Supplementary Materials for

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1 Supplementary Materials for RNA polymerase II associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II Ming Yu, Wenjing Yang, Ting Ni, Zhanyun Tang, Tomoyoshi Nakadai, Jun Zhu, Robert G. Roeder* *Corresponding author. roeder@rockefeller.edu Published 11 December 2015, Science 350, 1383 (2015) DOI: /science.aad2338 This PDF file includes: Materials and Methods Supplementary Text Figs. S1 to S11 Tables S1 to S5 References

2 Materials and Methods Cell culture and treating cells with chemicals The human AML THP1 cells and the human ALL CCRF-CEM cells were cultured in RPMI % FBS + 2% Penicillin/Streptomycin + 2 mm L-Glutamine + 55 µm β- mercaptoethanol. Flavopiridol (Sigma, cat. no. F3055) treatment was performed as previously described (2). THP1 cells were cultured in standard medium supplemented with DMSO or 1 µm flavopridol in DMSO for 1 hour before fixation for ChIP. Chromatin immunoprecipitation (ChIP) and ChIP-sequencing (ChIP-seq) ChIP assays were performed as previously described with minor modifications (24). Normally, cells were fixed with 0.4% (v/v) formaldehyde at room temperature for 10 min. To improve the ChIP efficiency, double fixation was used. For double fixation with EGS (Thermo, cat. no ) and formaldehyde, cells were fixed initially with 1.5 mm EGS at room temperature for 30 min, and subsequently with 0.4% formaldehyde at room temperature for 10 min. For double fixation with DMA (Thermo, cat. no ) and formaldehyde, cells were fixed initially with 25 mm DMA at room temperature for 1 hour, and subsequently with 0.4% formaldehyde at room temperature for 10 min. For sonication, fixed cells were washed twice with PBS and resuspended in ice-cold RIPA-0.3 buffer (10 mm Tris-HCl, 1 mm EDTA, 1% Triton X-100, 0.1% SDS, 0.1% NaDOC, and 0.3 M NaCl, ph 7.4) supplemented with Protease Inhibitor Cocktail (Millipore, cat. no ) at a concentration of 40 million cells/ml; genomic DNA was disrupted to a size range of 100 to 500 bp. For immunoprecipitation, on day 1 antibodies were diluted in RIPA-0.3 and bound to Dynabeads protein A (Thermo, cat. no D) by incubating at 4 C for 3 hours. Afterwards, the bead-antibody complexes were washed twice with RIPA-0.3 and then incubated with sonicated chromatin at 4 C overnight. On day 2, after 2 washes with RIPA-0.5, 1 wash with RIPA-0.3, 1 wash with RIPA-0, 2 washes with LiCl buffer (10 mm Tris-HCl, 1 mm EDTA, 0.25 M LiCl, 0.25% NP-40, and 0.25% NaDOC, ph 7.4), and 2 washes with TE buffer, bound protein-dna complexes were resuspended in elution buffer (10 mm Tris-HCl, 1mM EDTA, 0.2 M NaCl, and 1% SDS, ph 7.4) supplemented 2

3 with 10 µg/ml RNase A for both elution and RNA digestion, and incubated at 55 C for 1 hour. Then Proteinase K was added to a final concentration of 200 µg/ml, and after 30 min incubation the temperature was increased to 65 C for crosslink reversal. After incubation for 4 to 6 hours, DNA was purified by ChIP DNA Clean & Concentrator (Zymo Research, cat. no. D5205). For qpcr, a site 2 kb downstream of the TSS of the NANOG gene was used as an internal control, and fold-enrichment was calculated by the 2 ΔCt method. The antibodies and the corresponding fixation conditions that were used for ChIP assays are listed in table S4. Real-time PCR primers for ChIP are listed in table S5. ChIP-seq libraries were constructed with 2 10 ng immunoprecipitated DNA. After endrepair, A-tailing, and barcode ligation, barcoded DNA was amplified by 16- to 18-cycle PCR. Libraries were sequenced on Illumina Genome Analyzer II, HiSeq 2000, or HiSeq 2500 by following the manufacturer's protocols. After decoding, raw reads were mapped to reference genome (hg19) using BWA through GALAXY. ChIP-seq analysis HOMER (25) was used to generate BedGraph files and for calculating peak genomic distribution. ChIP-enriched regions were identified using SICER (26), and plots of binding-onan-average-gene were generated as previously described (2). RNA interference plko.1 TRC control and mission shrna clones were purchased from Sigma Aldrich. For lentivirus production and transduction, 60-90% confluent 293T cells in antibiotic-free medium were transfected on day 1 with TRC control or gene-specific shrna clones along with packaging plasmids pspax2 and pmd2.g. On day 2 in the morning, medium containing transfection reagent was replaced by fresh medium containing 2% Penicillin-Streptomycin (Sigma Aldrich, cat. no. P0781). On day 3 in the afternoon, THP1 or CCRF-CEM cells were resuspended in virus-containing medium, and spun at 2,000 rpm at 20 C for 1 hour. After spin infection, virus-containing medium was removed and cells were resuspended in fresh medium and cultured overnight. On day 4 in the morning, cells were washed twice with PBS and 3

