Identification of the functional elements in the promoter region of human DNA topoisomerase IIIbeta gene.
In this study, we have isolated and characterized the promoter region of the human DNA topoisomerase IIIbeta (hTOP3beta) gene. The 5' RACE assay showed a short exon 1 encoding only the 35-bp untranslated region and suggested the presence of multiple transcription initiation sites. The hTOP3beta gene promoter lacks a canonical TATA box or initiation element and is moderately high in GC content. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequence identified an activator element between -141 and -119 upstream of the transcription initiation site and a second regulatory element between -91 and -71. On the basis of scanning mutations of triple nucleotides, we demonstrated that a 5'GGAACC3' element between -117 and -112 plays a critical role in the up-regulation of the basal transcription activity. Changing the 5'GGAACC3' sequence leads to markedly reduced promoter activity. Gel mobility shift assays revealed that the 5'GGAACC3' element is required for DNA binding by the transcription factor complex. These observations lead to the conclusion that the positive regulatory region including the 5'GGAACC3' core element is essential for efficient expression of the hTOP3beta gene as well as for the binding of as yet unidentified regulatory factor(s).
Cho
Young Hoon
YH
Department of Biology, Molecular Aging Research Center, and Protein Network Research Center, College of Science, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, South Korea.
Park
Jee Young
JY
Han
Sang Youp
SY
Chung
In Kwon
IK
D024506:5' Flanking Region; D020121:5' Untranslated Regions; D001483:Base Sequence; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site
D020121:5' Untranslated Regions; D007527:Isoenzymes; D011994:Recombinant Proteins; D014157:Transcription Factors; D008156:Luciferases; D004264:DNA Topoisomerases, Type I