Establishing the role of the «zona pellucida» binding protein 2 (ZPBP2) in the asthma-associated locus using a mouse model

Childhood asthma is a common chronic condition with increasing incidence in many developed countries, including Canada. Sex stratification indicates prepubescent boys are more susceptible than girls and adult males. Childhood asthma has a complex etiology involving the contribution of both genetic and environmental factors. One of the best replicated genome-wide association study (GWAS) hits for childhood asthma localizes at human chromosomal region 17q12-q21 (17q21). This region harbors several genes including, IKAROS zinc finger 3 (Aiolos) (IKZF3), zona pellucida binding protein 2 (ZPBP2), and the putative causal asthma gene, Orosomucoid-like 3 (ORMDL3).Genetic variants associated with higher ORMDL3 expression are also associated with increased asthma susceptibility. Several studies assessing functional single nucleotide polymorphisms (SNPs) in this region point to ZPBP2 as a potential regulatory locus for ORMDL3. We hypothesized that the ZPBP2 locus contributes to asthma predisposition through regulatory elements(s) controlling gene expression in cis, including ORMDL3 and/or the function of the ZPBP2 protein. If the first hypothesis is true, we expect to find gene expression changes in cis and if the second hypothesis is correct we expected no effect on the expression of neighboring genes. In both cases, we expect to detect effects on gross mouse phenotypes and trans differential gene expression identifying relevant biological pathways. The human asthma-associated 17q21 region has an ortholog on mouse chromosome 11 and is largely conserved. To test our hypothesis, we examined gene expression in mice with a targeted deletion of Zpbp2 (Zpbp2ko). These mice also show increased airway hyperresponsiveness. To better understand the role of Zpbp2 locus we explored the developmental expression dynamics of genes in the asthma-associated region in B6 mice and the effect of deleting the Zpbp2 5' region on 1) gross phenotypes, 2) global gene expression in the mouse genome (trans), and 3) the expression of neighboring genes (cis), including Ormdl3 and Ikzf3. We found that the expression of asthma-related genes is age- and sex-dependent with the highest Ormdl3 and Ikzf3 expression observed at 4 weeks old in males. Our data show reduced expression of genes involved in adipocytokine signaling i.e. PPAR pathway, and statistically significant increase in Ormdl3 expression compared to B6 controls. In addition, there was an overall age-dependent down-regulation of Ormdl3 expression in B6 mice that is lost in Zpbp2ko males, suggesting the observed increase in Ormdl3 expression is due to the absence of age-dependent downregulation. Ikzf3 expression differences were not statistically significant for both males and females although mutants generally showed higher mRNA levels. The role of Ikzf3 in inflammatory responses suggests the effect of the deletion may be more pronounced in allergen-challenged mice. Our data supports our hypothesis that Zpbp2 plays a role in the regulation of Ormdl3 expression, however we cannot refute that either the Zpbp2 locus or the protein regulates Ormdl3 expression because we observe both cis and trans effects of the deletion. We cannot also refute the possibility that Zpbp2 regulates other neighboring genes in addition to Ormdl3 and Ikzf3 due to low mRNA levels in lung and spleen tissues. Finally, we posit that the Zpbp2 5' locus harbors repressor element(s) that influence changes in AHR in male mice potentially due to the maintenance of prepubertal Omdl3 expression levels.

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