Nucleic Acids Research, 2020 1 doi: 10.1093/nar/gkaa666

SURVEY AND SUMMARY

Tumor suppressor p53: from engaging DNA to target Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 gene regulation Morgan A. Sammons 1,*,†, Thuy-Ai T. Nguyen 2,*,†, Simon S. McDade 3,*,† and Martin Fischer 4,*,†

1Department of Biological Sciences and The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, NY 12222, USA, 2Genome Integrity & Structural Biology Laboratory and Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences/National Institutes of Health, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA, 3Patrick G Johnston Centre for Cancer Research, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7AE, UK and 4Computational Biology Group, Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstraße 11, 07745 Jena, Germany

Received May 24, 2020; Revised July 24, 2020; Editorial Decision July 28, 2020; Accepted July 30, 2020

ABSTRACT INTRODUCTION The p53 transcription factor confers its potent tumor The TP53 gene encoding the tumor suppressor p53 is the suppressor functions primarily through the regula- most frequently mutated gene in human cancers (1,2). p53 tion of a large network of target genes. The recent ex- is also deactivated or repressed in a large proportion of tu- plosion of next generation sequencing protocols has mors containing wild-type p53 through diverse mechanisms enabled the study of the p53 gene regulatory network including aberrant degradation, deregulation of activators, effectors, or repressors such that most, if not all, cancers cir- (GRN) and underlying mechanisms at an unprece- cumvent the p53 signaling pathway. p53 is the eponymous dented depth and scale, helping us to understand member of the p53 transcription factor (TF) family that precisely how p53 controls gene regulation. Here, we evolved from an ancestral p63/p73 gene that can be found in discuss our current understanding of where and how most invertebrates (3,4). Functionally the ancestral p63/p73 p53 binds to DNA and chromatin, its pioneer-like role, gene protects organismal integrity and the germ line by in- and how this affects gene regulation. We provide an ducing cell death in cells with genome damage. In higher overview of the p53 GRN and the direct and indirect vertebrates all three p53 family members can be found and mechanisms through which p53 affects gene regula- their function has diversified. While p53 largely functions as tion. In particular, we focus on delineating the ubiqui- a tumor suppressor, p63 and p73 play important develop- tous and cell type-specific network of regulatory ele- mental roles in addition to context-dependent tumor sup- ments that p53 engages; reviewing our understand- pressive and tumor promoting activities. p53 functions as a sequence-specific TF, which is acti- ing of how, where, and when p53 binds to DNA and vated in normal cells in response to a diverse range of stress- the mechanisms through which these events regu- induced stimuli, in particular DNA damage, to regulate a late transcription. Finally, we discuss the evolution large network of target genes through which it exerts the of the p53 GRN and how recent work has revealed re- majority of its tumour suppressive functions (5,6). The tran- markable differences between vertebrates, which are scriptional output of the complex p53 gene regulatory net- of particular importance to cancer researchers using work determines whether cells pause until stress/damage is mouse models. resolved or repaired, terminally arrest, or die. Consistent with its tumor suppressive TF function, cancers most fre-

*To whom correspondence should be addressed. Tel: +49 3641 656876; Fax: +49 3641 656255; Email: [email protected] Correspondence may also be addressed to Thuy-Ai T. Nguyen. Email: [email protected] Correspondence may also be addressed to Simon S. McDade. Email: [email protected] Correspondence may also be addressed to Morgan A. Sammons. Email: [email protected] †The authors wish it to be known that, in their opinion, all authors should be regarded as Joint First Authors.

C The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Nucleic Acids Research, 2020 quently harbor mutation in the region of TP53 that encodes p53 functions mostly as an activator of transcription recon- the DNA binding domain (DBD) (7). ciling many years of research and debate. In the following paragraphs we discuss what we know The largest fraction of genes indirectly regulated by p53 about p53 binding to DNA and chromatin and how this is represented by cell cycle genes, which are repressed in relates to gene regulation. Particularly, we focus on recent response to p53 activation. These genes are largely reg- advances in our understanding of how and where p53 binds ulated through the p53–p21–DREAM/RB pathway (16– to DNA and the mechanisms through which these events 19), which is highly conserved between mouse and hu- Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 regulate transcription. man (20) and may control up to one thousand cell cy- cle genes (8). This has been complemented through studies that challenged multiple trans-repression mechanisms pro- THE p53 GENE REGULATORY NETWORK––A UNI- posed for p53 and rectified how p53 regulates several genes VERSE OF TARGETS (13,14,16,21,22). Additional mechanisms of indirect gene regulation through p53 include the direct activation of non- A TF gene regulatory network (GRN) comprises all genes coding RNAs such as micro RNAs. This includes mir-34a regulated by a given TF: through binding directly to its cog- that is a tumor suppressor in its own right (23), as well as nate gene regulatory DNA elements or indirectly through several long non-coding RNAs (24). Moreover, p53 activa- the effects of its direct targets on downstream signaling tion directly affects the mRNA levels of many other TFs pathways and transitional TFs. Importantly, myriad stud- and their regulators, including ATF3, BCL6, BHLHE40, ies over the last decade have demonstrated that the major- E2F7, HES1, GRHL3, SMAD3, STAT3, TEAD3, and ity of TFs, including p53 and its family members p63/p73, YAP1, which could cumulatively contribute to the p53- can affect their GRNs through binding to both proximal dependent regulation of a large number of genes (8). Such promoters or distal enhancers, the latter of which are con- indirect effects are particularly difficult to decipher, and nected to the respective gene by long-range interactions. the mechanisms that mediate p53-dependent expression of As described further below, recent meta-analyses of high- many genes remain to be uncovered (Figure 1). throughput datasets generated over the last decade sug- Our current understanding of gene regulation by p53 gest that p53 predominantly activates expression of di- binding events has been biased by a logical focus on proxi- rect targets through binding to proximal promoter regions, mal promoter binding events, since assigning longer-range and have begun to define the broader p53 binding land- regulatory events presents significant technical challenges. scape and disentangle the direct and indirect regulatory Hence, the information content required to recruit p53 to events within the p53 GRN. Surprisingly, integrating mul- DNA and to mediate p53-dependent gene regulation re- tiple gene expression and chromatin-immunoprecipitation mains elusive. For example, in silico motif searches in the (ChIP) datasets revealed that only ∼11% of the >3000 mouse and the human genome, using position weight ma- genes whose mRNAs were consistently altered in response trices without spacers, identified 124 313 and 98 553 po- to p53 activation exhibit reproducible p53 binding prox- tential canonical p53REs respectively (20). However, less imal to their transcriptional start sites (TSS) (thus pre- than 8% of these sites are found to recruit p53 across dis- dicting them as direct p53 targets) (6,8). Two websites parate datasets, with an even smaller subset of those p53- enable researchers to quickly query their gene of in- bound sites to reproducibly confer direct regulation of the terest for information on its p53-dependent regulation nearest gene. While a large proportion of direct p53-induced and p53 binding near the gene locus (www.targetgenereg. target genes are regulated through p53 binding to proxi- org;(8)) and https://www.niehs.nih.gov/research/resources/ mal p53REs, we are beginning to understand that expres- databases/p53/index.cfm;(6)). Notably, while p53 regulates sion of a number of p53-regulated genes may be directly in- a substantial set of genes through gene proximal regions, it fluenced by distal p53RE binding events12 ( ,25–29). A re- binds to many more distal locations, the function of which cent genome-wide screen for distal p53 binding events that is harder to discern, and may regulate even more genes. influence cell proliferation and survival after doxorubicin Canonically, p53 is recruited through a DNA motif called treatment identified productive p53 binding events upto the p53 response element (p53RE) comprising two de- 250 kb from the nearest gene (30). Moreover, we are begin- cameric half-sites of the consensus sequence RRRCWW- ning to understand that p53 can bind to distinct genomic GYYY (R = A/G, W = A/T, Y = C/T). Productive loci in a cell type-specific manner as exemplified by a sub- p53 binding leading to transcriptional activation predom- set of squamous-specific p53REs bound by its sibling p63 inantly occurs at p53REs within 2.5 kb of the TSS of the (31–33), to which p53 can bind to only in squamous cells regulated gene, which also can be situated in the first in- (12). Notably, our current understanding likely underesti- tron (8,9). Recent integrative ChIP-seq studies suggested mates p53 interactions with difficult to map repetitive re- that proximal p53 binding events are predominantly associ- gions of the genome, an important consideration since at ated with target gene up-regulation in response to p53 acti- least a subset of p53REs are thought to have evolved from vation (10–13), which is strongly supported by recent meta- retroviral elements (34–38). analyses (6,8,14,15). Conversely, these studies also indicate that the vast majority of genes down-regulated in response THE UBIQUITOUS DNA BINDING LANDSCAPE OF to p53 activation are not associated with proximal p53 bind- p53 ing events indicating they are either regulated through indi- rect mechanisms or distal binding events. While this does The last decade of genome-scale studies has provided deep not preclude p53 exerting context-specific direct repressive insights into the genomic locations and contexts in which effects on some genes, it strongly supports a model wherein p53 binds to DNA. To date, at least 132 (Table 1) human Nucleic Acids Research, 2020 3

The p53 gene regulatory network: The links and nodes we think we know...

SUPT7L DDR1 PRKAB1 SULF2 VWCE NDUFV3 NCEH1 APAF1 UACA BLM HIST1H4A FBXW7 IGDCC4 PHPT1 CREBZF TSEN15 PPIH TMEM97 MNS1 TOE1 TAF5 AK2 HAUS7 HSPE1 SAAL1 TRUB1 CYFIP2 PPM1D SLC46A1 AMZ2 ETV7 RAD1 TTF2 ACP1PAFAH1B3 SIVA1RNF219 ARHGEF39KIFC1 HSPB11 RNASEH2A PAICSARRB2 SMC3GTSE1 SEMA3B C12orf45 SYTL1 ARHGAP42 SMAD3 COBLL1 TRAF4 CDCA7L PIDD1 E2F7 TCP1 MEIS2 BYSL ING1NSMCE4ASFR1 CCDC18NUP155 LIMA 1 RALGAPB NADSYN1 PCNA UCHL5CIRH1AMARCKSC5orf34 PLK4 POLQ DHFR HNRNPA3ASF1BTRIM37 TMEM39B UTP15 YAP1 POU3F1 ICOSLG SNX2 CLCA2 FAM98C TUBB LCORLCENPO SASS6SNRPB TRIM59HIST1H2AL RRM2B ENTHD2 GNAS BTG1 BLOC1S2 CCNG1 TM7SF3 CFLAR EIF3B PASK SIN3A NUCKS1TIMELESS LYRM7 ACTA2 GSS CUL3 DTYMK POC5CHRAC1 TTI1 TOPBP1CDR2 KNTC1 TDP1 FANCG PPAT NUDT1 CDC25A TXNDC12 SERPINE1 NKAIN4 RGS16 RFT1 H2AFZ POLA1HNRNPD CCDC15 RQCD1FANCD2 ASTN2 SOCS4 KIAA1217 TMEM64 TXNIP NUPR1 TP53INP1 NRGN CDIP1 CMBL PMAIP1 ARID2 CNOT1 LIN52 CDCA2 UQCC1 CYSRT1 ZNF561 GDF15 HIST1H2AC XRCC4ABHD2 MCM7CHEK2 SRSF3 MIS12 ANP32ENCAPG PIGW PBRM1 PPFIBP1 TRIB1 GADD45A KIF24 CENPAHIST1H3GCEP295ORC3HIST1H1D RNF2 KIF23 SHMT2 MXD3SYNCRIPNUDCD2 POLH SMARCA5 TNFRSF10A PLK3 HRAS SLC25A45 RAD51C DYRK3 TCAIM PLXNB2 BCL2L1 ANKRA2 GPATCH11 H2AFV PCNT FOXM1 KPNA2UBE2R2 SP1 HIST1H2AH HELLS POLE SNRPD1 LCE1E PML EDN2 PANK1 LBR PRKDC TCF19FAM161ACOQ7 RPA2 CISD2 DCK PRKAA1 pk CCNE1 HSP90AA1 WDR81 PARD6B AGBL5 KRT15 AEN CEP78 ZNF146 HIST1H3BHIST1H3C RHEBCNTROB KRT8 CD82 GRHL3 MAST4 SYNC TUBB6 ABCB6 BHLHE40 SLC12A4 HIST2H2BE ZNF714 TCTN2 KPNB1 TFAP4 MTHFD1 RAD54B AURKATBC1D31ACTL6ACDKAL1 CKS2 DCPS EBAG9METTL16 KIAA0101 F5 MFAP3L ALDH1A3 NCAPD3 PHF19 GEN1 WHSC1YEATS4MAD2L1TRA2B PIM1 SRSF10CKAP5 BRCA1MIS18A CBX3 IER3 SLC9A1 EFNB1 RGL1 ZMAT3 WDR46 TSKU TEAD3 MLF2 MCC CEP55 ERI1 KHK DIAPH3HMGXB4 NUP54 BRMS1L VCAN ZNF79 SCN2A GPX1 POLA2 CLN6 CHEK1TMPO MTBP ESPL1 FAM111ASLC3A2 RASSF1 FOXRED1 PALB2PPAN−P2RY1NXT11 MRPL3CTDSPL2 UBE2CCCNB2DSCR3 FBXO4 RDX RMI1 EFCAB10 DUSP14 BBS2 ZNF473 MASTL PHLDA3 NTPCR EPS8L2 ISCU CDH8 KHDRBS1 DDX20 POLD2 SMAD5 TRIM35 SAC3D1 BUB1 CAD CENPF ILF3 ZNF100LAS1L SPAG9 ANKRD32WDR62DYRK1AGATAD2A ATF3 FBN2 ORAI3 ZNF423 USP15 MAP3K12 SNRNP40 LSM3CARHSP1TMEM18 BARD1HJURP KIAA1715KIF20A STRA13 FAM76B AC004381.6 HIST1H2AEECT2 CEP152PYCRL