4 resuspended in fresh medium. On day 5 in the morning, puromycin was added to a final concentration of 2 µg/ml. Usually, cells were cultured for additional 72 hours before being harvested for qrt-pcr, Western blot, and ChIP. For Western blots and ChIP assays comparing the protein levels or chromatin occupancies of total Pol II and CTD serine-phosphorylated Pol II in control and knockdown cells, cells were cultured for additional 120 hours before being harvested. Mission shrna clones used in this study are listed in table S5. RNA extraction, reverse transcription, real-time PCR, and RNA-seq RNA was extracted from cells using RNeasy Plus Mini Kit (Qiagen, cat. no ) or Quick-RNA MiniPrep Kit (Zymo Research, cat. no. R1054) by following the manufacturer s protocol. For reverse transcription, single-strand cdna was synthesized using iscript cdna Synthesis Kit (Bio-Rad, cat. no ). For real time PCR, GAPDH was used as the internal control. Relative abundance of transcripts was calculated by the 2 ΔCt method. Primers for real time PCR are listed in table S5. Libraries of strand-specific RNA-seq were constructed as previously described (27). Co-immunoprecipitation (co-ip) and pull-down assay Co-IP experiments were performed as previously described with minor modification (24). On day 1, antibodies were incubated with Dynabeads protein A at 4 C for 3 hours and then crosslinked to beads by 25 mm DMA at room temperature for 1 hour. Nuclear extract (NE) from THP1 cells was diluted 2 to 3 fold by NE dilution buffer (31 mm Tris-HCl, 1.6 mm EDTA, and 0.16 % NP-40 (v/v), ph 7.5); 0.5 to 1 mg NE was used for each co-ip, and incubated with beadantibody complexes at 4 C overnight. On day 2 in the morning, bead-antibody-protein complexes were washed with BC150 buffer (10 mm Tris-HCl, 0.2 mm EDTA, 150 mm KCl, 20% glycerol, and 0.1% NP-40, ph 7.9) for 4 times. Afterwards, protein complexes were eluted from beads by 50 mm glycine (ph 2.4), immediately neutralized in 1 M Tris-HCl, ph 7.4, and analyzed by Western blot. Pol II was purified as previously described (28). For pull-down assays, around 10 pmol tagged bait protein was bound to anti-affinity tag antibody-coated magnetic beads by incubation 4

5 at 4 C for 2 hours. After 4 washes with BC300 and 2 washes with BC150, purified prey protein was added, and incubated at 4 C for 2 hours. After 4 washes with BC150, the protein complexes were eluted by boiling beads for 2 min in 1 X SDS-PAGE loading buffer and analyzed by Western blot. Antibodies used for Western blot and Co-IP experiments are listed in table S4. P-TEFb release assay P-TEFb release assays were performed as previously described (29). Briefly, LARP7 antibody (Bethyl, A A) was used to immunoprecipitate 7SK snrnp complex. The 7SK snrnp complex was incubated with 4 µg RNase A (Life Technologies, cat. no. AM2270) or increasing amounts of purified PAF1C at 37 C for 15 min. After washing, CDK9 that remained on beads was analyzed by Western blot. 5

6 Supplementary Text Author Contributions M.Y. and R.G.R. designed the experiments. M.Y. performed the experiments and analyzed the data. W.Y., M.Y., T. Ni, and J.Z. performed the bioinformatics analyses. Z.T. purified PAF1C, and T. Nakadai purified Pol II. M.Y. and R.G.R wrote the paper. 6

