The long-range interaction between two GNAS imprinting control regions delineates pseudohypoparathyroidism type 1B pathogenesis
Yorihiro Iwasaki, Cagri Aksu, Monica Reyes, Birol Ay, Qing He, Murat Bastepe
Yorihiro Iwasaki, Cagri Aksu, Monica Reyes, Birol Ay, Qing He, Murat Bastepe
View: Text | PDF
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

×

Figure 1

Putative GNAS ICRs and microdeletions identified in patients with AD-PHP1B.

Options: View larger image (or click on image) Download as PowerPoint
Putative GNAS ICRs and microdeletions identified in patients with AD-PHP...
(A) Schematic locations of STX16, GNAS, and putative GNAS ICRs. Each box represents the targeted region to generate ICR-deleted hESC clones. White (unmethylated) and red (methylated) lollipops represent CpGs. Arrows show transcription from each exon, with a dotted arrow indicating silencing of Gsα expression in a tissue-specific manner. (B and C) Distribution of microdeletions in patients with AD-PHP1B with the NESP-ICR (B) or STX16-ICR (C) deletion. Each deletion is shown with a blue (B) or red (C) horizontal bar. Each ICR targeted by CRISPR/Cas9 is highlighted in light blue (GRCh37 chr20:57,414,216-57,418,552 and GRCh37 chr20:57,243,339-57,245,500 for the NESP-ICR and the STX16-ICR, respectively). The number on the left of each deletion corresponds to the number in Supplemental Tables 1 and 2, where the detailed information is described. For A and C, STX16 exon numbers are based on NCBI RefSeq NM_003763.6.