NINJ1 BCL6 EPM2AIP1 Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 KLHDC7A APOBEC3C TMEM68 KCTD1 CSF1 HAT1 LRRCC1 NT5C3A CDCA4 RAD51 MYC TARS ZWINTKIF22 HIST1H2AK SLC25A19SLC29A1KIF2C TEX30 XPC BBC3 SRA1 HMGN2 RACGAP1POLD1 ORC2 NCOA5 CDK2 SMC2C12orf65 FKBP5 EML2 MUC2 RAB1A LRP1 PTEN BCL10 WDR76 PTGES3 SHCBP1MCM2 ATAD2EIF3A TRIM22 LRPAP1 CPSF4 TIPIN FIGNL1 PADI4 HSPA4L FAM198B RETSAT EDA2R WBP11 BRCA2 FANCI PMF1 IFRD2 RECQL4 GRWD1L3MBTL2GTF3C2 CKS1B EXOSC9CDCA5KLHDC4 ATF2 ZNF184 CHAF1AFANCL NUPL1HCFC1 FAM178A CCNA2 IRF2BPL TANC1 PRKAB2 AARS RAD9A CCT5 CIT SRSF2SSRP1 C10orf32 PRKD2 TNFAIP8 NHLH2 KIAA0430 PTP4A1 DNA2 MDM1 GREB1 GCH1 LMNA DDX11 TMEM194A SMNDC1R3HDM1KAT7 PKP4 CLSPNDCLRE1BMUTYH MARCH8 ADCK3 DNAJB5 ZC3HAV1 CROT DARS2 MYO19RAD51AP1CENPMMRPS18BLSM4 CYP51A1CDKN2C THOC1 HMGB3 LIN54 HIST1H4ECWC27POLD3PRKRIRTROAP SKP2 HYLS1 ZNF385A CCDC90B GABARAPL2 C17orf89 MFGE8 SFN PRRG2 RABGGTA FBXO22 CEP192 BAZ1B SPATS2 NUP50TOMM40SPATA5 E2F1 TOP2A DNAJC9MCM10MYBL2 CPEB2 STAT3 TP53I3 SMC4 NASP AGPAT5 GGHHIST1H3JFZR1 KNSTRNC5orf24 GINS3 TRIP13 ALMS1 ZNF273 UTP3 DDB2 RFK SPATA18 ARAP1 NAA15 GGCT DTL RRM2 HMBSPARP2 NYNRIN CLP1 KCNN4 FCHSD2 SDPR IGFBP7 TP53 FCHO2 ISYNA1 HIST1H2BHCDK4 CMC2 RSRC1 ZNF138 SGOL1 CMSS1RNPS1 DBF4BSTAG1 ZNF85 JADE1ZNF367 ARHGAP11A SUZ12LRRC45 LIAS ZWILCH C2orf69 ZNF684 MON2 SLC30A1 TET2 EXOC4 FAM111B ZNF680SMARCC1DSN1 XRCC2 DLGAP5H2AFX RRAD SESN1 MDM2 EPHA2 CSNK1G1 DREAM RAD54L TSPAN11 GBE1 TMEM131 CEP72 PTTG1 DDIASNCAPG2 TGS1 ZNF37A POLR2H CHST14 COL7A1 NEFL DEPDC1 PRIM2GAS2L3 SMC6HIRIP3 C5orf30HS2ST1 MND1CDC25CTFDP1 SAP30 SLC38A1 NUP85 DDX21DCAF16CCDC77CKAP2TACC3 TAF1A HINT3 ALG10 MCM4SEC22CMARS DRAM1 LPXN KLHL12 LIN9 EID2B ZNF337 MICALL1 ZNF564 DHRS3 HEATR1HAUS5 VRK1 SFPQ KIF18B HMGB2 ASCC3 FDXR RNF144B MBD5 WDR4HIST1H4LSTK17BNEDD1 EWSR1 BCS1L AURKBCCDC150 HIST1H3D LIF RIN1 SARS ACER2 ITGA3 RB-E2F ELP5 TTLL4 APITD1MPHOSPHCEP579 GART DNMT1 ATAD5 SLBPCACYBP PFAS CENPC LMNB1 NIF3L1 AKAP9 RPS27L PARD6G CDKN1A OBSL1 SUSD6 IKBI P ZNF530 MCM8 USP37 LNPEP APOD TTF1 EXO1 CDKN2DREEP4RANGAP1PEX3 HIST1H1B EXOSC5GTPBP3C19orf40SPC24 TMEM243 C17orf82 MRPL49 AFMID IFT80TMEM138NUP205NUP107 E2F2 TUBD1 SNRPANUSAP1 ANAPC1 AK3 GPR87 HES1 DCP1B SESN2 MGRN1 CCHCR1 GPSM2CDKN3 PTBP1XRCC6BP1 GPC1 BTG2 PDE4C PHTF2 NEIL3 SLC1A5 ZNF219 FAS RPS19 METTL4 CDC25B EZH2 HLTFHIST1H2BIC4orf46 GABPB1 ATAD3ARNASEH2BVPRBP ACD FAM212B APOBEC3H SAPCD2 WDHD1MYBL1HIST1H2BE HSPD1 MAZ MMS22LMRTO4 SKA1 ARFGEF2 TLCD1 TMEM63B ZBTB14 RMI2 BORANUP43 KPNA3RAD18 CEL C12orf5 TRIM32 HHAT TRIP6 BAX GINS2 HIST1H2AJCAND1 RCBTB1 PPP1R3C CDC42SE1 RAD21 NAP1L1 CEP135 FUS CKLFRHNO1 ARL13B NFATC2IPRFWD3EHBP1 OIP5 SERPINB5 TNFRSF10D SCRIB HIST1H2AI SGOL2 DUT NEK2 DBP G2E3 KDM1AZNF207 POLR3K BLCAP FLRT2 CDC45 KIF15 HNRNPH1CDCA7 DDX39AHIST1H2BL PLK2 ABCA12 CNOT6 RNF19B STX6 IER5 PRDM1 PBLD POU2F2 PRPF4 PIGAHNRNPA2B1 KIAA1524 EME1 BRD8 C2orf43 HAUS3 PIF1 SLFN11 CWF19L1PGAP2 POLE3 DDX46 WDR63 TNFRSF10C ITPKC RFC2 WRAP53SUV39H1CENPE SCLT1HIST1H3H EXOSC2MZT1 BIRC5 RGMA REV3L RAP2B HEXIM1 FAM84B CENPH DDX10 AAASKHSRP FAR1CENPQ MRPL39 PSTPIP2 ADIRF BCL9L ATF7IP INIP DGKA EI24 GNAI1 SMEK1 TRIAP1 GPR19 C1orf112 UBR7RPL22L1HSPA9 HAUS6 CLIC4 NOLC1DNMT3B TMEM109 DPYSL2 RBL1TOR1AIP1MDC1 RIF1 CASC5HNRNPA0 PGP PIGK C14orf80 MSH3 ANXA4 SFXN5 SLC4A11 SLC25A40 IPO11 XRCC3SRSF7 NCAPD2C18orf54 BTBD10 FAM210B ZMYND19 TNFRSF10B TYMSOS S100A2 POLE2 NPAT MCM6 FEN1 NCAPHHIST1H2AB CENPN NUP98 TLR3 SERTAD1 ARHGAP19INTS7 CCNF PBKCASP8AP2STIL PSIP1 FKBP7 ZNF92FOPNL HMMR IQGAP3 STAT3 DHX15 ABHD10 PLK1 ASPM AHI1HIST2H3A SRRM1FKBPL SPC25 PTBP2 HNRNPAB CDCA3 THEM4 RFC5 ZNF107 SENP1 RFC4C17orf53PPP3R1 WRN RTTN TBRG4CENPWTWISTNBGRK6 STIP1 CEP83 SKA2HNRNPR CTCF UHRF1 TPX2 MCM3 HIST1H3FDCAF15 TTK KLHL23 BCL6 E2F7 THRAP3 BIRC6 MTF2 DSCC1 FN3KRP MIIP GTPBP2SNRNP25GMNN ZGRF1 AUNIPSLC16A1RNF26 NUP35 NUP93 PSRC1 POP7 NUFIP2 RDM1 LRR1 TUBA1CFANCE RCC1 GRHL3 MED30 DBF4 CCNB1 DEK HIST1H2BONUP160 TONSL ATF3 PRIM1 HIST1H4CZMYMHNRNPH3 1 SUV39H2CBX5 MCM5 CDK5RAP2HNRNPDLAHCTF1 IQCB1 BCLAF1PRR11NUP153 OPA1 USP1 HIST1H4ILDLR DHTKD1GNL3 FAM72D KIF14 YAP1 AZIN1 CEP97 BRIP1HAUS8 CCDC34PATL1 HES1 HADH ZNF724P BHLHE40 C1orf174 MIS18BP1 PBX3 SRSF1NDC80 NOC3LPLSCR1 STMN1 RAD23BTUBG1FAM64A AIM1 PRC1 ASXL1 SF3B3 CCSAP LSM5DEPDC1B GRPEL2EXOSC3 HDGF C9orf40 G3BP1XRCC1 ATF4CHTF18 FANCC MELK GINS4 HIST1H2BM FANCA RFC3 RRP9 TCERG1FAF1 NCBP1 FANCBSLC1A4RANBP1 NUF2 SMAD3 PKMYT1 CSE1L CDC6FAM83D KIF11 USP28 METTL2BINCENP COX20 HNRNPF SP4 CKAP2L NUP88HIST2H3D MRPL17 CASP2 BUB1B HMGB1 RAD51BTCOF1 RFC1 MKI67 MRE11ANCAPH2NUP62 TEAD3 ARL6IP1 DCLRE1AZRANB3HIST2H2ACMAGOHBDLEU1 MSH2 WDYHV1DGCR8 TCEA1 MTFR2 NF2 ZNF45IFRD1 EXOSC8ESCO2 FIGN GSG2 ANKRD17DOLPP1 NDC1HIST1H2BFPOC1A HAUS4MYADM TNPO3GPR137C AMD1MSH5−SAPCD1POLR1E USP39 NUDC ABCB10CENPJ RRM1 RMND1 BMPR1A HAUS2RAD23A EBP SETD8SMC1A SMCHD1HNRNPU RBBP4 ZNF695 PARPBPCHCHD3TOP3ASPAG5 KIF2A GSTCD ABCE1 GARS CDCA8 TK1 PRIMPOLTICRR SPDL1 ZNF551HNRNPA1 SUPT16HATAD3B CENPP ODF2 FBXO5 UBE2S CDT1 EMG1PDS5B ZW10 ORC1 DHX32 TBC1D8 SMPD4 UBE2T PRODH CCNK SERINC2 TMEM127 ISG15 ATP6V1A ZNF654 PAG1 NUAK2 PAPPA ITGB3BP GPR180 RTKN2ARL6IP6KNOP1 E2F8 CEP85 CENPL HAUS1 GINS1 MSH6 LONP1 CITED2 C16orf59 HIST1H4DCEP57L1ARHGAP11B OXNAD1WEE1 SMC5 BUB3 KIF18A MCF2L PGAP1 RDH10 STX5 F2RL2 ANLN CDC20ERCC6L RAB8ACENPU CYP4F11 POLR2L EIF2AK4 GOLGB1 DSC3 CTF1 ADAMTS13 MARK4 FOXO4 NCKAP5 ANKRD13B FANCMCCDC14 SAE1 C19orf48ACACA GPATCH4 MGST2 KCNK6 EGFR RIPK4 ABCA5 HPX WDR66 DIRC2 CDK1 BCL2L12HIST2H2ABCENPK NOP58 GPD2 HACD2 HBEGF RGS20 FAM13C CDH10 CTSL NAPEPLD TRAIP KIF4A BRIX1 LOC642846KIF20BTMEM209CDC7CDKN2AIPNLUNG PSMC3IP POU2F1 OCIAD2 HSD17B7 BTBD11 WRAP73 DOCK8 MYL5 TMX1 NET1 SKA3 ABCC3 BBS9 ADAMTS7 DHRS1 ERCC5 HMOX1 ULBP2 EBI3 PFKFB4 MYO15B CTSF NPEPL1 SPTSSA SPATA20 TSPAN2 MXD1 ZSWIM8 LAT ZNF33A EAF1 EMC3 ZDHHC9 STX12 SORBS1 ELL HSD17B14 ARHGAP22 NCKAP5L PLCD1 GM2A ZNF697 RELL2 AGTRAP UCN2 C9orf172 STAT1 ACP6 LCAT SMOX FUCA1 NTN4 GAD1 CD59 ADAP2 HSPA12A RHOC KRT17 CYP4F2 ARFGAP1 SLC48A1 GJD3 FADS3 ZNF616 DUSP13 TECPR2 MTFR1LARRDC2 C17orf103 FRMPD2 CTSK BBS12 FAM65C ARHGEF3 HSD17B7P2 MAN2B2 RALGDS C1orf226 DNHD1 DRAXIN LAPTM5 TSPAN1 FHL2 ZNF488 GPR108 CAV2 S100A11 EGR1 NEK8 EGFLAM UBR1 SLC9A3R1 ZNF750 MYO1E PLEKHG1 CCDC142 NQO1 AP1G2RC3H1 EIF2B2 AHSA2 ZNF433 CHCHD7 GALE ESPN VAMP8 CNFN SDHA TAP1 IL11 BMP7 SRGAP2 RASSF4 MSX2 SPAG1 NPR2 GRB10BCAP29SAP130 STX2 ILVBL AUTS2 FOXP1 HPS3TNFAIP8L1VLDL RZYG11A HOMEZ FERMT2VPS54 TXLNGZNF566TUBGCP3SH3PXD2CLASP2B SIK3 ENAH VEPH1 WDR5 BOP1 PSMG1 HSPB1 SIDT2 MBNL2 TMEM217 ELOVL4 TRIT1 RADIL EMC8 WDR45 ZNF746 NDUFA13 PITPNM1 PNRC1 SYTL2 ING4 DUSP4 PLAUSERINC5 SDSL ETNK1 HAS3 DOCK4 YPEL2 IFT20 MAPRE3 ITGA6 ACSF2 AK1 RBM38 LRP8 TRAPPC6A PLXNB1 PTPRU MBD4 FLT4DONSON NMT1 RIOK2KDELC1PPAP2C DHODH NFIA ICMT FAM72B OSCP1 TMEM132A BCL3 SLC12A8MGST1 BCORSESTD1 ZBTB2 CBX2HIST1H3I EHMT2PATZ1 MLKL GULP1 PFN2 PUS1 OSBPL2 FBXL2 PRDX5 PLTP SPECC1 TP53I11 TMEM216MTDH TUBGCP4 HES2 PPAP2A CRIP2 ZNF195 YBX3 IL1RN TINF2 AMPD3 UBASH3BKLHL24 COQ10B ZNFX1 EHD1 RIN2 CCDC92 IRF5 ANKEF1 PADI3 CCNDBP1 LACTB DENND2D KDM5B SLC7A1 CTDSP1 HDAC1 SMS GAA NTN1 POMZP3 UPF3B NF1 QSER1TENM2NR2C2APMSTO1 KATNAL 1 PUF60GXYLT2 TCF12 NMU ABL1OSGEPL1 DCUN1D5ARF6 TRIM38 SF1 ARHGAP5 CLMP RBPMSBCAT1 RAI14 ZZZ3 NEU1 PCNXL2 NIPAL1 WDR26 COQ10A FBXO33 SLC25A10CSPP1 MTO1 NFKBIZ ARL14 ATP10D TMEM134 SLC35G2 CSMD3 ARMCX3 ZDHHC11HES6 THBS3 MAB21L1 PDCD4 BACE1 NEURL2 STOX2 MTURN P4HTM MARCH2 AVPI1 EHD3 IGFBP2 PLEKHO1 PER3S100PBP NAV2 MBNL1EP400 PEX5 SHMT1 RNF4 RNF128 MAGEH1 KDM7A PRTFDC1 RPP25PPARG COQ3 PHGDH PIGU CDH2 NOP16IGF2BP1FAM102BDNAH14PMP22 FAM133A PYGL KIF5BMTHFD1L KIAA0020 C9orf142DDX55 HSPB8 CASP10 LAMB 3 UBE2H RFX7 CGRRF1 CS JUNB OPTN DFNA5 CAPN2 PI4K2A CHMP4C FAXDC2 SMPD1 GPRC5ACLK4 IRF1 HSD17B3 THNSL2 SULT1A1 LAMP3 CXCR2 VPS13C ADM ZNF441 NUDT18 ABCC10 CDKN2B EPB41L2 TRIO FBXO16SNTB1 HEY2 ATP11CCCBE1GUCY1B3LARP1 SCUBE2 AKAP8L NFAT5 MYH10 PPP1R9AGAR1 BRI3BPCCDC86 PKN3 NEDD4 NREP AXIN2 TELO2LOC28507IGFLR14 CBFB LDB2 TEAD1 TNFSF4 SLAMF7 SH3RF2 TAF15STARD8 TMEM19 PHYKPL ZFP36L2 DCLK1 PTK7 ZNF468 HAGH SERPINB8 ABCA1 DNAJC18 NR4A2 COL4A5 STAT2 SPINT1 DHRS7 SCIN ZBTB38 RGAG4 TTC39A FAM196A MOSPD1 CHRNA3 SGSH C12orf76 APTX CHMP1B IL12A CAPRIN1 PCGF6 PDK1SFMBT1 TRIM5 VPS39 OS9 ZNF862 TTC26 RAB2A TNS3RAP1GDSIMMP1L1 EMP2 LRCH1 CHAF1BLCLAT1 GEMIN2LMO4 HEATR3TBL1XR1 SREBF1PRELID1 XDH FKTN MPP6 TBL1X SLC19A1CTNNAL1 RNF145 CBX1 PRKACA GTF2B PLCD3 ACCS TRIM8 RRP15 MKS1 CAPN1 KLHL 7 CUEDC2 RILP S100A3 RBL2 TMEM88 PIK3R3 TCTAOVOL1 CHIC2 ARHGEF37 LTBP3 ATP6V1D CHPF2 ITM2B TYSND1 TEP1 ZNF667 UROS PCMTD2 SLCO4C1 MTFP1 DTD1 DMC1 TGFBRAP1 ZNF440 GPD1L FAM84A SIAE RPF2 PTAR1 UTP18CNTLNHIST1H4B SSB DHX37 QKI FARSB RAVER2SSBP3 EXOC6 DLG1 PRKAR2BRBM25 RP11−706O15.1 CNKSR3 SET C10orf2 UCK2SLC35F2 KLF11 C1orf21 ARHGAP30 BLVRB PIGG CPA4 GAL3ST4 FJX1 PRELID2 FSTL4 SAT2 PARP14 AIFM2 RIOK3 HOXC13 THSD4 LOXL4 EPB41L4BTRIM26 PPL SRC LGALS3 SLC25A15 SDC3 MAF ABTB1 ABHD4 SLC4A3 RAB5B ELL2 NAT10 DPYD DAZAP1 FXR1 MAMLD1PRKD3FAM20B CERK SCML2 PAQR6 LPIN3 EXOC7 SOS2 GCC2 FBXO17 IFT81BMPR1BFMNL1 RPP40 IFT122KHDRBS3 NR2C2 NAV1 RIBC2 APBB2ARHGAP33 COMMD10 GSDMB C20orf27PHLPP1 TPM1 SSX2IPALG13 RGS12 PCDH10 SCD5 GRAMD2 FAM214B PES1 TINAGL1 ABCA7 SYTL5 PLCL2 ATXN3 ALOXE3 PRKX CNIH4 BBS7TSPYL1 DYNLT3 ZER1 PORCN ZNF469 ABHD15 ARC SATB1 BBS4 ZNF540 GYG1 RP11−553A10.1 ZNF22 NSUN4RPS6KA3COCH ANKRD28THG1L THBS1SLC20A2ATG4C ZNF846 FITM2 CD70 MARC1 TSEN2KCTD20 CHN1 THNSL1 CEP70 TCHP YARSSLC25A13TIFA KDM3B HLCS CHD7 PEX6 CCDC186 PINK1 HIPK2 MTRR KLHL13 PDZD8 NSUN7 NCOA4 INO80B PNPO SLC46A3 REPS2 GTF2H2 SPRYD4 GLMP TUSC2 APBB3 CPE TBX3 GNS FAM43A PVRL4 CLU D4S234E PELI3 PTAFR CCRN4L LYRM4 MAMDC4 ANKRD1 GRN TMEM144 GLIPR2 CUL7 MCL1 P3H2 UGT8 UBE2E2ADIPOR2MID1 ARHGAP35DAXX PYCR1 NPM3 RAB37 ITPRIPL2 REXO2 CTGF MRPS2 DHX33TMEM177GJC1 TCF7L2GOPC RNFT2 THOP1 SRPK1UBIAD1 ATL2 SMAGPCCDC138 RABEPK WDR36 PCMTD1 TCF7L1 SLC43A3GFPT1 LRRC20 RNPEP MAFB CALCOCO1 LRRC37A3 ZNF641 ALDH4A1 MVP MYO5A C11orf96 RIBC1 HIPK3 SEC22A SIK1 C4orf3 PLAT NRG1 FXYD3 MXD4 ANGPTL2 TOM1 DNAJB2 DNAJB4 SLC7A8 GAS6 APOL2 CMTM4 JAZF1 FPGSMTERF3USP13 LRIG1 MAK16CEP170HIST1H4FRPA1 PLCXD2 TBCEL CEP170B PTK2 SEC63 FKBP4 NFIX PUS7 PLP2 HNRNPMMAP3K4 CSPG5 NFIC LRIG3 KLF6 DIAPH2 AMPH RBFA OSR2 PTPRE SIPA1L2 ROBO1 LYAR HHEX THADA SEC14L2 TREM2 PCBP4 FAM169A FIG4 MANBA HLA−DM A FBXO32 PLEKHM1 KHNYN MAP3K3 STOM DSE PVRL1 GPR155PROM2 C9orf89 IRS2 BCDIN3D C9orf9 C9orf47 PPAPDC2 MAN2B1 BMP1 BEND3 ZNF148ZNHIT6EIF4EBP1 MCMBPZNF827HIBADH PCK2 CBR3 PMEPA1 S1PR3 KIAA1324 KRT19 STON2 MKKS RNF138GNB1 SMAD2SLC36A4WARS FZD7 VPS72 POGZ BDH1 PLCE1LMNB2ZNF519 HS3ST3A1 CCNJL TMEM2 CDH3 ARRDC4 KDELC2 PTCH1 OCRL DNPEP C5AR1 ZFPM1 CD46 GRAMD3 FANK1 RHBDF1 KIAA2026 TMEM129 S100A13 NOTCH1 SESN3 CEP85L LYNX1 PRRG4 TEX9TSPYL2 HLA−E SAT1 PIFO ADA ATP6AP2 RELB FAM49A CYB5D2 DLAT PCDH18KIAA0586EML4 SACS BAG2 ZNF395ZNF711SPATA33 TMBIM1 SUGCT TRIM29 CPEB4 SWT1 OSBPL6 MAGI3RNF216UBE2K GJB2 RRP7A PLEK2 TRAF2 TMCO3ZNF511 TCF4 XPOT NETO2 IL1R1 SLC6A15 PERP ZNF436 IDS NOB1 PHF13 IMP4 TENM3 SFRP4 HIC2 IL32 CCND2 FAM214A LYPLA2 FLYWCH1 APLP1 PRICKLE2 PPP1R14C CCL2 TNFSF9 HLA−F KDM4A PPM1AESYT2 MRI1 UFM1 MLYCD VAMP1 KSR1 GPRC5C MSX1 PRIMA1 TMEM194BUAP1 SCMH1 RIOK1 RBBP8 PGBD1 SFXN1 ETV1 PYROXD2 CST3 GJB5 HMGCL CEP68 CSRNP2 LRRC8C NR2F2CABLES2CHD1 C17orf58HSD17B11 TYRO3MLLT10 RALGPS2DBN1TRERF1 ELP4 ENOSF1 LRRC58 SCD ANKRD46 IPO13 PPP2CB TUBB2B CCDC58 CDV3 PGAM5 PODNL1 TNFAIP1 GLRX PNPLA3 ARVCF GGT6 PROCR CD44 SERPINE2 AKR1B10 NEDD9 CTNNBIP1 NFKBIE PSMB8 ITGA2PIK3IP1 MIB2 RASAL1 KAT2B NUAK1 TAX1BP3 MERTK COL17A1 CCDC85C AGPAT9 JDP2BHLHE41 ING5 KLF12HMGCS1DGKE MTMR2 SLC38A9 GALNT11 ARRDC1 AMT RIT1 HSPBAP1 RAN ADNP NRF1 GNB4WHSC1L1GLI2 POLR3GOSBPL10 VEZF1TMEM218TFCP2PLCH1SLC29A2 INO80 SPAST ATP6V1H CHST2 WIPI2 ANKRD27 TAF4B NIFK PTTG2 USP20 NABP1 BDNF MAPK13 LAMC1 DUSP11 TMEM40 IL7 C1RL SERPINF1 RHOD INPP1 DNAJC21 ELF3 PLEKHA1TP53RK CASP1 TMEM27 FBXO25 JUP GSN EZH1 MEX3D H1FX PHLPP2ATP2B1 SAMD11CMTM8PLEKHA2DNAJC15RAPH1 SLC29A3 PLEKHM1P RPP25L RPS6KL1 SLC35E3 PITPNC1 TNFAIP6 EXT1DCTPP1POLRMT TCAF1 GLI3 BNIP1ZDHHC13 CCDC127NEK3 APPL1 FXN RBBP7 NTHL1 KMT2A ERAP2 SPATA7 LYRM5 RNF130 SERTAD4VANGL1 CEP128 CLDN1 CCDC51 TRAFD1 DBT MMP14 RAB40B TFEC SNN ACYP2 GPCPD1 TCP11L1 GABARAPL1 MYO6 RABL2B TFPI2 ZNF658ABAT OLFM2 ITGAV GATS ARG2 UNC13B YPEL5 WDR3 TLE1 PDCD2LSTAG2 PDCD11TEAD4SEC23IRPGRIP1LP COLGALT1 TMOD1 KRT7 SGK223 RND3 LIMK 2 MOAP1 SPRED1 LETM1UTP20 SEH1L DIP2B HPSE NIN ZMYM 3 C11orf49 IPO9 NUDT21ZNF496LMAN2L DDX18 CHD4 BAG3 UXS1 MOV10L1 TCF3 ZEB1 PRPF4B MYBBP1A BVES IGF1R TMTC3 CERS5 SLC6A8 PREPL ACHE KDM4B KIAA0513 CIB1 ZNF20 LIPH MKNK2 ACBD4 ZFHX2 MAFFMICALL2 AQP3 PGBD2 TCEA3 MAST3 DUSP3 SPATS2L PPIF STRBPGEMIN4FOXRED2 ROCK2 INSIG1 MAP4 PTP4A2KIF26A ZNF596 EPPK1 RSPH3 NARF ARHGAP27 DNAJB9 ZNRF3 RTN4IP1NOL10 XYL B QTRTD1 PTX3 FNDC3A DLG3 GJA1DNAJC1 TSR1HP1BP3NONO CBS ST7 XG CDC42EP3 PAQR7 CDC27 IFT172 SLMO1 ERF ABCB9 PPP2R5B RAB33B LYSMD2 FIBCD1 SLC16A5 TSPAN10 SDC4 CLSTN3 RP2 TMEM184C SUOX TACSTD2 PLXNA2 TMEM87B SRPX XPR1 ETS2 RDH5 EXO5 METTL8 PGPEP1 LAMA 3 CXorf23 IRS1 TSHZ1 AGPS DHCR7PPP1R14BBICC1 ATOH8 KCNK5TBC1D16SH3D19C1QTNF3 SEMA3F ARSD ATOX1 IL4R CPT2 ROM1 BIK ABCC4 MUM1 FZD2 RIMKLB DIS3L CLGN CTSC EIF2B3DPYSL3MANEAHIATL1 CNTRL GPX8 SLC35D1 CRIPT VPS37D CLUH NRARP KIF4B CPD PARP10 TMEM173 ADGRG1 ZNF383 PHLDB3 TSPAN6 ANK1 ALKBH6 HLA−B CLN8 ZNF425 ATP7A C10orf54 NDRG4PRSS23 PAXIP1UBE2E3 LCE1B CSAD RFX5 CADM1 TAF3 C6orf89 PRKD1ZFAND3MTMR12 SLC44A1FAM20C PAFAH1B1 GMPS MT1F LARSTMEM180HIST4H4 CDC42BPAGTDC1DRAM2 GCNT4 CHADL TM7SF2 TOB1 PGF TSPAN31 TMEM150A CEP112 CENPV MPDZ TUBE1 PPRC1PIP4K2A EZR NRXN3 ZNF609 AREG ABCC5 S100A6 POLR1BTTLL12 MAD2L2 NICN1 DAPK1 SLC26A11 AHNAK DUSP5 RFNG EAPP PRKCD CCND1 DEPDC7 HSDL2 SLC40A1 APOBEC3F SQSTM1 CAPN8STARD4 DIAPH1SMAD1 DQX1 CRISPLD2 CES2 LCOR CASP6 LACC1 MAPK9PAK1IP1 WAC CCNY CHRNA5 NLE1 IQCC ARNTL2 SUN2 EXTL2CHST10 NBN IGF2BP2REEP3 CEP164 TMEM30A CREBRF CSTA ZNF319 SHISA4 CYHR1 CMTM7 EFNA5 PAPD7 PARN TAF5LMRPL15PHIP MEX3A BUD13 KRBA1 KIFC3 PODXL DPH5 ALKBH2 TERT DFNB31 CYGB THSD1 SLC44A5 MVB12B CSGALNACT1 KITLG LRP10 PPCS TNKS2 ZNF600 RNF122 TCP11L2 HLA−DOA ARL15ZFYVE1 PRKACB IVNS1ABP FAM57AZNF618 KIAA1671 C14orf28 DKK1 LPCAT3 TGFA EPHX1 CEACAM1 BSDC1 CTDSPLGEMIN5SPIN4C7orf50 ARSJ NDUFAF4 SYNPO2 PDS5APRPS2 DOT1L CENPI ABI2 SLC16A7 NARS2 BRE TCEAL7 ADAMTS5 DMD AHNAK2 ADCY6 FAM72AGKAP1 FBL TLK1 NAA40 ID1 CPSF6 PRRG1 SLC52A1 STARD5 PBX2 AFAP1 SLC31A2 FAM46A MAOA ASB1 TRIM2 CST6 SMPDL3B PLXNB3 STXBP3 SERINC1 WIPI1 GXYLT1 TRANK1 TPP1 INTU EPHB3 NEK1 YLPM1 GOT1 TLE3 DSEL LITA F PEX1 HS1BP3 ARFGAP3 LAMB 2 MAK ADAMTS1ADAMTS3MRGBP JPH1 FGFRL1 SVIP PPTC7 NUP188SFXN4 MPHOSPH6TPR NUFIP1 BCL2 BNC2 SORT1 STAMBP DPYSL4 CHRNA9 TOM1L2 TLDC1 PHLDA2 NTNG1 CSF1R UBL4A AKT3 NES TMX2 REPS1 AMN1 MIEF2 SRRM3 CTH SLC30A6 CEACAM19 YPEL3 MPZL2 GAMT CCNG2 TRAF3IP2 ZNF563 SLC7A6 PCED1A SRGAP3 RHOB PLEKHO2 FOSL2 PRPSAP2 RABL2AHBP1 KPNA4 CNP DPF1TMEM106C GTF2IRD1 FTO LANCL1INHBEMEF2C MAP2K3 S100A10 DTX4 SPG7 PPP1R3B FBXL18 HSPG2 SLC43A1 RAB27ACCNE2ARID1A NIP7 FABP5 PNPT1 PPP4R2HIST1H1APFKPALDH18A1SPTBN1 ZNF449 PRKCZ PAPLN SERPINA1 KANK3 P4HA2 FAM162A SORD LRRC8D SLC1A3 EPRS AKAP12 PHF11 WDR59 SLC2A12 MAP3K5 LTBP2 EPHA4 TMPRSS2 RNASEL PHLDA1 BTBD19 FERMT1 DDX58 POLD4 MXI1 RASGRP1 NMNAT2 BBS1 PMEL TNFRSF14GABBR2 NCOA6 HPRT1ARHGEF2PCID2 STAMBPL1MTUS1EIF1AXANP32BCCDC12 PLLP PLEKHF1 CES3 GPR37 SLU7 UBE3B BMP2 ENC1 KCTD15 GNPNAT1LDLRAD3TMEM38BPDSS1 CHAC2 ACTR5HIST1H1E MMP16CAMKK1SURF6RHOBTB3TMEM51 H1F0 CCDC91 F11R CD109 FBXO44 C1S MAGED2 HPDL ZC3H14CLOCK GPT2 HGSNAT TMEM59 CYLD RASL11A FAM188A TMEM229B VPS33B INPP5D FEZ1 STAT4 TST JAG1 SPRY1 TBC1D9 LSR ITPR2 MORC4 SMIM14NIPAL3 PRKCA CEBPZCCDC88AEPB41L3 SLC7A11NOP14ASRGL1SLC2A4RGPARP1 IKBKB PTPRB NLRP1 ITIH5 OSTM1 GLS2 TGFBR1 ADNP2 SEPHS1PHF23 NCOA7ELOVL6GPRC5B RBM27 NOL9 SKIV2L2STK32C NFIB SCFD2SLC39A14 HOMER1 ZNF639 KIAA1462 SNAI2 IDUA ARSA RTN1 ZNF610 LIMS1 THOC6 SQLE CEBPG FAM83H TAB3 C21orf91 SERPINB1 SLC12A6 ERCC6 MOSPD3 NFATC4 LURAP1L RAB3B TRIML2 BEX4 MED23 TMEM8B BAK1 AMOTL1 RSRP1 ARL8A SYK TBL2 MANEALTBC1D4FAM210A POT1 KCNS3 LOXL3 RGMB PEG10 TIMP1 LTB4R REEP2 SGTB C19orf66 KLHDC2 MXRA7 WWC1 ME1 KLF16 PHF6 KCMF1 RRP1 OXCT1 RRP1B DHRS13RNF220MBOAT1IMPA2SLC35G1 CLCN5 EXPH5 PLA2G6 ZNF324 VAMP5 TIMP3 TRAK1 PPP1R15A URB2 SRR BCR MYB KDSR ABTB2 ADAM23 DYRK1B EPAS1 MUC19 NMRK1 IP6K2 POLR3GL GP1BB NACC2 ZBTB43 NR4A1 FRMD8 ATG4A SMYD5 GRIN2C WSB1 APOE CLK1 XPO7 CDKN1BGNAZ GPER1 TRIB3 KANK2 SSH2 SLC4A7MRPS34 SERINC3 FAM46C AGRN HMOX2 HIP1R STK17A ZFP90 ERLIN1 PAQR4 HK2 TM2D3SLC25A12C8orf33 CALML4 ANO6 DKC1ATP6V0A2YWHAHKCTD3 GCNT1 VKORC1L1 TNFSF15 KYNU GALNT5 CABYR IL1B CSRNP1 POGK NR3C2 SDC2 CD320 MR1 SMAP2 BLNK DUSP1 DUSP10 ZNF623 RHBDF2 EDEM1 ALS2CL MYCT1 FDX1 TRIM21 C15orf57 OSBPL3 PPM1J ERBB3 WBSCR27 TRIM3 TCF7 FGF18 XPO4ALDH1L2 CMTM1SMARCA4LIMCH1RNF144A ADAMTS6ADCY3SH3KBP1PHF14 ANKRD33B PHYH STARD10 SDC1 CCS MARCH4 RINL RIMKLA LSM2 WDR12GALNT14BCAT2 CCDC109BLSM6 TIMM44CFAP97VKORC1MRPL35 SLC20A1 TRAP1 SLC39A13 SMTN TBC1D2 ESM1 SHANK3 NSD1 LRRFIP1 GPRIN1NOP56 SLC11A2 ALOX5 TSPAN14 SHC4 ATPIF1 SLC43A2 NDRG1 ORMDL2 CYP4F3 PLA2G15 NLRX1 C10orf10 COL27A1 TP53INP2 MOCS3 GLTPPDGFA PTCHD4ERO1LB CHRD PSG9 SCN3BKCTD11 TTC27RANBP6RAB3IL1 PBXIP1 IL1RAP RBM41 MAPKBP1 UBA2 NLN ACSL3 NRM JMY F8 ENDOD1 C3orf52 MAPK11 RBM24 ASPA BAG1 GRAMD4 ... as well as p53-regulated genes with unknown links to p53.

