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Supplementry Figure 1 Ncor1 Expression 2. 1.5 1..5. Muscle Liver Lmi Ppropi Kindey Pncres Lung Testis Bone Mrrow Thymus Spleen Peripherl Lymph nods Smll Intestine Ncor1 Expression 1.5 1..5. DN DP SP CD8 SP Supplementry Figure 1. Ncor1 expression profiles in different tissues nd thymocyte susets. () Quntittive RT-PCR nlysis of Ncor1 mrna expression in vrious tissues(n=3) nd () in the indicted thymocyte susets (n=3, repet 2 times) sorted using flow cytometry. DN, - CD8 - doulenegtive thymocytes; DP, + CD8 + doule-positive thymocytes; SP, + single-positive thymocytes; CD8 SP, CD8 + single-positive thymocytes.

Supplementry Figure 2 Ncor1 f/f 5.64 86.9 1.59 CD8 Lck-Cre 4.91 87.7 1. Ncor1 f/f Lck-Cre 2.86 87..74 Thymocytes (%) 1 8 8 4.4 DP SP CD8 SP Ncor1 f/f Lck-Cre Ncor1 f/f Lck-Cre 3.49 6.82 4.25 2.94 6.19 3.94 1.6 2.1 1.21 Thymocytes (%) 8 6 4 2.3 ** TCRβ P3 P4 P5 c d #5 5.8 #4 3.89 5 Ncor1 /f #5 5.81 #4 3.43 #3 2.87 2.5 dull CD8 dull (%) 4 3 2 ** Lck-Cre #3 3.72 22. 1 Ncor1 f/f Lck-Cre Ncor1 f/f Lck-Cre Ncor1 f/f Lck-Cre #5 1.89 #4.99 MHCI #3 1. 33.1 CD8 Supplementry Figure 2. Thymocyte profiles in Ncor1 flox/flox, Lck-Cre nd Ncor1 flox/flox Lck-Cre mice. () Surfce expression of versus CD8 (up) nd versus TCRβ (down) on totl thymocytes (n=4 for ech group) detected y flow cytometry. () Quntifiction of different thymocyte susets in (). (c) Flow cytometry nlysis of 5 su-popultions (left) y MHCI nd expression on totl thymocytes (n=4 for ech group, respectively). The right pnel shows nd CD8 expression on gted popultion #3. (d) The proportion of dull CD8 dull susets in popultion #3. Sttisticl significnce ws nlysed using the two-tiled Student s t test (, not significnt; ** P<.1; P<.1).

Supplementry Figure 3 7.82 85.4 2.82 CD8 4KO 3.15 93.2.83 % of Thymocytes 1 8 6 3 DP SP CD8 SP Supplementry Figure 3. Thymocyte profiles in Ncor1 flox/flox -Cre mice. () Surfce expression of nd CD8 on thymocytes from Ncor1 flox/flox -Cre (4KO) mice nd Ncor1 flox/flox () mice (n=3 to 5). () Quntifiction of different thymocyte susets in (). DP, + CD8 + doule-positive thymocytes; SP, + single-positive thymocytes; CD8 SP, CD8 + single-positive thymocytes. Dt re representtive of three independent experiments. Sttisticl significnce ws nlysed using the two-tiled Student s t test ( P<.1).

Supplementry Figure 4 % of Mx CD5 + (%) 3 ** 2 1 CD5 MFI (*1 3 ) 15 * 1 5 CD5 lo hi c % of Mx MFI 4 ** 3 2 1 Supplementry Figure 4. CD5 nd expression in NCoR1-deficient thymocytes. () Flow cytometry nlysis of CD5 expression (left) on totl thymocytes from NCoR1-deficient () nd wild type () mice (n=3 to 5) nd quntifiction of the CD5-positive su-popultion (right). () Men fluorescence intensity of CD5 on lo nd hi DP + CD8 + doule-positive) thymocytes from (n=4) nd (n=4) mice. (c) The histogrm plot (left) nd the men fluorescence intensity (right) of expression on totl thymocytes from nd OT-II TCR-trnsgenic mice (n=3) detected y flow cytometry. Dt re representtive of two independent experiments. Sttisticl significnce ws nlysed using the two-tiled Student s t test (, not significnt, ** P<.1).

