The long-range interaction between two GNAS imprinting control regions delineates pseudohypoparathyroidism type 1B pathogenesis
Yorihiro Iwasaki, … , Qing He, Murat Bastepe
Yorihiro Iwasaki, … , Qing He, Murat Bastepe
Published February 28, 2023
Citation Information: J Clin Invest. 2023;133(8):e167953. https://doi.org/10.1172/JCI167953.
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Research Article Endocrinology Genetics

The long-range interaction between two GNAS imprinting control regions delineates pseudohypoparathyroidism type 1B pathogenesis

Abstract

Genetic defects of GNAS, the imprinted gene encoding the stimulatory G protein α-subunit, are responsible for multiple diseases. Abnormal GNAS imprinting causes pseudohypoparathyroidism type 1B (PHP1B), a prototype of mammalian end-organ hormone resistance. Hypomethylation at the maternally methylated GNAS A/B region is the only shared defect in patients with PHP1B. In autosomal dominant (AD) PHP1B kindreds, A/B hypomethylation is associated with maternal microdeletions at either the GNAS NESP55 differentially methylated region or the STX16 gene located approximately 170 kb upstream. Functional evidence is meager regarding the causality of these microdeletions. Moreover, the mechanisms linking A/B methylation and the putative imprinting control regions (ICRs) NESP-ICR and STX16-ICR remain unknown. Here, we generated a human embryonic stem cell model of AD-PHP1B by introducing ICR deletions using CRISPR/Cas9. With this model, we showed that the NESP-ICR is required for methylation and transcriptional silencing of A/B on the maternal allele. We also found that the SXT16-ICR is a long-range enhancer of NESP55 transcription, which originates from the maternal NESP-ICR. Furthermore, we demonstrated that the STX16-ICR is an embryonic stage–specific enhancer enabled by the direct binding of pluripotency factors. Our findings uncover an essential GNAS imprinting control mechanism and advance the molecular understanding of PHP1B pathogenesis.

Authors

Yorihiro Iwasaki, Cagri Aksu, Monica Reyes, Birol Ay, Qing He, Murat Bastepe

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Figure 3

Generation and characterization of STX16-ICR–deleted hESCs and HCT116 cells.

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Generation and characterization of STX16-ICR–deleted hESCs and HCT116 ce...
(A) Experimental workflow for generating STX16-ICR–deleted hESCs. An SNP (rs2296524, A/G) was used to distinguish A allele–deleted (ΔA) or G allele–deleted (ΔG) clones. (B and C) Baseline A/B methylation levels at UP (B) and DOWN (C) amplicons were calculated by MSRE-qPCR in WT hESCs, 3 STX16-ICR ΔA hESC clones, and 4 ΔG hESC clones. (D) Sequencing of a GNAS exon 5 SNP (rs7121) in A/B transcripts. Three ΔA and 4 ΔG hESC clones were analyzed: 1 representative ΔA clone and 2 representative ΔG clones are shown. (E–J) Following the treatment with 2 μM GSK3484862 for 2 days, A/B methylation levels were calculated at the indicated time points by MSRE-qPCR. Time courses of the methylation levels at UP (E) and DOWN (F) amplicons in WT hESCs, 2 ΔA hESC clones, and 2 ΔG hESC clones. UP (G) and DOWN (H) amplicons on day 23 in WT hESCs, 3 ΔA hESC clones, and 4 ΔG hESC clones. Time courses of the methylation levels at UP (I) and DOWN (J) amplicons in WT and STX16-ICR–/– HCT116 cells. (K) Expression levels of GNAS transcripts in WT, ΔA, and ΔG hESCs, quantified by qRT-PCR and normalized to β-actin. For B, C, G, H, and K, each dot represents an independent hESC clone. WT hESCs versus ΔA or ΔG hESC clones were compared using a 1-sample t test with Bonferroni correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.