Figure 1. The p53 gene regulatory network. Many genes are frequently and reproducibly up-regulated (n = 1392, green nodes) or down-regulated (n = 1707, red nodes) by p53. The common p53-regulated genes include a subset of genes directly regulated by p53 (n = 311, upper left cluster). In these cases, p53 binds to the gene’s proximal promoter within 2.5 kb from the TSS. The best-known indirect regulatory mechanism by p53 involves its direct target CDKN1A that encodes for p21 and leads to reactivation of the cell cycle trans-repressor complexes DREAM and RB-E2F. Following their activation by p21, DREAM and RB-E2F are particularly important to down-regulate cell cycle genes (n = 888, upper right cluster). For many other genes differentially regulated by p53 the underlying regulatory mechanism remains to be uncovered (bottom clusters). Green and red nodes are respectively up- and down- regulated by p53. Saturation indicates the p53 Expression Score (threshold ±5), which is calculated as the number of datasets reporting significant gene up-regulation minus the number of datasets reporting significant gene down-regulation upon p53 activation. Thus, high saturation indicatespendent p53-de regulation across cell types and treatments. Green and red edges represent respective direct target gene up- and down-regulation through proximal promoter binding by p53 or DREAM/RB. Distance contains no information. Data from (8). p53 wild-type ChIP-seq datasets have been produced by in- datasets (6), which are highly concordant with those found dependent labs examining chromatin binding of p53. While in earlier meta-analyses (8,15). Similar efforts to catalogue efforts to robustly integrate and reconcile the large amounts p53-binding sites in mouse cells, also identified a ‘default of ChIP-seq and gene expression data generated have been set’ of p53 binding sites regardless of activating stimulus or limited by the diversity of cell types, duration, and nature cellular context (20,39), providing strong evidence for the of stimuli analyzed to date, important patterns have be- existence of a conserved ubiquitous group of p53:DNA in- gun to emerge from these meta-analyses (6,8,9,14,15). The teractions that are commonly induced when p53 is stabi- largest meta-analysis to-date compared similarly processed lized. Despite this, significant differences were observed be- genomic data and identified a reproducible subset of >1000 tween the DNA binding landscapes of human and mouse genomic locations bound by p53, present in at least 20 of 41 p53 (20). 4 Nucleic Acids Research, 2020

