Evidence from AD PHP B patients as well as

Evidence from AD-PHP1B patients as well as mouse models indicates that the expression levels of the two transcripts, exon A/B and Gsα, are oppositely regulated in cis in imprinted tissues (Plagge and Kelsey 2006; Williamson et al., 2004). Absence of paternal Gsα transcript expression is attributed at least in part to the prevention of Gsα expression by the expressed A/B transcript. As expected, we detected biallelic expression of Gsα in all hiPSCs and fibroblasts, independently of A/B DMR methylation and transcription. Regarding the results in progenies, tissue-specific silencing of paternal Gsα has been described in brown fat ptio manufacturer (Williamson et al., 2004) of mesenchymal origin) and specific neurons (Chen et al., 2009); however, we do not observe Gsα allelic silencing in the MSC and NSC studied. The MSC and NSC analyzed here have not reached the differentiation status of brown fat cells and imprinted neurons, likely explaining our results and supporting the requirement of “terminal” cell differentiation for Gsα allelic silencing to occur, as previously shown in the kidney (Turan et al., 2014; Zheng et al., 2001). In most tissues, XL DMR has a maternal-specific germline methylation; thus, XL DMR methylation, absence of maternal XLsα transcription, and monoallelic paternal expression are expected in hiPSCs and progenies. Surprisingly, the paternally expressed imprinted XLsα transcript showed biallelic expression in all hiPSC clones from the two parental fibroblast lines but, as expected, monoallelic expression in all progenies (except one, also unstable for A/B). Intriguingly, this biallelic expression of XLsα in all hiPSC clones was observed in spite of a maintained XL DMR methylation and thus was independent of a change in allele-specific DNA methylation. This indicates that imprinting mechanism of XLsα transcript expression is not methylation dependent (at least mostly). Correlation between allelic expression of imprinted genes including NESP55 and methylation of identified DMR has been previously reported in hESCs (Adewumi et al., 2007; Kim et al., 2007; Rugg-Gunn et al., 2007). An association between the variability observed in inter-cell line allelic expression status and the DMR DNA methylation was present in one study (Kim et al., 2007), but not others in which monoallelic NESP55 expression associated with maintenance in NESP DMR methylation in hESCs (Adewumi et al., 2007; Rugg-Gunn et al., 2007). We detected the maternally expressed imprinted transcript NESP55 in three hiPSC samples. In contrast to A/B and XLsα transcripts, expression was monoallelic in these hiPSCs. This stability in the monoallelic expression of NESP55 in hiPSCs raises the possibility that the process that maintains methylation at NESP DMR (or protect the unmethylated allele against aberrant methylation) might differ for NESP whose imprint is acquired postfertilization. In summary, our studies indicate that (1) methylation at the GNAS locus DMRs is DMR and cell line specific, (2) methylation at the A/B DMR is correlated with A/B transcript expression, and (3) changes in allelic transcript expression can be independent of a change in allele-specific DNA methylation. The study of parental, reprogrammed, and differentiated cells should provide a model for studying the mechanisms controlling GNAS methylation, such as hydroxymethylation (Smallwood and Kelsey 2012); factors involved in transcript expression; and possibly mechanisms involved in the pathophysiology of PHP1B. This model will benefit from the possibility of differentiating PSCs in cell types in which Gsα is paternally silenced, such as BAT (Elabd et al., 2009) or proximal tubule (Montserrat et al., 2012) as shown for Angelman and Prader-Willi syndromes, two neurodevelopmental disorders of genomic imprinting (Chamberlain et al., 2010).
Experimental Procedures
Acknowledgments
Introduction