Assessment of the Relevance of a CD14 Gene Polymorphism on RNA Polymerase II DNA Binding by Haplotype-Specific Immunoprecipitation (HaploChIP)

Aim: Genetic background may influence susceptibility and progression of various diseases. A polymorphic site within the CD14 gene, rs2569190 / C-159T, was described to be associated with soluble CD14 levels (sCD14) in hepatitis C virus (HCV) infected patients. Both, sCD14 and the membrane-associated form of CD14 (mCD14) are important compounds in mediating endotoxin sensing on myeloid and non-myeloid cells. In vitro experiments suggest the T allele of the SNP to sensitize the host for exogenous or endogenous lipopolysaccharide (LPS) via an enhanced CD14 expression. This study aimed at analysing the in vivo relevance of that SNP on RNA polymerase II DNA binding as a surrogate for transcriptional activity.

Methods: rs2569190 genotyping was performed by TaqMan PCR-based allelic discrimination in 47 healthy blood donors. Peripheral blood mononuclear cells (PBMC) from heterozygous donors were subjected to haplotype-specific chromatin immunoprecipitation (HaploChIP) using antibodies directed against phosphorylated RNA polymerase II. Within freshly prepared intact cells, formaldehyde-fixed protein/DNA complexes were enzymatically sheared and captured with specific antibodies. Non-immunoprecipitated fragments were used as an input control. After reversion of cross-linking, fragments were subjected to quantitative restriction fragment length polymorphism analyses (RFLP).

Results: Samples from 8 heterozygous individuals were examined by HaploChIP. Whereas input chromatin was found to contain about the same amount of C and T variant fragments, the chromatin that had been captured by anti-RNA polymerase II antibodies was found to contain about twice the numbers of T variant fragments.

Conclusion: The enrichment of T allele fragments in anti-RNA polymerase II captured material argues for a functional relevance of the rs2569190 variation in terms of a stronger RNA polymerase II binding to T allele gene variants in PBMC.