Xq26.3 duplications in X-linked acrogigantism (X-LAG) disrupt a topologically associating domain (TAD) and rewire GPR101-enhancer interactions

M. Franke^a (Dr), A. Daly^b (Prof), L. Palmeira^b (Dr), A. Tirosh^c (Dr), A. Stigliano^d (Prof), E. Trifan^e (Ms), F. Faucz^f (Dr), D. Abboud^b (Dr), P. Petrossians^b (Prof), J. Tena^a (Dr), E. Vitali^e (Dr), A. Lania^e (Prof), J. Gomez-Skarmeta^a (Prof), A. Beckers^b (Prof), C. Stratakis^f (Dr), G. Trivellin*^e (Dr)

^a Centro Andaluz de Biologia del Desarrollo (CABD), Seville, SPAIN ; ^b University of Liege, Liege, BELGIUM ; ^c Tel Aviv University, Ramat Gan, ISRAEL ; ^d Sapienza University of Rome, Rome, ITALY ; ^e Humanitas Research Hospital, Milan, ITALY ; ^f Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, UNITED STATES

* giampaolo.trivellin@humanitasresearch.it

X-linked acrogigantism (X-LAG) is the most severe form of pituitary gigantism and is characterized by aggressive growth hormone (GH)-secreting pituitary tumors that occur in young infants. X-LAG is associated with chromosome Xq26.3 duplications (the X-LAG locus typically includes the VGLL1, CD40LG, ARHGEF6, RBMX, and GPR101 genes) that lead to massive pituitary tumoral expression of GPR101, a novel regulator of GH secretion. The mechanism underlying GPR101 over-expression was previously unknown.

The genome has recently been shown to be compartmentalized into discrete regulatory units termed topologically associating domains (TADs). TADs are submegabase-size chromatin domains with high levels of internal interaction that are insulated from each other by regions of low interaction (TAD borders). TAD insulation, therefore, favors the specificity of enhancer-promoter interactions.

Using chromosome conformation capture techniques (Hi-C and 4C-seq), we characterized the normal chromatin structure at the X-LAG locus. We showed that GPR101 is located within a TAD delineated by a tissue-invariant and evolutionary conserved border that separates it from centromeric genes and cis-regulatory sequences (CREs). All duplications reported to date in X-LAG patients involve GPR101, the strong TAD border, and centromeric sequences. RNA-seq analysis in X-LAG tumors and normal pituitary tissue showed that gene misexpression predominantly affects GPR101 (12 log-fold increase). Next, using 4C-seq with GPR101, RBMX, and VGLL1 viewpoints, we showed that the duplications in X-LAG patients disrupt the invariant TAD border and produce ectopic interactions that form a novel chromatin domain (neo-TAD). We then identified several pituitary active CREs within the neo-TAD and demonstrated in vitro that an enhancer located close to the RBMX gene significantly enhanced reporter gene expression. At the same time, we showed that the GPR101 promoter permits the incorporation of this new regulatory information both in pituitary and embryonic cell lines.

In conclusion, our results identify X-LAG as a new TADopathy; to our knowledge this is the first TADopathy to be described in endocrinology. Xq26.3 duplications disrupt the local chromatin architecture by forming a neo-TAD and this rewiring of GPR101-enhancer interaction causes the marked over-expression of GPR101 in X-LAG pituitary tumors, which in turn drives tumoral GH hypersecretion and gigantism in affected children.

The author has declared the following conflict(s) of interest:

Giampaolo Trivellin, Adrian Daly, Constantine Stratakis, Albert Beckers, and Fabio Faucz hold a patent on the GPR101 gene and its function (US Patent No. 10,350,273, Treatment of Hormonal Disorders of Growth).

Adrian Daly and Albert Beckers received research funding from Pfizer Inc. and Novo-Nordisk.

Constantine Stratakis received research funding from Pfizer Inc. for investigations on growth-hormone producing pituitary adenomas. He also has consulted within the last 12 months with Lundbeck Pharmaceuticals and Sync, LLC, and is currently employed by ELPEN Pharmaceuticals.

The authors declare that they have no conflicts of interest with the contents of this abstract.