GATA-4 binds to an upstream element of the human alpha2(I) collagen gene (COL1A2) and inhibits transcription in fibroblasts.
GATA-4 binds to an upstream element of the human alpha2(I) collagen gene (COL1A2) and inhibits transcription in fibroblasts.
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DOI：
10.1016/j.matbio.2005.06.001
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发表时间：
2005-08
期刊：
影响因子：
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通讯作者：
Lu Wang;Shizuko Tanaka;F. Ramirez
中科院分区：
文献类型：
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作者：
Lu Wang;Shizuko Tanaka;F. Ramirez
染色质分析已鉴定出两个 DNase I 超敏感位点（HS1 和 HS2），其距人和小鼠 α2(I) 胶原基因转录起始位点的距离相当（-130 bp 和 -2.3 kb）。尽管包含 HS1 的 DNA 区域已通过蛋白质结合和功能测定进行了广泛的表征，但关于 HS2 序列对 α2(I) 胶原蛋白基因转录的贡献尚不清楚。在这里，我们报道人类 α2(I) 胶原 (COL1A2) 基因的 HS2 序列是成纤维细胞中转录抑制因子的结合位点。 DNase I 足迹识别了 HS2 周围的单个核酸酶保护位点，该位点对应于可能能够形成 13 bp 长发夹结构且中间有 4 bp 中断的序列。凝胶迁移率变化分析显示，发夹内嵌入的两个 GATA 共有序列与成纤维细胞中的 GATA-4 特异性结合。他们还表明，HS2 蛋白复合物的形成需要整个发夹序列的完整性。成纤维细胞中荧光素酶报告基因构建体的瞬时转染将 HS2 元件与 -2.3 kb 启动子序列的转录抑制相关联。通过显示在培养的成纤维细胞中强制过度表达 GATA-4 导致共转染的 -2.3 kb 启动子/报告基因构建体的转录减少以及内源胶原基因的表达减少，进一步证实了最后的观察结果。最后，染色质免疫沉淀测定证明了 GATA-4 在体内结合 HS2 元件的能力。因此，这些结果首次表明 GATA-4 参与调节成纤维细胞中的组成型 COL1A2 基因表达。
Chromatin analyses have identified two DNase I hypersensitive sites (HS1 and HS2) at comparable distances (−130 bp and −2.3 kb) from the transcription start site of the human and mouse α2(I) collagen gene. Whereas the DNA region encompassing HS1 has been extensively characterized using protein binding and functional assays, nothing is yet known about the contribution of the HS2 sequence to α2(I) collagen gene transcription. Here we report that the HS2 sequence of the human α2(I) collagen (COL1A2) gene is a binding site for a transcriptional repressor in fibroblasts. DNase I footprinting identified a single site of nuclease protection around HS2, which corresponds to a sequence potentially capable of forming a 13 bp long hairpin structure with a 4 bp interruption in the middle. Gel mobility shift assays revealed that two GATA consensus sequences embedded within the hairpin are specifically bound by GATA-4 in fibroblasts. They also showed that formation of the HS2 protein complex requires the integrity of the whole hairpin sequence. Transient transfections of luciferase reporter gene constructs in fibroblasts correlated the HS2 element with transcriptional repression of the −2.3 kb promoter sequence. This last observation was further corroborated by showing that forced overexpression of GATA-4 in cultured fibroblasts leads to decreased transcription from the co-transfected −2.3 kb promoter/reporter construct, as well as reduced expression of the endogenous collagen gene. Finally, a chromatin immunoprecipitation assay documented GATA-4 ability to bind to the HS2 element in vivo. These results are therefore the first to implicate GATA-4 in regulating constitutive COL1A2 gene expression in fibroblasts.