Regulatory mechanism of transcriptional factors, RX and CRX.

组织特异性的cys元件PCE-1结构域（CAATTAA/G）和OTX结构域（TGATTAA）存在于视网膜特异性蛋白的启动子部分，如视紫红质、IRBP和抑制蛋白，并且已知同源盒型转录因子CRX与OTX结构域结合。我们的一系列研究发现了与PCE-1结构域结合的转录因子，并适合于研究它们如何参与视网膜特异性基因的表达。以PCE-1结构域为探针，经计算机同源性检索，鉴定出一个与RX相同的同源盒基因。Western blot和免疫组化表明RX仅存在于视网膜中，并且存在于整个视网膜中，不仅存在于光感受器中，还存在于内层颗粒层和神经节细胞层中。此外，通过EMSA方法分析了RX与PCE-1结构域的结合。此外，利用突变探针的EMSA方法发现，PCE-1或OTX结构域的核心结构域（ATTAA）前的两个碱基对（CA或TG）决定了RX和CRX类似转移因子的结合亲和力。随后，我们在CAT实验中研究了RX和CRX对视网膜特异性启动子活性的影响。RX和CRX增加抑制素和IRBP启动子活性呈剂量依赖性。当抑制蛋白启动子的PCE-1结构域发生突变时，只有RX的活性降低。在tkCAT基因前插入PCE-1和OTX两个序列，构建了新的构建体。CAT检测显示RX激活PCE-1结构体，CRX激活OTX结构体PCE-1和OTX结构域都是视网膜特异性基因表达所必需的，RX和CRX传递因子以特定方式与PCE-1和OTX结构域结合，激活视网膜特异性启动子。

The tissue specific cys-element called PCE-1 domain (CAATTAA/G) and OTX domain (TGATTAA) existed in the promoter part of retina specific proteins, such as rhodopsin, IRBP and arrestin, and it was known that the homeobox type transcription factor called CRX bound to the OTX domain. A series of our researches identified the transcription factor combined with PCE-1 domain, and suited examining how they are involved in the expression of retina specific genes. We identified a homeobox gene which using PCE-1 domain as a probe by south Western method and computer homology search revealed the gene was same as RX. Next, performing the Western blot method and the immunohistochemistry, it was shown that RX exists only in retina, and exists in the whole retina, not only in photoreceptors but also an inner granular layer and a ganglion cell layer. Furthermore, it was analyzed that RX combined with PCE-1 domain by the EMSA method. Moreover, the EMSA method using mutated probes revealed that two base pairs (CA or TG) before the core domain (ATTAA) of PCE-1 or OTX domain decided the binding affinity of similar transfer factors of RX and CRX. Subsequently, we investigated the retina specific promoter activity by RX and CRX in CAT assay. RX and CRX increased arrestin and IRBP promoter activity in dose dependence. When PCE-1 domain of the arrestin promoter was mutated, only the activity by RX decreased. Furthermore, new constructs, two tandems of PCE-1 or OTX was inserted in front of tkCAT gene, was created. CAT assay revealed RX activated to PCE-1 construct, CRX to OTX construct Both PCE-1 and OTX domain were required for expression of a retina specific gene, a transfer factor called RX and CRX combined with the PCE-1 and OTX domain in specific manner, and activated retina specific promoters.