SAT-032 In Vitro Characterization Of GPR101 Transcriptional Regulation

Background: X-linked acrogigantism (X-LAG) is a novel syndrome of early childhood-onset gigantism caused by duplications of GPR101, a gene that is strongly upregulated in the pituitary adenomas (PAs) of the patients. Aim: To study the human GPR101 promoter. Methods:GPR101 promoter annotation was carried out in silico using the Genomatix and MPromDb softwares and by querying specific tracks in the UCSC Genome Browser. The identified sequences were cloned into reporter vectors and transiently transfected into HEK293 cells. Promoter activity was assessed by luciferase assays and quantitative PCR (qPCR) under normal growth conditions and after treatments with forskolin (10 μM, 6h) and β-estradiol (E2, 100 nM, 24h). Putative binding sites for cAMP responsive elements (CREs) were functionally evaluated by mutagenesis studies. GPR101-specific primers were used for an open chromatin assay. Results:In silico characterization of the GPR101 promoter revealed two putative promoter regions, each overlapping a CpG island. The proximal sequence partially extends into the coding sequence (CDS), is marked by a promoter-specific histone modification (H3K4me3), and harbors one CRE; the distal sequence is located 2 Kb upstream of the start codon and harbors three CREs. In vitro reporter assays confirmed that both sequences were functional (3-fold change over mock for both, P<0.001) and that activation of the cAMP signaling pathway further stimulates GPR101 transcription only via the distal promoter (1.8-fold change over basal, P<0.05). Mutation of two CREs within the distal promoter significantly reduced basal transcription rates and forskolin stimulation could not restore normal expression. Treatment with E2 inhibited basal transcription rates of both promoter sequences (0.3 and 0.4-fold change, respectively, P<0.01). qPCR in cells transfected with a promoterless vector containing only GPR101 CDS showed strong GPR101 levels, suggesting that sequences within the CDS can drive its transcription. However, mutation of a CDS-located CRE did not affect basal or forskolin-stimulated transcription. No endogenous GPR101 transcripts were detected in HEK293 cells. We determined that the lack of correlation between reporter activities and endogenous expression was due to the location of the GPR101 locus in a heterochromatin region. Conclusions: These results show that GPR101 is a gene endowed with a complex promoter that likely allow varying levels of expression as required. Unraveling the transcriptional regulation of GPR101 is an important step towards understanding how this gene is regulated during physiological (development) and pathological (X-LAG) states. The finding that cAMP (a pathway commonly deregulated in PAs) stimulates GPR101 expression and that mutations disrupting CREs negatively impact this expression warrants screening of GPR101 promoter in patients with PAs.