title: Functional characterization of genetic polymorphisms in the H2AFX distal promoter.
abstract: Due to the critical role of the H2AX histone variant in double-strand break repair, genetic variants in the H2AX gene, H2AFX, may influence cancer susceptibility. Genetic association studies have correlated H2AFX upstream variants with cancer risk; however it is unclear if any are causal. H2AFX has at least two alternate transcripts that encode the same reading frame; a short 0.6kb transcript that lacks an intron or poly-A tail and is predicted to be highly expressed during the replication stage of the cell cycle, and a long 1.6kb poly-A tailed transcript that is expressed in a replication-independent manner. To examine the functional impact of the rs643788, rs8551, rs7759, and rs2509049 upstream variants, we characterized their influence on gene expression, cell survival after DNA assault, and transcription factor binding. Analysis of allelic imbalance using quantitative sequencing of cDNA from lymphoblast cell lines did not reveal any difference in expression of the 1.6kb polyadenylated transcript between the common H2AFX upstream haplotypes. We did, however, identify a previously unreported 197 base pair intron in the H2AFX 3'untranslated region that appears to be present regardless of haplotype. Assessment of cell survival after irradiation treatment did not show any difference in survival between cell lines of different haplotypes. Gel shift assays revealed that the rs643788 C allele disrupts YY1 transcription factor binding and the rs2509049 C allele binds more strongly to a protein complex than does the rs2509049 T allele. Though we did not identify any differences in expression or survival between haplotypes, differential protein binding at two of the polymorphisms suggests further functional analyses may reveal a role for these variants in influencing gene expression at specific points of the cell cycle or in specific tissues.
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, 675W 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada; Department of Medical Genetics, University of British Columbia, C201 - 4500 Oak Street, Vancouver, British Columbia V6H 3N1, Canada.
authors.forename: Karla L
authors.initials: KL
authors.lastname: Bretherick
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, 675W 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada.
authors.forename: Stephen
authors.initials: S
authors.lastname: Leach
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, 675W 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada; Department of Medical Genetics, University of British Columbia, C201 - 4500 Oak Street, Vancouver, British Columbia V6H 3N1, Canada; Department of Biomedical Physiology and Kinesiology, Simon Fraser University, K9625 - 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada. Electronic address: abrooks-wilson@bcgsc.ca.
authors.forename: Angela R
authors.initials: AR
authors.lastname: Brooks-Wilson
mesh_terms: D022981:Allelic Imbalance; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D004252:DNA Mutational Analysis; D005720:Gamma Rays; D006367:HeLa Cells; D006657:Histones / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: C468783:H2AX protein, human; D006657:Histones
keywords: H2AFX; Intron; Promoter; Single nucleotide polymorphism; Transcription factor; YY1