Table 1. 132 ChIP-seq datasets of human wild-type p53 published using multiple cell lines cultured with various conditions Cells Treatment public accession Reference A498 IR (4 Gy, 2 h) GSM2677375 (52) A549 IR (4 Gy, 2 h) GSM2677380 (52) A549 Nutlin (5 ␮M, 2 h) GSM3771330 (52) ␮

A549 Nutlin (5 M, 2 h) GSM3771331 (52) Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 A549 TGF␤ (2.5 ng/ml, 5 days) + Nutlin (5 ␮M, 2 h) GSM3771332 (52) A549 TGF␤ (2.5 ng/ml, 5 days) + Nutlin (5 ␮M, 2 h) GSM3771333 (52) A2780 Cisp (2 ␮M, 3 days) GSM3720408 (53) A2780 vehicle GSM3720407 (53) BJ IR (10 Gy, 6 h) GSM1348340 (54) BJ RasV12+control vector GSM508793 (55) BJ untreated GSM1348339 (54) CAL51 IR (5 Gy, 1 h) ERR375899 (56) CAL51 IR (5 Gy, 2 h) ERR375900 (56) CAL51 untreated ERR375898 (56) Calu-1 DMSO (0.01%, 48 h) GSM3682106 (57) Calu-1 Belinostat (0.1 ␮M, 48 h) + Cisp (10 ␮M, 48 h) GSM3682107 (57) FSF DXR (0.2 ␮g/ml, 12 h) GSM1342488 (47) FSF DXR (0.2 ␮g/ml, 12 h) GSM1342494 (47) GM06993 + GM11992 DXR (0.5 ␮M, 18 h) GSM1142696 (58) GM06993 + GM11992 untreated GSM1142697 (58) GM12878 IR (10 Gy, 4 h) GSM1142702 (58) GM12878 Nutlin (10 ␮M, 18 h) GSM1142700 (58) H460 IR (4 Gy, 2 h) GSM2677379 (52) HCT116 5-FU (350 ␮M, 12 h) GSM1412744 (59) HCT116 5-FU (350 ␮M, 6 h) SRR1343581 (60) HCT116 5-FU (375 ␮M, 6 h) GSM1417250 (61) HCT116 5-FU (50 ␮g/ml, 24 h) - (62) HCT116 Camptothecin (CPT) (1.5 ␮M, 4h) SRR2817469 (63) HCT116 Camptothecin (CPT) (5 ␮M, 8 h) SRR2967009,SRR2967010 (64) HCT116 DMSO (0.05%) control SRR1343582 (60) HCT116 DMSO (0.2%, 12 h) SRR4090090 (9) HCT116 DMSO (4 h) SRR2817470 (63) HCT116 DXR (1.6 ␮M, 6 h) SRR1343583 (60) HCT116 IR (4Gy, 2 h) GSM2677381 (52) HCT116 IR (8 h) SRR1539836 (65) HCT116 IR (8 h) SRR1539837 (65) HCT116 Negative control siRNA (3 days) GSM3103907 (66) HCT116 Nutlin (10 ␮M, 6 h) SRR1343584 (60) HCT116 Nutlin (10 ␮M, 12 h) SRR4090091 (9) HCT116 Nutlin (12 h) GSM3103906 (66) HCT116 siRNA against iASSP (3 days) GSM3103908 (66) HCT116 Untreated GSM1412743 (59) HCT116 Untreated SRR1539838 (65) HCT116 Untreated SRR2967011,SRR2967012 (64) HCT116 Untreated GSM3103905 (66) HDF DMSO (48 h) SRR3125899 (67) HDF Nutlin (10 ␮M, 48 h) SRR3125901 (67) HepG2 control AdV (48 h) + UVC (24 h) GSM1581946 (68) HepG2 Hepatitis B (HBx)-expressing AdV (48 h) + GSM1581947 (68) UVC (24 h) hESC DXR (6 h) GSM981236 (51) hESC RA (1 ␮M, 48 h) GSM981237 (51) hESC Untreated GSM981235 (51) HFK Cisp (25 ␮M, 24 h) GSM1366691,GSM1366697 (12) HFK DXR (350 nM, 24 h) GSM1366690,GSM1366696 (12) HFK Untreated GSM1366689,GSM1366695 (12) HuSkFib Nutlin (5 ␮M, 6 h) GSM3020116, GSM3378524 (48) HuSkFib DMSO (6 h) GSM3020115, GSM3378522 (48) IMR90 5-FU (375 ␮M, 6 h) GSM783262 (69) IMR90 DMSO control GSM1418969 (25) IMR90 Etop (100 ␮M, 6 h) SRR7357223 (70) IMR90 Etop (100 ␮M, 24 h) GSM1294880,GSM1294881,GSM129 (71) 4893,GSM1294882,GSM1294883 IMR90 Nutlin (5 ␮M, 6 h) GSM1418970 (25) IMR90 O/EE1A/RasG12V GSM1294879,GSM1294885, (71) GSM1294891 IMR90 O/E RasG12V GSM1294877,GSM1294878, (71) GSM1294890 IMR90 Senescent (HRasV12) GSM1048851 (72) IMR90 Untreated GSM1294876,GSM1294884 (71) IMR90 Untreated GSM1048850 (72) Nucleic Acids Research, 2020 5

