Settleis综合征(SS)是一种罕见的局灶性面部真皮发育不良,由碱性螺旋-环-螺旋(bHLH)转录因子的隐性突变引起,TWIST2.表达微阵列分析显示,在三名具有Q119XTWIST2突变的SS患者的真皮成纤维细胞中,脊索蛋白样1(CHRDL1)基因上调。
方法:在CHRDL1基因的上游区域发现了推定的TWIST结合位点,并通过电泳迁移率变化(EMSA)和报告基因测定进行了检查。
结果:EMSAs显示TWIST1和TWIST2同二聚体的特异性结合,以及具有E12的异二聚体,到更远的E盒。相邻的E盒被ADD1/SREBP1c绑定。EMSA分析表明TWIST2和ADD1/SREBP1c可以竞争结合。荧光素酶(luc)报告基因分析显示,CHRDL1基因上游区域驱动其表达,而ADD1/SREBP1c使其比基础水平增加2.6倍。TWIST2,但不是TWIST2-Q119X突变体,ADD1/SREBP1c阻止激活,但TWIST2-Q119X的过表达增加了luc基因的表达。此外,EMSA竞争分析显示,TWIST2,而不是TWIST1,与ADD1/SREBP1c竞争DNA与同一位点的结合。
结论:在TWIST2Q119X和Q65X突变蛋白与ADD1/SREBP1c之间形成无活性复合物可能阻止阻遏物结合,并允许其他调节因子结合以激活CHRDL1基因表达。
Setleis syndrome (SS) is a rare focal facial dermal dysplasia caused by recessive mutations in the basic helix-loop-helix (bHLH) transcription factor, TWIST2. Expression microarray analysis showed that the chordin-like 1 (CHRDL1) gene is up-regulated in dermal fibroblasts from three SS patients with the Q119X TWIST2 mutation.
METHODS: Putative TWIST binding sites were found in the upstream region of the CHRDL1 gene and examined by electrophoretic mobility shift (EMSA) and reporter gene assays.
RESULTS: EMSAs showed specific binding of TWIST1 and TWIST2 homodimers, as well as heterodimers with E12, to the more distal E-boxes. An adjoining E-box was bound by ADD1/SREBP1c. EMSA analysis suggested that TWIST2 and ADD1/SREBP1c could compete for binding. Luciferase (luc) reporter assays revealed that the CHRDL1 gene upstream region drives its expression and ADD1/SREBP1c increased it 2.6 times over basal levels. TWIST2, but not the TWIST2-Q119X mutant, blocked activation by ADD1/SREBP1c, but overexpression of TWIST2-Q119X increased luc gene expression. In addition, EMSA competition assays showed that TWIST2, but not TWIST1, competes with ADD1/SREBP1c for DNA binding to the same site.
CONCLUSIONS: Formation of an inactive complex between the TWIST2 Q119X and Q65X mutant proteins and ADD1/SREBP1c may prevent repressor binding and allow the binding of other regulators to activate CHRDL1 gene expression.