7 Fig. S1. PAF1C occupancy positively correlates with gene expression level. (A) to (C) Metagene analyses showing positive correlation between the occupancies of CDC73 (A), LEO1 (B), and CTR9 (C) and gene mrna levels (High, Medium, Low, Silent). (D) to (F) IGV browser snapshots comparing the occupancy of the PAF1, CDC73, LEO1, and CTR9 subunits of PAF1C, total Pol II, and CTD serine 2-phosphorylated Pol II (ser-2p) within the RUNX1 (D), RUNX2 (E), and MEF2C (F) loci in THP1 cells. 7

8 Fig. S2. PAF1 knockdown increases promoter-proximal pausing of Pol II in THP1 cells. (A) and (B) IGV browser snapshots comparing the occupancies of PAF1, total Pol II, and CTD serine 5- phosphorylated Pol II (ser-5p) within the c-myc (A) and the CTNNB1 (B) loci in control and PAF1 knockdown THP1 cells. 8

9 Fig. S3 PAF1C is a critical regulator of promoter-proximal pausing release of Pol II in human cells. (A) Comparison of PAF1 levels in control and PAF1 knockdown CCRF-CEM cells by Western blot. PAF1 shrna #2 was used for the knockdown, and β-actin was used as a loading control. (B) and (C) Comparison of the occupancies of PAF1 (B) and Pol II (C) on an average gene in control and PAF1 knockdown CCRF-CEM cells. (D) and (E) IGV browser snapshots comparing the occupancies of PAF1 and Pol II within the RUNX1 (D) and the BCL11A (E) loci in control and PAF1 knockdown CCRF-CEM cells. (F) and (G) Comparison of the occupancies of PAF1 (A) and Pol II (B) within the c-myc locus in control and PAF1 knockdown CCRF-CEM cells. Error bars indicate mean ± SD (N=3). 9

10 Fig. S4 Flavopiridol treatment increases promoter-proximal pausing of Pol II but decreases the chromatin occupancy of LEO1 in THP1 cells. (A) and (B) IGV browser snapshots comparing the occupancies of Pol II and LEO1 within the c-myc (A) and the c-myb (B) loci in DMSOand flavopiridol-treated THP1 cells. 10

11 Fig. S5 Knockdown of the SPT5 subunit of DSIF reduces the occupancy of both Pol II and PAF1. (A) Comparison of the levels of SPT5 in control and SPT5 knockdown THP1 cells. Two shrnas were able to efficiently reduce the level of SPT5. β-actin was used as a loading control. (B) to (D) ChIP-qPCR data comparing the occupancies of SPT5 (B), Pol II (C), and PAF1 (D) within the c-myc locus in control and SPT5 knockdown THP1 cells. Error bars indicate mean ± SD (N=3). 11

12 Fig. S6 CDK9 knockdown preferentially reduces the CDK9 fraction in 7SK snrnp. (A) Comparison of Pol II occupancies on an average gene in control and CDK9 knockdown THP1 cells. (B) CDK9 knockdown preferentially reduces the CDK9 fraction in 7SK snrnp. 12

13 Fig. S7 PAF1C does not effect the release of P-TEFb from 7SK snrnp. PAF1C is incapable of extracting P-TEFb from 7SK snrnp in release assays. RNase A was used as a positive control of P-TEFb release. 13

14 Fig. S8 PAF1 knockdown reduces Pol II CTD serine-2 phosphorylation but increases Pol II CTD serine-5 phosphorylation. (A) Comparison of the levels of PAF1, Cyclin K, CDK9, total Pol II, Pol II (ser-5p), and Pol II (ser-2p) in control, PAF1 knockdown, CDK9 knockdown, and Cyclin K knockdown THP1 cells by Western blot. β-actin was used as a loading control. (B) and (C) ChIP-qPCR data comparing the occupancies of Pol II (ser-5p) (B) and Pol II (ser-2p) (C) within the c-myc and the CTNNB1 loci in control and PAF1 knockdown THP1 cells. Error bars indicate mean ± SD (N=3). 14

