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 U2Os-wtGR vs. U2Os-GR3KR BIRC3

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) was analyzed by Illumina BeadChips as described for HEK293 cells in Figure 1. (A) Venn-diagrams showing comparison of dex-regulated genes between wtGR- (green) and GR3KR-expressing U2Os cells (purple), and additionally dex-up- and -down-regulated genes in separate diagrams. Values inside the diagram indicate the number of dex-regulated genes shared and unique to each cell line. (B) The dex-regulated genes were clustered by unsupervised hierarchical clustering, which resulted in 6 clusters shown as heat map: clusters 1, 3, 4, 5 and 6 contain genes that were dex-regulated differently between the U2Os-wtGR and the U2Os-GR3KR cells. (C) The genes that were differently expressed between the cell lines were analyzed by Ingenuity Pathway Analysis. The table shows the top 6 molecular and cellular functions that were the most significantly enriched for the genes differently expressed between the U2Os-wtGR and the U2Os- GR3KR cells. (D) RT-qPCR validation of genes that affect to cell growth and are differently expressed between the cell lines. Cells were analyzed as described in Figure 1 for HEK293 cells. Bars represent the mean ± SD of three experiments. ***, p<0.001; **, p<0.01, *, p<0.05 for the difference of dex treatment between U2Os-wtGR and U2Os-GR3KR cells. (E) Cells were treated with dex as indicated and cell proliferation was measured by Cell Titer96 Aqueous cell proliferation assay reagent at the indicated time points. - dex 40

35

30

25 10 e g n

a 8 h c d l 6 o f

4

2

0

R R R R R R R R R R R R G K G K G K G K G K G K t 3 t 3 t 3 t 3 t 3 t 3 -w -w -w -w -w -w s R s R s R s R s R s R O -G O -G O -G O -G O -G O -G 2 s 2 s 2 s 2 s 2 s 2 s O O O O O O U 2 U 2 U 2 U 2 U 2 U 2 U U U U U U

Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 Cluster 6 TSC22D3 SNAI2 IL8 MAP2K1 PMEPA1 CD34

Figure S4. Validation of expression of one gene from each U2Os cell hierarchical cluster (shown in Fig. S1B) 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), SNAI2 (cluster 2), IL8 (cluster 3), MAP2K1 (cluster 4), PMEPA1 (cluster 5) and CD34 (cluster 6). Bars represent the mean ± SD of three experiments. A dex-up-regulated genes B dex-down-regulated genes with or without GRBs ± 100 kb from TSS with or without GRBs ± 100 kb from TSS with GRBs in wtGR with GRBs in GR3KR with GRBs in wtGR with GRBs in GR3KR

73% 27% 79% 21% 43% 57% 58% 42%

without GRBs in GR3KR without GRBs in wtGR without GRBs in wtGR without GRBs in GR3KR C D 6000 10000 wtGR 8 786 GR3KR

5 115 s s B 8000 B R R G

G 4000

R 6000 R K G 3

t 2 578 4 298 R w

4000 G f

2000 f o

o .

. o 2000 o n 1 0 0 0 n 1 0 0 1 0 0 1 2 3 4 5 6 1 2 3 4 5 6 no. of genes to one GRB is associated with no. of genes to one GRB is associated with E F dex-up-regulated genes dex-down-regulated genes associated with GRBs associated with GRBs

GR3KR GR3KR

wtGR wtGR

1 250 31 6 118 55

Figure S5. Association of GRBs with the dex-regulated genes in HEK293 cells. (A, B) Exploded pie charts showing the number of (A) dex-up-regulated and (B) dex-down-regulated genes in the wtGR- or the GR3KR-expressing cells harbouring at least one GRB (blue/red portion) within 100 kb up- or downstream of gene's TSS. Values inside the pie charts indicate the percentage of genes with or without (grey portion) GRBs. (C, D) Graphs showing the number of genes to GRBs are associated with in (C) wtGR or (D) GR3KR cells. (E, F) Venn-diagrams showing comparison of (E) dex-up- or (F) dex-down-regulated genes associated with at least one GRB. A B dex-up-regulated genes dex-down-regulated genes with or without SUMOBs ± 100 kb from TSS with or without SUMOBs ± 100 kb from TSS

with SUMOBs in wtGR with SUMOBs in GR3KR with SUMOBs in wtGR with SUMOBs in GR3KR

66% 34% 57% 43% 43% 57% 36% 64%

without SUMOBs in wtGR without SUMOBs in GR3KR without SUMOBs in wtGR without SUMOBs in GR3KR C D 8000 wtGR 5000 GR3KR s s B B

6 236 O 3 936

O 4000

6000 M M U U S

S 3000

3 981 R R 4000 K 2 241 G 3 t 2000 R w

G f

f o 2000

o .

