此外，用SHH刺激培养中的原代人软骨细胞可使内源性SOX9表达增加3倍。电泳迁移率变动分析（EMSA）研究表明GLI1转录因子与SOX9cre1内的一个假定结合位点之间存在物理相互作用，以及报告基因构建体与GLI1共转染的实验，提示GLI1与SOX9cre1之间存在直接相互作用。在染色质免疫沉淀实验中验证了GLI1 - SOX9cre1相互作用。这些数据支持Hh信号通路与SOX9调节之间存在直接分子联系，其中SHH通过其介质GLI1刺激SOX9，并且与通过远端染色质相互作用调节SOX9的机制一致。
