Regulation of transcription of the murine gamma-glutamyl hydrolase gene. Delineation of core promoter A and the role of LYF-1, E2F and ETS-1 in determining tumor-specific expression.
Our earlier studies (Gene 268 (2001) 183) showed that transcription of the mouse gamma-glutamyl hydrolase (gamma GH) gene is under the control of two separate promoters widely distributed within the genome. We now report on further studies examining the functional characteristics of the more efficient promoter (promoter A) which is contiguous with an exon 1 (exon A1a) alternate within the main body of the gene. Functional deletion analysis of promoter A in pGL3 transfected in NIH3T3 cells defined a 189 bp region of sequence 26 bp upstream of exon A1a as mediating core promoter transcriptional activity. Further functional deletion analysis and site-directed mutagenesis attributed the activity of this core region to the presence of five SP1 sites within the most upstream 130 bp of sequence and the presence of three cis-active elements, Ets-1, Lyf-1 and E2F in the remaining downstream 59 bp of sequence. Electrophoretic gel-mobility shift assays showed that differences in the binding of trans-acting factors to these three elements accounts for the tumor-specific expression of this gene. Finally, analysis by fluorescent in situ hybridization revealed that promoter A and the main body of the mouse gamma GH gene are located between A3 and A5 on chromosome 4. This is an interesting finding in light of our earlier results which located promoter B and two associated alternates of exon 1 of this gene on chromosome 17.
Masumoto
Naoko
N
Program in Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Chen
Jing
J
Sirotnak
F M
FM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology; D050684:E2F Transcription Factors; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D017404:In Situ Hybridization, Fluorescence; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000502:physiology; D050783:Proto-Oncogene Proteins c-ets; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; C494042:Ets1 protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases; D011623:gamma-Glutamyl Hydrolase