Down-regulation of the cyclin A promoter in differentiating human embryonal carcinoma cells is mediated by depletion of ATF-1 and ATF-2 in the complex at the ATF/CRE site.
The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of N,N'-hexamethylene-bis-acetamide (HMBA). After treatment with HMBA, the level of cyclin A transcript decreased steeply, reaching less than one-tenth of the original level by 48 h. The promoter elements concerned with this down-regulation were studied by using reporter genes and by analyzing DNA-protein complexes. The deletion of the sequence between -608 and -259 containing three GC boxes decreased the promoter activity to about a half, and further deletion up to -194, eliminating the ATF/CRE site, resulted in a decrease to about a tenth of the original level in undifferentiated NEC14 cells. These sequences were involved in down-regulation of the promoter activity in differentiation-induced NEC14 cells. DNA-protein complexes formed at the ATF/CRE site with extracts prepared from undifferentiated and differentiation-induced cells gave the same footprint, but showed different electrophoretic mobilities. The supershift assay with specific antibodies against ATF-1 and ATF-2 indicated that both factors were depleted in the complex after induction of NEC14 cell differentiation. Both the ATF/CRE site and GC boxes seemed to be also involved in up-regulation of the cyclin A promoter in growth-stimulated human fibroblasts at the G1/S boundary.
Nakamura
T
T
Biomedical R & D Department, Sumitomo Electric Industries, Yokohama, Japan.
Okuyama
S
S
Okamoto
S
S
Nakajima
T
T
Sekiya
S
S
Oda
K
K
D000081:Acetamides / Q000494:pharmacology; D051698:Activating Transcription Factor 2; D051696:Activating Transcription Factors; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism*; D018236:Carcinoma, Embryonal; D002453:Cell Cycle; D002454:Cell Differentiation / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics*; D004273:DNA, Neoplasm / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
C497105:ATF2 protein, human; D000081:Acetamides; D051698:Activating Transcription Factor 2; D051696:Activating Transcription Factors; D001798:Blood Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D016213:Cyclins; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C014026:hexamethylene bisacetamide