1000 . o o n 11 0 0 1 7 0 0 1 n 0 0 1 2 3 4 5 6 1 2 3 4 5 6 no. of genes to one SUMOB is associated with no. of genes to one SUMOB is associated with E F dex-up-regulated genes dex-down-regulated genes associated with SUMOBs associated with SUMOBs

wtGR wtGR GR3KR GR3KR

44 192 10 39 90 18

Figure S6. Association of SUMOBs with the dex-regulated genes in HEK293 cells. (A, B) Exploded pie charts showing the number of (A) dex-up-regulated genes and (B) dex-down-regulated genes in the wtGR or the GR3KR-expressing cells harbouring at least one SUMOB (blue/red portion) within 100 kb up- or downstream of gene's TSS. Values inside the pie charts indicate the percentage of genes with or without (grey portion) SUMOBs. (C, D) Graphs showing the number genes to SUMOBs are associated with in (C) wtGR or (D) GR3KR cells. (E, F) Venn-diagrams showing comparison of (E) dex-up- or (F) dex-down-regulated genes associated with at least one SUMOB. A GRBs and SUMOBs associated with dex-up-regulated genes

Hormone-nuclear bba -zinc finger Grainyhead Receptor ETS domain p-value: 1.0x10-12 p-value: 2.2x10-3 p-value: 1.8x10-3 p-value: 9.2x10-3

GR3KR-GRBs GR3KR-SUMOBs wtGR-GRBs wtGR-SUMOBs Homeodomain 301 Hormone-nuclear 41 Receptor -3 p-value: 1.2x10 246 14 90 p-value: 1.6x10-3 28 117 26 16

1 238 10 1 129 9

Hormone-nuclear bba -zinc finger Receptor Homeodomain p-value: 1.9x10-6 p-value: 4.9x10-4 p-value: 7.8x10-3

B GRBs and SUMOBs associated with dex-down-regulated genes

Hormone-nuclear Leucine zipper Receptor ETS domain p-value: 3.3x10-7 p-value: 7.5x10-3 p-value: 8.1x10-4

GR3KR-GRBs GR3KR-SUMOBs wtGR-GRBs wtGR-SUMOBs 184 31 Hormone-nuclear 95 6 79 Receptor 28 79 p-value: 3.4x10-4 14 6

0 38 3 0 33 3

Figure S7. Enrichment of motifs in GRBs with or without overlapping SUMOBs in dex-regulated genes. (A) Quadruple Venn-diagram shows comparison between the GRBs (blue/red) and the SUMOBs (light blue/red) associated with dex-up- regulated genes between the wtGR- and the GR3KR-expressing HEK293 cells. Values inside the diagram indicate the unique GRBs and the SUMOBs to each cell line and different intersections the GRBs and the SUMOBs shared between each other and between each cell line. Motif enrichment analysis was performed from the sets indicated by arrows. Only the GRBs of the GR3KR cells without SUMOBs harbored in addition to canonical GRE, ETS domain, bba-zinc finger (IKZF1) and grainyhead (TFCP2L1) motifs. Only the GRBs of the wtGR cells without SUMOBs harbored homeodomain (IRX3) motif. The canonical GRE was enriched in sites shared by all four groups. The GRBs/SUMOBs of the wtGR cells, but only the GRBs of the GR3KR cells, harbored bba-zinc finger (ZNF304) and homeodomain (HOXA13) motif in addition to the canonical GRE. The p-values of the motif enrichment are shown. (B) The same analyses as in (A) but for the dex-down-regulated genes. Only the GRBs of the GR3KR cells without the SUMOBs harbored in addition to the canonical GRE, ETS domain and leucine zipper (CEBPA) motifs. The GRBs/SUMOBs of the wtGR cells, but only the GRBs of the GR3KR cells, harbored the canonical GRE. CCND2 A EtOH dex *** ns 1.5 *** e