15 Fig. S9 PAF1 knockdown reduces the chromatin occupancy of CDK12 but not that of BRD4. (A) and (B) ChIP-qPCR data comparing of the occupancies of CDK12 (A) and BRD4 (B) within the c-myc locus in control and PAF1 knockdown THP1 cells. Error bars indicate mean ± SD (N=3). 15

16 Fig. S10 Cyclin K knockdown does not affect the chromatin occupancy of PAF1C. (A) Comparison of Cyclin K levels in control and Cyclin K knockdown THP1 cells by Western blot. (B) to (D) ChIP-qPCR data comparing the occupancies of CDK12 (B), PAF1 (C) and Pol II (D) within the c-myc locus in control and Cyclin K knockdown THP1 cells. Error bars indicate mean ± SD (N=3). 16

17 Fig. S11 Model for PAF1C function in the release and phosphorylation of promoter-proximal paused Pol II. The model emphasizes (i) the previously established (1) role of DSIF and NELF in the establishment of Pol II (CTD ser-5p) pausing and the role of P-TEFb in paused Pol II release through phosphorylation of NELF (leading to its dissociation), DSIF (leading to its conversion to a positive regulator and the creation of PAF1C binding sites on the SPT5 subunit) and Pol II CTD (ser-2), (ii) the herein established role of PAF1C as a positive regulator of pausing release, with PAF1C recruitment and Pol II association facilitated by direct interaction with promoter-associated P-TEFb, and (iii) the herein established recruitment of CDK12/CycK to the elongating Pol II-PAF1C complex, through a direct PAF1C CDK12/CycK interaction, for consequent downstream Pol II CTD (ser-2) phosphorylation. Not shown are factors implicated in P-TEFb recruitment and PAF1C interactions implicated in histone modifications and transcription-coupled RNA processing events (6). Also, as discussed in the text, recent and current studies have documented both positive and negative functions for PAF1C that are dependent on the genetic background and physiological states of the cells. The asterisks in the figure represent phosphorylation events and the short solid black bars indicate direct interactions that are newly described in the current study. SEC refers to the super elongation complex comprised of P-TEFb (CDK9+CycT), AF9, AFF1/AFF4, and ELL 17

18 Table S1. Lists of direct target genes of PAF1 in THP1 cells 18

19 table S1. Direct taget genes of PAF1 Downregulated (FC >2) #GeneName FC(WT/KD) P- value PDR 6- Sep E AARS E E- 06 ACAT E E- 05 ACOT E ACOT E E- 05 ADCK E E- 05 ADM E E- 05 AES E AIG E E- 07 AKR7A E E- 05 ALKBH E ANKLE E E- 07 ARL6IP E E- 08 ARMC E ASF1B E E- 05 ATF E E- 14 ATIC E E- 05 ATP8B E E- 05 AXL E B3GNT E E- 06 B4GALT E E- 08 BAG E BATF E BATF E E- 06 BDH E E- 07 BEND E BRCA E E- 05 BRIP E E- 06 BTBD E E- 06 C12orf E C14orf E E- 06 C14orf E C16orf E E- 07 C16orf E E- 05 C19orf E C19orf E E- 05 C1orf E E- 08 C1orf E

20 C1orf E E- 08 C20orf E E- 06 C20orf E C21orf E E- 05 C5orf E E- 06 C5orf E C6orf E E- 10 C6orf E C9orf E C9orf E E- 06 CA E E- 05 CACNA2D E E- 06 CAMK2B E E- 06 CAMKV E CBR E CBS E E- 09 CCDC E E- 11 CCDC E CCDC28B E CCDC E E- 07 CCDC E CCDC90B E E- 05 CD E CD E E- 06 CDC42EP E E- 07 CDC E E- 07 CDK E E- 05 CDT E E- 06 CENPH E E- 06 CENPM E E- 05 CHAF1A E E- 05 CHAF1B E E- 06 CHCHD E CHST E E- 05 CHSY E E- 05 CKAP E E- 09 CKS1B E E- 05 CLEC3B E CNTLN E E- 05 COMTD E CREB3L E E- 05 CRELD E E

