FP25-6 Methylation and Transcripts Expression of GNAS Locus in Human iPSC and Their Differenciated Derivatives

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP25-Signaling Originating from Membrane Receptors
Basic/Translational
Sunday, June 16, 2013: 10:45 AM-11:15 AM
Presentation Start Time: 11:10 AM
Room 133 (Moscone Center)

Poster Board SUN-389
Virginie Grybek*1, Mathilde Girard2, Stéphanie Maupetit-Mehouas3, Christine Baldeschi4, Marc Peschanski5, Agnes Linglart6 and Caroline Silve7
1INSERM U986 , Hôpital Bicêtre, Le Kremlin-Bicêtre, France, 2CECS, I-STEM, AFM, Institute for Stem cell Therapy and Exploration of Monogenic Diseases, Evry cedex, France, 3INSERM U986, Hôpital Bicêtre, Le Kremlin-Bicêtre, France, 4UEVE U-861, I-STEM, AFM, Institute for Stem cell Therapy and Exploration of Monogenic Diseases, Evry cedex, France, 5INSERM U-861, I-STEM, AFM, Institute for Stem cell Therapy and Exploration of Monogenic Diseases, Evry cedex, France, 6INSERM U986, Service d'Endocrinologie Pédiatrique, Hôpital Bicêtre, Bicêtre, France, 7INSERM U986, Hôpital Bicêtre, Le Kremlin Bicêtre, France
Objective

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.

Methods

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).

Results

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.

Conclusion

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

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

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