Regulatory mechanism of transcriptional factors, RX and CRX.

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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.