21 CRIP E E- 06 CRISPLD E E- 11 CRNDE E CSPG E E- 09 CSPG E E- 06 CTSG E E- 07 CYB5R E E- 16 CYB5R E DACH E E- 05 DDB E E- 05 DDIT E E- 05 DFNA E DHRS E DLEU E DLEU E DNAH E E- 06 DPH E DTYMK E E- 06 DUT E E2F E EAF E EIF4EBP E E- 05 EME E EMILIN E E- 06 EML E E- 05 EP400NL E E- 05 ETFB E E- 05 ETV E E- 12 EXOSC E E- 06 FADS E E- 16 FAM116B E E- 05 FAM151A E FAM189B E E- 06 FAM54A E E- 06 FAM69B E E- 09 FAM82B E E- 05 FAM92A E E- 05 FANCG E FARSB E FBF E E- 06 FBXO E E- 05 FBXO E

22 FDPS E FKBP E E- 08 FNTB E E- 06 G6PC E E- 08 GALE E GALK E E- 07 GAMT E E- 07 GBAP E E- 06 GCAT E E- 06 GCHFR E GDF E E- 13 GGCT E E- 05 GHDC E GINS E GINS E E- 06 GLYCTK E GMNN E E- 05 GNA E E- 05 GNG E E- 06 GPR E E- 09 GPR E GPR E GPT E E- 11 GSTZ E E- 06 GTPBP E HAAO E HAGHL E E- 05 HIP1R E HIST1H4E E E- 06 HMBS E E- 08 HMMR E E- 05 HSH2D E E- 07 IFRD E E- 05 IGFBP E E- 05 IL21R E IMPA E E- 05 ING E INTS E E- 08 ITGB E E- 05 KAT2A E E- 11 KCNQ E KCNQ E E

23 KHK E E- 05 KREMEN E LAMP E E- 07 LAT E LETMD E LOC E LOC E E- 06 LOC E LOC E E- 05 LOC E LOC E E- 07 LONP E LRRC E E- 07 LRRC E E- 07 LRRC E E- 07 LRRCC E E- 06 LTBP E LTK E LYPLAL E E- 05 MACROD E E- 07 MANF E E- 06 MAP4K E E- 05 MAPK E MCM E E- 06 MCM E E- 06 MCM E MCM E E- 05 MEI E E- 11 MELK E METTL7B E MGAT4B E E- 05 MGC E E- 05 MGST E E- 05 MLH E MLST E E- 05 MMAB E E- 05 MND E E- 07 MPDU E MRM E E- 07 MRPL E E- 06 MRPL E E- 05 MS4A6A E E

24 MTHFD E E- 06 MYBL E E- 07 MYBPH E E- 07 MYH E E- 05 N6AMT E NAPSB E E- 07 NCF E E- 10 NCS E E- 06 NDUFA E E- 09 NFU E NIT E E- 06 NPM E E- 05 NPRL E E- 09 NUDT E E- 05 OBFC2B E E- 05 OIP E E- 06 OSGEPL E PACSIN E E- 06 PAF E E- 63 PAFAH1B E E- 08 PAQR E PCCB E E- 09 PCDH E PCDH E E- 05 PCK E E- 06 PHF E E- 08 PHF E E- 05 PHGDH E E- 05 PIH1D E PKMYT E E- 05 PLAC E E- 07 POLA E E- 15 POLE E E- 05 PPIB E E- 05 PPP1R14B E PRDX E PRPS E PRR E E- 08 PRTN E E- 05 PSAT E E- 05 PSPH E E- 05 PTPRF E E

25 PYCR E E- 05 R3HDM E E- 05 RAC E E- 13 RAD23A E E- 05 RAD E E- 05 RAD54L E E- 12 RANGRF E E- 05 RFC E E- 05 RHBDL E E- 05 RIBC E E- 07 RIPK E RLTPR E RNASEH2B E ROBO E E- 05 RPA E E- 07 RPL26L E RPS E E- 07 RRP7A E RTKN E RUVBL E SARS E E- 11 SERPINB E SHF E SIRT E E- 05 SLC17A E SLC24A E SLC25A E E- 05 SLC2A4RG E SLC37A E SLC39A E SMC1B E E- 05 SNHG E SORBS E E- 06 SPNS E E- 08 SPTB E E- 08 ST3GAL E STRA E STYXL E TCF E E- 09 TEC E E- 05 TERT E E- 07 TFB1M E

