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 αsubunit, 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 PHP1B patients. 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 ~170 kb upstream. Functional evidence is meager regarding the causality of these microdeletions. Moreover, the mechanisms linking A/B methylation and these 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. Using this model, we showed that NESP-ICR is required for methylation and transcriptional silencing of A/B on the maternal allele. We also found that SXT16-ICR is a longrange enhancer of NESP55 transcription, which originates from maternal NESP-ICR. Furthermore, we demonstrated that 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 the PHP1B pathogenesis.

GNAS AS2 methylation status enables mechanism-based categorization of Pseudohypoparathyroidism Type 1B

“…Remarkably, AS2 methylation levels were significantly lower in the absence of either the STX16- or the NESP-ICR, specifically on the maternal allele but not on the paternal allele ( Figure 4, B and C ). The methylation levels at the flanking AS1 and XL DMRs were not reduced, as we have shown previously ( 18 ). These findings demonstrate that STX16- and NESP-ICRs are indispensable for AS2 methylation in an early embryonic period and, furthermore, suggest that STX16 enhancer–driven NESP55 transcription specifically affects the AS2 DMR within the AS-XL region on the maternal allele.…”

“…Based on the result that AS2 methylation levels are almost entirely lost in patients with PHP1B with deletions in either the STX16 or NESP55-AS exons 3/4 regions ( Figure 3B ), we hypothesized that these regions are involved in a previously uncharacterized mechanism regulating AS2 methylation. Two ICRs, NESP-ICR and STX16-ICR, are required for A/B methylation on the maternal allele in an early embryonic period and possibly during oogenesis ( 18 , 21 ). Accordingly, we used hESCs with either STX16-ICR or NESP-ICR deletion ( 18 ) to test which ICR affects AS2 methylation levels.…”

“…Two ICRs, NESP-ICR and STX16-ICR, are required for A/B methylation on the maternal allele in an early embryonic period and possibly during oogenesis ( 18 , 21 ). Accordingly, we used hESCs with either STX16-ICR or NESP-ICR deletion ( 18 ) to test which ICR affects AS2 methylation levels. WT hESCs showed lower AS2 methylation levels (~3.6%) by MSRE-qPCR compared with those observed in the leukocyte DNA from unaffected controls (3.73%–46.7%) ( Figure 3B and Figure 4, A–C ).…”

“…The inserted sequences of both kindreds share substantial homology (~93% identity) over a ~600 bp region, followed by several tandem consensus polyadenylation signals at the telomeric end ( Figure 5B and Supplemental Figure 2 ). To examine if these sequences might prematurely truncate the NESP55 transcript, we cloned the polyadenylation signals and the flanking sequences (from kindred #1 in Figure 5A ) into luciferase reporter plasmids driven by the NESP55 promoter with STX16 enhancer ( 18 ) and tested its effect in hESCs. Remarkably, including these patient-derived sequences suppressed the luciferase activity driven by the NESP55 promoter.…”

“…Until recently, the GNAS methylation pattern in patients with deletions affecting STX16 was thought to be limited to isolated A/B hypomethylation. Mechanistically, STX16 was shown to operate as an early embryonic stage–specific enhancer for NESP55 transcription, which is essential for methylating the maternal A/B DMR ( 18 ). However, a recent study identified an additional DMR between AS exon 1 and the XL exon, referred to as AS2, that shows hypomethylation in patients with PHP1B with STX16 deletions ( 19 ).…”