Fig. 3
Identification of Fkbp6 as a novel imprinted gene. a The Fkbp6 gene region with the location of ZFP57 binding within the promoter regions. PSQ1 and PSQ2 denote regions analyzed by bisulphite pyrosequencing shown in (c). b Fkbp6 expression levels and allele-specificity in mouse tissues and ES cells from quantitative RT-PCR and SNP pyrosequencing. The height of each bar represents Fkbp6 mRNA levels normalized to 18 s rRNA and shown relative to liver expression levels. Blue and red stacked bars show relative contribution of BL6 and Cast alleles, respectively, to overall Fkbp6 levels. Upper and lower error bars correspond to standard deviation between biological replicates for PCR and pyrosequencing analysis, respectively (n = 8 replicates per each tissue: 4 BC and 4 CB; n = 4 for ES cell lines (ESCs). c The Fkbp6 promoter is not a somatic DMR. Quantitative bisulphite pyrosequencing analysis of the Fkbp6 promoter region, which assays overall methylation level in the population of cells in a non-allele-specific manner. Relative proportions of black (methylated) and white (unmethylated) filled areas in circles depict average percentage methylation for individual CpGs (n = 4; 2 BC and 2 CB bioreplicates for each tissue type). Numbers adjacent show median percentage methylation values across CpGs covered by PSQ1 and PSQ2 sequencing primers. Grey circles denote CpGs not covered in the assay. Black arrow denotes Fkbp6 transcription start site and direction of expression. Grey arrow underneath indicates position of the Cast-specific CpG (SNP rs13487942), quantified in (d). d Left: Bar chart showing methylation status of the CpGCast when inherited on paternal (BC cross, blue bars) and maternal (CB cross, pink bars) chromosomes. Data from pyrosequencing of adult testis, E16.5 brain and placenta, n = 2 × BC and 2 × CB samples per tissue. Right: Representative bisulphite pyrograms from individual BC and CB placenta tissues. CpGCast region is framed in red, methylation level is calculated from the relative height of ‘T’ (unmethylated, Um) and ‘C’ (methylated, Me) peaks. e Top: Relative enrichment of H3K4 and H3K9 tri-methylation in E16.5 tissues. Data shown relative to input and normalized to a reference non-enriched genomic location. Bottom: Allele ratios from SNP pyrosequencing of ChIP-enriched DNA. Error bars reflect standard deviation between three technical replicates for each sample shown