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Background: The therapeutically important DNA repair gene O 6 -methylguanine DNA methyltransferase ( MGMT ) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT 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 [ Int. J. Mol. Sci.  2021 , 22 (5), 2492], we addressed its significance in MGMT transcription. Methods: 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 MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT 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 MGMT -E1 expression in MGMT -proficient SF188 and T98G GBM cells, compared to active transcription of MGMT -E2. In the MGMT -deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT -E2 and MGMT -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 MGMT -E2, respectively. Conclusions : The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT 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 MGMT transcription in glioma and other cancer types.

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