g 1.0 n a h c d l

o 0.5 f

0.0 wtGR GR3KR B IL8 * 2.0 ns

1.5 e g

n *** a

h 1.0 c d l o f 0.5

0.0 wtGR GR3KR

C ZIC2 ns 1.5 *** ns e

g 1.0 n a h c d l

o 0.5 f

0.0 wtGR GR3KR D CXXC4

1.5 *** ns ** e

g 1.0 n a h c d l

o 0.5 f

0.0 wtGR GR3KR

E DMRT3 * ns 1.5 ** e

g 1.0 n a h c d l

o 0.5 f

0.0 wtGR GR3KR F SPRY1 2.0 * * 1.5 ns e g n a

h 1.0 c d l o f 0.5

0.0 wtGR GR3KR

Figure S8. RT-qPCR analysis of select dex-down-regulated genes differently expressed between wtGR and GR3KR cells and associated with unique GRBs. Cells were treated with vehicle or dex for 6 h, RNA was isolated and analyzed with specific primers for CCND2 (A), IL8 (B), ZIC2 (C), CXXC4 (D), DMRT3 (E) and SPRY1 (F). Bars represent the mean ± SD of three experiments. ***, p<0.001; **, p<0.01; *, p<0.05 for the difference of vehicle and dex treatment in either the wtGR- or the GR3KR- expressing cells, or for the difference of dex treatment between wtGR- and GR3KR-expressing cells. ns, no significance. A mRNA expression chr11 : 2,890 kb 2,910 kb 2,930 kb - - - - - CDKN1C _ 15.27

_ 125 R _ _ _ G _ t 20x _ w _ _ _ 0- q

_ - _ e R

_ 125 s _ R K - _ _ 3 _ G P 4x _ I R a _ _ h G _ 0

C -

_ - _

_ 125 _ T _ _ R _ 0.5x _ F _ _ _ 0-

KCNQ1DN CDKN1C SLC22A18AS SLC22A18

B chr14 : 35,840 kb 35,860 kb 35,880 kb - - - - _ 656 NFKBIA _ R

_ 31.26

_ 44.91 _ G _ t 18.1 12.83 29.72

_ 21x _ w _ 0 _ - q _

e -

_ 53.77 R s

_ 656 R - _ _ K _ G P 3 _ I 23.04 8x a _ R 26.99 18.25 h _ _ 12.44 G _ C 0- _ _ -

_ 656 _ T _ _ R _ 1x _ F

_ 13.94 _ _ 0-

NFKBIA C chrX : 47,490 kb 47,500 kb 47,510 kb 47,520 kb - - - - _ - ELK1 _ 381 R _ _ _ G t _ 4x _

_ 26.19 w _

_ 0 q - _ e _ R s

_ - R 53.26 - _

_ 381 K _ G P 3 8x _ I a _ R h _ _ 19.72 G _ C 0 _ _ -

_ - _

T 381 _ _

R 1x _ _ F _ _ _ 0-

CFP ELK1 UXT NR_028119

chr2 : 112,660 kb 112,700 kb 112,740 kb 112,780 kb - D - - - - MERTK _ 573 _ R _ _ _ G 32.92 1x t _ 20.87 _

_ 9.66 11.54 w 15.87 _ 0

_ - q _

e - 51.19 _ R s 573 _ R - _

_ 47.76 K 3x

_ 32.42 32.27 G P 3 _ I 32.21 a _ R h _ _ G _ C 0-

_ - _

_ 573 _ T 1x _ _ R _ _ F _ _ _ 0-

MERTK

E chr17 : 17,380 kb 17,400 kb 17,420 kb - - - - RASD1

_ 343 _ R _ _ 40.31 34.41 _

G 91x _ t _ _ w _ 0- _ q _ - 51.9 e

_ 343 R s _ R - _ _ K 31.05 321x _ G P 3 _ I a _ R h _ _ G _ 0 C - _ _ 343 _ _

T 1x _ _ R _ _ F _ _ _ 0-

MED9 RASD1 PEMT

F chr20 : 39,270 kb 39,290 kb 39,310 kb 39,330 kb - - - - MAFB _

_ - R

_ 493 _ 64.45 _ G _ t 4x _ w _ _ 18.23 _ 10.01 q 0 _ - _ e

R - _ s _

R 493 K - _ _ 3 _

G 45.84 P 1x _ I R a _ _ h G _ 10.23

C 0

_ - _

_ -

_ 493 T _ _ R _ 1x _ F _ _ _ 11.37 0-

MAFB

Figure S9. Peak tracts showing the occupancy of GR in the regulatory regions of CDKN1C (A), NFKBIA (B), ELK1 (C), MERTK (D), RASD1 (E) and MAFB (F) in wtGR- (blue), GR3KR- (red) and empty FRT-expressing (green) HEK293 cells. The numbers above the GRBs depict the FE value. Fold-induction values of the genes as measured by RT-qPCR are shown on the right. A mRNA expression chr12 : 4,300 kb 4,400 kb 4,500 kb CCND2 - - - - _ 165 16.6 9.3 15.12 _ R _ _ _

