Developmental regulation of specific protein interactions with an enhancerlike binding site far upstream from the avian very-low-density apolipoprotein II gene.

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Expression of the avian very-low-density apolipoprotein II (apoVLDLII) gene is completely dependent on estrogen and restricted to the liver. We have identified binding sites for nonhistone nuclear proteins located between -1.96 and -2.61 kilobases. One of these sites, located at -2.6 kilobases (designated site 1), was found to span an MspI site that becomes demethylated between days 7 and 9 of embryogenesis, the stage of development at which competence to express the apoVLDLII gene begins to be acquired. Levels of the factor(s) involved were high at day 7 of embryogenesis, decreased two- to threefold by days 9 to 11, and continued to decline more slowly until hatching. Furthermore, the mobility of the complex formed underwent a well-defined shift between days 11 to 13 embryogenesis. Methylation interference studies showed that modification of the outer guanosines of the MspI site resulted in marked inhibition of the formation of the protein-DNA complex. Competition studies, fractionation of nuclear extracts, and tissue distribution indicated that the factor was not the avian homolog of hepatocyte nuclear factor 1, nuclear factor 1, or CCAAT/enhancer-binding protein (C/EBP). However, site 1 could complete for binding to an oligonucleotide, previously shown to be recognized by C/EBP, in a nonreciprocal fashion. These studies demonstrate that the sequence recognized by the protein includes a C/EBP consensus sequence but that elements in addition to the core enhancer motif are essential for binding.

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