Multiple protein binding sites within the ovalbumin gene 5′-flanking region: Isolation and characterization of sequence-specific binding proteins

To understand the molecular basis of the steroid hormone regulated expression of the ovalbumin gene, we sought to identify and isolate nuclear factors froa chicken oviduct which interact specifically with the ovolbumin promoter. Using DNase I footprinting and mobility shift assays, we have defined at least four distinct protein binding sites, OV-150, OV-220, OV-250 and OV-330, in the promoter region between -100 to -400. Binding competition and protein fractionation studies revealed the existence of two distinct proteins, each recognizing two promoter sites: Both OV-330 and OV-250 are recognized by one protein factor which is distinct from the one binding to both OV-220 and OV-150. The location of the DNase I footprints coincides with those of in vivo chromatin hypersensitive sites (1). The OV-330 site is located in a sequence area required for the repression of the gene in the absence of hormone (2). The factor binding to OV-330 has been substantially purified and renaturation experiments indicate that the binding activity is associated with a polypeptide(s) of Mr 40K.