Supplementry Figure 5 Foxp3.27.14 + Foxp3 + (%).5 **.4.3.2.1. Foxp3 + (%) SP 5 4 3 2 1 c CD1d Tetrmer TCRβ.32.14 CD1d Tetrmer + TCR + (%).5.4.3.2.1. Supplementry Figure 5. Development of oth Treg cells nd inkt cells is defective in the thymus of NCoR1-deficient mice. () Foxp3 expression in totl thymocytes from NCoR1-deficient () (n=7) nd wild type () (n=6) mice y intrcellulr stining (left) nd quntifiction of Foxp3 + cells (right). () Quntifiction of Foxp3 + cells in + single-positive ( SP) nd thymocytes in (). (c) CD1d Tetrmer nd TCRβ expression in totl thymocytes from (n=6) or (n=7) mice (left) y flow cytometry nd quntifiction of CD1d Tetrmer + TCRβ + cells (right). Sttisticl significnce ws nlysed using the two-tiled Student s t test (, not significnt; ** P<.1; P<.1)..

Supplementry Figure 6 1.5 3 5 1 1.5 3 5 1(min) p-plcγ1 PLCγ1 p-erk1/2 Erk p-p38 p38 β-ctin 155 kd 149 kd 44 kd 42 kd 44 kd 43 kd 4 kd 45 kd Supplementry Figure 6. TCR signling in NCoR1-deficient thymocytes. Immunolot nlysis of phosphorylted (p)-plc-γ1, p-erk1/2, p-p38 nd totl protein of PLC-γ1, Erk nd p38 in lystes from wild type () nd NCoR1-deficient () thymocytes without or with stimultion in vitro for the indicted mounts of time. β-ctin served s loding control. Dt re representtive of there independent experiments.

Supplementry Figure 7 MOCK.44..11 TCRβ+CD28 1.37 1.24 36.9 MFI 2 ** Act. Csp3 - ** Act. Csp3 + 15 1 5 Act. Csp3 2.76.2.1 8.15 6.78 23.1 c MOCK TCRβ+CD28 d Annexin V 7.17 15.9 PI 14.5 4.7 Annexin V + PI - 5 4 3 2 1 ** 3(hr) 6(hr) 12(hr) ** 24(hr) Supplementry Figure 7. NCoR1-deficient thymocytes re prone to poptosis. () The surfce mrker nd intrcellulr stining of the ctive cspse-3 (Act.Csp.3) detected y flow cytometry in totl NCoR1-deficient () (n=3) nd wild type () (n=3) thymocytes stimulted or not with TCR β nd CD28 for 6 hr. () The men fluorescence intensity of expression in Act.Csp3 + nd Act.Csp3 - thymocytes in (). (c) Flow cytometric stining of Annexin V nd propidium iodide (PI) on nd thymocytes stimulted in vitro for 6 hr (n=3 to 5). (d) Quntifiction of the poptotic thymocytes shown in (c). Dt re representtive of two independent experiments. Sttisticl significnce ws nlysed using the two-tiled Student s t test (** P<.1; P<.1).

Supplementry Figure 8 CD3e(µg/ml) CD28(µg/ml) NCoR1 Bim Tuulin.1.1.1.1.1.1 1 1 1 1 1 1 1 1 25 kd 25 kd 5 kd Bim Reltive Expression 5 ** 4 3 2 1 p1+2 p4+5 Supplementry Figure 8. Bim expression in TCR signlling stimulted thymocytes in vitro nd in sorted non-ctivted nd ctivted thymocytes in vivo. () Bim protein levels in NCoR1-deficient () nd wild type () thymocytes stimulted with different concentrtions of nti-tcrβ. () Non-ctivted thymocytes (popultion 1 + 2, p1+2) nd ctivted thymocytes (popultion 4 + 5, p4+5) were sorted y the surfce expression of nd TCRβ on totl (n=7) nd (n=7) thymocytes ccording to the gting in Fig. 2. Bim mrna levels in these sorted thymocytes were nlysed y quntittive RT-PCR. Sttisticl significnce ws nlysed using the two-tiled Student s t test (, not significnt, ** P<.1; P<.1).

