Resident National Institutes of Health Bethesda, Maryland, United States
Introduction: Pituitary adenomas causing Cushing’s disease are largely (60%) mutationally bland. PPP1R17 is known to be an endogenous inhibitor of tumor suppressor phosphatase PP2A. Here, we found the underpinnings of epigenomic dysregulation of PPP1R17.
Methods: We performed single nucleus assay for transposase accessible chromatin (snATACseq, 10X Genomics) in adenomas from 4 CD patients, and two syngeneic adenoma-normal pairs alongwith parallel single nucleus RNAseq (snRNAseq). Transcription factors bound to open chromatin were identified by reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin (Millipore Sigma) and promoter-specific synthetic guide RNAs (Invitrogen TrueGuide). Bound transcription factors were quantified using mass spectrometry (SimulTOF 300).
Results: Using snATACseq, we mapped out the chromatin accessibility landscape at single nucleus resolution in CD adenomas compared to adjacent normal pituitary glands. We identified coordinated chromatin accessibility (snATACseq) and transcriptional upregulation (snRNAseq) at the R17 locus. Using rChIP, we identified several peptides enriched at the R17 promoter, including the RNA polymerase II transcriptional coactivator p15 (SUB1), as well as the histone variant H1.4, indicating active chromatin remodeling.
Conclusion : We identified increased R17 expression as an underlying mechanism of tumor hyperproliferation in CD adenomas. We found chromatin accessibility underlying PPP1R17 overexpression. Our study identifies a novel targetable mechanism of CD pathogenesis.