Table 1. Continued

Cells Treatment public accession Reference LOXIMVI IR (4 Gy, 2 h) GSM2677373 (52) Lymphocyte BS104 DMSO (0.1%, 24 h) GSM2988942 (6) Lymphocyte BS104 DXR (0.3 ␮g/ml, 24 h) GSM2988944 (6) Lymphocyte BS104 Nutlin (10 ␮M, 24 h) GSM2988946 (6) Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 Lymphocyte BS116 DMSO (0.1%, 24 h) GSM2988948 (6) Lymphocyte BS116 DXR (0.3 ␮g/ml, 24 h) GSM2988950 (6) Lymphocyte BS116 Nutlin (10 ␮M, 24 h) GSM2988952 (6) Lymphocyte BS45 DMSO (0.1%, 24 h) GSM2988930 (6) Lymphocyte BS45 DXR (0.3 ␮g/ml, 24 h) GSM2988932 (6) Lymphocyte BS45 Nutlin (10 ␮M, 24 h) GSM2988934 (6) Lymphocyte BS90 DMSO (0.1%, 24 h) GSM2988936 (6) Lymphocyte BS90 DXR (0.3 ␮g/ml, 24 h) GSM2988938 (6) Lymphocyte BS90 Nutlin (10 ␮M, 24 h) GSM2988940 (6) MALME3 IR (4 Gy, 2 h) GSM2677378 (52) MCF7 5-FU (100 ␮M, 8 h) SRR287799 (73) MCF7 Decitabine (2 ␮M, 5 days) GSM2740046 (52) MCF7 DMSO (0.2%, 12 h) SRR4090093 (9) MCF7 IR (4 Gy, 2 h) GSM2677372 (52) MCF7 IR (10 Gy, 1 h) SRR5690016 (74) MCF7 IR (10 Gy, 2.5 h) SRR5690017 (74) MCF7 IR (10 Gy, 4 h) SRR5690018 (74) MCF7 IR (10 Gy, 5 h) SRR5690019 (74) MCF7 IR (10 Gy, 7.5 h) SRR5690020 (74) MCF7 IR (10 Gy, 7.5 h), then sequential Nutlin SRR5690021 (74) MCF7 neocarzinostatin (NCZ) (400 ng/ml, 3 h) SRR5857009,SRR5857012 (75) MCF7 Nutlin (5 ␮M, 2 h) GSM2677384 (52) MCF7 Nutlin (5 ␮M, 24 h) GSM1146168 (76) MCF7 Nutlin (10 ␮M, 8 h) SRR287800 (73) MCF7 Nutlin (10 ␮M, 12 h) SRR4090094 (9) MCF7 RITA (0.1 ␮M, 8 h) SRR287797 (73) MCF7 RITA (1 ␮M, 8 h) SRR287798 (73) MCF7 Untreated SRR287796 (73) MCF7 Untreated SRR5690015 (74) MCF7 Untreated SRR5857010,SRR5857013 (75) MCF7 Untreated GSM2740045 (52) MCF7 Untreated GSM1429753 (77) MCF10A Nutlin (5 ␮M, 6 h) GSM3020136, GSM3378513 (48) MCF10A DMSO (6 h) GSM3020135, GSM3378510 (48) MDA-MB-175VII Untreated GSM1429754 (77) PBMC 5-FU (50 ␮g/ml, 24 h) - (62) SaOS-2 O/E GFP ERR206782,ERR206794,ERR206795 (11) SaOS-2 O/E p53-wt (18 h) ERR206781,ERR206779,ERR206784 (11) SaOS-2 O/E p53-wt (24 h) GSM501691,GSM501692 (78) SaOS-2 O/E p53-wt (24 h) GSM1241481 (79) SJSA DMSO (0.2%, 12 h) SRR4090096 (9) SJSA Nutlin (10 ␮M, 12 h) SRR4090097 (9) SKMEL5 IR (4 Gy, 2 h) GSM2677377 (52) SW480 Empty vector SRR1920910 (80) SW480 p53 wt O/E SRR1920909 (80) UACC62 IR (4 Gy, 2 h) GSM2677382 (52) UACC257 IR (4 Gy, 2 h) GSM2677383 (52) UACC257 Nutlin (5 ␮M, 2 h) GSM2677385 (52) U2OS ActD (5 nM, 24 h) GSM545807 (81) U2OS DMSO (0.1%, 24 h) control GSM1133482 (82) U2OS DXR (0.6 ␮g/ml, 24 h) GSM1133484 (82) U2OS Etop (10 ␮M, 24 h) GSM545808 (81) U2OS IR (4 Gy, 2 h) GSM2677376 (52) U2OS Nutlin (10 ␮M, 24 h) GSM1133486 (82) U2OS Untreated SRR1344509,SRR1343579 (60) U2OS Untreated GSM1133488 (82) U2OS Untreated ERR359700,ERR359705 (83) U2OS UV (50 J, 6 h) SRR1344510,SRR1343580 (60) U2OS UVC (20 J/m2, 16 h) ERR359704,ERR359706 (83) U2OS UVC (20 J/m2, 8 h) ERR359699,ERR359702 (83) UO31 IR (4 Gy, 2 h) GSM2677374 (52)

Abbreviations: 5-FU, 5-fluorouracil; ActD, actomycin D; Cisp, cisplatin; DXR, doxorubicin; Etop, etoposide; IR, ionizing radiation; nutlin,Nutlin-3; RITA, reactivation of p53 and induction of tumor cell apoptosis; UV, ultraviolet radiation; p53 O/E, p53 overexpression; RA, retinoic acid; Ras O/E, Ras overexpression; DMSO, dimethyl sulfoxide; TGF␤, Transforming growth factor beta; AdV, adenovirus. 6 Nucleic Acids Research, 2020

Despite the identification of a ubiquitous group ofp53 such, p53 falls into a functionally defined group of pro- binding sites, the molecular mechanisms that facilitate these teins called pioneer factors, which can interact with and common p53 binding events across experimental conditions recognize specific DNA information even when the DNA and cell lines remain unclear. Analysis of genomic context resides in the context of nucleosomes (85). Pioneer factors of p53 binding sites based on enrichment of particular his- can use this nucleosome binding activity to initiate a lo- tone modifications or accessibility based on DNAse-I hy- cal chromatin remodeling cascade, ultimately establishing persensitivity or Assay for Transposase-Accessible Chro- DNA accessibility at cognate transcriptional regulatory re- Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 matin followed by sequencing (ATAC-seq) as a proxy to gions (Figure 3C) (86). This ability is uncommon amongst identify active regulatory regions (40–46) indicates that sequence-specific TFs, with analyses from ENCODE show- p53 binds within at least three observable genomic con- ing that DNA binding for most TFs is strongly inhibited by texts: (i) accessible DNA, (ii) constitutive nucleosome- the presence of nucleosomes (40,41). rich regions, and (iii) nucleosome-rich regions that dis- A key question regarding p53’s pioneer factor func- play some level of nucleosome eviction in a p53-dependent tion relates to how nucleosomal positioning and occu- manner (25,27,47). p53 binding events within accessible pancy affect p53 binding and transcriptional activity. Ini- DNA are also enriched for histone modification patterns tial evidence for p53 pioneer factor activity comes from in that indicate an active transcriptional regulatory region, vitro studies demonstrating direct binding to the p53RE such as an enhancer or promoter (25,26,47,48). In agree- in the CDKN1A promoter even when the DNA template ment with this prediction, examination of the chromatin is within a nucleosome (87). Biochemical reconstitution of context of ubiquitous p53 binding events derived from p53RE DNA into mono-nucleosomes confirmed the di- chromHMM (chromatin Hidden Markov Model) across rect p53 binding to DNA within chromatin (88). Both 127 unique cell types (49) shows that a median of approx- an updated, highly-parallel sequencing approach (89)and imately one-third of ‘ubiquitous’ p53 binding sites reside DNase I-mediated footprinting of the native CDKN1A pro- within transcriptionally-associated genomic regions across moter sequence came to similar conclusions about preferred all cell types (Figure 2A, TSS, transcribed regulatory, en- p53:nucleosome orientations (90). p53 prefers binding to hancer, or weak enhancer). The chromatin context of p53 nucleosome orientations where the p53RE is near the nu- binding sites varies significantly between cell types, even at cleosome edges and disfavors binding near the nucleosome binding locations that are ubiquitous across all cell types dyad or core (Figure 4A) (89,90). Whether this preference is tested to date (15). These cell type differences are not limited due to dynamic unwrapping/‘breathing’ of DNA near nu- to transcriptionally-associated chromatin features, as com- cleosome edges or a specific recognition mechanism of p53 mon p53 binding sites across embryonic stem cells (hESC) for DNA at nucleosome edges remains to be directly exper- and primary fibroblasts are enriched for constitutive and imentally validated (91). facultative (polycomb-mediated) heterochromatin features Binding of p53 to nucleosomal DNA is also influenced (Figure 2B). CDKN1A/p21 expression is repressed in hESC by the rotational position of the p53RE relative to the nu- through polycomb-mediated H3K27me3 at the promoter cleosome dyad (Figure 4B). Strongest p53 binding in vitro which can be alleviated by H3K27me3 methyltransferase in- was observed when the DNA minor groove of the p53RE hibition (50). Other p53-bound gene promoters were also half site was exposed to solvent (88). Within a nucleo- decorated with this repressive modification in hESC, sug- some, this would happen when the p53RE is centered 5bp gesting that the chromatin state of a p53 binding site can from the dyad, repeating every 10bp after that. Rotational alter p53-dependent transcriptional activation (51). Consis- position does not supersede location around the nucleo- tent with previous work (25,27), the majority of common some, as nucleosome edge binding, even in disfavored ro- p53 binding events (median of 59% across all cell types) ac- tational positions, was stronger than any rotational posi- tually fall within quiescent chromatin (Figure 2A). These tion within the nucleosome core (89). p53REs at some pro- chromatin regions are devoid of active and repressive hi- apoptotic p53 targets appear to exist in a non-preferred nu- stone modifications and lack features of open or accessi- cleosome rotational position relative to some cell cycle ar- ble chromatin. The potential function of these regions is a rest targets (92) and this has been proposed to explain dif- rich area for future investigation given the total number and ferential transcription activation kinetics between these two broad conservation of these binding events across cell types gene classes (92,93). This possibility has not been directly and treatment conditions. tested on a broad scale, although the advent of both high- throughput in vitro nucleosome binding assays and more sensitive genome-scale chromatin structure measurements DNA BINDING IN THE CONTEXT OF CHROMATIN: should allow such analyses in the future. PIONEER-LIKE p53 The binding of p53 to native nucleosomal DNA se- DNA wrapped around a histone octamer is called a nucleo- quences within cells is supported by examination of p53 some. DNA in a nucleosomal context is generally unable to binding using ChIP coupled to highly parallel analysis ap- be recognized and bound by sequence-specific TFs. There- proaches (Figure 3A and E). Using ChIP-coupled microar- fore, nucleosomes often act as direct barriers to TF bind- ray (ChIP-chip), p53 was shown to bind to high-affinity ing and biochemical reactions on DNA, such as transcrip- p53REs within approximately 2,000 nucleosome-rich re- tion and DNA replication/repair (84). Extensive evidence gions in cells (94). Subsequent ChIP-seq studies confirmed now indicates that p53, in addition to canonical binding to that p53 can bind to genomic locations with high nucle- accessible promoter and enhancer regions (Figure 3A, B), osome occupancy (25,27), and that many p53:nucleosome also can bind to nucleosomal DNA with high affinity. As interactions take place at nucleosome edges, similar to in Nucleic Acids Research, 2020 7

