Supplementary material SUMOylation Regulates the Chromatin Occupancy and Anti-Proliferative Gene Programs of Glucocorticoid Receptor Ville Paakinaho, Sanna Kaikkonen, Harri Makkonen, Vladimir Benes, and Jorma J. Palvimo A FRT wtGR GR3KR clone wt-1 wt-2 wt-3 3KR-1 3KR-2 3KR-3 - - - - - - - dex + + + + + + + kDa a-GR - 100 a-GAPDH - 35 A549 + - - HEK293-wtGR - + - - - HEK293-GR3KR + kDa a-GR - 100 a-GAPDH - 35 B wtGR GR3KR 43°C - - + + - - + + dex - + - + - + - + kDa - 170 a-GR - 130 - 100 a-GAPDH - 35 - 170 a-SUMO-1 - 130 R - 100 G - a : P I a-SUMO-2/3 - 170 - 130 - 100 Figure S1. Expression and SUMOylation of GR in stable isogenic HEK293 cells. (A) Upper panel, immunoblot analysis of three HEK293 cell clones expressing either wtGR (wt-1-3) or GR3KR (3KR-1-3). The clones and background HEK293 (FRT) cells were exposed to vehicle or dex for 17 h, and cell samples were immunoblotting with anti-GR (Santa Cruz, sc-1003) and anti- GAPDH (Santa Cruz, sc-25778) antibodies. Lower panel, comparison of the GR level in A549 cells (from ATCC) with that of the selected clones from isogenic HEK293 cells (wt-3 and 3KR-3) by immunoblotting. (B) To analyse GR SUMOylation, wtGR- and GR3KR-expressing cells were grown with of without dex for 2 h and exposed for 30 min to 43°C. Immunoprecipitation (IP) with anti- GR antibody and immunoblotting with anti-SUMO-1 (Invitrogen, 33-2400) or anti-SUMO-2/3 (MBL M114-3) antibody were performed essentially as described (Rytinki et al. 2011, Methods Mol. Biol.). A total dex- sensitivity induced for dex expression ADARB1 HLA-DRAADARB1 APPL2BBS12 ADARB1CHN2 GRHL1PLEKHA9 CHMP1BFBXO21 ZBTB16PPP2R3B HPCAL1TNFRSF10D TMEM87A GRHL1SCAND3 AKTIPJAK2 NKD1AP3S1 UPF2ACVR1B SLC25A4SNX33 ZBTB7CKLHL36 IGSF3GTPBP2 KIAA0513RPRM LIPGSTK35 SLC36A1 TMX4FIS ARNTLS1PR3 INHBBNEXN SLC9A2SATB2 MRFAP1L1OSBPL5 C9orf61OGFRL1 FOXO4OSBPL5 TXLNACLEC16A PRKAB2C5orf62 FAM105ASLC41A1 SEPX1PTTG1IP TSHZ1SFXN5 PTCHD1HIVEP1 SQLE POU3F2 ANKRD50PDE5A HOXD11HIST2H2BE TSC22D3WDR7 FAM49AURB2 MID1GCOM1 CRKMED30 ZBTB46C16orf80 MID1THAP10 SSTR2 AENPDZD2 TMEM43KBTBD11 MBOAT2PPP1R1A C17orf80MT1F RASSF4ZDHHC9 GCH1CCDC107 FAM100AMAP2K4 EIF4A3GUCY1A3 GCH1GREB1 GOT1ARCN1 PXNADAMTS8 NBNTSC22D3 TSC22D3 BIRC2COG1 PDK4KCNS3 C7orf57ZNF688 LINS1ERN1 RAD51CRHPN1 CCDC126CLPTM1L TSPAN3MN1 ZNF341ALAD GNA12SLC39A11 FKBP5STAT3 NOL3GNA12 ETNK2SPSB1 LPLNET1 NPHP4HPCAL1 XKZNF364 B PTTG1IPGJB2 TOX3 FAM104AE2F3 