G 0.6x _ t _ _ w _ 0

_ - q _

e - 26.71 _ R s

_ 165 R - _ _ K _

G 14.96 P 3

_ 1x I a _ R h _ _ G _ C 0- _ _ -

_ 165 _ T _ _ R

_ 1x _ F _ _ _ 0-

CCND2 C12orf5 FGF23 FGF6

B chr4 : 74,530 kb 74,570 kb 74,610 kb

- - - - _ IL8 _ 75 9.78 R _ _ _ G _ t _ 0.2x w _ _

_ 0 q - _

e - _ R

_ 75 s R _ - K _ _ 3 G P

_ 1x I _ R a h _ _ G _

C 0-

_ - _

_ 75 _ T _ _ R _ _ F 1x _ _ _ 0-

IL8

C 100,620 kb 100,640 kb 100,660 kb

chr13 : - - - - 12.3 _ ZIC2

_ 96 R _ _ _ G _ t

_ 0.5x w _ _

_ 0 q - _

e - _ R

_ 96 s R _ - K _ _ 3 G 1x P _ I _ R a h _ _ G _

C 0-

_ - _

_ 96 _ T _ _ R _ _ F 1x _ _ _ 0-

ZIC5 ZIC2 chr4 : 105,340 kb 105,400 kb 105,460 kb - - - - _ 275 D _ CXXC4 R _ _ _ G _ t _ _ w 1x _ 0 _ - q _

e - 44.19 _ R s

_ 275 R - _ _ K _ G P 3 26.51 _ I a _

R 0.4x h _ _ G _ C 0- _ _ -

_ 275 _ T _ _ R _ _

F 1x _ _ _ 0-

CXXC4

E chr9 : 900 kb 1,000 kb 1,100 kb - - - - _ 179 DMRT3 _ _ _ R

_ 1x G t _ _ _ w 0

_ - _ q _ e - _ s 33.31 R R -

_ 179 31.05

_ 27.42 _ K

G 20.01 P 0.6x _ I 3 _ a h _ R _ _ C G _ 0- _

_ -

_ 179 _ T _ _ R _ 1x _ F _ _ _ 0-

DMRT1 DMRT3 DMRT2

F chr4 : 124,280 kb 124,320 kb 124,360 kb - - - - _ 147 SPRY1 _ _ _ R _ G t _ 1x _ _ w 0

_ - _ q _ e - 14.91 _ s R R -

_ 147

_ 16.07 _ K

G 0.6x P _ 13.73 I

3 13.45

_ 10.61 a h _ R _ _ C G _ 0- _

_ -

_ 147 _ T _ _ R _ _ F 1x _ _ _ 0-

SPRY1

Figure S10. Peak tracts showing the occupancy of GR in the regulatory regions of CCND2 (A), IL8 (B), ZIC2 (C), CXXC4 (D), DMRT3 (E) and SPRY1 (F) in wtGR- (blue), GR3KR- (red) and empty FRT-expressing (green) HEK293 cells. The numbers above the GRBs depict the FE value. Fold-induction values of the genes as measured by RT-qPCR are shown on the right. A all wtGR peaks wtGR GRBs and SUMOBs associated wtGR GRBs and SUMOBs associated with dex-up-regulated genes with dex-down-regulated genes

GRBs SUMOBs GRBs SUMOBs GRBs SUMOBs

6 836 4 208 7 911 301 377 233 137 74 167 ) ) ) % %

% 65 65 ( 65 ( ( 60 60 p 60 p p a a l a 55 l l 55 55 r r r

e 50

e 50 e 50 v v v 45 45 45 o o o

t 40 t t 40 40 e e e 35 35 s 35 s s

k 30

k 30 k 30 a a a 25

25 e

25 e e p

p 20 p 20 20 E

E 15 E 15 15 D D D 10 10 10 O O O 5 5 5 C C C

N 0

N 0

N 0 E E E 3 3 3 3 3 3 3 3 3 S S S S S S S S S e e e e e e e e e H H H H H H H H H m m m m m m m m m D D D D D D D D D 4 4 4 4 4 4 4 4 4 K K K K K K K K K 3 3 3 3 3 3 3 3 3 H H H H H H H H H

T T T T T T T T T 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 K K K K K K K K K K K K K K K K K K E E E E E E E E E E E E E E E E E E H H H H H H H H H H H H H H H H H H