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Figure 2. The local chromatin modification state of core p53 binding sites. (A) Integration of ubiquitous (core) p53 binding sites identified by (15) with ChromHMM-assigned chromatin features across 127 unique cell types (ChromHMM = chromatin Hidden Markov Model). These results are qualitatively similar when the ubiquitous p53 binding sites from the Nguyen et al. meta-analysis are used (6). Each dot represents the percent of p53 binding sites in each cell type that have the listed local chromatin environment. The 25-state ChromHMM model was collapsed into 10 distinct groups similar to (252). Red lines represent the median percentage of p53 binding sites with the given chromatin state across the 127 cell types assayed. (B) The percent of core p53 binding sites with the given chromatin state for primary fibroblasts, epithelial cell types, T cell types and embryonic stem cells (ESC). vitro observations (89). Taken together, these biochemical The molecular mechanisms of other pioneer TFs may and genomic observations demonstrate the capacity of p53 provide some clues into p53 family pioneer activity. For ex- to bind to nucleosomal DNA, and that this is influenced ample the pioneer factors SOX2 and KLF4 display exten- by the context of the p53RE. Context-dependent p53 bind- sive non-specific nucleosome binding that may aid sliding- ing based on nucleosome positioning provides intriguing based DNA motif searching and binding to exposed par- avenues for studying the biology of p53, including how tial DNA motifs within nucleosomal DNA (99). Pioneer p53RE positioning within a nucleosome influences differ- factor binding to nucleosomal DNA is aided by the abil- ential transcriptional activation and p53-dependent pheno- ity to differentially recognize full and optimal or degen- types. erate sequence motifs (100). p53 binding preferences dis- Another key question regarding p53’s pioneer activity re- play defined periodicity from the nucleosome dyad, as has lates to the molecular mechanisms allowing p53 to recog- been demonstrated for both forkhead and homeobox pio- nize and bind to nucleosomal DNA. Sequence-specific nu- neer factors (46). Thus, the mechanisms used by other well- cleosomal DNA binding activity of p53, perhaps unsur- studied pioneer factors may provide context into specific prisingly, is dependent on the p53 DBD. The p53 DBD molecular functions needed for p53-dependent nucleosome alone can bind to p53RE in many of the same contexts as binding. Understanding the mechanisms used by p53 to full-length p53 (88). Notably, full-length p53 is capable of recognize nucleosomal DNA and how differential binding non-specific binding to nucleosomal DNA lacking a rec- of p53 to various nucleosomal DNA contexts affects tran- ognizable p53RE (88), whereas deletion of the disordered scriptional activation and other biological processes are key and posttranslational modification-rich C-terminal domain questions for future research. (CTD) diminishes this non-specific binding (88,89,95). The CTD appears critical for binding to p53RE that deviate from the consensus sequence (96), a finding that has been CHROMATIN-ENGAGED COFACTORS OF p53 further confirmed by SELEX (systematic evolution of lig- To enact its chromatin modifying roles, p53 interacts ands by exponential enrichment)-based high-throughput with an extensive list of transcriptional cofactors and can analysis (97). Non-specific DNA binding has also been mediate their recruitment directly to p53REs. Amongst proposed to allow p53 to scan DNA for high affinity these cofactors are a number of chromatin modifying p53REs (98); however, the ultimate functional relevance and remodeling enzymes with known co-activator and of the CTD mediating non-specific binding to both naked co-repressor roles, involved in nucleosome eviction, reg- and nucleosomal DNA is unclear and requires additional ulation of chromatin structure, and the activity of tran- study. scriptional regulatory regions. This includes key modu- 8 Nucleic Acids Research, 2020

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Figure 3. Chromatin binding states of p53. (A) A general schematic of the range of chromatin states with observed p53 binding. (B) Binding of p53 to gene proximal promoters represents the canonical model for p53 engagement with gene regulatory elements. p53-bound promoters are generally characterized by accessible (nucleosome-free) chromatin and histone modification-based hallmarks such as H3K4 trimethylation (H3K4me3). (C) p53 can bind to DNA in the context of a nucleosome using pioneer factor-like activity. Pioneer factor activity reflects two related functions: the recognition of aTF motif in nucleosomal DNA and the facilitation of nucleosome remodeling to create an accessible DNA element. p53 recognizes its RE across multiple nucleosomal contexts, although binding is strongly disfavored when the p53RE is at the nucleosome dyad. The majority of nucleosome binding sites remain nucleosome-enriched (closed), although this may be context or treatment-specific. p53 binding to closed chromatin in fibroblasts, for example, leadsto a subset of regions that become accessible and correlate with transcriptional enhancer activity. (D) p53 also binds to genomic regions with hallmarks of transcriptional enhancers, which includes accessible DNA, the presence of H3K4me1 and H3K27ac, and the absence of H3K4me3. p53 binding to these regions elicits transcription of bidirectional enhancer RNA (eRNA). Although very limited data currently exists regarding p53, one mechanismfor distal gene regulatory element activity is through chromatin looping, which brings these elements in physical proximity to target gene promoters. (E)The final class of binding events is to condensed/closed chromatin, which can either be characterized as actively repressed heterochromatin (containing the histone modifications H3K9me3 or H3K27me3) or quiescent. Quiescent chromatin is condensed but does not have the stereotypical histone modifications associated with heterochromatin. Quiescent chromatin represents the largest individual chromatin state bound by p53, although their function is unknown (Figure 2A). lators of histone modification state, such as CBP and translationally modify p53 itself (122) to affect changes in p300 (mediating H3K27ac) (101,102), TIP60 and hMOF p53 DNA binding, stability, and interactions with cofactors. (H4K16ac) (103,104), GCN5/PCAF (H3K9ac) (105), and Acetylation of multiple lysine residues in p53’s DNA- the H3K27me3 demethylase KDM6B (54). p53 can also binding and oligomerisation domains by histone acetyl- directly interact with components of chromatin remodel- transferase (HAT) p300, potentiates DNA binding in vitro ing complexes, such as RSF1 (106), BRG1 (107), ARID1A (102), which in turn elicits localized effect on histones, both (108), BRD7 (55), and TRRAP (109). These interactions of which are competitively removed by the activities of are not limited to those supporting p53-dependent trans- HDACs. HDACs have been shown to both interact with activation, as p53 also associates with known transcrip- MDM2 to enhance p53 degradation and directly deacety- tional repressors, such as histone deacetylases (HDACs) late the p53 C-terminus to influence its transcriptional ac- (110) as well as modulators of repressive chromatin and tivity (123–127). These C-terminal lysine residues are tar- heterochromatin including PCL1 (111), KMT5A/SETD8 geted by additional HATs, such as CBP and PCAF, to fa- (112), LSD1 (113), SMYD2 (114), SUV39H1 (115), USP7 cilitate p53 activity, whereas they are also validated sub- (116), as well as EHMT1 and EHMT2 (117). Such in- strates for lysine methyltransferases and ubiquitin ligases, teractions are mediated through both transactivation do- whose activity can variably repress or activate p53 func- mains (TADs) of p53 as well as the post-translational tion in vivo (113,117,128,129). Delineating the importance modification-rich CTD, whose modification state (similar of specific post-translational modifications (PTM) and the to a histone tail) can be read by chromatin regulators such residues on which they are found has been extremely com- as SET (118), 53BP1 (119), and PHF20 (120,121). While plex. However, recent data suggest that the overall net the list of known chromatin regulators interacting with p53 charge of the p53 C-terminus may play an important role is extensive, key questions remain regarding their roles in in repressing its activity, with an un-acetylated ‘acidic’ C- modulating p53 activity. Many of the above factors post- terminus being read by SET, much like a histone, inhibiting Nucleic Acids Research, 2020 9

