Fig 2: CDKN1C mRNA expression is linked to chromatin accessibility of CERES and GR levels in clinical samples.a Biopsies of human primary non-lymphoid solid cancers were subjected to whole-transcriptome and chromatin accessibility analysis by the TCGA (n = 404). b (top) Representative samples showing chromatin accessibility signal at CERES enhancer. b (bottom) Expression of CDKN1C, MKI67, PCNA, and FOXM1 sorted based on CERES accessibility. Survival events (alive = white, dead = green), SWI/SNF mutation status (WT = white, mutated = green), and cancer types are displayed below. c Histogram depicting correlation value counts for CDKN1C expression levels and 306 up- and downstream enhancers in its genomic vicinity. Red line shows the correlation of CERES accessibility with CDKN1C expression. d Histograms showing correlation of four proximal genes with the accessibility of 306 up- and downstream enhancers surrounding CDKN1C. Red lines show correlations of these genes with CERES accessibility. e Line plot showing Spearman’s correlation of accessibility of CERES (red) or 306 (blue) surrounding enhancers with CDKN1C mRNA levels, and NR3C1 mRNA levels (green).

Fig 3: GR Inhibition Has Significant Antitumor Effect on Antiandrogen-Resistant Tumors with CHD1 Loss(A) Relative gene expression of NR3C1 and GR target genes in tumors collected from LNCaP/AR xenografts, all normalized and compared with shNT + Veh group. Mean ± SEM is presented. p values were calculated using two-way ANOVA, and numbers of biological replicates are presented.(B) Western blot showing AR, GR, and their downstream target genes in xenografted LNCaP/AR tumors. For (A) and (B), Enz denotes enzalutamide at 10 mg/kg from day 1 of grafting. Veh denotes 0.5% CMC + 0.1% Tween 80.(C) Histograms of representative FACS-based competition assay showing the distribution of shCHD1-XE-1 cells (RFP-negative) versus shCHD1-XE-1 cells transduced with shGR (RFP-positive). The distributions on different days are presented in different colors.(D) Relative cell number of shCHD1-XE-1 cells transduced with annotated inducible shRNAs, normalized to shCHD1-XE-1 + Veh. Cells were treated with 250 ng/mL doxycycline for 48 h, and then 7 days of 10 µM enzalutamide (Enz) or DMSO (Veh) before cell numbers were counted. Mean ± SEM is presented, and p values were calculated by two-way ANOVA, with three biological replicates in each group.(E) Model depicting the chromatin dysregulation (plasticity) and antiandrogen resistance in mCRPC due to CHD1 loss.For all panels, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. See also Figure S8.

Fig 4: Nitro-alkylation of glucocorticoid receptor NR3C1. (A) Percent inhibition of [3H]dexamethasone binding to human NR3C1-LBD by different compounds. Compounds were screened at 10 µM in duplicates and the description of significant response (=50% maximum inhibition) is an arbitrary criterion used in primary high-throughput screening assays. (B) Dose response curves of 9-NO2-OA and 10-NO2-OA were conducted in duplicates. The compound concentration that caused 50% inhibition of [3H]dexamethasone binding to human NR3C1-LBD (IC50 values) were determined from these curves. Dexamethasone was included as a control. (C) The most stable complex obtained for 9-NO2-OA in the NR3C1-LBD (1NHZ). 9-NO2-OA and relevant residues are colored. (D) Binding site analysis for 9-NO2-OA (Cyan) with NR3C1-LBD. 9-NO2-OA forms a covalent bond with Cys643, and hydrogen bonds with Tyr640 and Ala618 (orange residues). Hydrophobic effect was contributed by Leu647 and Met639 (yellow residues). (E) Labeling of wildtype (wt) NR3C1 and indicated mutants in HEK293T cells at 2.5 µM alk-9-NO2-OA for 1 h (2 independent biological repeats). 5C>S refers to the mutant with all 5 cysteines (C622, C638, C643, C665, C736) in the LBD mutated to serine. 4C>S(Cxxx) refers to mutants with 4 of the 5 cysteines in the LBD mutated to serine, but with the indicated cysteine remaining intact. Anti-NR3C1 and Coomassie blue (CB) stain act as loading controls for the fluorescence gel. Selected protein molecular weight markers are indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Characterization of the neuroendocrine effects of the chronic stress paradigm. a Schematic diagram of experimental design. b Body weight was recorded before the beginning and at the end of the chronic stress procedure. Chronically stressed mice gain less body weight than unstressed control animals. C: n = 18, CS: n = 17. Unpaired t test. t = 5.262, ***p < 0.001. c, d Chronic stress increases the weight of the adrenal glands. C: n = 18, CS: n = 17, AS: n = 10. One-way ANOVA F(2,42) = 40.14, p < 0.001 with Tukey’s multiple comparison test: ***p < 0.001 CS versus C, #p < 0.001 CS versus AS. e Corticosterone plasma levels were measured at the beginning of the light cycle. C: n = 18, CS: n = 17, AS: n = 10. One-way ANOVA F(2,42) = 26.20, p < 0.001 with Tukey’s multiple comparison test: *p < 0.05 CS versus C, ***p < 0.001 AS versus C, #p < 0.001 CS versus AS. f Hypothalamic mRNA transcription of genes associated with corticosteroid signaling. Nr3c1, nuclear receptor subfamily 3 group C member 1. Nr3c2, nuclear receptor subfamily 3 group C member 2. Fkbp4, FK506 binding protein 4. Fkbp5, FK506 binding protein 5. Crh, corticotropin releasing hormone. Relative mRNA expression is reported as the value normalized to tripeptidyl peptidase 2 (Tpp2) for each sample. C: n = 8, CS: n = 9. Mann-Whitney U test. Nr3c1: U = 27, p = 0.423. Fkbp5: U = 12, *p < 0.05. Crh: U = 14, *p < 0.05. Unpaired t test. Nr3c2: t = 1.016, p = 0.326. Fkbp4: t = 1.214, p = 0.244. g Representative Western blots of GR and FKBP5 protein expression in hypothalamic homogenates. GR, glucocorticoid receptor. FKBP5, FK506 binding protein 51. ACTB, ß-actin. h Densitometric analysis. Values were normalized to ß-actin. C: n = 8, CS: n = 9. Unpaired t test. GR: t = 0.029, p = 0.977. FKBP5: t = 2.173, *p < 0.05. C, unstressed control mice. CS, chronically stressed mice. AS, acutely stressed mice