STAG2 inactivation increases tumor-related metabolic processes and cell differentiation. (a) Schematic of tumor initiation in KT and KT;Stag2^flox mice. Mice grew tumors until 8 wk to represent large atypical adenomatous hyperplasia and small adenomas and 16 wk to represent larger solid adenomas and early adenocarcinomas (Marjanovic et al., 2020). Outline of tumor cell sorting and sample preparation for bulk RNA-seq and bulk ATAC-seq. (b) Upregulated and downregulated genes (n = 2,545 genes) in KT relative to KT;Stag2^flox tumors (absolute value of log₂ fold change (|log₂FC|) > 1, FDR < 0.01). (c and d) GSEA pathways enriched in KT;Stag2^flox relative to KT tumors (c) and enriched in KT relative to KT;Stag2^flox tumors (d). (e and f) GO Term Gene Count Analysis with ClusterProfiler and EMBL-EBI GO:Term Category Analysis established pathways enriched from upregulated genes for KT;Stag2^flox/KT mice (e) and from down-regulated genes for KT;Stag2^flox/KT mice (f). (g and h) ES versus NKX2-1 gene expression (TPM) from single sample GSEA (ssGSEA) for KPT and KT samples (Chuang et al., 2017) for the gene sets from genes upregulated in KT;Stag2^flox versus KT (g) and genes downregulated in KT;Stag2^flox versus KT (h). (i) Rank correlation of chromatin accessibility across KT and KT;Stag2^flox samples. Samples cluster into two distinct groups. (j) Differential accessibility across 3,576 significant peaks in KT and KT;Stag2^flox mice (three mice/group). The x-axis represents the log₂ mean accessibility per peak and the y-axis represents the log₂ fold change in accessibility. Colored points are significant (|log₂FC| > 1, FDR < 0.05). Red points are increased chromatin accessibility in KT;Stag2^flox accompanied by transcription factor hypergeometric motif enrichment in KT;Stag2^flox, and blue points are decreased chromatin accessibility in KT;Stag2^flox accompanied by transcription factor hypergeometric motif enrichment in KT tumors. (k) ATAC-seq signal tracks for the Sftpd gene locus in neoplastic cells from KT and KT;Stag2^flox mice. (l) Comparison of log₂ fold change for KT and KT;Stag2^flox mice for top genes from both bulk RNA-seq and bulk ATAC-seq. r = 0.311, P < 2.2 × 10^–16.