Annual Report

33. Analyses of Transcriptional Control Region of NPAT and ATM Genes

Masashi Sagara, Yasuharu Ninomiya, Hiroko Ito and Takashi Imai

Keywords: NPAT, ATM, transcription, Promoter

NPAT has been identified as a gene closely linked to the ATM gene on human chromosome 11q23. Because these two genes are transcribed in opposite directions and share a 0.5kb 5' upstream sequence, we investigated the nucleotide sequence essential for the expression of ATM and NPAT genes. Both the 5'-flanking region (approximately 2.8kb) of NPAT and that (approximately 2.2kb) of ATM were amplified with each specific primer set from human genomic DNA by PCR. The PCR-amplified DNA fragments were subcloned and the structure of the clones was confirmed by sequencing. These plasmids covered the region containing the sequence of exon from 1a to 4 and intron from 1 to 4 of the ATM gene and exon 1 and a portion of the first intron of NPAT gene. The cloned fragments were sequentially deleted and fused with the coding region of the luciferase gene in the promoterless reporter plasmid pGL3-basic. Each of these recombinant DNAs was then used for transient gene expression experiments by transfection into HeLa cells and the resultant luciferase activity was measured. The data indicated that the region from -350 to +10bp upstream of NPAT gene is essential for NPAT transcription and that the region from -709 to +11bp upstream of ATM gene is required for ATM transcription. Previous nucleotide sequence analysis showed that the region essential for both gene transcriptions contains potential binding sites for multiple transcription factors such as Sp1, CCAAT-box binding protein, E2F and CREB. To investigate the significance of the CRE-like site and two E2F-like sites, these sites were mutated and the fragments were fused to the luciferase gene in sense or antisense orientations relative to both NPAT and ATM genes. The NPAT promoter activities decreased from 100% (wild-type) to 82% (E2F-like site I), to 57% (E2F-like site II), to 42% (E2F-like site I and E2F-like site II) and to 10% (CRE-like site). The ATM promoter activities also decreased from 100% (wild-type) to 57% (E2F-like site I), to 75% ((E2F-like site II), to 47% (E2F-like site I and E2F-like site II) and to 24% (CRE-like site). Therefore, the two E2F-like sites and the CRE-like site in the common franking region of the genes are required for both transcriptions. These results suggested that the 0.5kb nucleotide sequence flanked by the two genes has bi-directional promoter activity.

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