Dorval Kimberley M, Bobechko Brian P, Fujieda Hiroki, Chen Shiming, Zack Don J, Bremner Rod
Toronto Western Research Institute, University Health Network, Ontario, Canada.
J Biol Chem. 2006 Jan 13;281(2):744-51. doi: 10.1074/jbc.M509470200. Epub 2005 Oct 18.
The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.
同源框基因CHX10在视网膜发育早期对于视网膜祖细胞增殖是必需的，随后对于双极神经元分化也是必需的。为了阐明CHX10所采用的分子机制，我们试图鉴定其靶基因。在酵母单杂交试验中，Chx10与光感受器特异性基因视紫红质的Ret1位点相互作用。使用体外翻译蛋白进行的凝胶迁移试验证实CHX10与Ret1结合，但不与相似的视紫红质位点Ret4和BAT-1结合。使用视网膜核裂解物，我们观察到Chx10与其他光感受器特异性元件之间的相互作用，包括PCE-1（视杆细胞 arrestin/S-抗原）和视锥视蛋白基因座控制区（红/绿视锥视蛋白）。然而，染色质免疫沉淀试验表明，在体内，Chx10与视杆细胞 arrestin和光感受器间类视黄醇结合蛋白基因上游的位点结合，但不与视紫红质或视锥视蛋白结合。因此，在染色质环境中，Chx10与它在体外以相当的表观亲和力结合的特定元件子集相关联。我们的数据表明，CHX10可能靶向这些基序以抑制双极细胞中视杆光感受器基因的表达。
