CHARACTERIZATION OF PROTEIN INTERACTIONS WITH POSITIVE AND NEGATIVE ELEMENTS REGULATING THE APOVLDLII GENE

Synthesis of avian apo very-low-density lipoprotein (apoVLDL)II is est rogen dependent and liver specific. Competence to express the apoVLDLI I gene is not acquired until days 7-9 of embryogenesis and thus lags 5 -6 days behind appearance of the liver primordial bud. It is not known whether the delayed ability to activate the gene is attributable to h epatic estrogen receptor profiles, or a requirement for other transcri ption factors not expressed at earlier stages of embryogenesis. The la tter possibility is supported by developmental alterations in nuclease hypersensitivity flanking the gene that occur independently of estrog en administration. We have examined the influence of these hypersensit ive regions on expression from the apoVLDLII promoter and have charact erized novel protein-DNA interactions at two of them. One is located i n a copy of the CR1 family of middle repetitive elements approximately 3.0 kb upstream from the start of the gene. We demonstrate by DNase I footprinting that the site contains an element which matches a predic ted consensus silencer sequence. The other site contains no previously identified binding motifs. It is located between nucleotides -228 and -245 and is adjacent to an imperfect estrogen response element (ERE) that we demonstrate acts additively with a canonical ERE 30 nucleotide s downstream. We have identified ubiquitous and liver-specific factors that display overlapping DNA contacts with the site. Mutation of G re sidues contacted by these proteins decreases hormone-inducible express ion from the promoter 5- to 8-fold. Hepatic levels of the liver-enrich ed factor interacting with this site increase abruptly between days 7 and 9 of embryogenesis, suggesting that it may be an important determi nant of the ability to express the apoVLDLII and possibly other liver- specific genes.