Session: FP25-Signaling Originating from Membrane Receptors
Room 133 (Moscone Center)
Poster Board SUN-389
Genomic imprinting is an epigenetic process whereby one allele undergoes a partial or total loss of expression. The GNAS locus exhibits a complex pattern of reciprocal genomic imprinting. It produces several transcripts comprising Gsa (the alpha stimulatory subunit of the G-protein), XL, A/B (also referred as 1A), NESP and the antisense transcript AS. Due to differential methylation of their promoters (DMRs), XL, A/B, AS and NESP transcripts originate from one parental allele only. The promoter of Gsa is not differentially methylated and, therefore, Gsa expression arises from both alleles in most tissues. However, due to a yet incompletely understood imprinting mechanism, Gsa is expressed from the maternal allele only in several tissues. The objective was to determine whether stem cells (SC) reprogrammed from human fibroblasts (iPSC) then differentiated, constitute a model for studying the mechanisms of establishment and maintenance of the imprinting of the GNAS locus.
We studied the methylation at the 4 GNAS DMRs and the allelic expression of GNAS transcripts by pyrosequencing in 4 iPSC lines reprogrammed from human fibroblasts and differentiated (3/4) in mesenchymal SC (MSC) and in neural SC (NSC). The mono or biallelic expression of transcripts was determined by the polymorphism rs7121 present in exon 5 and common to all GNAS transcripts (except AS).
Methylation was normal (~ 50%) in 4/4 iPSC lines at the XL and NESP DMRs, and in 3/4 iPSC lines at AB and AS DMRs. In all iPSC lines, expression of Gsa was biallelic, NESP expression was monoallelic, XL and AB expression was either mono or biallelic. After differentiation, XL and NESP methylation remained stable (50%) in 3/3 MSC and 1/3 NSC; AS methylation was increased) in 3/3 MSC and 3/3 NSC, and AB methylation was normal in 3/3 MSC and altered in 2/3 NSC. In MSC and NSC, Gsa expression was biallelic, XL and AB expression was monoallelic, NESP transcript was undetectable. Only the mono or biallelic expression of AB was correlated with methylation profile of its associated DMR.
Our results indicate that reprogramming and differentiation of iPSC alter the methylation pattern of the GNAS locus and AB and XL transcript expression.
Nothing to Disclose: VG, MG, SM, CB, MP, AL, CS
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