Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the Gene Expression in Human Glioblastoma Cells.

The therapeutically important DNA repair gene O-methylguanine DNA methyltransferase () is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [, (5), 2492], we addressed its significance in transcription. RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the gene in glioblastoma cells. Bioinformatic characterization of the predicted exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak -E1 expression in -proficient SF188 and T98G GBM cells, compared to active transcription of -E2. In the -deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that -E2 and -E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and -E2, respectively. : The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of transcription in glioma and other cancer types.