Multiple Nuclear Regulatory Proteins Bind a Singlecis-Acting Promoter Element to Control Basal Transcription of the Human α4 Integrin Gene in Corneal Epithelial Cells

摘要

ABSTRACTExpression of the fibronectin-binding integrin α4β1 has been postulated to be an important event in the process of corneal epithelial wound healing. In a previous study, we identified upstream positive and negativecis-acting regulatory elements that are needed to modulate the transcriptional activity of the human α4 integrin subunit gene promoter in primary cultures of rabbit corneal epithelial cells. We have shown that most of the basal activity directed by this promoter was dependent on the presence of acis-acting DNA sequence designated the α4.1 element, centered at position −45 relative to the human α4 mRNA start site. Here, we demonstrate that five distinct nuclear regulatory proteins (designated Bp1 to Bp5) from rabbit corneal epithelial cells possess the ability to bind the α4.1 element in a specific mannerin vitro. However, when they are combined together, only two of them (Bp2 and Bp5) retained their ability to interact with their specific target sequence inin vitroassays. The apparent molecular 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 ability to bind the α4.2 element, a site homologous to α4.1 which plays a minor role in α4 gene expression. Despite the presence of three Ets binding sites in the immediate vicinity of α4.1, competition experiments in EMSA clearly indicate that Bp1, Bp2, Bp4, and Bp5 do not belong to the Ets family of transcription factors. Insertion of both α4.1 and α4.2 upstream from the basal promoter of the mouse p12 gene provided evidence that both elements have the ability to modulate basal expression driven from a heterologous promoter. α4.1 was shown to function as an activator, whereas α4.2 acted as a repressor in a manner that is dependent on its orientation, further stressing the critical regulatory function played by these two elements on α4 gene