26 TGM E TIFAB E TIMM E E- 06 TIPIN E TLCD E TMBIM E TMEM194A E E- 05 TMEM38B E TOE E TRAP E TRIM E E- 05 TRPT E E- 07 TYSND E E- 05 UBE2T E E- 06 UXT E E- 05 VEGFB E E- 08 VILL E E- 08 WDR E YEATS E ZCWPW E E- 08 ZNF E ZNF E E- 05 ZNF E ZNF E ZNF E ZSWIM E

27 Upregulated (FC >2) #GeneName FC(WT/KD) P- value PDR ABCA E E- 05 ABCA E E- 07 ABL E ABTB E E- 07 ABTB E E- 15 ADAM E E- 09 ADAMTS E ADAMTS E E- 11 ADRB E E- 05 AFF E E- 06 AHNAK E E- 08 AHNAK E E- 13 AKAP E ALDH8A E E- 06 ANKRD36BP E ANPEP E AOC E E- 14 AOC E E- 09 AP4S E E- 06 APC E E- 05 APOL E ARHGAP E ARID3B E E- 05 ARID3C E E- 09 ARID4A E ARID4B E ARRDC E E- 09 ATG E ATP6V0A E ATP7A E E- 07 ATXN1L E AVPR E E- 05 B3GNT E E- 08 B3GNT E BAIAP E E- 07 BCAS E E- 05 BCL2L E E- 10 BCL E E- 05 BCL9L E E

28 BMF E E- 16 BTG E E- 33 C10orf E E- 05 C10orf E C12orf E E- 05 C12orf E E- 05 C17orf E C1orf E E- 05 C21orf E C3orf E E- 07 C3orf E E- 07 C5orf E E- 06 C8orf E E- 05 C9orf E CA E E- 05 CCDC144NL E E- 07 CCNG E E- 05 CCR E E- 06 CD E CDC42BPB E CDC42EP E E- 06 CDKN1A E E- 11 CEBPD E E- 06 CEBPE E E- 05 CELSR E CITED E E- 05 CLDN E E- 08 CNTNAP E E- 06 CPD E CRB E E- 05 CREG E E- 07 CSF2RB E CSGALNACT E CSRNP E E- 05 CTSB E E- 05 CX3CR E CYP1B E E- 10 DAAM E E- 06 DCUN1D E E- 07 DICER E E- 09 DLL E E- 16 DMPK E

29 DNAJB E E E- 07 DUSP E E- 08 DYNLL E E- 07 EGR E E- 05 EHD E E- 05 ENC E E- 10 ENO E EOMES E E- 10 EPHB E EPM2AIP E E- 06 EVI2B E E- 09 EVI E F11R E E- 06 FAM20C E FAM43A E E- 06 FAM53C E E- 06 FBXL E FBXO E FLJ E E- 06 FLNB E FLT E FMNL E E- 05 FNDC3A E E- 06 FOS E E- 07 FOSL E E- 08 FOXO E E- 09 FOXO E E- 09 FRS E E- 05 FSCN E E- 05 FTH E E- 06 FUT E E- 06 GAB E E- 06 GAL3ST E GALNT E E- 05 GGN E E- 05 GJC E E- 12 GLS E E- 05 GNAT E E- 14 GP1BB E GPX E E- 06 GRIN3B E E- 05 H1F E E

30 HBEGF E HIC E HIPK E E- 05 HIST1H2AC E E- 10 HIST1H2AG E E- 05 HIST1H2BD E E- 06 HIST2H2BE E E- 07 HIST2H2BF E E- 08 HOXA E E- 05 HRH E IER E IER5L E E- 09 IFI E E- 05 IL10RA E E- 08 IL6ST E E- 07 IQCD E E- 05 ITGA E E- 06 ITGAV E E- 05 ITGAX E E- 07 JMY E JUN E E- 06 KBTBD E E- 06 KCND E E- 13 KCNG E KDM5B E E- 05 KIAA E E- 05 KIAA E E- 05 KIAA E E- 06 KLF E E- 05 KLF E E- 06 KLF E E- 14 KLF E KLHL E KLHL E KLHL E E- 05 LAMP E LFNG E E- 05 LGMN E E- 05 LMTK E LOC E E- 05 LOC E LOC E E

