Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site.
We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the -40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, -10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5' flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at -25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5'-UTR (5'-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5'-UTR may co-operate with the 5'-flanking region to achieve the optimal promoter activity.
Pearse
Ian
I
Department of Medicine, Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, 840 South Wood Street, Chicago, IL 60612, USA.
Zhu
Ying X
YX
Murray
Eleanor J
EJ
Dudeja
Pradeep K
PK
Ramaswamy
Krishnamurthy
K
Malakooti
Jaleh
J
D020121:5' Untranslated Regions / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D018899:CpG Islands; D004330:Drosophila / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic; D050436:Regulatory Elements, Transcriptional; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*; D024363:Transcription Initiation Site / Q000502:physiology*
D020121:5' Untranslated Regions; C081517:SLC9A2 protein, human; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor