Abstract 2084: Genetic variation at a cis-acting C/EBPG binding site is associated with allele-specific ERCC5 transcript expression

Abstract

Abstract Background: CCAAT/enhancer-binding protein gamma (C/EBPG) transcription factor expression is correlated with that of ERCC5 and other key DNA repair genes in normal bronchial epithelial cells (NBEC) suggesting a regulatory role. In prior studies, ERCC5 transcript expression was increased in a human lung carcinoma cell line H23 following CEBPG overexpression and in NBEC from 81 subjects, A allele at putative ERCC5 cis-regulatory SNP (rSNP) rs751402 and T allele at rSNP rs2296147 were associated with higher expression of ERCC5 marker SNP rs1047768 T allele transcript. rs751402 is located in open chromatin region identified by FAIRE-seq in NBEC and variation at rs751402 is predicted to alter binding of C/EBP. These studies support the hypothesis that allelic differential affinity to C/EBPG at rs751402 contributes to hereditary inter-individual variation in regulation of ERCC5 either directly or through interaction with complexes bound at rs2296147. The purpose of this study was to further investigate the role of C/EBPG in ERCC5 cis-regulation in an independent cohort of subjects and lung cancer cell lines. Methods: We knocked-down C/EBPG transcript level by C/EBPG siRNA transfection in human non-small cell lung carcinoma cell line H1703. Total and allele-specific expression (ASE) at rs1047768 was measured through multiplex competitive PCR-based amplicon sequencing library preparation followed by Illumina HiSeq next generation sequencing (NGS). This NGS controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. The genotype at rs751402 and rs2296147 in NBEC from 78 subjects and 14 human lung carcinoma cell lines was determined by TaqMan SNP genotyping assays. Direct assessment of the syntenic relationship of alleles in gDNA from poly-heterozygous individuals was assessed by allele-specific PCR followed by sequencing. Results: CEBPG transcript expression was knocked-down by 93% in H1703 cells and this was associated with 4-fold reduction in the ERCC5 transcript level at rs1047768. ERCC5 displayed significant inter-individual variation in allele specific expression (ASE) in NBEC from 85 subjects. Thirty nine out of 92 subjects including 3 cell lines were heterozygous at rs751402 and rs2296147 rSNPs and rs1047768 marker SNP and will be assessed for haplotypes comprising those sites. Conclusions: Results are consistent with CEBPG regulation of ERCC5 in the cell line H1703. The results obtained will enable us to test the hypothesis that haplotypes comprising particular alleles syntenic between rs1047768 and rs751402 are associated with higher allele-specific ERCC5 transcript abundance. Cell lines heterozygous at three sites will be subjected to CEBPG up and/or down regulation to assess effect on allele-specific ERCC5 expression at rs1047768. Citation Format: Xiaolu Zhang, Jiyoun Yeo, Erin Crawford, James C. Willey. Genetic variation at a cis-acting C/EBPG binding site is associated with allele-specific ERCC5 transcript expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2015-2084