Genomic Space of in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression.

BACKGROUND

The molecular regulation of increased expression in human brain tumors, the associated regulatory elements, and linkages of these to its epigenetic silencing are not understood. Because the heightened expression or non-expression of plays a pivotal role in glioma therapeutics, we applied bioinformatics and experimental tools to identify the regulatory elements in the and neighboring gene loci.

RESULTS

Extensive genome database analyses showed that the MGMT genomic space was rich in and harbored many undescribed RNA regulatory sequences and recognition motifs. We extended the 's exon-1 promoter to 2019 bp to include five overlapping alternate promoters. Consensus sequences in the revised promoter for (a) the transcriptional factors CTCF, NRF1/NRF2, GAF, (b) the genetic switch MYC/MAX/MAD, and (c) two well-defined response elements in intron-1, were identified. A putative protein-coding or non-coding RNA sequence was located in the extended 3' UTR of the transcript. Eleven non-coding RNA loci coding for miRNAs, antisense RNA, and lncRNAs were identified in the region and six of these showed validated potential for curtailing the expression of both and genes. ChIP analysis verified the binding site in promoter for CTCF which regulates the genomic methylation and chromatin looping. CTCF depletion by a pool of specific siRNA and shRNAs led to a significant attenuation of MGMT expression in human GBM cell lines. Computational analysis of the ChIP sequence data in ENCODE showed the presence of NRF1 in the promoter and this occurred only in MGMT-proficient cell lines. Further, an enforced NRF2 expression markedly augmented the mRNA and protein levels in glioma cells.

CONCLUSIONS

We provide the first evidence for several new regulatory components in the gene locus which predict complex transcriptional and posttranscriptional controls with potential for new therapeutic avenues.