IER5, a DNA damage response gene, is required for Notch-mediated induction of squamous cell differentiation

  1. Li Pan
  2. Madeleine E Lemieux
  3. Tom Thomas
  4. Julia M Rogers
  5. Colin H Lipper
  6. Winston Lee
  7. Carl Johnson
  8. Lynette M Sholl
  9. Andrew P South
  10. Jarrod A Marto
  11. Guillaume O Adelmant
  12. Stephen C Blacklow
  13. Jon C Aster  Is a corresponding author
  1. Department of Pathology, Brigham and Women’s Hospital, and Harvard Medical School, United States
  2. Bioinfo, Canada
  3. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, United States
  4. Department of Dermatology and Cutaneous Biology, Sidney Kimmel Medical College, Thomas Jefferson University, United States
  5. Departmentof Oncologic Pathology and Blais Proteomics Center, Dana FarberCancer Institute, HarvardMedical School, United States
7 figures, 3 tables and 10 additional files

Figures

Figure 1 with 3 supplements
Notch activation induces growth arrest and differentiation of squamous carcinoma cells.

(A) Strategy used to activate Notch in a tightly regulated fashion. (B) Notch-induced suppression of SC2 cell growth in standard cultures is abrogated by DN-MAML, a specific inhibitor of canonical …

Figure 1—figure supplement 1
Notch activation induces differentiation and growth arrest of the squamous carcinoma cell lines IC8 and SCCT2.

(A, B) Effects of GSI and sham washout on the growth of IC8 and SCCT2 cells transduced with either empty vector (pBABE) or ΔEGF-L1596H. Cell numbers at various times post-GSI washout (DMSO) or sham …

Figure 1—figure supplement 2
Characterization of clones derived from single IC8 cells transduced with ΔEGF-L1596H.

(A) Western blot showing ICN1 levels 4 hr post-GSI washout in IC8-ΔEGF-L1596H clones. (B) Effects of Notch activation on growth of IC8-ΔEGF-L1596H clones. Cell numbers at various times post-GSI …

Figure 1—figure supplement 3
Immunohistochemical assessment of p63, BCL6, keratin5 (KRT5), and filaggrin (FLGR) protein levels in S2 cells grown in the Notch-off (GSI) or Notch-on (WO) states in 3D rafts.
Figure 2 with 2 supplements
Identification of Notch-induced genes in squamous carcinoma cells.

(A) Volcano plots showing changes in RNA transcript read counts induced by Notch activation in SC2 cells for 4, 24, and 72 hr as compared to control cells treated with sham GSI washout. RNA-seq for …

Figure 2—figure supplement 1
GSI treatment has little effect on gene expression in IC8 squamous carcinoma cells.

Duplicate cultures of IC8 cells were treated with GSI or vehicle (DMSO) for 24 hr and transcript abundance was assessed by RNA-seq in two biological replicates. A volcano plot shows no …

Figure 2—figure supplement 2
Venn diagram showing the overlap in Notch target genes (defined as log2 change >1 and FDR < 0.05 following GSI washout) in IC8 squamous carcinoma cells (S2 subclone), MB157 triple negative breast carcinoma cells (Petrovic et al., 2019), REC1 mantle cell lymphoma cells (Ryan et al., 2017), and DND-41 T-ALL cells (Petrovic et al., 2019).

The lists of overlapping genes are provided in Supplementary file 6.

Characterization of Notch transcription complex (NTC) binding sites in IC8-ΔEGF-L1596H cells.

(A) Number and overlap of RBPJ and MAML1 binding sites determined by ChIP-Seq of chromatin prepared 4 hr after Notch activation. (B) Genomic distribution of RBPJ/MAML1 co-binding sites 4 hr after …

Figure 4 with 1 supplement
IER5 is a direct Notch target gene.

(A) Chromatin landscapes around IER5 in IC8-ΔEGF-L1596H cells. ChIP-Seq signals for RBPJ, MAML1, and H3K27ac for cells maintained in and 4 hr after GSI washout (WO) are shown. (B, C) Activities of a …

Figure 4—figure supplement 1
IER5 is regulated by Notch in non-transformed keratinocytes.

(A, B) TERT-immortalized NOK1 keratinocytes transduced with either empty MigRI retrovirus or MigRI encoding dominant negative MAML1 (DN-MAML) were grown in low Ca2+ medium (Day 0) or were shifted to …

Effect of IER5 on Notch-dependent changes in gene expression in SC2 cells and NOK1 cells.

