Nucleic Acids Research, 1992, Vol. 20, No. 13 3427-3433
© 1992
MOLECULAR BIOLOGY |
Characterization of the factors binding to a PEPCK gene upstream hypersensitive site with LCR activity
Departments of Cell Biology and Molecular Physiology and Biophysics, Vanderbilt University Nashville, TN 37232, USA
* To whom correspondence should be addressed
Received March 3, 1992. Revised June 3, 1992. Accepted June 3, 1992.
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 super-shift 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.
+Present address: Department of Biology, University of California at San Diego, La Jolla, CA 92093, USA
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