A major cis activator of the IRBP gene contains CRX-binding and Ret-1/PCE-I elements

Abstract

Purpose: Interphotoreceptor retinoid binding protein (IRBP) is expressed exclusively and to high levels in photoreceptive cells. This study was an attempt to delineate the minimal regulated control region of the murine IRBP promoter involved in this expression pattern.

Methods: Fragments of the mouse IRBP 5' flanking region were tested for promoter activity in transient transfections of embryonic chick retina cells in primary culture. Electrophoretic mobility shift assays were used to identify specific cis-acting DNA elements within these fragments.

Results: Nested deletion analysis of a 1783 bp fragment of the murine IRBP 5' flanking region shows that high promoter activity is maintained with truncated fragments as short as 70 bp 5' to transcription start, but is lost with truncation to 45 bases. The 1783 bp promoter is active in cultures of retina cells but not brain cells or fibroblasts. The 70 bp fragment is active in retina and brain cells but not fibroblasts. Within retina cell cultures, the 1783 bp fragment is active in photoreceptor-like and amorphous or unidentifiable cells whereas the 70 bp is additionally active in multipolar neuron-like cells. The -70 to -45 interval contains Ret-1/PCE-I (AATTAG in the IRBP gene), a proposed retina-specific consensus sequence cis element, and a same-strand reversed copy of this sequence, GATTAA, the consensus binding element of the photoreceptor-specific trans-acting factor CRX. Mutation of either element suppresses promoter activity. Paralleling promoter tissue-specificity, the -70 to -45 fragment binds a sequence-specific protein complex found in retina and brain extracts but not fibroblasts. Mutation of both or either element inhibits this binding.

Conclusions: These data suggest that a trans-acting complex binds a cis-element in the -70 to -45 sequence. This binding fully activates transcription but confers only partial tissue-specificity to IRBP gene expression.