(A) Western blots showing IER5 and ICN1 protein levels in SC2 cells, a single-cell clone derived from SC2-IER5 knockout cells (I5KO), I5KO cells transduced with empty virus (I5/con), and pooled I5KO …

IER5 binds to B55α.

(A) Polypeptides identified by mass spectroscopy in immunoprecipitates prepared from I5 cells expressing tandem-tagged IER5. (B) Cartoon showing the structure of tandem-tagged IER5 polypeptides. FH, …

PPP2R2A is epistatic to IER5.

(A) Western blot showing IER5 and B55α protein levels in single (KO) and double (DKO) PPP2R2A and IER5 knockout clones in the presence of GSI (-) and 4 hr after GSI washout (+). (B) PPP2R2A knockout …

Tables

Table 1
Sequence variants, IC8* and SCCT2** squamous cell carcinoma cell lines.
GeneVariantVariant allele frequency
IC8 cell
CASP8c.971T > C(p.M324T)66% of 411 reads
FBXW7c.1633T > C(p.Y545H)33% of 195 reads
KMT2Dc.7412G > A(p.R2471Q)42% of 255 reads
MGAc.5599G > A(p.V1867I)39% of 710 reads
MTORc.4828G > A(p.E1610K)56% of 280 reads
NOTCH1c.5059C > T (p.Q1687*)100% of 412 reads
PAXIP1c.2023C > T(p.H675Y)17% of 384 reads
PMS1c.566_567delTCinsAT(p.V189D)36% of 108 reads
RIF1c.658G > A(p.E220K)62% of 251 reads
ROS1c.1144T > G(p.Y382D)87% of 169 reads
ROS1c.1164+2_1164+8delTTAGTCC ()19% of 191 reads
SDHAc.1627T > C(p.Y543H)56% of 668 reads
SF3B1c.2549T > C(p.I850T)31% of 246 reads
TERTCC242-243TT promoter mutation50% of 26 reads
TP53c.451C > T(p.P151S)100% of 366 reads
WHSC1c.2185C > T(p.R729C)66% of 410 reads
WWTR1c.551T > G (p.V184G)64% of 256 reads
ZNF217c.2590C > T(p.L864F)39% of 835 reads
ZNF217c.1162delC(p.H388Tfs*77)55% of 822 reads
SCCT2 Cell
ALKc.2854G > A (p.G952R)50% of 441 reads
ASXL1c.3959C > T (p.A1320V)31% of 930 reads
BRD3c.533C > T (p.S178F)49% of 281 reads
BRD4c.3915_3917dupTGC (p.A1306dup)45% of 170 reads
CDH4c.1801C > T (p.L601F)30% of 447 reads
CDKN2Ac.*151–1G > A ()100% of 172 reads
CDKN2Ac.212A > T (p.N71I)100% of 184 reads
CREBBPc.5842C > T (p.P1948S)74% of 77 reads
CREBBPc.2116G > A (p.G706R)45% of 172 reads
DDB1c.327+6G > A ()47% of 451 reads
DICER1c.775C > T (p.P259S)42% of 301 reads
DOCK8c.185T > A (p.V62E)100% of 597 reads
EGFRc.1955G > A (p.G652E)48% of 518 reads
EGFRc.298C > T (p.P100S)49% of 595 reads
ERCC2c.886A > T (p.S296C)48% of 165 reads
ERCC5c.264+1G > A ()50% of 442 reads
ETV4c.1298C > G (p.P433R)45% of 302 reads
FANCFc.494C > T (p.T165I)50% of 644 reads
FANCLc.155+1G > A ()51% of 220 reads
FAT1c.9076–1G > A ()49% of 367 reads
FHc.681G > T (p.Q227H)4% of 756 reads
FLT4c.2224G > A (p.D742N)49% of 346 reads
GALNT12c.1035+5G > A ()52% of 523 reads
GLI2c.1859C > A (p.T620K)45% of 351 reads
HNF1Ac.1640C > T (p.T547I)54% of 392 reads
JAZF1c.477C > T (p.I159I)47% of 606 reads
JAZF1c.328C > T (p.P110S)44% of 211 reads
KMT2Dc.10355+1G > A ()49% of 622 reads
LIG4c.1271_1275delAAAGA (p.K424Rfs*20)40% of 659 reads
MAP2K1c.568+1G > A ()53% of 239 reads
MED12c.2080G > A (p.E694K)100% of 269 reads
MYBc.1461+5G > A ()41% of 430 reads
NF1c.2608G > A (p.V870I)50% of 615 reads
NF2c.813T > G (p.F271L)48% of 168 reads
NOTCH1c.1226G > T (p.C409F)44% of 519 reads
NOTCH1c.1406A > G (p.D469G)50% of 912 reads
NOTCH1c.1245G > T (p.E415D)42% of 495 reads
NOTCH2c.5252G > A (p.G1751D)44% of 459 reads
NOTCH2c.1298G > A (p.C433Y)50% of 484 reads
NOTCH2c.1108+1G > A ()53% of 305 reads
NSD1c.7669G > A (p.G2557R)49% of 743 reads
PDGFRBc.2586+2T > A ()43% of 380 reads
PHOX2Bc.181A > T (p.T61S)52% of 222 reads
POLQc.6565G > A (p.A2189T)27% of 462 reads
POLQc.1634G > A (p.S545N)33% of 667 reads
PPARGc.819+6T > C ()100% of 134 reads
PRKDCc.6436G > A (p.A2146T)42% of 471 reads
RAD51Cc.996G > A (p.Q332Q)45% of 302 reads
RHEBc.443C > T (p.S148F)46% of 120 reads
ROS1c.6871C > T (p.P2291S)45% of 605 reads
ROS1c.3342A > T (p.Q1114H)48% of 274 reads
ROS1c.137A > T (p.D46V)42% of 215 reads
RPTORc.2992G > A (p.V998I)48% of 352 reads
RUNX1T1c.1039G > A (p.D347N)45% of 715 reads
SDHAc.1151C > T (p.S384L)53% of 446 reads
SLC34A2c.1700T > A (p.I567N)50% of 460 reads
SMARCA4c.3947T > G (p.F1316C)55% of 431 reads
SMARCE1c.395C > T (p.A132V)51% of 587 reads
STAT3c.1852G > A (p.G618S)47% of 527 reads
TDGc.166+4G > A ()48% of 329 reads
TP53c.375+1G > T47% of 173 reads
TP53c.832_833delCCinsTT (p.P278F)46% of 418 reads
UIMC1c.971T > C (p.V324A)48% of 745 reads
XPCc.571C > T (p.R191W)100% of 219 reads
  1. *Based on analysis of 16,131,317 unique, high-quality sequencing reads (mean, 406 reads per targeted exon, with 98% of exons having more than 30 reads).

