MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression.
BACKGROUND
The O6-methylguanine-DNA methyltransferase (MGMT) protein removes O6-alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes are located within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentially affect transcription factor binding and, subsequently, alter MGMT promoter activity.


METHODS
In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively.


RESULTS
Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p ≤ 0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system.


CONCLUSIONS
The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.
Department of Obstetrics and Gynecology, Maternal-Fetal Pharmacology and Biodevelopment Laboratories, University of Texas Medical Branch, Galveston, TX, 77555-1066, USA.
Meixiang
M
Xu
Department of Obstetrics and Gynecology, Maternal-Fetal Pharmacology and Biodevelopment Laboratories, University of Texas Medical Branch, Galveston, TX, 77555-1066, USA.|A.T. Still University School of Osteopathic Medicine, 5850 E. Still Circle, Mesa, AZ, 85206, USA.
Courtney E
CE
Cross
Department of Obstetrics and Gynecology, Maternal-Fetal Pharmacology and Biodevelopment Laboratories, University of Texas Medical Branch, Galveston, TX, 77555-1066, USA.
Jordan T
JT
Speidel
Department of Obstetrics and Gynecology, Maternal-Fetal Pharmacology and Biodevelopment Laboratories, University of Texas Medical Branch, Galveston, TX, 77555-1066, USA. sabdelra@utmb.edu.
Sherif Z
SZ
Abdel-Rahman
D045744:Cell Line, Tumor; D015254:DNA Modification Methylases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes
MGMT P/E haplotypes; Promoter activity; Transcription factor binding; siRNA regulation