Control of allelic Gsα expression for regulating hormone signaling

Abstract The α-subunit of the stimulatory G protein (Gsα) mediates the signaling of many hormones, autocrine/paracrine factors, and neurotransmitters. Gsα and its gene, the GNAS complex locus, are central to many human disorders with perturbed hormone actions and skeletal development. A distinctive way Gsα signaling is regulated involves

the control of allelic Gsα expression. While Gsα is produced from both parental GNAS alleles in most cells, including growth plate chondrocytes and osteoblasts, it is derived predominantly from the maternal allele in specific cells/tissues, most prominently renal proximal tubules and thyroid cells. This monoallelic expression

plays a crucial role in patients with maternal loss-of-function GNAS mutations, causing severe Gsα deficiency and impaired hormone signaling in those tissues already with monoallelic expression, most prominently affecting parathyroid hormone (PTH) actions, particularly in the renal proximal tubule, and thyroid-stimulating hormone

(TSH) action in the thyroid. Patients with this disorder, pseudohypoparathyroidism type-1A (PHP1A), also show additional phenotypes, including brachydactyly and short stature due, at least partly, to Gsα haploinsufficiency

in tissues with normally biallelic Gsα expression. Thus, allelic expression of Gsα critically regulates Gsα signaling in specific tissues and dictates the phenotypes associated with GNAS mutations. To better understand the regulation of Gsα-mediated hormone signaling and to develop curative treatments for these diseases, an

improved knowledge of the mechanisms governing allelic Gsα expression is required. We focus on PHP1B, a pseudohypoparathyroidism subtype characterized by PTH and TSH resistance without the additional phenotypes in PHP1A. GNAS is a methylated gene, and all patients with PHP1B show maternal hypomethylation

in the GNAS exon A/B differentially methylated region (DMR) adjacent to Gsα-coding exons, suggesting that the A/B DMR is involved in allelic Gsα expression. However, the role of A/B methylation and the underlying mechanisms have remained poorly understood. We have recently developed human embryonic stem cell models

of PHP1B by introducing the genetic deletions identified in patients into the GNAS locus. We will employ these unique tools, as well as induced pluripotent stem cells (iPSCs) derived from healthy and PHP1B patients, to elucidate the mechanisms by which GNAS methylation regulates allelic Gsα expression. Based on our recently

published findings, we hypothesize that exon A/B methylation, controlled by the upstream NESP55 transcription in the early embryo, is crucial for allelic Gsα expression and hormone signaling in specific differentiated tissues. Aim 1 will determine whether inducing NESP55 transcription can increase A/B methylation in cis to rescue Gsα

deficiency due to different GNAS defects. Aim 2 will determine whether the methylation of the GNAS exon A/B DMR is needed for Gsα expression and hormone signaling in a tissue-specific manner. Our study will establish the role of GNAS methylation in regulating Gsα expression and signaling in specific tissues and contribute to a

deeper understanding of the disease mechanisms responsible for end-organ resistance to PTH and TSH.