Repression of the AFP gene in newborn mouse liver is achieved by a decrease in transcription and is regulated by the raf gene, which is an unlinked, regulatory hepatocyte-autonomous locus. Gluocorticoids enhance this repression by suppressing transcription suggesting a direct negative action of the glucocorticoid receptor protein complex on the AFP gene. Repression of the AFP gene is also exhibited by somatic cell hybrids in which liver specific functions are extinguished. Exinction is mediated by the tissue specific extinguisher locus (Tse), an unlinked regulatory gene that functions in the non- hepatocyte parent. In all 3 cases it is posulated that trans- negative factors interact with cis-acting sequences of the AFP gene. Our goal is to elucidate the mechanisms of down regulation mediated by (a) the hepatocyte autonomous trans-acting factor of the raf gene; (b) the heterologou trans-acting factor(s) of Tse- gene and (c) the glucocorticoid-receptor complex. Attempts will be made to characterize the cis-acting sequences and trans-acting factors. ExoIII, DNase I and methylation protection analyses will be done to localize the DNA-protein binding sites of these factors. Upon identification of these sites DNA fragments or oligonucleotides will be used to isolate trans-acting factors by DNA-affinity chromatography and other procedures. Oligonucleotides of the protein-binding sequences will be used in in vitro competition studies to determine whether the trans- acting factors bind to the same or different sequences. In vivo competition studies will be done to determine if competition for these sites can alter AFP gene expression. These studies involve the insertion of cis-acting DNA sequences into polyma (PY-ori) or SV40 (SV-ori) amplification vectors and their amplification in AFP+ or AFP-cells. Since amplification vectors are extrachromsomal, they will be isolated as nucleoprotein complexes and used as a source of protein in the fractionation of DNA-binding factors. Both raf and Tse-AFP gene products, as well as the Tse-AFP DNA sequences will be isolated. The Tse- AFP locus will be identified in human fibroblast chromosomes transferred into mouse hepatoma cells. The gene will be isolated by insertional mutagenesis in which the M-MLV, carrying amber suppressor DNA sequences (M-MLV in31SuIII) will be used as a mutagenizing agent and for the subsequent isolation of Tse-AFP sequences in a Charon 30A genomic library. The long range goal of these studies is to elucidate the mechanism of trans-negative control reactions associated with distinct developmental events in eukaryotic cells.
Showing the most recent 10 out of 12 publications