    Based on analysis of 20,972,158 unique, high-quality sequencing reads (mean, 413 reads per targeted exon, with 99% of exons having more than 30 reads).

Table 2
Copy number variants, squamous cell carcinoma cell lines.
ChromosomeTypeGenes affected
IC8 cell line
1qGainMCL1, GBA, RIT1, NTRK1, DDR2, PVRL4, SDHC, CDC73, MDM4, PIK3C2B, UBE2T, PTPN14, H3F3A, EGLN1, AKT3, EXO1, FH
2LossXPO1, FANCL, REL, MSH6, EPCAM, MSH2, SOS1, ALK, BRE, DNMT3A, GEN1, MYCN, TMEM127, GLI2, ERCC3, CXCR4, RIF1, ACVR1, ABCB11, NFE2L2, PMS1, CASP8, SF3B1, CTLA4, ERBB4, IDH1, BARD1, XRCC5, DIS3L2
3pLossMITF, BAP1, PBRM1, COL7A1, RHOA, SETD2, CTNNB1, MLH1, MYD88, XPC, PPARG, RAF1, FANCD2, OGG1, VHL
3qGainNFKBIZ, CBLB, POLQ, GATA2, MBD4, TOPBP1, FOXL2, ATR, MECOM, PRKCI, TERC, PIK3CA, SOX2, ETV5, BCL6
4LossPHOX2B, RHOH, SLC34A2, FGFR3, WHSC1, KDR, KIT, PDGFRA, FAM175A, HELQ, TET2, FBXW7, NEIL3, FAT1
5GainRICTOR, IL7R, SDHA, TERT, MAP3K1, PIK3R1, XRCC4, RASA1, APC, RAD50, CTNNA1, PDGFRB, ITK, NPM1, TLX3, FGFR4, NSD1, UIMC1, FLT4
6GainCCND3, NFKBIE, POLH, VEGFA, CDKN1A, PIM1, RNF8, FANCE, DAXX, HFE, HIST1H3B, HIST1H3C, ID4, PRDM1, ROS1, RSPO3, MYB, TNFAIP3, ESR1, ARID1B, PARK2, QKI
7GainEGFR, IKZF1, JAZF1, ETV1, PMS2, RAC1, CARD11, SBDS, CDK6, SLC25A13, CUX1, RINT1, MET, POT1, SMO, BRAF, PRSS1, EZH2, RHEB, XRCC2, PAXIP1
8pLossKAT6A, POLB, FGFR1, WHSC1L1, NRG1, WRN, NKX3-1, PTK2B, GATA4, NEIL2
8q11.21-q21.11LossPRKDC, MYBL1, TCEB1
8q21.3-q24.3GainNBN, RUNX1T1, RAD54B, RSPO2, EXT1, RAD21, MYC, RECQL4
9p13.2-p21.3LossPAX5, FANCG, RMRP, CDKN2A, CDKN2B, MTAP
9p24.1-p24.3GainCD274, JAK2, PDCD1LG2, DOCK8
11p11.2-p13GainEXT2, LMO2
13q33.1LossERCC5
15qGainFAN1, GREM1, BUB1B, MGA, RAD51, TP53BP1, B2M, USP8, MAP2K1, PML, NEIL1, FAH, NTRK3, BLM, FANCI, IDH2, IGF1R
16p13.3LossCREBBP, SLX4
19LossBABAM1, CRTC1, JAK3, KLF2, MEF2B, BRD4, NOTCH3, CALR, KEAP1, SMARCA4, ELANE, GNA11, MAP2K2, STK11, TCF3, CCNE1, C19orf40, CEBPA, AKT2, AXL, CIC, XRCC1, ARHGAP35, ERCC1, ERCC2, BCL2L12, PNKP, POLD1, PPP2R1A
20GainMCM8, ASXL1, BCL2L1, MAFB, AURKA, ZNF217, GNAS, CDH4
SCCT2 Cell Line
1q32.1LossUBE2T
1q42.12-q42.2GainH3F3A, EGLN1
1q43LossAKT3, EXO1
1q43GainFH
3 p Arm levelLossMITF, BAP1, PBRM1, COL7A1, RHOA, SETD2, CTNNB1, MLH1, MYD88, XPC, PPARG, RAF1, FANCD2, OGG1, VHL
3q Arm levelGainNFKBIZ, CBLB, POLQ, GATA2, MBD4, TOPBP1, FOXL2, ATR, MECOM, PRKCI, TERC, PIK3CA, SOX2, ETV5, BCL6
8q Arm levelGainPRKDC, MYBL1, TCEB1, NBN, RUNX1T1, RAD54B, RSPO2, EXT1, RAD21, MYC, RECQL4
9q Arm levelGainGNAQ, NTRK2, FANCC, PTCH1, GALNT12, XPA,
KLF4, TAL2, ENG, ABL1, TSC1, BRD3, NOTCH1
18q11.2GainGATA6, RBBP8
18q11.2-q21.33GainSS18, SETBP1, SMAD2, SMAD4, BCL2
20GainMCM8, ASXL1, BCL2L1, MAFB, AURKA, ZNF217,
GNAS, CDH4
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyRabbit monoclonal anti-MAML1Cell Signaling TechnologyCat. #: 12166ChIP, 1 ml per1 × 106 cells
AntibodyRabbit monoclonal anti-RBPJCell Signaling TechnologyCat. #: 5313ChIP, 2.5 ml per1 × 106 cells
AntibodyRabbit polyclonal anti-histone H3 acetyl K27AbcamCat. #: ab4729ChIP, 9 ml per1 × 106 cells
AntibodyMouse monoclonal anti-involucrinSigmaCat. #: I9018IF, 1:500; IHC, 1:10,000
