Characterization of the factors binding to a PEPCK gene upstream hypersensitive site with LCR activity.
A previously described upstream hypersensitive site (HS) in the PEPCK gene at -4800 bp, termed HS A (1), has been characterized and determined to bind at least two factors. One of these is a member of the ubiquitous CREB/ATF family, and the second is a novel tissue specific protein, pep A. A construct carrying HS A and the PEPCK proximal promoter was tested in transgenic mice and its CAT activity compared to the proximal promoter alone. The HS A was shown to drive tissue-specific, position-independent transcription of the CAT reporter gene 2-3 fold more effectively than the proximal promoter alone, with a concommitant 4-5 fold higher expression of CAT. Protein binding activity has been localized to a 33 bp region. This region contains a CRE (2) which is shown to bind a member of the CREB/ATF family through competition assays with an oligo containing a CRE from the proximal promoter and by the appearance of a supershift when the factor/oligo complex was exposed to CREB polyclonal antibody. Through restriction enzyme digests and competition of protein binding with an oligonucleotide homologous to HS A with a mutated CRE we have characterized a putative binding site for a liver-specific factor. In vitro and 'in vivo' footprinting studies complement each other, as well as, mobility shift assay data in designating the binding site of the proteins. The CREB/ATF factor and Pep A bind independently of each other during short term incubations, however, both factors can be accomodated on the DNA substrate as a function of extended time of incubation. Preliminary biochemical analysis defines the subunit molecular mass of the CREB/ATF like proteins at 55, 42, and 35 kD, while the tissue specific material exists as a single homogeneous subunit polypeptide in SDS of molecular mass = 49 kD.
Cheyette
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Department of Cell Biology, Vanderbilt University, Nashville, TN 37232.
Ip
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Faber
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Matsui
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Chalkley
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D011494:Protein Kinases; C069767:phosphoenolpyruvate carboxylase kinase; D017346:Protein Serine-Threonine Kinases