519 Single Nucleus ATAC Sequencing Reveals Increased PPP1R17 Chromatin Accessibility Driving Cushing’s Disease

Abstract

INTRODUCTION: Epigenetic drivers of pituitary adenomas causing Cushing’s disease (CD) are virtually unknown. Here, we investigate the epigenetic mechanisms underlying gene dysregulation in CD. METHODS: We isolated single nuclei from 3 CD adenoma – normal gland pairs and performed single nucleus RNA sequencing (snRNAseq) and assays for transposase-accessible chromatin (snATACseq, 10x Genomics). We integrated these data with prior paired single cell data (scRNAseq, n = 3) and bulk RNAseq (CD = 3, non-CD = 3). We performed bulk DNA methylation assays on independent adenomas (CD = 3, non-CD = 3; Illumina Infinium MethylationEPIC). CD adenomas were examined by multiplexed immunohistochemistry (mIHC). We generated murine corticotroph (mCort) cell lines stably transfected with lentiviral expression vectors (Origene) and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). RESULTS: snATACseq and snRNAseq data identified increased chromatin accessibility with upregulated PPP1R17 expression in single nuclei from CD adenomas, in agreement with our prior scRNAseq and bulk RNAseq data (16-fold in paired analysis, FDR < 0.01). DNA methylation failed to identify global or local differential methylation at the PPP1R17 locus. mIHC confirmed PPP1R17 protein overexpression in human CD tumors. PPP1R17 overexpression in mCort cells accelerated the cell cycle with fewer cells accumulating in S-phase (16.4% vs 24.1%, P < 0.01), and increased cell proliferation (two-way ANOVA P < 0.01). CONCLUSIONS: We identified coordinated chromatin accessibility, transcriptional activation and protein expression of PPP1R17 in human CD adenomas compared to surrounding normal tissues, and recapitulated hyperproliferation in-vitro by stable PPP1R17 overexpression. Our findings uncover targetable epigenetic mechanisms in CD pathogenesis.