GCNT1JAK1 UNC84BSLC25A4 STOMITGB2 STOMEP400 KIAA0232NET1 EDG7KIAA0182 TPM2PPP1R3F ARID5BB4GALT2 PRKCQFGFRL1 TACC2TPM2 KIAA0355ANO6 RPS6KA2TUBA3D EDNRAC10orf10 TMX4ZNF784 SFRP2KAL1 RNF115CYR61 C3orf58HOXA4 8 ASB7C1QTNF1 FBXO2KRCC1 ASB7ZDHHC8P TAOK2 FLJ31568RASD1 RAD51C GPERPNMT NSDHLMSX2 - ACPL2FAM55C RNF14ACPL2 MAP2K4C6orf81 VPS37ACAMK1G TUBA3ETHSD1 COL23A1RASGRP1 ASB7BIRC3 C16orf5MRFAP1L1 PSD4SGSM1 FGF18HSD11B2 SBK1ENOX2 NET1RGS2 DGCR6LERN1 NAGKAMOT APOLD1AIFM1 ZNF259UPF2 FLJ31568ISG20 FAM65BMMP28 dex TUBA3C PGCGSTM3 ZNF18FBXO32 CLN8TUBA3D SHISA2ATP4A PHLDA1RAB20 APOL2SNN AGRP APOL2SSX2IP LIPGMTNR1A FBXO32CLN8 ZNF189ELK1 KCNA5OLFML2B C18orf54PAQR8 APODTRAF5 IGSF3B3GAT2 CHN2HMGCS1 ENPP5MERTK C18orf1BBS1 6 PHF19 OGFRL1SESN1 INPP1C10orf140 ZSWIM5TMEM91 MERTKFZD4 HIST1H1CMOCOS IGFBP5 ASB9 FUT4SLC6A3 CDH7HOXD10 HIST1H2AGHIST2H2AA3 PCDH7C4orf31 CCND1GAD1 TET1SOX12 HIST1H2ACMICB HIST1H2BDHIST1H2BJ DMDDMRT3 CDKN1ALRRTM4 DDX26BZNF503 C19orf23CXXC4 HSPA1LHIST1H2BD TMEM100HSPA6 S100PNFKBIA e HOXA9HOXA13 NTF3HOXA9 ZIC2NOS2A MID1IP1 TRIB3 g HES1CDKN1C MYC ATF3ID3 PPP1R15APRR15L RGS16 n OKL38RRS1 IRF8DDIT3 IER3SAT1 BHLHB2SGK3 EGFLAMWWP2 a STC1HOXC4 SVILSLAIN1 ZNF217SCHIP1 KLF10TGIF2 ALG3 h RFESDIRF2BP2 HMOX1C15orf39 4 PPRC1 TSC22D1HOXA10 C10orf2 c TESSTC2 FAM90A1RASSF1 OSGIN1SPRYD5 PI15MEX3B FOXA2JUN BCORSPEN GADD45AHRK PAG1IRF2BP2 d CDADC1NMI OSBPL11TF l GPERC1QTNF1 NBN CAPN7WDR60 PPP1CBING1 GTF2E1 o FAM163AELMO2 SFRS5C8orf51 FAM134BARRDC4 f CSTF3ZFP36 B3GAT1 MYOM2KLHL26 ARRDC4RP1L1 SELS TRIOBPSOX2 C3orf52OPRL1 APPBP2KHDC1 HSD17B1HOXC9 PHLDA1F3 SLC25A18ZNF57 SNAI2RGMB CRKSEPT4 SOX2HOXC6 MBSGK1 GIPC1MECP2 SGK1SGK CRKEYA1 NR2F2C17orf100 BCL11BSELS HSPA2 PCDH7PLEKHF2 DDX24 SLC39A14MB S100A13PPP3CC 2 ABCC8 PTGESABHD6 SOX18SLC44A2 TSC22D1GDF15 WDR43EGR1 HMGB3PLEK2 HOXA11ASFOXN4 RGS19TIPARP KLF15ADRB2 KLF10RFESD DUSP5KCNG1 HOXA11MME PKP2POU3F2 NOL3CNIH3 FHDC1 PROX1FOXF2 DLX1BMP6 CDKN1ARSC1A1 HOXB8GLIS3 IDI1CXXC6 HIST2H4BGFOD1 ULK1NME2 ST8SIA2SOX4 ULK1FOXQ1 RHOUSMAD6 FAM46CRBMS3 ARHGAP20ZDHHC14 NOTCH1CABLES1 PRDM8HIST2H3C DBPSLC2A14 SLC2A3NRP1 HIST2H4AMYLIP TLCD1BRD2 SOBPZNF467 HEY2IRS4 CBX2USP3 ZFXBAMBI HOXA2EFNA1 RNF14LMCD1 BTBD3NR2F1 TLE4HOXD13 MNTDDX3X C21orf69SOBP PDLIM1PITPNA 0 TLE4 GALNT10 ZNF462FZD8 PLAURIER5 ZDHHC9TSC22D3 HSPA1B wtGR GR3KR wtGR GR3KR wtGR GR3KR wtGR GR3KR wtGR GR3KR wtGR GR3KR wtGR GR3KR wtGR GR3KR CHST3 FAM43BSERTAD4 FANCBHOXC8 PLAURLYPD1 SMG1C1orf26 