Supplementry Figure 9 1 5.777 Fsl Expression 8 6 4 2 * Tnf Expression 4 3 2 1 UD UD *.5 1 3 6 (hr).5 1 3 6 (hr) Tril Expression 4 3 2 1 Bk Expression 1 5.5 1 3 6 (hr).5 1 3 6 (hr) Supplementry Figure 9. Expression of propoptotic genes in enriched DP thymocytes. Quntittive RT-PCR nlysis of Fsl, Tnf, Tril, nd Bk mrna levels in enriched NCoR1-deficient () nd wild type () DP ( + CD8 + doule-positive) thymocytes ctivted in vitro for the indicted time points. Dt re representtive of two independent experiments with four mice per genotype. The numer indictes the p vlue. Sttisticl significnce ws nlysed using the two-tiled Student s t test

Supplementry Figure 1 Cells (%) 8 6 4 2 Cells (%) 8 6 4 2 Bim KO dko CD62L hi lo CD62L lo hi CD62L hi CD24 lo CD62L lo CD24 hi Supplementry Figure 1. Proportions of mture (CD62L hi lo or CD62L hi CD24 lo ) nd immture (CD62L lo hi or CD62L lo CD24 hi ) susets in (n=4), (n=4), Bim KO (n=4) nd dko (n=5) thymocytes. Flow cytometry nlysis nd gting of mture or immture susets were performed s shown in Fig. 6c., wild type;, NCoR1-deficient; dko, Ncor1 nd Bim doule knockout.sttisticl significnce ws nlysed using the two-tiled Student s t test (, not significnt).

Supplementry Figure 11 18.7 CD8 13.6 9.68 5.49 Cells (%) 25 2 15 1 5 CD8 Cells (*1 6 ) 4 3 2 1 CD8 c d 2 ** Foxp3 SSA 8.86 11.8 Foxp3 + (%) 15 1 5 Supplementry Figure 11. Loss of NCoR1 reduces peripherl nd CD8 T cells in the spleen ut increses the proportion of Tregs. () The surfce expression of nd CD8 on NCoR1-deficient () (n=4) nd wild type () (n=4) splenocytes detected y flow cytometry. () The percentges (left) or numers (right) of nd CD8 T cells gted in (). Dt were generted from three independent experiments. (c) Intrcellulr expression of Foxp3 in + TCRβ + (n=6) nd (n=6) splenocytes detected y flow cytometry. (d) Quntifiction of the Foxp3 + + T cells in (c). Dt re representtive of three independent experiments with more thn three mice per group. Sttisticl significnce ws nlysed using the two-tiled Student s t test (** P<.1; P<.1).

Supplementry Figure 12 CD62L live cells singel cells lymocytes DAPI FCS-W FCS-A CD8 #4 #3 lo Vα2+ hi CD5+ #5 #1 CD24 #2 TCRβ Vα2 CD5 #2 #1 DAPI FCS-W Act. Csp3 lymocytes FCS-A #4 #5 dullcd8dull live cells MHCI singel cells #3:hiMHCI- #3 CD8 lymocytes FCS-A FCS-W Annexin V singel cells Act. Csp.3 c PI live cells 5.1 singel cells d lymocytes FCS-A CD1d Tetrmer live cells Foxp3 e singel cells lymocytes FCS-A FCS-W DAPI CD8 5.2 DAPI FCS-W TCR β Supplementry Figure 12. Gting strtegies for flow cytometry nlyses. Representive FACS plots showing: () Gting strtegy for, CD8,, TCRβ, CD5, nd Vα2 (for OTII ckground) expression. () Gting strtegy for intrcellulr Act.Csp.3 in himhci-dullcd8dull thymocytes. (c) Gting strtegy to detect poptosis of thymocytes stimulted in vitro.(d) Gting strtegy to nlyze thymocytes profiling in one mrrow chimers. (e) Gting strtegy to nlyze T regultory cells nd invrint nturl killer T cells in thymus.

