Transcriptional regulation of the vacuolar H(+)-ATPase B2 subunit gene in differentiating THP-1 cells.
Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions.
Lee
B S
BS
Department of Medicine/Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Underhill
D M
DM
Crane
M K
MK
Gluck
S L
SL
D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007951:Leukemia, Myeloid; D046911:Macromolecular Substances; D008264:Macrophages / Q000201:enzymology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014617:Vacuoles / Q000201:enzymology*
D015342:DNA Probes; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D003850:Deoxyribonuclease I; D006180:Proton-Translocating ATPases; D013755:Tetradecanoylphorbol Acetate