Allelic expression and DNA methylation profiles of promoters at the parental Oct4 and Nanog genes in Mus musculus ES cell/Mus caroli splenocyte hybrid cells

Abstract

Expression of the parental Oct4 and Nanog alleles and DNA methylation of their promoters were studied in a set of Mus musculus embryonic stem (ES) cell/M. caroli splenocyte hybrid cells containing a variable ratio of parental chromosomes 6 and 17. The transcripts of the reactivated splenocyte Oct4 and Nanog genes were revealed in all hybrid cell clones positive for M. caroli chromosomes 6 and 17. We found that 11 CpG sites in the Oct4 promoter were heavily methylated in M. caroli splenocytes (>80%), whereas M. musculus ES cells were essentially unmethylated (<1%). Analysis of the methylation status of the Oct4 promoter in seven hybrid cell clones showed that the splenocyte-derived promoter sequence lost DNA methylation so that its methylation level was comparable with that of the ES cells. Additionally, no preferential de novo methylation was seen in the Oct4 promoters of M. musculus and M. caroli in teratomas developed from two independent hybrid clones. The upstream region of Nanog was heavily methylated in mouse embryonic fibroblasts (66%) and less methylated in M. caroli splenocytes (24%). The Nanog promoter region was completely unmethylated in M. musculus ES cells. We found that both parental alleles of the Nanog gene promoter were essentially unmethylated in five examined hybrid clones. Thus, we have demonstrated that (1) the Oct4 and Nanog genes of splenocytes are activated, and their promoters undergo demethylation in ES cell hybrids; (2) these events are independent of the number and ratio of parental chromosomes carrying these genes.

Bidirectional reprogramming of mouse embryonic stem cell/fibroblast hybrid cells is initiated at the heterokaryon stage

“…Using inter-specific hybrid cells obtained by fusing M. musculus ES cells and M. caroli embryonic fibroblasts, we were able to employ the differences in DNA sequence to distinguish the methylation status of 12 CpG sites (from -470 bp to -110 bp) in the promoter of the Oct4 allele of M. musculus and 11 CpG sites (from -458 bp to -111 bp) in the promoter of the Oct4 allele of M. caroli (Battulin et al 2009). Figure 6 illustrates the methylation status of CpG sites in the promoter of parental Oct4 alleles in M. musculus ES cells and M. caroli fibroblasts.…”

“…The regions containing five CpG sites in the M. musculus (-301 bp to +1 bp) and five CpG sites in the M. caroli (-306 bp to +1 bp) Nanog promoter were analyzed by bisulfite DNA sequencing. The outer and inner primers for the Oct4 and Nanog promoters were used as previously described (Battulin et al 2009). Recombinant clones were only accepted with ≥90% cytosine conversion and all possible clonalities were excluded.…”

“…Bisulfite DNA methylation analysis Isolation of genomic DNA and bisulfite mutagenesis sequencing analysis were conducted as previously described (Battulin et al 2009). The regions containing 12 CpG sites in the M. musculus (-470 bp to -110 bp) and 11 CpG sites in M. caroli (-458 bp to -11 bp) Oct4 promoter were examined for DNA methylation.…”

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

“…The region containing 12 CpG sites in the M. musculus Oct4 promoter (−470 bp to−110 bp) and the region containing 5 CpG sites in the Nanog promoter (−301 bp to +1 bp) were examined for DNA methylation. The outer and inner primers for the Oct4 and Nanog promoters were used as previously described (Battulin et al 2009). …”

“…Bisulfite DNA methylation analysis The extraction of genomic DNA and bisulfite mutagenesis sequencing analysis were conducted as previously described (Battulin et al 2009). The region containing 12 CpG sites in the M. musculus Oct4 promoter (−470 bp to−110 bp) and the region containing 5 CpG sites in the Nanog promoter (−301 bp to +1 bp) were examined for DNA methylation.…”