Interaction of nuclear factors with the regulatory region of the N-myc gene during differentiation of human embryonal carcinoma cells.
The human embryonal carcinoma cell line, NEC14, can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). During the early stage of HMBA-induced differentiation, the level of N-myc expression decreased steeply and transiently, and then quickly returned to its original level after reaching a minimal level at 18 h after addition of HMBA. Nuclear run-on experiments indicated that this transient decrease is regulated at the transcription start point. To investigate the mechanism of this down-regulation, the 5'-flanking region of the human N-myc gene was cloned and sequenced. Computer analysis of the sequence revealed high homology with the 5'-flanking region of the mouse N-myc gene, especially (greater than 80%) in the region of nt positions -1777 to -1732, nt positions -763 to -501 and nt positions -260 to + 1. The patterns of protein binding to the upstream region during the early stage of NEC14 cell differentiation were analyzed by gel retardation assay. The DNA fragments VIII and X, containing the sequences of nt positions -1437 to -1237 and nt positions -1863 to -1710, respectively, formed the DNA-protein complexes which were greatly reduced in quantity in the cell extract prepared 18 h after the addition of HMBA. This reduction, however, was not observed with an extract similarly prepared from the NEC14 derivative cell line, H10, expressing the N-myc gene constitutively. These results suggest a causal connection between the complex formation and the high-level transcription of the N-myc gene.
Takehana
K
K
Department of Applied Biological Science, Science University of Tokyo, Chiba, Japan.
Nakada
S
S
Hara
E
E
Taya
Y
Y
Sekiya
S
S
Oda
K
K
D000081:Acetamides / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D003001:Cloning, Molecular; D015536:Down-Regulation / Q000502:physiology; D016259:Genes, myc; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics* / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000502:physiology; D014407:Tumor Cells, Cultured
D000081:Acetamides; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; C014026:hexamethylene bisacetamide