Cis-regulatory variations: a study of SNPs around genes showing cis-linkage in segregating mouse populations

Abstract

Background: Changes in gene expression are known to be responsible for phenotypic variation and susceptibility to diseases. Identification and annotation of the genomic sequence variants that cause gene expression changes is therefore likely to lead to a better understanding of the cause of disease at the molecular level. In this study we investigate the pattern of single nucleotide polymorphisms (SNPs) in genes for which the mRNA levels show cis-genetic linkage (gene expression quantitative trait loci mapping in cis, or cis-eQTLs) in segregating mouse populations. Such genes are expected to have polymorphisms near their physical location (cis-variations) that affect their mRNA levels by altering one or more of the cis-regulatory elements. This led us to characterize the SNPs in promoter (5 Kb upstream) and non-coding gene regions (introns and 5 Kb downstream) (cis-SNPs) and the effects they may have on putative transcription factor binding sites.

Results: We demonstrate that the cis-eQTL genes (CEGs) have a significantly higher frequency of cis-SNPs compared to non-CEGs (when both sets are taken from the non-IBD regions, i.e. regions not identical by descent). Most CEGs having cis-SNPs do not contain these SNPs in the phylogenetically conserved regions. In those CEGs that contain cis-SNPs in the phylogenetically conserved regions, enrichment of cis-SNPs occurs both within and outside of the conserved sequences. A higher fraction of CEGs are also seen to harbor cis-SNP that affect predicted transcription factor binding sites, a likely consequence of the higher cis-SNPs density in these genes.

Conclusion: This present study provides the first genome-wide investigation of the putative cis-regulatory variations in a large set of genes whose levels of expression give rise to cis-linkage in segregating mammalian populations. Our results provide insights into the challenges that exist in identifying polymorphisms regulating gene expression using bioinformatic sequence analysis approaches. The data provided herein should benefit future investigations in this area.

Figure 1

Determining nIBD-BXH regions from IBD blocks between mouse strains. B6 refers to C57BL/6J, DBA refers to DBA/2J, and C3H refers to C3H/HeJ. Horizontal bars represent genomic sequence. Regions that are in the same color between two or more strains represent the IBD blocks between those strains. nIBD-BXH (indicated with a box) are regions that are IBD between C3H/HeJ and DBA/2J, but nIBD between C3H/HeJ and C57BL/6J, and nIBD between C57BL/6J and DBA/2J, as explained in the text.

Figure 2

An example of a putative candidate cis-regulatory SNP affecting transcription in mouse F₂ population. A. cis-acting LOD scores for Casc4 on chromosome 2 in multiple tissues and sample sets (male, female or combined/all) in the BXH cross. x-axis – genomic location in Mb, y-axis – LOD score from interval mapping. Physical location of the gene is indicated with a red arrow-head. Only LODs scores >10 are shown. B. Association of expression levels of Casc4 with genotypes of the promoter SNP, mCV23866990. The distribution of the expression levels in brain (left) and adipose (right) is shown according to the genotypes of this SNP in the F₂ animals. A_A represents the DBA and C3H allele, and C_C the B6 allele. C. A binding site for transcription factor Hand1 is affected by SNP mCV23866990. The polymorphism changes a highly conserved base in the binding site (T→C change on the reverse strand, boxed and shaded). The frequency matrix and a sequence logo of the profile representing the binding site are shown. D. Scatter plot of Casc4 (x-axis) versus Hand1 expression levels in the body atlas data set [49, 50]. Hand1 expression is correlated to that of Casc4 with a p-value of < 10^-6 (Spearman rank order correlation -0.58).