Supplementry Figure 13 Fig. 5d nti-ncor1 Suppl. Fig. 6 nti-p-plcγ1 nti-erk Suppl. Fig. 8 nti-ncor1 nti-bim nti-plcγ1 nti-p-p38 nti-bim nti-p-erk1/2 nti-p38 nti-tuulin nti-tuulin nti-β-ctin Supplementry Figure 13. The uncropped scns of western lots from Figure 5, Supplemenry Figure 6 nd 8.

Supplementl Tle 1. Genes expressed differentilly in thymocytes y RNA-seq In stedy stte thymocytes Gene / (ex vivo) Gene / (ex vivo) log2(fold_chnge) log2(fold_chnge) Gm7125 infinite Adcy7 -.81285 Gm6594 infinite Itg7 -.82898 AC163653.5 infinite Cxxc5 -.833748 Gh infinite Als2cl -.847795 AC158672.3 infinite Mllt6 -.866141 Sfn 1.41297 Cnn2 -.91838 Sps1 1.2823 Pdcd1 -.949894 St3gl6 1.25667 Cd53 -.96488 Wnt8 1.24523 St3gl1-1.81 Gm8186 1.164 Slc619-1.84 Dusp4 1.865 Shis5-1.1297 Ifngr1 1.5791 Sem7-1.856 Tl1xr1.954922 AB124611-1.8824 Cdk5r1.926474 H2-K1-1.1853 Bcl6.918982 Fgf13-1.1645 Mnl1.85341 Pdi4-1.1741 Dusp5.83168 AC165246.1-1.2128 Slc99.798935 Ccr7-1.26486 Scd2.78573 Slfn1-1.3495 Cd74.72575 H2-Q7-1.31484 Pik3r1.725251 Ephx1-1.32313 Pik3cg.7227 Ctsw -1.32528 Gimp6.692412 Nkg7-1.32796 Ctsl.683771 Xrr1-1.36339 Lig4.6834 Jup -1.4723 Tesp1.67143 Phld1-1.4258 Tu2.627266 Pqr7-1.57394 Acg1.595489 Ccnd2-1.5918 Esyt1 -.632325 Lfng -1.6718 Lt -.645846 Cd7-1.67425 Gpr56 -.67893 Gm1499-1.73999 Rpl23 -.674669 Sdc4-1.93547 Nrp1 -.728366 n-r5-8s1-2.25768 Ntn1 -.742279 Cd2-3.59722 Smoc1 -.791651 Gm11223-4.56491

In ctivted thymocytes Gene KO/ (3h ctivtion) Gene KO/ (3h ctivtion) log2(fold_chnge) log2(fold_chnge) Gm7125 infinite B4glnt1 -.8943 Gm6594 infinite Myc -.83976 Ighv1-49 infinite Gm1485 -.814945 AC16982.1 2.3594 Slc619 -.82457 Sfn 1.334 Ccr7 -.823115 Acoxl 1.14459 H2-K1 -.839568 Tl1xr1 1.6335 Fm134 -.856552 Scd2 1.6322 Ador2 -.886581 Il12r1.956431 Tnf -.95543 Dusp4.91335 Cxxc5 -.955899 ERDR1.896455 Evl -1.4317 Gimp9.8911 Pdcd1-1.7871 Bcl6.852289 H2-Q7-1.11737 Acg1.841595 Cd83-1.25182 Ptger4.739682 Ifi27l2-1.3544 Ldlr.732622 Cited4-1.38267 Gimp8.719569 Ld1-1.43564 Grmd3.7789 Tgp -1.43739 Bcl2l11.673482 Egr3-1.63327 Mr1.651132 Xcl1-1.71541 Mnl1.638582 Lfng -1.89111 Ccnd3.63751 Tnfrsf9-2.2836 Ctsl.612296 Rgs16-2.9375 Ccr4.592412 Ccnd2-2.23925 Irf4 -.655463 n-r5-8s1-2.32324 Cd53 -.67139 Sh3gl3-2.4747 Ntn1 -.739517 Ap2-3.47654 Icos -.7512 Gm11223-3.8225 Gpr56 -.835

