Imprinting status of Galpha(s), NESP55, and XLalphas in cell cultures derived from human embryonic germ cells: GNAS imprinting in human embryonic germ cells

Abstract

GNAS is a complex gene that through use of alternative first exons encodes signaling proteins Galpha(s) and XLalphas plus neurosecretory protein NESP55. Tissue-specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune-Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of Galpha(s), NESP55, and XLalphas prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of Galpha(s) was biallelic (maternal allele: 52.6%+/- 2.5%; paternal allele: 47.2%+/- 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%+/- 10%; paternal allele: 18.1%+/- 10%; p= 0.002) and XLalphas was preferentially expressed from the paternal allele (maternal allele: 2.7%+/- 0.3%; paternal allele: 97.3%+/- 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5-11 weeks postfertilization, and that imprinting of XLalphas occurs very early postfertilization. By contrast, imprinting of Galpha(s) most likely occurs after 11 weeks postfertilization and after tissue differentiation.

Figures

Figure 1

The GNAS gene complex located at 20q13.3 consists of 13 exons that encode the signaling protein Gαs. Upstream of exon 1 are three alternative exons, labeled exon 1A, XLαs, and NESP55. These three alternative first exons are spliced to exons 2–13 to produce unique transcripts. NESP55 is transcribed exclusively from the maternal allele; XLαs and exon 1A are transcribed exclusively from the paternal allele. RT‐PCR using an upstream primer specific for Gαs, XLαs, and NESP55 first exons of GNAS and a common downstream primer within exons 6, 8, and 13 of GNAS enabled us to genotype the alleles using a highly variable single nucleotide polymorphism in codon 131 (T/C) of exon 5, codon 185 (T/C) of exon 7, and codon 371 (T/C) of exon 13. The primers are indicated by thin black arrows; direction of transcription are indicated by thick arrows (thick gray arrows indicate transcription from maternal allele; black arrows indicate transcription from paternal allele).

Figure 2

(A) Sequence analysis of the product of RT‐PCR of XLαs, NESP55, and Gαs exon 5 RNA derived from the two EBD cell cultures (LVEE and SLRC) that were heterozygous for the polymorphism in codon 131 (T/C). (Polymorphism indicated by arrow; order of base pairs: GATC) (B) Sequence analysis of the product of RT‐PCR of Gαs and NESP55 exon 7 RNA derived from three EBD cell cultures (BBEP, EDEC, SCEC) that were heterozygous for the polymorphism in codon 185 (T/C). (Polymorphism indicated by arrows; order of base pairs: GATC).

Figure 2

(A) Sequence analysis of the product of RT‐PCR of XLαs, NESP55, and Gαs exon 5 RNA derived from the two EBD cell cultures (LVEE and SLRC) that were heterozygous for the polymorphism in codon 131 (T/C). (Polymorphism indicated by arrow; order of base pairs: GATC) (B) Sequence analysis of the product of RT‐PCR of Gαs and NESP55 exon 7 RNA derived from three EBD cell cultures (BBEP, EDEC, SCEC) that were heterozygous for the polymorphism in codon 185 (T/C). (Polymorphism indicated by arrows; order of base pairs: GATC).

Figure 3

Relative expression of Gαs, NESP55, and XLαs alleles in EBD cell cultures. Quantification of band intensity of the polymorphic alleles was performed with the Phosphorimager system (Bio‐Rad, Hercules, CA, USA) and corrected for variability of sample loading between lanes. In all five cell cultures heterozygous for the exon 5, 7, or 13 polymorphism, the expression of Gocs was biallelic (maternal allele = 52.6%± 2.5%; paternal allele = 47.2%± 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele = 81.9%± 10%; paternal allele = 18.1%± 10%; p= 0.002) and XLαs was preferentially expressed from the paternal allele (maternal allele = 2.7%± 0.3%; paternal allele = 97.3%± 0.3%; p= 0.007). Error bars indicate SEM.