ARID1A and PI3-kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion

Abstract

ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. Here we show that monoallelic loss of ARID1A in the mouse endometrial epithelium is sufficient for vaginal bleeding when combined with PI3K activation. Sorted mutant epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT). We further show that ARID1A is bound to promoters with open chromatin, but ARID1A loss leads to increased promoter chromatin accessibility and the expression of EMT genes. PI3K activation partially rescues the mesenchymal phenotypes driven by ARID1A loss through antagonism of ARID1A target gene expression, resulting in partial EMT and invasion. We propose that ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates, and that coexistent ARID1A and PI3K mutations promote epithelial transdifferentiation and collective invasion. Broadly, our findings support a role for collective epithelial invasion in the spread of abnormal endometrial tissue.

ATAC-seq normalization method can significantly affect differential accessibility analysis and interpretation

“…In this in vivo study, a chromatin remodeler protein, ARID1A, was disrupted along with induced expression of a constitutively active oncoprotein, PIK3CA H1047R , resulting in myometrial [17] and peritoneal [36] invasion in LtfCre 0/+ ; (Gt)R26 Pik3ca*H1047R ; Arid1a fl/* mutant mice. Mutant and wild-type endometrial epithelial cells were positively selected by a surface marker, EPCAM, and purified by magnetic bead separation [17]. ATAC-seq was selected as a suitable method for analyzing chromatin accessibility changes in sorted cells due to feasibility of supplying low input material.…”

“…We recently reported an ATAC-seq data set in which chromatin accessibility was compared between sorted mutant and control mouse endometrial epithelial cells following disruption of a common tumor suppressor and oncogene [17]. In this in vivo study, a chromatin remodeler protein, ARID1A, was disrupted along with induced expression of a constitutively active oncoprotein, PIK3CA H1047R , resulting in myometrial [17] and peritoneal [36] invasion in LtfCre 0/+ ; (Gt)R26 Pik3ca*H1047R ; Arid1a fl/* mutant mice. Mutant and wild-type endometrial epithelial cells were positively selected by a surface marker, EPCAM, and purified by magnetic bead separation [17].…”

“…2e). We previously reported a role of ARID1A and PIK3CA mutations in EMT-related processes through multiple molecular and cell-based in vitro and in vivo assays [17].…”

“…Using our recently reported in vivo sorted mouse ATAC-seq data set, we show that different tools and normalization methods for calculating significant DA regions yield distinct results following disruption of a chromatin remodeler [17]. We show that qualitative techniques like MA plots (as applied in microarray analysis) can be used to identify and address global accessibility patterns which may or may not be technical in nature.…”

“…However, ATAC-seq offers many benefits over comparable assays including a lower input material requirement, shorter assay time, in situ library preparation, and further protocol adaptation to fresh-frozen tissue [11]. These advantages have permitted precise in vivo regulatory genomic assays on small populations of sorted cells [12][13][14][15][16][17].…”