Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3.
Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentially active during growth and development. Promoter 3 (P3) is the most active promoter during fetal development as well as in most adult tissues. P3 is also the most active promoter in tumour tissues and cell lines expressing IGF-II. Transient transfections of HeLa and Hep3B cells with truncated promoter constructs revealed that the region between -289 and -183 relative to the transcription start site supports basal promoter activity in both cell lines. Footprint experiments showed that the region between positions -192 and -172 (P3-4) is the only element bound by nuclear proteins. P3-4 is bound by five proteins, of which three proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility shift assays and differential footprint experiments revealed the presence of two protein-binding regions within the P3-4 element. Proteins 4 and 5 bind box A (-193 to -188), whereas box B (-183 to -172) is bound by protein 3. From transcription experiments in vitro it can be concluded that Box A is essential for P3 activity. Box A is part of a region 11 dG residues long and is protected by proteins 4 and 5 that bind a contiguous set of six dG residues. DNA-binding of proteins 4 and 5 to box A requires the presence of Zn2+ ions. Thus structural and functional analysis reveals that the P3-4 element is a key regulatory element of P3 that contains two separate binding sites for proteins essential for the basal activity of IGF-II P3.
Rietveld
L E
LE
Laboratory for Physiological Chemistry, Utrecht University, Stratenum, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
Holthuizen
P E
PE
Sussenbach
J S
JS
D000818:Animals; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D029721:Drosophila Proteins; D050980:GATA Transcription Factors; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D015032:Zinc / Q000378:metabolism
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D050980:GATA Transcription Factors; D014157:Transcription Factors; C103965:srp protein, Drosophila; D007335:Insulin-Like Growth Factor II; D003850:Deoxyribonuclease I; D015032:Zinc