| Popis: |
Additional file 5: Fig. S5. A subset of maternally expressed imprinted genes are downregulated after Kdm6a KO in BC but not in CB clones. (A) Scatter plot of average expression changes for canonical imprinted genes, either maternally (purple) or paternally (blue) expressed, in Kdm6aΔ/Y (Kdm6aΔE1, Kdm6aΔE3, and Kdm6aΔP4) versus wt (average from 2 clones) based on diploid RNA-seq analysis in male BC derived clones. Only imprinted genes with > 1TPM in at least one sample are shown. (B) RT-PCR analysis confirms decreased expression of Meg3 and Rian in all male BC KO clones (Kdm6aΔE1, Kdm6aΔE3, Kdm6aΔP4). CRISPR + /– labels indicate presence/absence of transfection with CRISPR/Cas9 and Kdm6a sgRNAs, while a ± for Kdm6aΔP and Kdm6aΔE clones indicates editing result. Non-edited CRISPR control clones (Kdm6aE14, Kdm6aP2) maintain expression of these genes. (C) Sanger sequencing in wt male BC clones confirmed the presence of SNPs (gDNA) and mono-allelic expression (cDNA) of Meg3 and Rian. (D, E) UCSC Genome browser (GRCm38/mm10) views of allele-specific RNA-seq profiles at the maternally expressed Dlk1/Mirg polycistron region and at a subset of imprinted Xlr genes. Allelic read profiles on the maternal chromosome (purple) and the paternal chromosome (blue) are based on SNP analysis in male BC wt clone (KO-), compared to male BC Kdm6aΔE1 clone (KO +). (F) UCSC Genome browser (GRCm38/mm10) view of allele-specific RNA-seq, ATAC-seq and H3K27me3 ChIP-seq profiles generated in male wt and Kdm6aΔE1 at the imprinted genes H19 (maternally expressed) and Igf2 (paternally expressed). The DMR (differentially methylated region) is denoted by a green bar. (G) TPM expression fold change measured by RNA-seq for H19 and Igf2 in Kdm6AΔE1 versus wt shows H19 downregulation concordant to the expected upregulation of Igf2. (H) Same analysis as in (A) for male CB Kdm6a Δ/Y clones (Kdm6aΔE2.5, Kdm6aΔE2.7, and Kdm6aΔP2.1). (I, J) Venn diagrams to compare the number of allele-biased genes in BC male wt, BC female wt, and CB male wt. Allelic bias was defined as a ≥ twofold difference between alleles. BC derived cells show more commonality in allele-biased genes than with CB derived cells. |
