MULTIPLE NUCLEAR REGULATORY PROTEINS BIND A SINGLE CIS-ACTING PROMOTER ELEMENT TO CONTROL BASAL TRANSCRIPTION OF THE HUMAN ALPHA-4 INTEGRINGENE IN CORNEAL EPITHELIAL-CELLS

Expression of the fibronectin-binding integrin alpha 4 beta 1 has been postulated to be an important event in the process of corneal epithel ial wound healing, In a previous study, we identified upstream positiv e and negative cis-acting regulatory elements that are needed to modul ate the transcriptional activity of the human alpha 4 integrin subunit gene promoter in primary cultures of rabbit corneal epithelial cells, We have shown that most of the basal activity directed by this promot er was dependent on the presence of a cis-acting DNA sequence designat ed the alpha 4.1 element, centered at position -45 relative to the hum an alpha 4 mRNA start site, Here, we demonstrate that five distinct nu clear regulatory proteins (designated Bp1 to Bp5) from rabbit corneal epithelial cells possess the ability to bind the alpha 4.1 element in a specific manner in vitro, However, when they are combined together, only two of them (Bp2 and Bp5) retained their ability to interact with their specific target sequence in in vitro assays, The apparent molec ular masses of the Bp1 to Bp5 proteins were determined and found to be of 91, 74, 59, 45, and 39 kD, respectively, Electrophoretic mobility- shift assays (EMSAs) indicated that only Bp2 also possesses the abilit y to bind the alpha 4.2 element, a site homologous to alpha 4.1 which plays a minor role in alpha 4 gene expression, Despite the presence of three Ets binding sites in the immediate vicinity of alpha 4.1, compe tition experiments in EMSA clearly indicate that Bp1, Bp2, Bp4, and Bp 5 do not belong to the Ets family of transcription factors, Insertion of both alpha 4.1 and alpha 4.2 upstream from the basal promoter of th e mouse p12 gene provided evidence that both elements have the ability to modulate basal expression driven from a heterologous promoter, alp ha 4.1 was shown to function as an activator, whereas alpha 4.2 acted as a repressor in a manner that is dependent on its orientation, furth er stressing the critical regulatory function played by these two elem ents on alpha 4 gene basal expression.