Regulatory mechanism of transcriptional factors, RX and CRX.

组织特异性CyS元件称为PCE-1结构域(CAATTAA/G)和OTX结构域(TGATTAA)，存在于视紫红质、IRBP和arrestin等视网膜特异性蛋白的启动子部分，已知同源盒型转录因子CRX与OTx结构域结合。我们的一系列研究确定了转录因子与PCE-1结构域结合，并适合研究它们如何参与视网膜特异性基因的表达。我们用Southwest方法鉴定了一个以PCE-1结构域为探针的同源框基因，计算机同源性搜索表明该基因与RX基因完全相同。接下来，通过Western印迹和免疫组织化学的方法发现，RX只存在于视网膜中，并且存在于整个视网膜中，不仅存在于光感受器中，还存在于内颗粒层和神经节细胞层中。并用EMSA方法分析了RX与PCE-1结构域的结合。此外，使用突变探针的EMSA方法显示，PCE-1或OTX结构域核心区(Attaa)之前的两个碱基对(CA或TG)决定了相似转移因子RX和CRX的结合亲和力。随后，我们在CAT实验中用RX和CRX研究了视网膜特异性启动子的活性。RX和CRX可剂量依赖地增加arrestin和IRBP启动子的活性。当arrestin启动子的PCE-1结构域发生突变时，只有Rx的活性降低。此外，在tkCAT基因前插入了两个PCE-1或OTX的新构建体。CAT分析表明，RX激活PCE-1结构域，CRX激活OTX结构域表达视网膜特异基因，RX和CRX以特定方式与PCE-1和OTX结构域结合，并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.