AntibodyRabbit polyclonal anti-plakophilin-1SigmaCat. #: HPA027221IF, 1:300; IHC, 1:500
AntibodyRabbit monoclonal anti-activated NOTCH1 (ICN1)Cell Signaling TechnologyCat. #: 4147IHC, 1:50; WB, 1:1000
AntibodyRabbit monoclonal anti-keratin-1AbcamCat. #: ab185628IHC, 1:1000
AntibodyRabbit monoclonal anti-Ki-67BiocareCat. #: CRM325IHC, 1:100
AntibodyMouse monoclonal anti-B55aCell Signaling TechnologyCat. #: 5689WB, 1:1000
AntibodyRabbit monoclonal anti-NOTCH2Cell Signaling TechnologyCat. #: 5732WB, 1:1000
AntibodyRabbit monoclonal anti-NOTCH3Cell Signaling TechnologyCat. #: 5276WB, 1:1000
AntibodyRabbit monoclonal anti-GADD45ACell Signaling TechnologyCat. #: 4632WB, 1:1000
AntibodyRabbit monoclonal anti-ID3Cell Signaling TechnologyCat. #: 9837WB, 1:1000
AntibodyHorse polyclonal anti-mouse Ig linked to HRPCell Signaling TechnologyCat. #: 7076WB, 1:1,000-1:20,000
AntibodyGoat polyclonal anti-rabbit Ig linked to HRPCell Signaling TechnologyCat. #: 7074WB, 1:1000
AntibodyMouse monoclonal anti-actinSigmaCat. #: A1978WB, 1:10,000
AntibodyMouse monoclonal anti-FLAGSigmaCat. #: F3165WB, 1:1000
AntibodyRabbit polyclonal anti-IER5SigmaCat. #: HPA029894WB, 1:1000
AntibodyMouse monoclonal anti-filaggrinSanta Cruz BiotechnologyCat. #: sc-66192IHC, 1:100
AntibodyMouse monoclonal anti-p63Biocare MedicalCat. #: CM163AIHC, 1:250
AntibodyRabbit polyclonal anti-loricrinBioLegendCat. #: 905103IHC, 1:800
AntibodyMouse monoclonal anti-BCL6Cell Marque Tissue DiagnosticsCat. #: 227 M-95IHC, 1:500
AntibodyRabbit monoclonal anti-keratin5Cell Signaling TechnologyCat. #: 71536IHC, 1:2000
AntibodyChicken polyclonal anti-keratin14BioLegendCat. #: 906004IHC, 1:800
AntibodyChicken polyclonal anti-SUMOLifesensorsCat. #: AB7002WB, 1:2000
AntibodySheep polyclonal anti-rabbit Ig linked to DynabeadsThermoFisher ScientificCat. #: 11203DChIP, 100 μl beads per 20 × 106 cells
AntibodyMouse monoclonal anti-FLAG epitope linked to magnetic beadsSigmaCat #: M8823Tandem purification,40 μl to 1 ml beads
Cell line (Homo sapiens)IC810.1038/s41467-018-06027-1Dr. Andrew South (Thomas Jefferson University)
Cell line (H. sapiens)SCCT210.1038/s41467-018-06027-1Dr. Andrew South (Thomas Jefferson University)
Cell line (H. sapiens)NOK1Piboonniyom et al., 2003; 63:476–83Dr. Karl Munger (Tufts University)
Commercial assay or kitCellTiter BluePromegaCat. #: G8080
Commercial assay or kitChIP Assay KitMilliporeCat. #: 17–295
Commercial assay or kitNext Ultra II DNA Library Prep KitNew England BioLabsCat. #: E7645
Commercial assay or kitNext Ultra II RNA Library Prep KitNew England BioLabsCat. #: E7775
Commercial assay or kitQuickChange II KitAgilent TechnologiesCat. #: 200523
Commercial assay or kitDual Luciferase KitPromegaCat. #: E1910
Chemical compound, drugCompound ETocrisCat. #: CAS 209986-17-4
Recombinant DNA reagentpL-CRISPR.
SFFV.GFP
AddgeneCat. #: #57827
Recombinant DNA reagentpL-CRISPR.SFFV.tRFPAddgeneCat. #: #57826
Recombinant DNA reagentlentiCRISPRv2 neoAddgeneCat. #: 98292
Recombinant DNA reagentlentiCRISPRv2 hygroAddgeneCat. #: 98291
Recombinant DNA reagentpVL1392Expression SystemsCat. #: 91–012