31 LOC E E- 05 LOC E E- 08 LOC E LOC E LONRF E LSP E LUZP E LYN E E- 05 MAML E E- 08 MAP1A E E- 07 MAP1LC3A E E- 06 MAP3K E MCL E E- 05 MGEA E E- 05 MICALL E MKNK E E- 05 MMP E E- 08 MT2A E E- 12 MTSS E MYO1E E E- 05 MYSM E NCOA E NCOA E E- 09 NEAT E E- 07 NFAT E NOTCH E E- 06 NOXO E E- 07 OLIG E E- 16 OR2B E E- 05 OTP E OVGP E E- 06 PALLD E E- 05 PAQR E PAQR E E- 07 PBXIP E E- 05 PDE7B E E- 05 PGAM E E- 11 PGAP E PHLDA E E- 06 PHLDA E E- 06 PHTF E E- 05 PIGW E E

32 PLAU E E- 05 PLEKHO E E- 09 PLIN E E- 05 PNRC E E- 05 PPP1R3F E E- 06 PRDM E PRDM E E- 05 PRIC E E- 10 PRICKLE E PSPN E E- 08 PTAFR E PTGDS E E- 08 PURB E E- 05 RAB11FIP E E- 10 RAB3D E E- 05 RBM E E- 06 RC3H E RGAG E E- 05 RGL E RHOB E E- 16 RNF E E- 05 RNF E E- 05 SASH E E- 07 SEC23A E E- 08 SECISBP2L E E- 06 SERPINI E E- 09 SERTAD E E- 15 SESN E E- 09 SETD E SGK E E- 06 SH2B E E- 06 SH3BP E E- 07 SKIL E SLC12A E E- 06 SLC30A E E- 05 SLC34A E E- 05 SLC37A E SLC4A E SOCS E E- 12 SPOPL E SPRED3 9.65E E E- 06 SPSB E E

33 SPTBN E E- 10 SPTY2D E SSBP E SSPO E E- 06 STAB E STAT E E- 06 STK17B E E- 05 SYNPO E E- 05 SYT E E- 05 TAGAP E E- 05 TAS1R E TBC1D E E- 05 TBC1D E E- 05 TCHH E TCTE E E- 05 TEP E E- 06 TG E E- 07 TGFBR E E- 10 THBD E E- 05 THBS E THSD E E- 06 TLR E E- 06 TMCO E TNFRSF10B E E- 05 TNFRSF10D E E- 05 TNFRSF1B E E- 05 TNFSF13B E E- 05 TNFSF E E- 11 TNRC6A E TOB E E- 07 TOB E E- 07 TP53I E TP53INP E E- 13 TPP E E- 05 TREML E TRERF E E- 06 TRHDE E E- 05 TRIB E E- 11 TRIM E E- 05 TSPYL E TSSK E E- 06 TTC E

34 TULP E E- 06 TWF E E- 06 TXNIP E E- 05 UBASH3B E E- 06 UBFD E E- 06 UBXN E UHMK E E- 06 VAMP E VASN E E- 06 VSIG E E- 37 WASL E WDR E E- 05 WDR E WNK E E- 06 YOD E E- 05 YPEL E E- 06 ZAR E E- 07 ZBED E E- 06 ZC3HAV E ZFAND E E- 05 ZFP E ZFP36L E E- 15 ZFYVE E E- 05 ZNF E E- 05 ZNFX E E- 05 ZSWIM E

35 Downregulated (FC >1.5) #GeneName FC(WT/KD) P- value PDR 5- Sep E Sep E AAAS AARS E E- 06 AARSD ABCB ACAT ACAT E E- 05 ACBD ACCS ACD ACOT E ACOT ACOT E E- 05 ADCK E E- 05 ADM E E- 05 ADSL AES E AHCY E AIG E E- 07 AIMP E AKR7A E E- 05 ALDH7A ALKBH E ANKLE E E- 07 ANKRD APRT E ARHGAP ARHGAP ARHGEF ARHGEF ARL6IP E E- 08 ARMC ARMC E ARNTL ARPC ARRDC ASF1A E ASF1B E E

36 ASPHD E ASS ATAD3A ATF E E- 14 ATF E ATIC E E- 05 ATP5G ATP5I ATP8B E E- 05 AXL E B3GNT E E- 06 B3GNT B3GNTL B4GALT E E- 08 BAD BAG E BAI BANF BATF E BATF E E- 06 BCAT BDH E E- 07 BEND E BLM BLVRA E BRCA E E- 05 BRIP E E- 06 BTBD E E- 06 C12orf E C12orf C14orf E E- 06 C14orf E C14orf E C16orf E C16orf C16orf E C16orf E E- 07 C16orf E E- 05 C17orf C19orf C19orf E C19orf