Supplementl Tle 2. Antiodies Informtion Antiodies for Flow cytometry nlysis Antiody Clone Dilution Compny GK15 1:2 ebioscience CD8 53-6.7 1:2 ebioscience H1.2F3 1:2 ebioscience TCR H57-597 1:2 ebioscience MHCI AF6-88.5.5.3 1:2 ebioscience CD5 53-7.3 1:2 ebioscience CD24 M1/69 1:2 ebioscience CD62L MEL-14 1:2 ebioscience 5.1 A2 1:2 BD Bioscience 5.2 14 1:2 BD Bioscience Foxp3 FJK-16s 1:2 ebioscience V 2 2.1 1:2 ebioscience Antiodies for Immunolot nlysis Antiody Code Dilution Compny nti-rit-ncor1 5948S 1:1 Cell Signling Technology nti-rit-bim 2933S 1:1 Cell Signling Technology nti-rit-p-plc 1 2821S 1:1 Cell Signling Technology nti-mouse-plc 1 76155 1:1 Acm nti-mouse-p- Erk1/2 sc-7383 1:5 Snt Cruz Biotechnology nti-rit-erk sc-93 1:2 Snt Cruz Biotechnology nti-rit-p-p38 9211S 1:1 Cell Signling Technology nti-rit-p38 sc-728 1:2 Snt Cruz Biotechnology nti-rit- -ctin 497S 1:2 Cell Signling Technology nti-rit-tulin CP6 1:2 EMD Millipore Got nti-rit- IgG(H+L) 111-35-3 1:3 Jckson Immuno&Reserch Donkey ntimouse-igg(h+l) 715-35-15 1:3 Jckson Immuno&Reserch Antiodies for CHIP nlysis Antiody Code Dilution Compny nti-ncor1 5948S 1 g per IP Cell Signling Technology nti-h3c 6-599 1 g per IP Millipore nti-h4c 6-598 1 g per IP Millipore nti-h3k27c 4729 1 g per IP Acm nti-h3k4me1 858 1 g per IP Acm nti-h3k4me3 62 1 g per IP Acm IgG 39S 1 g per IP Cell Signling Technology

Supplementl Tle 3. Primer for Quntittive RT-PCR nlysis Ncor1 Forwrd 5'-TGCGTCAGCTTTCTGTGA TTCCACC-3' Reverse 5'-TGA TTTCTGCCTCTGCGTTTTCCA-3' Bim Forwrd 5'-CGACAGTCTCAGGAGGAACC-3' Reverse 5'-CCTTCTCCATACCAGACGGA-3' Fsl Forwrd 5'-TGAATTACCCATGTCCCCAG-3' Reverse 5'-AAACTGACCCTGGAGGAGCC-3' Tnf Forwrd 5'-CATCTTCTCAAAATTCGAGTGACAA-3' Reverse 5'-TGGGAGTAGACAAGGTACAACCC-3' Tril Forwrd 5'-ATGATGGTGATTTGCATAGTGCT-3' Reverse 5'-AGCTGCTTCATCTCGTTGGTG-3' Bk Forwrd 5'-AATGGCATCCGGACAAGGAC-3' Reverse 5'-TGTTCCTGCTGGTGGAGGTA-3' Act Forwrd 5'-GGCTGTATTCCCCTCCATCG-3' Reverse 5'-CCAGTTGGTAACAATGCCATGT-3'

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