Abstract
Transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from the rat is acutely regulated by a number of hormones, including glucagon (acting via cAMP), glucocorticoids, and insulin. In this study we demonstrate by DNase I footprinting that a region of the PEPCK promoter, extending from -460 to +73, contained eight protein binding domains. Two nuclear proteins protected adjacent sites from -121 to -99 and -96 to -77, which have been previously shown to be involved in maintaining the level of basal gene transcription and conferring cAMP responsiveness, respectively. Oligonucleotide competition studies suggested that the protein(s) binding to the cAMP-responsive element (CRE) occupies a second site at -147 to -130, which has a high degree of sequence homology to the CRE, and also binds to two other elements that show partial sequence homologies. The protein(s) which bound to these four elements copurified through oligonucleotide affinity chromatography, suggesting that the PEPCK promoter has four binding sites for the CRE-binding protein(s). Potential tissue-specific elements in the PEPCK promoter were identified by footprinting with nuclear extracts prepared from rat liver, kidney, brain, and spleen. The multiple protein-binding sites in this promoter-regulatory region reflect the complex transcriptional regulation that is characteristic of this gene.
Highlights
While CRE-2 contains a 7 out of 8 bp match with CRE-1 and site P4 a 6 out of 8 match, site P3 has only a 50% homology, yet has a higher affinity for CREB than CRE-2 and Psi4t.eThe likely reason for this greater affinity is that sitPe3 contains the sequence ACGT, whichforms the central fonuurcleotides of the CRE consensus sequence,while CRE-2 and site P4 each have mutations in thisregion
G at position -140 to a C, making it similar toCRE-1, the apparent affinity of CREB for this "corrected" CRE-2 becomes equal to CRE-l." An additional featureof these multiple binding sites is that CRE-1 and CRE-2 are exactly five turns of the helix apart, which orients them on the samfaece of the DNA
The first relates to the observation that spleen nuclear extracts displayed a n altered pattern of DNase I protection in this area of the promoter (Fig. 4)
Summary
DNA probe, 0.5 pg of poly[d(I.C).d(I.C)],20 mM Hepes, pH 7.6, 0.1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 2% (w/v) polyvinyl alcohol, and mM NaC1. We unequivocally established the P1 element as a n N F - l / CTF-binding siteby footprinting the PEPCK promoterwith NF-1/CTF purified from HeLa cells (Fig. 3) This purified protein protected site P1 from DNase I digestion but did not. A number of different binding reaction conditions were tested in the footprint studies, including those that closely mimicked conditions usedforinvitro transcription assays(removal of polyvinyl alcohol and addition of MgCI2), as well as the addition of 0.1 p~ ZnCl, These ingredient changes did not affect the pattern nor the bind to CRE-1(Fig. 3) or to any otheregion of the promoter extending to position-460 (data not shown), The patternof binding to the PEPCK promoteorf nuclear proteins extracted from a variety of rat tissues is shown, and a summary of thepresenceandthe relative abundance of the proteinswhich bind to thvearious promoter degree of proteinbinding to any of the domainsin the PEPCK promoter. Sites P3, the 3’ end of P4 and to CRE-2 (a region with a 7 out of 8 bp match with CRE-1) but did not affect binding to theother four sitesonthePEPCKpromoter
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