Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression
Abstract
:1. Introduction
2. Results and Discussion
2.1. Recapitulation of MGMT Genomic Space and the mRNA
2.2. New Features of 3′ Untranslated Region (UTR) of MGMT Transcript
2.3. New Regulatory Motifs in MGMT Promoter; ARE, ERE, and NRF1/NRF2 -Mediated Upregulation of MGMT Expression
2.4. Revised MGMT Promoter That Includes Alternate Promoters, CGI Status, and Other Promoter-Like Sequences in MGMT Genome
2.5. Recognition Motifs for GAF and MYC/MAD/MAX Switch within the MGMT Promoter
2.6. Identification of p53 Response Elements (PREs) in Intron 1
2.7. Presence of CTCF Recognition Motifs in the MGMT Promoter and Rest of the Gene: Evidence for CTCF as a Positive Regulator of MGMT Expression
2.8. Potential Involvement of RNA Regulatory Elements (Long Non-Coding RNA, Antisense RNA, Micro RNA) in the MGMT- EBF3 Region in the Regulation of MGMT Expression
3. Conclusions
4. Materials and Methods
4.1. Cell Culture
4.2. Western Blotting
4.3. siRNA and shRNA Transient Transfections and NRF2 Stable Transfection
4.4. Chromatin Immunoprecipitation (ChIP) Assay
4.5. Database Search
4.6. Regulatory Sequences and MGMT Alternative Promoters
4.7. Sequencing-Based Data from ENCODE and Bioinformatic Analysis of Non-Coding RNA Transcripts
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Source | Promoter ID | Map Locations at Chromosome 10/+ Strand | Span (bp) |
---|---|---|---|
PrESSTo/FANTOM | P1@MGMT | 129,466,944–129,467,344 | 401 |
P2@MGMT | 129,466,905–129,467,305 | 401 | |
Eukaryotic Promoter Database (EPD) | MGMT_1 | 129,466,745–129,467,344 | 600 |
NCBI/Nucleotide | X61657.1 | 129,466,183–129,467,339 | 1157 |
Ensembl 84 | ENSR00001428452 | 129,466,558–129,468,201 | 1644 |
Revised MGMT exon-1 promoter (this study) | MGMT-P1 | 129,466,183–129,468,201 | 2019 |
Transcriptional Regulatory Element Database (TRED) | TRED-5071 (MGMT-P2) | 129,535,540–129,536,539 | 1000 |
CGI Annotation | Nucleotide Position 5’–3’ | CGI Length (bp) | Map Location | %GC | Obs/Exp CpG |
---|---|---|---|---|---|
P1-CGI | 1–2019 | 2019 | chr10:129,466,183–129,468,201 | 63 | 0.63 |
US-CGI | 241–720 | 480 | chr10: 129,466,423–129,466,902 | 65.2 | 1.1 |
DS-CGI | 901–1440 | 540 | chr10:129,467,083–129,467,622 | 73.1 | 0.79 |
ncRNA | ncRNA Locus ID | ncRNA Transcript ID | The Similarity of Predicted miR with miRBase Mature miRNA Sequences | MRE Locations in MGMT and EBF3 mRNAs | No. of Identified MRE in MGMT and EBF3 mRNAs | Leftmost Position (5’→3’) of Predicted MGMT and EBF3 mRNAs Target Sites. |
---|---|---|---|---|---|---|
miRNA | ENSG00000266061 | ENST00000585165.1 | hsa-miR-574-5p | MGMT-3’-UTR | 1 | 908 |
EBF3-E8, E13, 3’-UTR | 5 | 759, 1309, 3500, 3821, 4101 | ||||
MIR4297 | ENST00000579857.1 | has-miR-4297 | MGMT-E4 | 1 | 406 | |
EBF3 | 0 | - | ||||
Antisense RNA | ENSG00000275005 | ENST00000614150.1 | hsa-miR-4645-3p | MGMT | 0 | - |
EBF3-E2, E4 | 2 | 353, 423 | ||||
ENSG00000275327 | ENST00000617939.1 | hsa-miR-942-5p | MGMT-3’-UTR | 1 | 1093 | |
EBF3-E5 & 3’-UTR | 2 | 499, 3989 | ||||
IncRNA | ENSG00000227374 | ENST00000428273.1 | hsa-miR-339-5p | MGMT | 0 | - |
EBF3-E1, E5, 3’-UTR | 3 | 186, 505, 3290 | ||||
ENSG00000283141 | ENST00000635764.1 | hsa-miR-6862-5p | MGMT-3’-UTR | 1 | 946 | |
EBF3 | 0 | - |
ncRNA Locus ID | Predicted miR by miRBase Search Tool | Predicted MRE by RNA22 v2 Tool | ||||
---|---|---|---|---|---|---|
Sequence | hsa-miR | Score | E-value | Heteroduplex | p Value | |
ENSG00000266061 | UGUGUGUGUGUGUGUGUGUGUGU | hsa-miR-574-5p | 100 | 0.009 | 0.36 | |
miRNA4297 | UGCCUUCCUGUCUGUG | hsa-miR-4297 | 88 | 0.22 | 0.14 | |
ENSG00000275005 | UAGUUCUUGCCUGG | hsa-miR-4645-3p | 70 | 4.2 | - | - |
ENSG00000275327 | ACAUGGCCAAAACAGAG | hsa-miR-942-5p | 67 | 3.6 | 0.072 | |
ENSG00000227374 | AGGUUCCCUCUGGCCGC | hsa-miR-4726-3p | 67 | 6.4 | - | - |
ENSG00000283141 | GCAUGCUGGGAGAGACU | hsa-miR-6862-5p | 67 | 2.4 | 0.225 | |
MIR127 | UCGGAUCCGUCUGAGCUUGGCU | hsa-miR-127-3p | 110 | 6 × 10−4 | 0.107 | |
MIR370 | GCCUGCUGGGGUGGAACCUGGU | hsa-miR-370-3p | 110 | 7 × 10−4 | 0.225 |
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Ibrahim Al-Obaide, M.A.; Arutla, V.; Bacolod, M.D.; Wang, W.; Zhang, R.; Srivenugopal, K.S. Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression. Int. J. Mol. Sci. 2021, 22, 2492. https://doi.org/10.3390/ijms22052492
Ibrahim Al-Obaide MA, Arutla V, Bacolod MD, Wang W, Zhang R, Srivenugopal KS. Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression. International Journal of Molecular Sciences. 2021; 22(5):2492. https://doi.org/10.3390/ijms22052492
Chicago/Turabian StyleIbrahim Al-Obaide, Mohammed A., Viswanath Arutla, Manny D. Bacolod, Wei Wang, Ruiwen Zhang, and Kalkunte S. Srivenugopal. 2021. "Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression" International Journal of Molecular Sciences 22, no. 5: 2492. https://doi.org/10.3390/ijms22052492