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Figure 4. The location and orientation of p53 response elements within nucleosomal DNA influences p53 binding affinity (A) 146 nucleotides of DNA wrap around a histone octamer (blue) with the nucleosome dyad normally denoted as position 0. In vitro nucleosome binding experiments and in vivo ChIP-seq analyses suggest p53 affinity for nucleosomal DNA is higher when the p53 response element is within linker DNA (non-nucleosome associated) or near the nucleosome edges (illustrated with a green box). p53 binding near the dyad or to the nucleosome gyre (opposite side when rotated 180˚) is disfavored relative to other positions (marked with red boxes). Of note, p53 binding to the disfavored positions still occurs at nanomolar Kd values and can also occur in the absent of a p53RE, suggesting intrinsic affinity of p53 for nucleosomal DNA.B ( ) p53 affinity for nucleosomal DNA depends partially on the rotational position of the p53RE relative to the nucleosome dyad. When the center of the 20bp p53RE is at rotational position 0 (middle), binding is disfavored relative to when the rotational position is shifted in increments of five nucleotides (left or right). This binding preference periodicity has been observed for other pioneer transcription factors, like the homeodomain and forkhead factors (46). its acetylation (118). Modulating the activities of regulators the combined activity of multiple TFs binding to accessi- of p53 PTMs, such as inhibitors of HDACs and other co- ble DNA elements (137,138) and can be characterized by factors, have shown potential to promote p53 induced cell their histone modification patterns and the presence of tran- death alone or in combination with DNA-damaging agents scribed enhancer RNA (139). p53 binds to regulatory re- (130–133), but the underlying mechanisms remain poorly gions with these characteristics, but also pervasively binds understood. to DNA that appears inaccessible and devoid of evidence In fact, direct evidence for roles of the modifying en- suggestive of transcriptional regulatory potential (Figure zymes in facilitating or impeding p53 interactions with the 3B–E). Thus, a key outstanding question centers on under- p53RE through remodeling and regulation of local chro- standing how local transcription factors and varying chro- matin structure are currently limited. One such example matin contexts of p53 binding relate to subsequent gene ac- are the two H4K16 targeting acetyltransferases hMOF and tivation. TIP60, which when bound to p53 acetylate adjacent nucleo- Recent work utilizing massively parallel reporter screens somes (25), and H4K16ac has been previously described as (MPRA) to assay the transcriptional activation potential one of the only histone modification that directly alters local of hundreds of p53 binding sites indicated that p53 alone chromatin compaction (134–136). The breadth and depth can activate transcription of plasmid-based reporters (15). of p53 interactions with chromatin regulatory proteins will These data suggest that any p53 genomic binding event can certainly require an extensive, but necessary, unraveling to lead to transcriptional activation as p53 would not need any better understand how these factors are related to poten- other local transcription factors. This interpretation is sup- tial pioneer factor activity versus canonical transcriptional ported by observation that the presence of other TF motifs co-activator and co-repressor activities. surrounding a p53RE are not predictive of transcriptional activation upon p53 binding (27). Further support is found FUNCTIONAL CONSEQUENCES OF p53 BINDING IN in the extensive evidence that p53 is a strong transcriptional VARIED GENOMIC CONTEXTS activator in yeast, where other cooperating TFs are presum- ably absent (140,141). This model is intriguing, as it would Generally, transcriptional regulatory regions such as en- suggest that p53 binding to all genomic contexts, includ- hancers and promoters are believed to function through ing to nucleosomal or closed chromatin, can support tran- 10 Nucleic Acids Research, 2020 scriptional activation. Additional experimental approaches colorectal cells p53 sites associated with bona fide p53REs are necessary to determine whether these p53 binding (CDKN1A, SFN) were accessible, both before and after p53 events are productive in their native genomic and chromatin activation and with no change observed in p53-null cell lines context. (142). p53 binding sites often have pre-established histone Considerable evidence, however, suggests that p53 is un- modification patterns before p53 activation and in cells with likely to act in the ‘single factor model’ at all genomic bind- genetic or biochemical depletion of p53 (25,26,48). More- ing sites. For example p53 frequently binds to regulatory re- over, the large majority of p53 binding sites in dermal or Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 gions such as enhancers and promoters, the canonical chro- lung fibroblasts were either accessible before p53 binding or matin features of which are established independent of p53 were constitutively inaccessible even after binding (25,27). activity (25–27,48,142). Furthermore, combinatorial activ- These data indicate that increased chromatin accessibility is ity of TFs with p53 has been extensively reported in the lit- not always a direct consequence of p53 binding and suggest erature (143–148). Another MPRA approach provided ad- a limited and context-dependent role for p53 in mediating ditional evidence for the role of other TFs in regulating p53 chromatin accessibility (25–27). transcriptional activity. (149). Screening the transcriptional Activation of transcription subsequent to p53 binding activity of thousands of variants of p53-bound regulatory to TSS proximal sites has been suggested to differ for spe- elements revealed that other motifs flanking a p53RE con- cific promoter elements87 ( ,154) at least in part due to vari- tribute to p53-dependent activation. One such example was ation between p53REs (155–157). Recent single-molecule binding of ATF3 and p53 to an enhancer element regulating and cryo-EM experiments indicate that p53 mediates re- expression of GDF15 (149). ATF3 and p53 have been pre- cruitment of the transcription pre-initiation complex mem- viously shown to co-occupy many genomic locations, with ber TFIID to central promoter elements that can be fur- ATF3 activity contributing to p53-dependent activation of ther affected by promoter sequence and thus alters stabiliza- numerous downstream targets (63,150). A novel CRISPR- tion of TFIID necessary for increasing transcription initia- based screen identified CEBPB as a key regulator of the tion (158). Interestingly, these studies also suggest that upon p53-responsive enhancer controlling CDKN1A expression successful interaction of TFIID with DNA elements in the and the establishment of a senescent cell state (151). Anal- promoter, p53 dissociates from the p53RE. Thus, the rate ysis of this CRISPR screen using multiple machine learn- of interaction and cycling of p53 interactions with different ing approaches identified additional TF motifs predicted p53REs and their capacity to engage TFIID recruitment to to contribute to the activity of enhancers bound by p53 cognate promoter sequences, likely plays an important role (152). p53 binding to nucleosome rich loci correlates with in differential binding and activation of p53 targets. In sup- a stronger adherence to a consensus p53RE, whereas p53 port of this model, promoters of p53 target genes show dif- binding to regions with high accessibility are linked to lower ferential transcriptional activation kinetics in response to scoring or non-canonical p53RE motifs (25,26). These ob- natural p53 protein pulses which may alter residence time servations might be related to the need for additional TFs and frequency of interaction with promoters (159). More re- to assist in establishing DNA accessibility and to facilitate cent work demonstrates that stochastic transcription of p53 binding of p53 to these elements to drive transcriptional target genes is controlled by transcription burst frequency, a activation. Additional evidence for the role of local chro- parameter controlled by transcription initiation rates (160). matin accessibility and co-occupancy of other transcription Live cell imaging suggests that elevated p53 protein ex- factors on p53 activity is still needed, but the integration pression increases the likelihood a gene target will be tran- of high-throughput biochemical and genetic methods with scribed (burst frequency), but does not influence the magni- computational approaches should help delineate the regu- tude of the transcriptional response (burst size) (161). These latory logic of p53-bound genomic loci. data also suggest that affinity-based models of promoter ac- Does p53, then, bind to regions with high nucleosome tivity are unlikely to fully describe p53 regulated transcrip- occupancy as a precursor to the eventual displacement tion, with p53 CTD modifications controlling some of the of nucleosomes and establishment of DNA accessibility observed kinetic differences between p53 target genes. It is (Figure 3C)? Activation and binding of p53 has indeed thus unclear to what degree transcriptional activation ki- been associated with reduced nucleosome occupancy at the netics are determined by p53 protein stability and modifica- CDKN1A promoter (90,153), and ChIP-chip analysis in tion state, intrinsic sequences at p53 binding sites, or other the MCF7 cell line and recent ATAC-seq in primary fi- promoter-associated factors, as all have the strong potential broblasts have demonstrated that p53-dependent nucleo- to alter p53 interaction frequency and activation of promot- some displacement occurs in additional locations (27,94). ers. Advances in single molecule imaging approaches and Together, these results suggest that p53 binding to nucleoso- single-cell RNA velocity measurements have the potential mal DNA can in certain contexts mediate chromatin acces- to advance our understanding of how multiple local factors sibility and can alter transcriptional activation. However, and sequence context alter p53’s ability to activate promot- analysis of the sequences encoding p53 regulated regions ers and drive the observed kinetics in mRNA synthesis. identified by ATAC-seq did not reveal any DNA sequence- In addition, there is also evidence to suggest a signifi- based information that might suggest the context required cant number of p53 binding sites may not be directly asso- for p53-mediated nucleosome eviction (27) consistent with ciated with regulatory elements of genes, long non-coding other computational approaches (15). Yet, while p53 nucle- RNAs, or micro-RNAs. The lack of traditional signifiers osome binding can lead to eviction and establishment of of transcriptional regulatory potential (such as particular accessible DNA elements, extensive data suggest that this histone modification patterns) might suggest these bind- activity is limited and context dependent. For example, in ing events are evolutionary remnants, could have context- Nucleic Acids Research, 2020 11 dependent transcriptional roles, or could function in non- opment or cellular reprogramming (86,179). Mice lacking traditional roles. One possibility is that p53 might act lo- p53, while cancer prone like the human p53 mutation car- cally to influence DNA repair and protect genome integrity, rying Li-Fraumeni syndrome patients (180), generally de- such as in the case where p53 binds to both human telom- velop normally, although variably penetrant developmen- eres and other fragile genetic sites to influence chromatin tal defects have been observed (181–183). This suggests structure that protects DNA from damage (64,162). Pro- that p53 and its putative pioneer factor activity are not tection of these regions from DNA damage requires local strictly required for the establishment of chromatin states Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 p53-dependent transcription to establish the appropriate re- and transcriptional networks needed for proper develop- pressive heterochromatin features, but does not appear to ment. In contrast, genetic evidence from mouse models require expression of any common p53 target gene. Further, and human genetically linked conditions/syndromes stud- p53 pervasively binds to p53REs derived from retrotrans- ies strongly indicate that the p53 family members p63 and posons or other mobile genetic elements (34–38,163)and p73 are key lineage determination factors for basal (p63) can suppress transcription of retroelements (164–167). The and ciliated (p73) epithelial cells (184–186). Recent studies viral origin of these elements may also lead to an under- suggest that the Np63 isoform certainly shares the ability count of p53 binding sites in the human genome as these to bind to inaccessible chromatin and exhibits extensive ca- regions tend to be highly repetitive and thus difficult to pacity for opening chromatin at these sites (187–190), but identify using traditional ChIP-seq approaches. The p53- unlike p53, this pioneer activity is critical for development mediated mechanisms conferring protection from retroele- (191). Specifically, p63 is required for the establishment of ments is also conserved through evolution, having recently enhancer accessibility and activation of an extensive squa- been identified within Drosophila (167). Certainly, p53 has mous cell-specific gene expression network (31,48,190,192). been shown to have other roles in DNA context, such as Moreover, p63-dependent chromatin remodeling is required playing an important role in replication restart (168)and for expression of key epithelial lineage-specific genes in two replication fork progression (169,170). The absence of these cellular reprogramming paradigms (33,188), and conversely p53-dependent processes can lead to further genomic insta- depletion of p63 in terminally differentiated epithelial cell bility. While these p53 binding events do not appear to in- types led to decreases in histone modification abundance at fluence p53-dependent gene regulation, they have key roles regulatory regions (48). In epithelial keratinocytes, loss of in the maintenance of genome fidelity in the germline and p63 activity led to a nucleosome sliding into normally ac- tumor suppression. cessible regulatory regions, similar to the chromatin book- marking activity previously proposed (187,192). Appropri- ate nucleosome structure at p63-bound regulatory regions PIONEER ACTIVITY OF p63 AND p73 AND THE IM- was related to p63 interactions with BAF chromatin remod- PLICATIONS FOR p53 eling complex (187), providing a direct biochemical mech- Full-length p63 (TAp63) functions as a haplo-insufficient anism for these observed activities. Although not fully in- tumor suppressor (171) that also ensures quality control in vestigated, these data strongly suggest that the pioneer fac- germ line cells (172). The shorter p63 isoform Np63 is tor activity of p63 is essential for epithelial lineage determi- overexpressed or amplified in almost all squamous cancers nation and that developmental phenotypes associated with and serves as master regulator of stratifying epithelia that is loss of p63 function may be due to decreased chromatin ac- crucial for limb, mammary gland, prostate, and epidermal cessibility at regulatory regions. development (173). Like TAp63, full-length p73 (TAp73) is Similarly, p73 is required for the establishment of cil- a haplo-insufficient tumor suppressor and it functions asa iated epithelial cell identity through control of cell type- key regulator of neuronal development, multi-ciliated cell specific regulatory elements and gene expression184 ( ,185), differentiation, and metabolism (171,174). The p53 family although direct binding to nucleosomal DNA has not yet shares highly-related DNA binding domains with similar, been demonstrated. Why then does p63 (and potentially but not identical, RE sequence preferences (88,175). Conse- p73) have the ability to modulate chromatin accessibility quently, the p53 TF family shares many binding sites; how- in specific cell types while the broadly expressed p53 hasa ever, all three family members also display substantial sub- more limited role? The ability of p63 to initiate and maintain sets of unique target genes (12,175,176). Understanding the chromatin accessibility requires an intact C-terminal sterile interplay between p53, p63, and p73 is complicated by the alpha-motif (SAM) domain as disease-associated mutants differential function of several N- and C-terminal isoforms in the p63 SAM domain fail to establish enhancer acces- of all three family members that exhibit overlapping targets sibility and activate downstream gene expression (33,191). with often antagonistic function within and between the Unlike p63 and p73, p53 lacks a SAM domain, suggesting family members (177,178). We are only beginning to under- this domain may be critical for the broad chromatin remod- stand how distinct binding and gene regulation by the p53 eling and pioneer activities of p63 and p73. What is clear is family members and their isoforms is coordinated, a bet- that the p53 family of TFs share a common ability to bind ter understanding of which may shed light on outstanding to nucleosomal DNA and mediate local chromatin remod- questions highlighted above in relation to full-length canon- eling. Continued investigation into the unique and shared ical TAp53␣. mechanisms used by members of the p53 family of TFs to Pioneer factors play crucial roles in organismal develop- facilitate chromatin remodeling is likely to reveal significant ment and differentiation events, with many pioneer factors insight into numerous biological processes and human dis- directing critical lineage determination events during devel- ease states. 12 Nucleic Acids Research, 2020

DISSECTING UBIQUITOUS AND CELL TYPE- ture of p53 binding events associated with canonical p53- SPECIFIC DIRECT p53 TARGET GENES induced genes. However, while many such events are de- tected in meta-analyses of binding sites alone (15) and their To date, identification of direct p53 target genes largely re- integration with gene expression studies (6,8,9,14), the ma- lies on correlating proximal p53 binding events, such as jority (∼70%) of p53 binding sites are detected in gene distal within 2.5 or 5 kb from the TSS, with genes significantly regions (Figure 3)(11,12,25,26,61,69,73,81). Recent analy- differentially expressed upon p53 activation. The identifica- Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 sis of the relationship between p53 binding sites and their tion is complicated by defining relevant thresholds for ro- distance to the nearest p53-regulated gene also suggested bust peak calling, distances to genes, and differential gene that p53 binding events within 2.5 kb of a TSS are much expression. Nonetheless, extensive genomic and transcrip- more likely to predict transcriptional activation and the tomic analyses have enabled the identification of common number of p53 binding events beyond this distance with human p53 target genes that exist across multiple cell con- experimentally verified trans-activation potential is low (9). texts, such as cell type, chromatin/epigenetic state, activat- Directly connecting distal elements to transcription is tech- ing signal, and time (6,8,9). These ubiquitous p53 target nically challenging and we have likely severely underesti- genes are all up-regulated in response to p53 activation sup- mated the contribution of distal p53 binding events in gene porting the view that p53 primarily functions in gene activa- regulation. Advances in computational and experimental tion (14). These ubiquitous targets could be examined in the approaches have improved our ability to make these con- context of the pioneering function of p53, especially since in nections. Genome-scale and targeted chromatin conforma- the absence of stress a number of these p53 target genes are tion capture approaches can identify potential distal p53 also pre-bound, and whether chromatin accessibility affects binding site interactions with target gene promoters. The gene transcription. Thus, ubiquitous p53 targets could pro- chromatin conformation capture (3C) approach identified vide a more effective assessment of p53 function in human a p53 binding element, which regulates the expression of cancers not based only on TP53 gene mutations, particu- three pro-apoptotic genes in Drosophila over a distance larly since p53 can be inactivated in cancers even though it is of nearly 330 kb (203). Use of the 4C (3C on chip) ap- not mutated (193). In support of this idea, it has been shown proach identified dynamic reorganization of chromatin con- that sensitivity of cell lines to an MDM2 inhibitor could be tacts across a 100kB region centered on CDKN1A after predicted by a set of 13 direct p53 target genes (194), most of p53 stabilization (204), suggesting that looping and struc- which represent ubiquitous p53 targets. Although the study ture of chromatin domains are involved in p53-dependent received critique for misclassifying the p53 status of sev- gene regulation. As previously discussed, the use of locus- eral cell lines (195), the authors amended their analyses and specific transcriptional repressors based on /CRISPR Cas9 provided evidence that after revising the annotation of cell technology revealed multiple distal p53 binding sites over lines’ p53 status, the gene set still was at least as predictive as 200kb from the nearest gene that were required for p53- the p53 status was (196). A more recent set of 175 genes pre- dependent cell cycle arrest (30), suggesting p53 can oper- dicting sensitivity to MDM2 inhibition (197) also contains ate over long genomic distances. These experimental ap- several ubiquitous p53 targets. In contrast to ubiquitous di- proaches, along with advancements in data curation such rect p53 target genes that are normally up-regulated, cell cy- as the GeneHancer, FOCS, InTAD, and HACER projects cle genes are commonly down-regulated by p53 and a recent (205–208), provide a rational basis for the future inquiry analysis of data from The Cancer Genome Atlas showed into the role of distal p53 binding sites in the regulation that, similar to most cancers (198), p53 mutant cancers dis- of gene expression. Recent efforts integrating TF binding, play up-regulation of cell cycle genes with a 4-gene subset histone modification, and gene expression profiling data promising prognostic value (193). The ubiquitous p53 tar- indicate that p53 may indeed function over long genomic get genes could also act as biomarkers to assess the effective- distances (209). Given the frequency of distal p53 binding ness of standard chemotherapeutics that lead to p53 activa- events and the general lack of information regarding their tion. The use of ubiquitous p53 target genes as biomarkers spatial configuration within the nucleus, the universe ofp53 will also be beneficial in establishing the potential efficacy target genes will inevitably become larger and its regulation of cancer therapies focused on reestablishing wild-type p53 more complex. Much work remains to understand poten- activity in cancers with mutant or aggregation-prone p53 tial longer-range regulatory events and regulation of non- (199,200). It is currently unknown the extent to which these coding RNA-species, including how topologically associat- drugs restore full, wild-type function and activate the same ing domains influence p53-dependent activities and pheno- wild-type target genes. types across cell types, tissues, and organisms. (210,211). In addition to TF binding events proximal to TSS, it is Beyond the ubiquitous p53 binding sites and target well-established that TF binding to enhancers affects the genes, unsupervised clustering demonstrates greater simi- expression of many genes (201). Within the ubiquitous set larity within one cell type than with a common treatment of p53 binding events, only ∼30% map to the proximal 5 for both DNA binding by p53 (Figure 5) and direct p53 kb surrounding annotated TSS (6). While a strong corre- target genes (6). Thus, while there are many shared binding lation between the presence of a gene proximal p53 bind- sites and target genes between cells and conditions, there are ing event and increased transcriptional activity of that gene also strong cell-type/fate differences. For example, in hu- has been reported (8,9,202), binding near a TSS appears to man embryonic stem cells (hESC) p53 binding and target be neither necessary nor sufficient for gene activation. Cer- gene activation differed between retinoic acid-induced dif- tainly, close proximity to promoters (either near the TSS or ferentiation and DNA damage conditions, suggesting the within the first intron) is a well-described and accepted fea- treatment affected cell fate to endow the p53-binding pro- Nucleic Acids Research, 2020 13 Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020

