Fig 2: CA induce discrete transcriptional changes by promoting the interaction of RARa and N-CoR1.a Heat map of the transcriptional changes in NIH3T3 cells cultured without additions (None) or with 20µM AR7, CA39, CA77, or BMS614 for 24 h. b–d Effect of CA in components of the CMA network13 (b). Changing components highlighted in bold. c AR7-induced changes in expression of CMA network components. n = 3 different experiments. d, e mRNA levels of the CMA network components by qPCR (d) and calculated CMA activation score (e) in cells treated as in a. n = 7 independent experiments. f Molecular docking of CA39 (orange) and CA77 (green) is compatible within the inactive (left) conformation of RARa (lilac) bound to N-CoR1 peptide (pink) (PDB ID: 3KMZ). RARa active (blue) conformation is shown for comparison. ATRA (yellow) binds only to the active conformation. Hypothetical binding poses of CA39 (orange) and CA77 (green) are not compatible within the active conformation of RARa due to steric clash (black dashed circle). g EC50 (µM) in fluorescence polarization assays with RARa and the N-CoR1 peptide incubated without additions (no ligand) or in the presence of 10 µ? of BMS394 (BMS), CA39, and CA77. n = 3 experiments. h Immunoblot for N-CoR1 and RARa of streptavidin pulldowns (top) or total cellular lysates (bottom) of NIH3T3 incubated without additions or with CA39 (10 µM) or biotin-CA (10 µM) for 24 h. IP: immunoprecipitation. This experiment was repeated 3 times. i CMA activity in NIH3T3 cells control (empty vector) or knock-down (KD) for N-CoR1 incubated without additions (none) or with 20 µM CA39 or CA77 for 24 h. Left: representative images. Nuclei are highlighted with DAPI. Inserts: higher magnification. Right: Quantification of the number of puncta per cell. n = 3 different experiments (>2,500 cells counted). Inset: immunoblot for N-CoR1. Individual values and mean + s.e.m are shown. One sample t and Wilcoxon test was used in c and d and one-way ANOVA (g) or two-way ANOVA (i) followed by Bonferroni’s multiple comparisons post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Uncropped blots are in Supplementary Fig. 10. Source data and exact p values are provided as a Source Data file.

Fig 3: Homeostasis and cytokine expression of NCOR1-deficient CD4+ T cells upon transfer. (A) Naïve WT or NCOR1 cKOCd4 CD4+ T cells were i.p. injected into Rag2-/- mice and 8 weeks later transferred CD4+ T cells in recipient mice were analyzed. Contour plots show TCRß expression vs. side scatter (SSC-A) of cells isolated from spleen, mLNs, small intestine IELs (SI-IELs) and lamina propria cells (SI-LPs) of recipient Rag2-/- mice. (B) Diagrams depict the percentages of TCRß+ cells (upper panel) and numbers of TCRß+ CD4+ cells (lower panel) in the indicated organs of Rag2-/- mice that have received either naïve WT or NCOR1 cKOCd4 CD4+ T cells. (C) Contour plots depict IL-17A vs. IFN? expression of cells isolated from spleen, mLNs, SI-IELs, and SI-LP cells of recipient Rag2-/- mice. (D) Diagrams indicating percentages of IFN? single-producing cells (IFN?+IL-17A-) (upper panel), IL-17A single-producing cells (IFN?-IL-17A+) (middle panel) and IFN?+IL-17A+ double-producing (lower panel) TCRß+ cells isolated from Rag2-/- mice that have received either naïve WT or NCOR1 cKOCd4 CD4+ T cells. (A,C) Representative plots for the various organs and tissues were taken from different experiments, however for each organ/tissue shown WT and NCOR1 cKOCd4 plots are from the same experiment. Numbers indicate the percentage of cells in the respective quadrants or regions. (B,D) Horizontal bars indicate the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (unpaired two-tailed t-test). Data are representative (A,C) or show summary (B,D) of 6–8 mice that were analyzed in 3 independent experiments.

