Proximal regulatory elements and nuclear activities required for transcription of the human Na+/H+ exchanger (NHE-1) gene.

We herein demonstrate competence of the 5' upstream region -1374 to +16 of the human growth factor-activatable Na+/H+ exchanger (NHE-1) gene to promote transcription of the chloramphenicol acetyltransferase gene in cells of hepatic origin (HepG2), vascular-smooth-muscle origin (VSM A7r5) and fibroblasts (3T3). We also describe the mapping of the regulatory elements required for such transcription. Sequential 5' end-deletions indicated that the 5' boundary of the positive regulatory elements of NHE-1 transcription is localized downstream of nucleotide -252 in both HepG2 and VSM A7r5 cells but downstream of nucleotide -654 in 3T3 cells. Footprinting analysis of the 0.25-kb promoter fragment using rat liver nuclear extracts identified 4 protected regions as follows: A, -31 to -9; B, -108 to -65; C, -124 to -111; and D, -239 to -215. Internal deletion and nucleotide substitutions within regulatory element D revealed its essential role for transcription of the human NHE-1 gene in HepG2 and VSM A7r5 cells. DNA binding and competition assays using rat liver nuclear extracts indicated that regulatory element D is recognized by 5 nuclear activities. Four of these activities (designated as NHE-1D1-4) are competed out completely by oligonucleotides containing the binding sites of transcription factors CREB, AP3, NFY, and other CCAAT box-binding proteins (C/EBP alpha or related proteins). This competition profile might be explained by the presence of homology between regulatory element D and the consensus sequence of C/EBP as well as the other competitor oligonucleotides. The actual relationship between these nuclear activities and the C/EBP family of proteins (or other transcription factors) remains to be determined.