MEX3CHOXB5 CKS2FGF9 TSPAN9FOXA1 C3orf70CMIP TP53BP2 ITGB2MEIS1 HEY1USP9X FOXC1 ZMIZ1BLM BHLHB3ZFAND5 C3orf70PDZD8 C14orf132MEIS2 TEX10FAM62B ISG20L1CTDSPL CDX2BTG1 ELL2KIAA1804 PIM1HOXA3 Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 Cluster 6 Cluster 7 Cluster 8 ACSL1 SLC7A2CKS2 RASEFFBXO5 TSPAN12ATP1A1 CNNM1C20orf177 ATP1A1 HEY1HOXA5 NDE1PIM2 KLF9PRICKLE1 TIMM8AGAS1 IGDCC3GAS1 TBX2HOXA6 TSC22D3 MAFB MERTK SGK1 IL8 JUN TSC22D1 FOXA1 LMO4 NR4A2 CDC42EP4KCTD15 BMFHCFC1 HOXA10 HELBBMF BCORINSIG1 BCORL1LAD1 TOB1HIST1H3B DLC1SOX8 FAM86ACEBPD HOXA10FAM86A KLF6PRAGMIN KLF6 BCORHIST2H3D INSIG1LMO4 PMAIP1ANKRD37 CXXC5SORBS1 HIST1H4B C14orf4PMAIP1 PRIC285CXXC5 RFESDTRIB1 KCNG1SH2B3 TMEM108SOX7 ARHGEF2PLAGL2 ZNF711KCNG1 NRP1PLD6 IL12AUNC5B NGFSOX21 KLHL15PHACTR3 SIX1HIST2H3A SOX7TRIM48 HOXC13LHX6 ZNF296GSK3B TNFRSF12AGADD45A LRRC8EPHACTR3 LDLRDDIT4 NEFLZFP64 FOXD1SPRY1 TFAP2AHCCS EFNA1AUTS2 SIX4MARVELD3 SAMD11EXT1 TUFT1IRX5 USP36PRICKLE3 FGFR3AVPI1 CNKSR3PNMA2 UBQLN2CRISPLD2 ZNF395FILIP1L NRINTNTFAP2A CALD1PER1 INHBEZNF22 ADAMTS1FOS HSPA1ACST1 R R K G 3 t R w G n i n i h h g i g i h h Figure S2. (A) Differences in dex sensitivity and total dex-induced gene expression between wtGR- and GR3KR-expressing HEK293 cells illustrated as heat maps. (Left column) Red color corresponds to higher fold-induction by dex in the GR3KR- expressing cells, blue color to that in the wtGR-expressing cells, and white color indicates that the sensitivity does not differ between cell lines. (Right column) Heat map depicting differences in total dex target gene expression level. (B) Validation of expression of one gene from each hierarchical cluster (shown in Fig. 1B) by RT-qPCR. Cells were treated with vehicle or dex for 6 h, RNA was isolated and analyzed by RT-qPCR with primers specific for TSC22D3 (cluster 1), MAFB (cluster 2), MERTK (cluster 3), SGK1 (cluster 4), IL8 (cluster 5), JUN (cluster 6), TSC22D1 (cluster 7) and FOXA1 (cluster 8). Bars represent the mean ± SD of three experiments. A B U2Os-wtGR U2Os-GR3KR SGK CACNA1I TXNIP dex-regulated MGC42367 DUSP1 MTE C20orf127 FKBP5 p<0.001 FC >1.5 and <0.7 up-regulated SGK1 SGK1 SPINK5L3 TSC22D3 SCNN1A s TUBA3D 1 TIPARP e TSC22D3 r ZFP36 n e ERRFI1 R t FOXO1 e KLF9 s g R TMEM100 K ZCCHC5 u l RHOB 6 NFKBIA G 3 c MT1G 3 t F3 LOC652968 R SLC46A3 MT1X U2Os-GR3KR w ZNF189 C2orf55 - U2Os-wtGR G MT1E 32 102 34 CEBPD - PDK4 s PHACTR3 PHACTR3 s O