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 4

Long-range interaction between the STX16-ICR and the NESP-ICR.

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Long-range interaction between the STX16-ICR and the NESP-ICR. (A) Genom...
(A) Genome Browser track showing H3K27Ac ChIP-Seq signals within the STX16 locus in H1 hESCs. Exon numbering is based on NCBI RefSeq NM_003763.6. (B) Schematic representation of cloned regions for luciferase assays and primer locations used in 3C assays. (C) Luciferase assay in WT hESCs using a negative control [promoter(–)] or NESP55 promoter (NESP55pr, blue box), NESP55pr plus STX16-ICR (red box,) or STX16–3′-UTR (light green box) constructs (left). Graph shows a representative result of 5 independent experiments. Intergroup comparisons were performed by 1-way ANOVA with Tukey’s post hoc test. ****P < 0.0001. (D–F) 3C PCR assay in hESCs. Proximity-ligated fragments were amplified using primers #1–#3, as depicted in B. (D) PCR results from 3C samples (Ligation+) and negative control templates, i.e., no ligation (Ligation–) and no digestion (Undigested) using primers #1 and #3 (upper gel), and primers #2 and #3 (lower gel, a negative control locus). Representative images from 5 independent experiments are shown. (E) Sequencing of the 3C-PCR product using primers #1 and #3 showing the ligation junction. (F) Percentages of A (maternal) and G (paternal) NGS reads at rs3787497 in 3C PCR products. Each dot represents an independent experiment. The interaction frequency was statistically compared with 50% (biallelic) by a 1-sample t test with Bonferroni correction. *P < 0.05.