Molecular cloning and characterization of human acid sensing ion channel (ASIC)2 gene promoter.
Acid sensing ion channel (ASIC)2 belongs to the amiloride-sensitive Na(+)-channel/ degenerin family. Our previous studies suggested that differential regulation of ASIC2 expression occurs between high-grade glial-derived tumor cells and normal astrocytes. To investigate the mechanisms involved in the regulation of ASIC2 gene expression, the human ASIC2 promoter region (-1551 to +117) was cloned and characterized. The ASIC2 promoter lacked a canonical TATA box, but contained one putative CCAAT box. Nucleotide sequencing of the promoter revealed the presence of a number of transcription factor-binding sites and a 404 bp CpG island upstream the transcription start site. Nested deletion mutants and transfection results showed that the construct between -133 and +117 base pairs conferred basal transcription specific activity. Mutation of Sp1 and CP2 sites in this region resulted in a 70 and 95% decrease in promoter activity, respectively. Gel shift assays demonstrated the existence of specific protein binding to the SP1 and CP2 elements. There was no mutation in the CpG island in six glioma cell lines, but methylation-specific PCR showed methylation in some of glioma cell lines and tumor tissues, and treatment with the methylation inhibitor 5-Aza-2'-deoxycytidine could partially restore ASIC2 expression in cell lines, suggesting that epigenetic mechanisms may contribute to dysregulated ASIC2 expression.
Xia
Jiazeng
J
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
Zhou
Zhen Hong
ZH
Bubien
James K
JK
Fuller
Catherine M
CM
Markert
James M
JM
Mapstone
Timothy B
TB
Gillespie
G Yancey
GY
Benos
Dale J
DJ
D062565:Acid Sensing Ion Channels; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005910:Glioma / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins; D018408:Patch-Clamp Techniques; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015222:Sodium Channels / Q000235:genetics* / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
C112231:ASIC3 protein, human; D062565:Acid Sensing Ion Channels; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D000077209:Decitabine; D004247:DNA; D008156:Luciferases; D001374:Azacitidine