37 C19orf E E- 05 C1orf E E- 08 C1orf E C1orf E E- 08 C1orf E C20orf E E- 06 C20orf E C20orf E C21orf E E- 05 C2orf C5orf C5orf E E- 06 C5orf C5orf C5orf E C6orf C6orf E E- 10 C6orf C6orf E C6orf C7orf C9orf E C9orf E C9orf C9orf C9orf E E- 06 C9orf CA E E- 05 CA CACNA2D E E- 06 CALR E CAMK2B E E- 06 CAMKV E CARD CARHSP CATSPER CBR E CBS E E- 09 CCDC E E- 11 CCDC E CCDC CCDC163P E

38 CCDC28B E CCDC E E- 07 CCDC E CCDC90B E E- 05 CCNB CCNE E CD CD E CD E E- 06 CD CDC42EP E E- 07 CDC E E- 07 CDC E CDCA E CDCA7L E CDK CDK E CDK E E- 05 CDK5RAP CDT E E- 06 CENPH E E- 06 CENPM E E- 05 CENPP CENPW CETN E CHAF1A E E- 05 CHAF1B E E- 06 CHCHD CHCHD CHCHD E CHST E E- 05 CHSY E E- 05 CIB CISD CKAP E E- 09 CKS1B E E- 05 CLEC3B E CLN E CMC CNTLN E E- 05 CNTROB E COMTD E

39 COPZ COQ COQ COX7A E CPNE CPSF CREB3L E E- 05 CRELD E E- 05 CRIP E E- 06 CRISPLD E E- 11 CRNDE E CSPG E E- 09 CSPG E E- 06 CTNNAL E CTSG E E- 07 CUL CXCR CYB5R E E- 16 CYB5R E CYP4V D2HGDH DACH E E- 05 DCLRE1B DCPS E DCTPP DDB E E- 05 DDIT E E- 05 DDX DEAF DERL DFNA E DHRS DHRS DHRS E DLEU E DLEU E DNAH E E- 06 DOK DPH E DSCC E DSN DTL

40 DTYMK E E- 06 DUS3L DUT E E2F E2F E EAF E EIF4EBP E E- 05 EME E EMG EMILIN E E- 06 EML E E- 05 EML E ENOPH ENOSF E EP400NL E E- 05 EPS8L ERCC ERMAP ETFB E E- 05 ETNK ETV E E- 12 EXOSC E E- 06 FADS E FADS E E- 16 FAF FAHD2A E FAM116B E E- 05 FAM151A E FAM162A FAM163A FAM189B E E- 06 FAM192A FAM54A E E- 06 FAM69B E E- 09 FAM82B E E- 05 FAM92A E E- 05 FANCD FANCG E FANCI E FARSA E FARSB E FBF E E

41 FBXO E E- 05 FBXO E FCHO E FDPS E FEN FH FHOD FKBP E E- 08 FNTB E E- 06 FUT E G6PC E E- 08 GALE E GALK E E- 07 GAMT E E- 07 GAS E GBAP E E- 06 GCAT E E- 06 GCHFR E GDF E E- 13 GGCT E E- 05 GHDC E GINS E GINS GINS E E- 06 GLDC GLTPD GLYCTK E GMNN E E- 05 GMPS GNA E E- 05 GNAL GNG E E- 06 GPM6B E GPR E E- 09 GPR E GPR GPR E GPSM GPT E E- 11 GRIP GSS GSTZ E E

42 GTPBP E GUF HAAO E HADH HADHA HAGHL E E- 05 HDDC E HHLA HINT HIP1R E HIRIP E HIST1H4A HIST1H4E E E- 06 HIST2H2AC HK HMBS E E- 08 HMMR E E- 05 HSD17B HSF2BP HSH2D E E- 07 HSPB E HSPE IAH IFRD E E- 05 IGFBP E E- 05 IL21R E IMPA E E- 05 IMPDH E ING ING E INTS E E- 08 INTS E INTS INVS IRAK ISOC E ITGB3BP ITGB E E- 05 ITPR KAT2A E E- 11 KATNAL KCNK