Mechanisms of differential expression of the CYP2A13 7520C and 7520G alleles in human lung: allelic expression analysis for CYP2A13 heterogeneous nuclear RNA, and evidence for the involvement of multiple cis-regulatory single nucleotide polymorphisms.
OBJECTIVE
To identify the mechanisms underlying the decreased allelic expression of a common CYP2A13 allele (7520C>G) in the human lung; CYP2A13 is expressed selectively in the respiratory tract, and is highly efficient in the metabolic activation of several chemical carcinogens.


METHODS
The 7520C/G alleles were compared for mRNA stability in cells and relative heterogeneous nuclear RNA (hnRNA) levels in human lungs. Promoter region single nucleotide polymorphisms (SNPs) were identified and analyzed through in-vitro reporter gene assays and gel-shift assays, to uncover the causative SNPs responsible for the decreased allelic expression.


RESULTS
(i) The 7520C>G SNP does not influence CYP2A13 mRNA stability in CYP2A13-transfected human lung or nasal epithelial cells; (ii) levels of the 7520G hnRNA were consistently lower (<10%) than the levels of the 7520C hnRNA in lung samples from nine heterozygous individuals; (iii) three SNPs (-1479T>C, -3101T>G, and -7756G>A) in linkage disequilibrium with the 7520C>G variation were found to cause altered interaction with DNA-binding proteins and decreases in promoter activity; (iv) the suppressive effects of the -1479T>C, -3101T>G, and -7756G>A SNPs on the CYP2A13 promoter were additive, whereas the negative effects of the -1479T>C SNP were enhanced by methylation of -1479C.


CONCLUSIONS
The decrease in the expression of 7520G allele was because of the cumulative suppressive effects of multiple SNPs, with each by itself having a relatively small effect on CYP2A13 transcription.
Wu
Hong
H
Wadsworth Center, New York State Department of Health and School of Public Health, State University of New York at Albany, Albany, New York 12201-0509, USA.
Zhang
Xiuling
X
Ling
Guoyu
G
D'Agostino
Jaime
J
Ding
Xinxin
X
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D002648:Child; D002675:Child, Preschool; D019175:DNA Methylation / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007223:Infant; D008168:Lung / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D020871:RNA Stability; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human