B GR3KR peaks GR3KR GRBs and SUMOBs associated GR3KR GRBs and SUMOBs associated with dex-up-regulated genes with dex-down-regulated genes GRBs GRBs GRBs SUMOBs SUMOBs SUMOBs

15 916 2 925 4 859 686 294 133 315 64 110 ) ) ) % % % 65 65 65 ( ( ( 60 60 60 p p p a a a l l 55 l 55 55 r r r

e 50 e 50 e 50 v v 45 v 45 45 o o o t t 40 t 40 40 e e e 35 35 35 s s s

k 30 k 30 k 30 a a 25 a 25

25 e e e p p 20 p 20 20 E E 15 E 15 15 D D 10 D 10 10 O O 5 O 5 5 C C C

N 0

N 0 N 0 E E E 3 3 3 3 3 3 3 3 3 S S S S S S S S S e e e e e e e e e H H H H H H H H H m m m m m m m m m D D D D D D D D D 4 4 4 4 4 4 4 4 4 K K K K K K K K K 3 3 3 3 3 3 3 3 3 H H H H H H H H H

T T T T T T T T T 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 K K K K K K K K K K K K K K K K K K E E E E E E E E E E E E E E E E E E H H H H H H H H H H H H H H H H H H

Figure S11. Overlap of the GRBs and the SUMOBs with ENCODE HEK293 cell H3K4me3 and HEK293T cell DHS dataset. The GRBs and the SUMOBs in wtGR cells (A) and GR3KR cells (B) were compared with the H3K4me3 ChIP-seq (GSM945288) and DNAse-seq (DHS) (GSM1008573) dataset. Colums show the percentage of overlap of the indicated GRBs and SUMOBs with the ENCODE datasets.

Supplementary Table S1. RT-qPCR primers used in the study

gene forward 5'-3' reverse 5'-3' CDKN1C CACTCGGGGATTTCGGGAC CGCTTGGAGAGGGACACG NFKBIA CACTTAGCCTCTATCCATGG ACACCAGGTCAGGATTTTGC ELK1 GAGCTGCCAACATTGCCAAC GGAGATGATGTGGCCATTGC RASD1 CACAGGAGACGTTTTCATCC TGTTCTTCTTGGCCGAGAT MAFB CCCGACCGAACAGAAGACAC GTGAGGGTGGTGGTGATGG MERTK AGCCTTCAACCTCACCTGTC CGGTCAGCCCTTTGTCATTG SGK1 ATGCCAACCCTTCTCCTCC TCAACAGAACATTCCGCTCC IL8 GACAAGAGCCAGGAAGAAAC TCCACTCTCAATCACTCTCA PMEPA1 CTTTGTTCCAGAGCATGGAG TTCTGAGGACAGGGCATCTT TSC22D3 GGACTTCAGGTTTCAGTGGACA AATGCGGCCACGGATG TSC22D1 GGACTGAAAACGCTTCTGTGA TGACCGCATACATCAAATGG FOXA1 CCGCCCGTCGCTTCGCACAG TGTTGCCGCTCGTAGTCATG JUN AGTCAGGCAGACAGACAGACAC AAAATAAGATTTGCAGTTCGGACTAT SNAI2 TCAACGCCTCCAAAAAGCCA TGATGGGGCTGTATGCTCCT MAP2K1 TCAAGCCCTCCAACATCCTA GCACAGAGTAATGAGTCCCC CD34 GCGCTTTGCTTGCTGAGT GGGTAGCAGTACCGTTGTTGT CCND2 GGACATCCAACCCTACATGC CGCACTTCTGTTCCTCACAG ZIC2 CCGAGAACCTCAAGATCCAC AGCCCTCAAACTCACACTGG CXXC4 CGTTGCAAATGGCAAATACTAA CCAAAGGTCTGCAGTGTTCA DMRT3 GTCCAGCCGATCCTCAGTC GGCAACGCTAGACTCTCAGG SPRY1 GGAAATCCACGGTGATCCT GGCATGCATCTGAAATCCTT

Supplementary Table S2. ChIP primers used in the study

gene forward 5'-3' reverse 5'-3' RASD1 (1) CGAATTAAGCCTGCACTCAT ACTCTTGGGCACTGACTGAG RASD1 (2) ACAATGCTCTGTGGGTCATT TTCTGTTAGGGAGCTTGTGG MAFB GGCAAAATGAAATGTCTTGG GAGTCTGCAACCTGGAAAGA