Fig 4: NCOR1 controls the survival of in vitro-activated CD4+ T cells. (A) Naïve WT and NCOR1 cKOCd4 CD4+ T cells were activated with anti-CD3e/anti-CD28 and cultured in the presence of recombinant hIL-2 for 3 days (= Th0 condition). Pseudocolor plots show side scatter (SSC-A) vs. forward scatter (FSC-A). Regions indicate T cell blasts. (B) Summary diagram shows the percentage of T cell blasts from all experiments performed as described in (A). (C) Representative histograms depict expression of CD25 (left) and CD69 (right) on WT and NCOR1 cKOCd4 Th0 cells before (0 h; upper histograms) and 24 h (lower histograms) after activation. (D) Representative contour plots of viability dye vs. side scatter (SSC-A) for WT and NCOR1 cKOCd4 Th0 cells. (E) Summary of all experiments performed as described in (D) shows percentages of viable WT and NCOR1 cKOCd4 Th0 cells. (F) Contour plots depict IL-2, TNFa, and IFN? vs. CD4 expression in WT and NCOR1 cKOCd4 Th0 cells. (G) Summary showing results of all experiments as described in (F). (H) Summary graphs depicting cytokine expression levels as mean fluorescence intensity (MFI). For each experiment, WT levels were set as 1 and relative MFI of IL-2, TNFa, and IFN? are shown. (I) MACS-purified naïve WT and NCOR1 RID cKOCd4 CD4+ T cells were activated as described in (A). Pseudocolor plots show side scatter (SSC-A) vs. forward scatter (FSC-A) for WT and NCOR1 RID cKOCd4 Th0 cells. (J) Summary diagrams of all experiments performed as described in (I) showing the percentages of WT and NCOR1 RID cKOCd4 Th0 blast cells. (K) Contour plots of viable WT and NCOR1 RID cKOCd4 Th0 cells. (L) Percentages of viable WT and NCOR1 RID cKOCd4 Th0 cells from all experiments as described in (K). (M) Representative histograms depict expression of CD44 on WT and NCOR1 RID cKOCd4 Th0 cells before (0 h, upper histograms) and 72 h (lower histograms) after activation. (B,L) The horizontal bars indicate the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (unpaired two-tailed t-test or paired t-test for E,G,J). (A,D,F,I,K) Numbers indicate the percentage of cells in the respective regions. (E,G,J) Lines indicate paired experiments. Data are representative (A,C,D,F,I,K,M) or show summary (B,E,G,H,J,L) of 7 (B), 3 (J,L), 8 (E), and 5–6 (G,H) samples that were analyzed in 3–8 independent experiments.

Fig 5: Enhanced IFN? expression in NCOR1-deficient Th1 cells. (A) Naïve WT and NCOR1 cKOCd4 CD4+ T cells were activated with anti-CD3e/anti-CD28 for 3 days in the presence of Th1-inducing cytokines. Contour plots depict T-bet and IFN? expression. (B) Summary of all experiments performed as described in (A). (C) Summary graph of mean fluorescence intensity of IFN? expression levels. For each experiment, WT levels were set as 1 and relative IFN? levels were calculated. (D) Representative histograms depict proliferation. Regions mark divided (left) and undivided cells (right). (E) Diagram showing the percentages of cells that underwent at least one cell division. (F) Contour plots in the upper panel show viability dye vs. side scatter (SSC-A) for WT and NCOR1 cKOCd4 Th1 cells with at least one cell division (left region in D). The lower contour plot panel depicts T-bet and IFN? expression within proliferated WT and NCOR1 cKOCd4 Th1 cells. (G) Summary shows percentages of viable cells (upper graph) and percentages of T-bet+IFN?+ cells (lower graph). (H) Contour plots show viability dye vs. side scatter (SSC-A) for WT and NCOR1 cKOCd4 Th1 cells of undivided cells (right region in D). (I) Summary shows percentages of viable undivided cells. (J) Integrative Genomics Viewer (IGV) (34, 35) screen shot displaying the Ifng gene locus. ImmGen consortium ATAC-seq peaks (GSE 100738) of splenic naïve CD4+ T cells are shown in the 1st row. The 2nd and 3rd rows display ChIP-seq tracks from WT and NCOR1 cKOCd4 naïve CD4+ T cells, respectively. Rows 4–7 display RNA-seq tracks from in vitro-generated WT and NCOR1 cKOCd4 Th1 and Th17 cells. (K) Diagrams show the summary of Ifng FPKM values obtained by RNA sequencing of naïve CD4+ T cells, and of in vitro-generated Th1 and Th17 cells. (B,E) The horizontal bars indicate the mean. *P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant (unpaired two-tailed t-test or paired t-test for G,I). (G,I) Lines indicate paired experiments. (A,D,F,H) Numbers indicate the percentage of cells in the respective quadrants or regions. Data are representative (A,D,F,H) or show summary of 6 (B,C) and 3 (E,G,I,K) samples that were analyzed in 6 (B,C) and 3 (E,G,I,K) independent experiments.