Additional files

Supplementary file 1

Differentially expressed genes after 4 hr of Notch activation, SC2 cells.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp1-v2.xls
Supplementary file 2

Differentially expressed genes after 24 hr of Notch activation, SC2 cells.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp2-v2.xls
Supplementary file 3

Differentially expressed genes after 72 hr of Notch activation, SC2 cells.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp3-v2.xls
Supplementary file 4

Unclustered GO annotations of Notch-sensitive genes, SC2 cells, at 72 hr of Notch activation.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp4-v2.xls
Supplementary file 5

Clustered GO annotations of Notch-sensitive genes, SC2 cells, at 72 hr of Notch activation.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp5-v2.xls
Supplementary file 6

Comparison of Notch-responsive genes in SC2 cells, MB157 triple-negative breast cancer cells, REC1 mantle cell lymphoma cells, and DND41 T-cell acute lymphoblastic leukemia cells.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp6-v2.xlsx
Supplementary file 7

Unclustered GO annotations of IER5-dependent Notch-sensitive genes, SC2 cells.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp7-v2.xls
Supplementary file 8

Clustered GO annotations of IER5-dependent Notch-sensitive genes, SC2 cells.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp8-v2.xls
Supplementary file 9

IER5-interacting proteins identified by tandem affinity purification.

https://cdn.elifesciences.org/articles/58081/elife-58081-supp9-v2.xlsx
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