Identification of functional cis-acting elements within the rat liver S14 promoter.
The structure of DNAase I hypersensitive site 1 (Hss-1), located adjacent to the 5' end of the rat liver S14 gene, is regulated by tissue-specific factors, and its formation correlates with the transcriptional activation of the S14 gene. We propose that tissue-specific trans-acting factors interacting with key cis-linked elements within this site function in the initiation of S14 gene transcription. To examine this hypothesis we used DNAase I footprint, gel shift and in vitro transcriptional analyses to identify cis-linked elements that function in the control of S14 gene transcription. Binding of rat liver nuclear proteins to the S14 promoter (from -8 to -464 bp) produced four DNAase I footprints (designated A-D). Gel shift studies showed that DNA-protein binding was tissue- and sequence-specific, differentially heat-sensitive, and abolished by proteinase K. The function of the four cis-acting elements was assessed by using an in vitro transcription initiation assay in which the S14 promoter was fused to a reporter gene (G-free cassette). Deletion studies showed that nuclear factors binding to regions A (-48 to -63 bp), B (-88 to -113 bp) and D (-286 to -310 bp) enhanced the rate of initiation of transcription, while proteins binding to region C (-227 to -244 bp) suppressed the rate of initiation of transcription. Based on oligonucleotide competition studies, we suggest that hepatic NF-1 (or a related protein) binding to the A region enhances the rate of initiation of S14 gene transcription. Since trans-acting factors interacting with regions B and D are found in liver but not in spleen or kidney, we suggest that the proteins interacting with these regions may be involved in the tissue-specific augmentation of S14 gene transcription.
MacDougald
O A
OA
Department of Physiology, Michigan State University, East Lansing 48824-1101.
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D B
DB
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation; D008099:Liver; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D012269:Ribosomal Proteins / Q000235:genetics*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D012269:Ribosomal Proteins; C039747:ribosomal protein S14; D003850:Deoxyribonuclease I