ZCCHC5 O 2 STK39 OR7A5 2 CHES1 FOXO3 U C10orf47 MT1E U RPS6KA2 CAV1 MT1A PRB1 TSC22D3 CA2 LRRC8A 39 120 67 CNKSR3 EDN3 IP6K3 e ADORA3 PLD5 g FAM90A7 GPR64 down-regulated n KIF13B NAV2 a CDO1 C10orf47 h RASSF4 PKP2 R c PRB2 s RGS2 2 R e d ZBTB16 r K l F3 PTGER2 n e o TSPYL2 G t e 3 GHR f t FOXO3 s g CRISPLD2 u R PNLIP l CDC42EP3 6 w SGSM1 c LAD1 6 - G SNAI2 7 18 33 DNER - SERPINA3 s PRB2 PROX1 s GALNTL4 O OGFRL1 OGFRL1 SLCO2A1 O 2 EYA2 CA2 2 CCND3 NT5DC3 U DDIT4 DKFZp451A211 U CTGF CTGF OLAH NAV2 IHPK3 ALOX5AP GPR1 TSPYL2 MUM1L1 SNAI2 PQLC3 GPIHBP1 CCL20 ALPL PROS1 TSPYL2 KCTD12 C TBC1D8 MT2A LASS6 SLC39A14 NEXN s ELF1 3 FUT4 e FSTL3 r IRF8 n e CLPTM1L t WDR60 e Ingenuity Pathway Analysis IL8 s g CREG1 SERPINE1 u l DUSP2 2 PRIC285 c CITED2 3 CAV1 KIF5C Genes dex-regulated whose expression differs; SP110 TNFSF4 ZBTB16 ZDHHC9 CXCR7 NAV2 BIRC3 U2Os-wtGR vs. U2Os-GR3KR NFIA ZFP36L2 PDZD8 NNMT PRRG1 TOP 6 Molecular and Cellular Functions NPTX2 DACT1 NFIL3 METTL7B PRB1 WNT5A C5orf62 s BNIP3 4 BCL2L1 e Pathw ay p-value r NDRG1 MAP2K1 n e SEPP1 t ITPR1 e FLRT3 s g Cellular Development 2,40E-13 - 1,27E-03 EDN2 CDO1 u l KLHL4 4 AP3S1 c NFATC1 3 Cellular Growth and Proliferation 1,86E-12 - 1,27E-03 SPRYD5 SLC2A14 MSX2 SCEL FAM90A1 ASB9 Cell Death and Survival 3,79E-12 - 1,28E-03 ING1 MCL1 GCOM1 Cellular Function and Maintenance 2,38E-09 - 1,04E-03 BIRC3 IER3 PRAGMIN PLEKHG3 ROR1 Cellular Movement 1,54E-08 - 1,14E-03 HAVCR2 GUCY1A2 JUN S100A3 EHD1 Cell Cycle 5,04E-08 - 1,20E-03 HES1 TMEM166 ZSWIM4 TIAM2 UPP1 s 5 LFNG COL16A1 e r MAP2K3 n PMEPA1 e PMEPA1 t e VEGFA s TUBA4A g CGN C15orf52 u l - GRK5 5 MARCH4 c 3 FOXS1 MYEOV SH3RF2 EPHA4 dex HBEGF RUNX1 TPST2 D CLDN1 FAM176A NFKBIA TCF4 40 25 SGK1 KRT80 *** FHL2 FHL2 ** LAMB3 CD34 20 ANKRD38 s PLAU 6 30 LCTL e e e LMCD1 r MAMLD1 n g g FRMD5 e t e n n RN7SK 15 SYNPO2L a a s DLC1 g h h SERTAD4 20 BAPX1 u l 3 c c C1orf133 c MYCN 2 d d 10 FHL2 l l ADAM19 o o TPST1 f f TGFBI 10 FRMD5 5 0 0 U2Os-wtGR U2Os-GR3KR U2Os-wtGR U2Os-GR3KR E 600 - IL8 PMEPA1 dex 10 * 2.0 *** 500 8 % 400 1.5 h e e t g g n 6 n w a a 300 h h 1.0 o c c r d 4 d l l 200 o o G f f 0.5 2 100 0 0.0 0 U2Os- U2Os- U2Os- U2Os- U2Os-wtGR U2Os-GR3KR U2Os-wtGR U2Os-GR3KR wtGR GR3KR wtGR GR3KR 48 h 72 h Figure S3. Dex target gene expression in wtGR- and GR3KR-expressing U2Os cells. RNA from U2Os cells that stably express either the wtGR (U2Os-wtGR) or the GR3KR (U2Os-GR3KR)