Figure 5. Hierarchical clustering of read coverage signal across p53 peaks that appear in ≥20 datasets was performed considering the Spearman correlation. Clustering results are displayed in a heatmap; the color of individual cells describes the correlation value between two datasets and the cluster distances between each sample shown using a dendrogram. Data from (6). file of a ‘different cell type’ (51). In addition, p53-dependent ceutical treatments can alter the binding and activity of p53 gene expression also differed between hESC and human (52). This study also showed that accessible chromatin dif- mesenchymal stem cells (hMSC), with the primary differ- ferences between cell types influenced p53 occupancy and ence attributed to chromatin modification patterns at p53- correlates with unique cell type-specific binding events (52). bound promoters (50). Cell type-specific chromatin accessibility is likely related to These differential effects of p53 activation are likely a the observed differential chromatin modification state at combination of tissue specific accessibility of p53 target and p53 binding sites. How chromatin accessibility and modifi- differential ‘priming’ of cell types for cell death (212), as cation state differ across cell types or after drug treatments has recently been shown with HDAC inhibitors that syner- and their influence on p53 occupancy requires more inves- gistically induce p53-dependent cell death in combination tigation. Future experiments utilizing ATAC-seq or NoMe- with Nutlin-3a or chemotherapy. This is mediated through seq (nucleosome occupancy and methylome sequencing) in HDAC inhibitors blocking activation of a subset of p53 these cells may provide more information to identify cell targets including the pro-survival caspase-8 inhibitor FLIP, type- and fate-specific features213 ( ,214). Together, these which releases the brake on ‘priming’ of pro-apoptotic tar- data suggest that p53 recruitment may be affected by ‘avail- gets TRAILR2, BAX, and PUMA (130). Similarly, treat- ability’ of p53REs defined by tissue-specific regulatory el- ment of MCF7 cells with the DNA methylation inhibitor ements expanding the repertoire of genes that can be af- decitabine led to de novo chromatin accessibility and p53 fected by p53 binding events, as has been shown for p63 binding, suggesting that changes in cell state and pharma- (12). Notably, differential gene regulation irrespective of 14 Nucleic Acids Research, 2020 p53 binding shows treatment-specific instead of cell type- pathway and to inform anti-cancer strategies (183,227,228). specific clusters (8). This is likely influenced by biological Gene regulatory networks of many TFs, however, have di- phenomena related to additional signals as well as tempo- verged substantially between mouse and human (229). For ral and treatment-specific effects. TFs investigated in the ENCODE project only an average In vivo studies in Drosophila and mice demonstrate of 44% of regulatory TF to gene associations have been tissue-specific transcriptional responses after exposure to identified as conserved between mouse and human (224). similar p53-activating stimuli (215,216). The overlap in p53- Consistently, early studies comparing p53REs of several di- Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 dependent gene expression is minimal between Drosophila rect p53 target genes identified only limited conservation tissues (215), suggesting a permissive role for cell type in across species (230,231). The small number of genes re- the p53 response. Similarly, p53 activation in disparate tis- ported to differ in their p53-dependent regulation between sue types in the developing mouse embryo yielded different mice and humans included the well-established p53 targets cell fates, such as the classical choice between apoptosis and GADD45A, RRM2B, DDB2, APAF1, XPC, and PCNA, cell cycle arrest, but regulates the same set of genes in dif- which were identified as direct p53 targets in human (231– ferent mouse tissues (216). Intriguingly, the magnitude of 234) but not in mouse (231,234). Another example includes p53-dependent gene induction or repression appears to be the LIF gene that functions in reproduction and that is a a major difference between the tissues and may play a role direct p53 target gene in human (235), but not in elephants in the disparity in cell phenotypes. Developmental timing (236). Instead, elephant p53 directly regulates the LIF pseu- can also alter p53-dependent target gene expression and cell dogene LIF6, which functions in apoptosis rather than in fates. Pre-migratory and migrating neural crest are highly reproduction (236). Elephants also possess up to 18 copies sensitive to p53-dependent apoptosis, whereas those cells of p53 pseudogenes which are under positive selection, sug- that reached their final destination in the pharyngeal arch gesting functionality in the evolution of cancer resistance in were highly resistant to apoptosis triggered by p53 (216). this long-lived mammal (237,238). In blind mole rats, p53 These data suggest that some of the differences between carries an R174K substitution identical to known tumor- p53-dependent outcomes may be due to developmental tim- associated mutations and is unable to induce multiple pro- ing and differences in p53 target gene activation between cell apoptotic genes, such as APAF1, but hyperactivates cell cy- types, both of which are likely influenced by gene regulatory cle arrest genes. Presumably, this enables blind mole rat cells elements and local chromatin structure. to favor a reversible cell cycle arrest over hypoxia-induced In a similar fashion to chromatin structure, DNA methy- apoptosis (239). A recent study provided a meta-analysis lation likely plays a regulatory role in the control of p53- of genome-wide p53 binding data and p53-dependent tran- dependent transcription. Among the occupied sites that scriptome regulation data from mouse and compared the are common across cell types and near TSS, 40% are mouse data to similar data of human. Strikingly, >1000 not associated with differential expression of the nearest genes have been identified to differ substantially in their re- gene, suggesting that p53 binding alone in many cases is sponse to p53 activation and these genes largely function not sufficient to drive expression of the nearest gene but in DNA damage response, metabolism, and cell cycle reg- that additional information and regulatory mechanisms can ulation (20). Thus, the p53-dependent regulation of these be required (6). These might include mechanisms regu- processes may at least partially differ between mouse and lating mRNA half-life, protein degradation, and protein human. folding/activation. For example, the Toll like receptor-8 Mechanistically, the comprehensive comparison of the (TLR8) gene, which is a direct p53 target, is undetectable p53 GRN between mouse and human revealed that evo- in cancer cells possibly because of promoter methylation lutionary turnover of p53REs underlies broad variation in (personal observation). Other direct p53 targets such as p53 binding profiles that are causative for different p53- IGFBP7 (217), 14–3-3 σ (also known as SFN)(218,219), dependent gene up-regulation (20). Concordantly, evolu- and the long non-coding RNA TP53TG1 (220) have also tionary turnover of p63REs and p63 binding was found been shown to be hypermethylated in cancer. Additionally, to substantially alter the p63 GRN (240). Taking only ge- viruses may also use this mechanism to silence downstream nomic regions into account that are present in both hu- p53 targets (221), suggesting that methylation or other epi- man and mouse, the meta-analysis found less than 13% of genetic modifications might affect differential cell type re- p53 binding sites to be conserved between the two species sponses to p53 activation. (20). This number is even smaller when also mouse- and human-specific genome regions are considered. For exam- ple, it was reported that p53REs can be shaped by long ter- EVOLUTION OF THE p53 GENE REGULATORY NET- minal repeats from endogenous retroviruses (34,35), long WORK IN VERTEBRATES interspersed nuclear repeats (LINEs; (37)), and ALU re- All p53 protein family members contain a highly conserved peats (36,38) in humans and fuzzy tandem repeats in mice DBD (222), and p53 homologs from multiple species have (163,241). It was shown that p53 oscillates faster in mouse been shown to specifically trans-activate common p53REs and rat cells than in cells from human, monkey, or dog. (223). Similar to p53 family proteins, most TFs display It is hypothesized that faster p53 oscillations in mouse evolutionary conservation of their sequence-specific DNA cells are triggered by a stronger negative feedback loop be- binding (224–226). Conservation of protein function is key tween p53 and MDM2, which in turn may be caused by a when animal models are employed to understand mecha- subtle change in the p53RE controlling the expression of nisms that underlie human diseases. In case of p53, mouse MDM2 (242). Different p53 oscillation patterns influence models are frequently used to unravel the p53 signaling p53-dependent gene expression (74),buttowhatextentthe Nucleic Acids Research, 2020 15 different p53 oscillation patterns between mouse and hu- edly a tumor suppressor across mammals, the specific genes man contributes to differences in the p53 GRN remains un- and regulatory networks required for this broad activity are clear. Interestingly, p53 and p63 binding sites have under- quite different. Consequently, single nucleotide variations gone more evolutionary turnover as compared to binding in p53REs and p53 mutants could have very different ef- sites of the DREAM complex (20) or most TFs that have fects in humans than in mice. In turn, a drug acting on the been investigated by ENCODE (224). A possible explana- p53 signaling pathway could affect a mouse very differently tion for the high turnover could be the exceptionally large than a human. If we want to translate findings from ani- Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 size of the p53 and p63 REs, which contain two decameric mal models to humans more efficiently, we have to under- half sites totaling 20 bp. TF recognition motifs that exceed stand their differences more precisely. The marked differ- 10 base pairs show substantially decreased evolutionary sta- ences that have been revealed encourage the use of advanced bility (243,244), and thus the long p53 and p63 REs may be animal and cell culture models. While in clinical research altered more rapidly during evolution. patient derived xenografts and humanized mouse models The lack of evolutionary conservation of a number of help to narrow the gap between animal and human, in ba- important direct p53 target genes suggests that regulating sic research organoids are promising tools to generate more these individual genes may not be critical or can be com- meaningful data. pensated by alternative p53-regulated genes in the same pathway to conserve p53’s tumor suppressor function. For CONCLUSION example, trans-activation-mutants of p53 that could up- regulate only subsets of direct p53 target genes were suffi- The last decade of research has greatly expanded our un- cient to suppress spontaneous tumor development in mice derstanding of how p53 functions as a transcription factor (245). Similarly, mice expressing a triple acetylation mutant and shed light on the composition of its GRN, but as high- form of p53 (p533KR), which is unable to up-regulate many lighted above, it is clear that much remains to be uncovered direct p53 target genes, are also protected from spontaneous to fully understand the molecular mechanisms that shape tumor development (246). Interestingly, a triple-knockout the p53 GRN in a given cell or tissue. Further investigation of the conserved key p53 target genes p21 (also known as of the role of distal p53 binding sites in the regulation of Cdkn1A), Puma (Bbc3), and Noxa (Pmaip1) was not suffi- gene expression, especially the role of local and long-range cient to impair p53’s tumor suppressor function (247). In enhancer:gene interactions, will be necessary to fully assess addition to the set of 86 evolutionary conserved direct p53 the direct transcriptional effects elicited upon p53 binding target genes that has been identified (20), species-specific to DNA (Figure 3D). Future work on identifying TFs coop- direct p53 targets may contribute substantially to the tu- erating with p53 to establish transcriptional networks, such mor suppressor function of p53. Particularly noteworthy as those working downstream of p53 activation, will expand are mouse- and human-specific direct p53 target genes that our understanding of the expansive gene regulatory reper- can serve a similar function in the p53 response. The Y- toire (Figure 1). family translesion DNA synthesis polymerases POLH and Despite the vast body of work, it is still unclear how POLK can both allow for DNA replication despite the pres- p53 activation induces differential cell fates at a single cell ence of DNA damage (248). In human solely POLH and level and how this is affected by cellular identity, stage of in mouse solely Polk was identified as direct p53 target cell cycle, and concurrent activation of other stress induced gene (20). Together the findings suggest that many species- survival/death programs. This will inevitably be enlight- specific direct p53 target genes may be dispensable forthe ened by studies using cutting edge technologies to evalu- tumor suppressor function of p53, and in some cases p53 ate single-cell transcriptional dynamics. Such studies cou- employs different species-specific targets to serve a similar pled with other high throughput modalities like modern function. However, it is important to note that only limited 3D organoid culture will be crucial to dissect cell/tissue analyses of epithelial tissue and in tumor initiating contexts and condition/treatment-specific p53 activity. This will in have been conducted to date in mouse and our assumptions turn help us to understand how p53, its isoforms, and fam- of target conservation may as a result be slightly underesti- ily members function as pioneer factors and in other non- mated. canonical roles associated with their interaction with the Mouse models serve as the gold standard in basic can- genome. Cumulatively, these insights will allow us to better cer research, but it is known that their translation po- understand what these diverse activities of p53 might mean tential to human is limited (249–251). For example, mice for its tumor suppressor and non-tumor suppressor role, en- expressing p533KR displayed impaired apoptosis, cell cy- able us to exploit p53 de-regulation in cancer; as well as pro- cle arrest, and senescence but retained the ability to regu- viding a more complete picture on emergent roles for the late metabolic genes, which may have protected these mice p53 family in cell lineage commitment, cell lineage transi- from spontaneous tumor development (246). However, p53- tions in cancer, stem cell reprogramming, and developmen- regulated genes involved in metabolic control have been par- tal disorders. ticularly subjected to alterations in their p53 response be- tween mouse and human (20). Thus, p53’s ability to con- ACKNOWLEDGEMENTS trol metabolic processes is different in human compared to mouse and may not be sufficient to suppress tumor forma- We thank Carl W.Anderson and Daniel Menendez for crit- tion in humans. ical feedback on the manuscript. We thank Sara A. Grimm The assumption with mouse models has been that p53 and C. Andy Lavender of the Integrative Bioinformatics functions largely similar to human. While p53 is undoubt- core at NIEHS for assistance with Figure 5 and Table 1. 16 Nucleic Acids Research, 2020

FUNDING its direct targets and unexpected regulatory mechanisms. Elife, 3, e02200. M.A.S. is supported by the National Institutes of Health 14. Fischer,M., Steiner,L. and Engeland,K. (2014) The transcription (NIH) [R15 GM128049]; T.T.N. and her research was factor p53: Not a repressor, solely an activator. Cell Cycle, 13, supported by the Intramural Research Program of the 3037–3058. 15. Verfaillie,A., Svetlichnyy,D., Imrichova,H., Davie,K., Fiers,M., NIH, National Institute of Environmental Health Sci- Atak,Z.K., Hulselmans,G., Christiaens,V. and Aerts,S. (2016) ences (NIEHS) [Z01-ES065079, Z01-ES100557, in part]; Multiplex enhancer-reporter assays uncover unsophisticated TP53 Downloaded from https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa666/5892750 by University at Albany user on 14 August 2020 S.S.M. is supported by Cancer Research UK (CRUK) enhancer logic. Genome Res., 26, 882–895. [C11884/A24367]; Biotechnology and Biological Sciences 16. 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