A variant in the CHEK2 promoter at a methylation site relieves transcriptional repression and confers reduced risk of lung cancer.
Checkpoint kinase (CHEK) 2, a tumor suppressor gene, plays an essential role in the DNA damage checkpoint response cascade. We first investigated two polymorphisms in the proximal promoter of the CHEK2 gene and evaluated their associations with the risk of lung cancer in a case-control study using 500 incident lung cancer cases and 517 cancer-free controls. We found that CHEK2 rs2236141 -48 G > A was significantly associated with lung cancer risk (P = 0.0018). Similar results were obtained in a follow-up replication study in 575 lung cancer patients and 589 controls (P = 0.042). Quantitative polymerase chain reaction showed that individuals with the G allele had lower levels of CHEK2 transcripts in peripheral blood mononuclear cells and normal lung tissues. The -48 G-->A variant eliminated a methylation site and thereby relieve the transcriptional repression of CHEK2. Therefore, this polymorphism affected downstream transcription through genetic and epigenetic modifications. Luciferase reporter assays demonstrated that the major G allele significantly attenuated reporter gene expression when methylated. Electrophoretic Mobility shift assays and surface plasmon resonance revealed that the methylated G allele increased transcription factor accessibility. We used in vivo chromatin immunoprecipitation to confirm that the relevant transcription factor was Sp1. Using lung tissue heterozygous for the G/A single-nucleotide polymorphism, we found that Sp1 acted as a repressor and had a stronger binding affinity for the G allele. These results support our hypothesis that the CHEK2 rs2236141 variant modifies lung cancer susceptibility in the Chinese population by affecting CHEK2 expression.
Zhang
Shuyu
S
The State Key Laboratory of Genetic Engineering and The MOE Key Laboratory of Contemporary Anthropology, School of Life Science, Fudan University, Shanghai 200433, China.
Lu
Juan
J
Zhao
Xueying
X
Wu
Wenting
W
Wang
Huibo
H
Lu
Jun
J
Wu
Qihan
Q
Chen
Xin
X
Fan
Weiwei
W
Chen
Hongyan
H
Wang
Feng
F
Hu
Zhibin
Z
Jin
Li
L
Wei
Qingyi
Q
Shen
Hongbing
H
Huang
Wei
W
Lu
Daru
D
D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000209:etiology / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D016329:Sp1 Transcription Factor / Q000502:physiology; D014158:Transcription, Genetic
D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases