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

Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in long- range enhancers. Among the direct Notch target genes are multiple DNA damage response genes, including IER5, which is regulated by Notch through several enhancer elements. We show that IER5 is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic to PPP2R2A, which encodes the B55α subunit of PP2A, and IER5 interacts with B55α in cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers. Impact Our findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.


Abstract
Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in longrange enhancers. Among the direct Notch target genes are multiple DNA damage response genes, including IER5, which is regulated by Notch through several enhancer elements. We show that IER5 is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic to PPP2R2A, which encodes the B55a subunit of PP2A, and IER5 interacts with B55a in cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers.

Introduction
Notch receptors participate in a conserved signaling pathway in which successive ligandmediated proteolytic cleavages by ADAM10 and g-secretase permit intracellular Notch (ICN) to translocate to the nucleus and form a Notch transcription complex (NTC) with the DNA binding factor RBPJ and co-activators of the Mastermind-like (MAML) family (for review, see (Bray, 2016)). Outcomes of Notch activation are dose and cell context-dependent, in part because most Notch response elements lie within lineage-specific enhancers (Castel et al., 2013;Ryan et al., 2017;Skalska et al., 2015;H. Wang et al., 2014). As a result, Notch-dependent transcriptional programs vary widely across cell types.
The context-dependency of outcomes produced by Notch signaling is reflected in the varied patterns of Notch mutations that are found in different cancers (for review, see (Aster, Pear, & Blacklow, 2017)). In some cancers oncogenic gain-of-function Notch mutations predominate, but in human cutaneous squamous cell carcinoma (SCC) (South et al., 2014; N. J. Wang et al., 2011) loss-of-function mutations are common, early driver events, observations presaged by work showing that loss of Notch function promotes skin cancer development in mouse models (Nicolas et al., 2003;Proweller et al., 2006). The mechanism underlying the tumor suppressive effect of Notch appears to involve its ability to promote squamous differentiation at the expense of self-renewal, a function that is operative in other squamous epithelia (Alcolea et al., 2014), where Notch also has tumor suppressive activities (Agrawal et al., 2011;Agrawal et al., 2012;Loganathan et al., 2020). In line with this idea, conditional ablation of Notch1 in postnatal mice results in epidermal hyperplasia and expansion of proliferating basal-like cells (Nicolas et al., 2003;Rangarajan et al., 2001). Moreover, murine and human b-papilloma viruses express E6 proteins that target MAML1 and inhibit Notch function (Meyers, Uberoi, Grace, Lambert, & Munger, 2017;Tan et al., 2012), thereby causing epidermal hyperplasia and delayed differentiation of infected keratinocytes. Conversely, constitutively active forms of Notch enhance keratinocyte differentiation in vitro and in vivo (Nickoloff et al., 2002;Rangarajan et al., 2001;Uyttendaele, Panteleyev, de Berker, Tobin, & Christiano, 2004).
While these studies delineate a pro-differentiation, tumor suppressive role for Notch in squamous cells, little is known about the Notch target genes that confer this phenotype. Work to date has focused on candidate genes chosen for their known activities in keratinocytes or their roles as Notch target genes in other cell types. These include CDKN1A/p21 (Rangarajan et al., 2001), which has been linked to cell cycle arrest and differentiation (Missero, Di Cunto, Kiyokawa, Koff, & Dotto, 1996); HES1, which represses basal fate/self-renewal (Blanpain, Lowry, Pasolli, & Fuchs, 2006); and IRF6, expression of which positively correlates with Notch activation in keratinocytes (Restivo et al., 2011). However, dose-and time-controlled genomewide studies to determine the immediate, direct effects of Notch activation in squamous-lineage cells have yet to be performed.
To this end, we developed and validated 2D and 3D culture models of malignant and non-transformed human squamous epithelial cells in which regulated Notch activation produces growth arrest and squamous differentiation. We find that immediate, direct Notch target genes are largely keratinocyte-specific and are associated with lineage-specific NTC-binding enhancers enriched for the motifs of transcription factors linked to regulation of keratinocyte differentiation, particularly AP1. Among these genes are multiple genes previously shown to be upregulated by DNA damage and cell stress, including IER5, a member of the AP1-regulated immediate early response gene family (Williams et al., 1999). Here we show that IER5 is required for Notch-induced differentiation of human SCC cells and TERT-immortalized human keratinocytes, and that this requirement is abolished by knockout of the B55a regulatory subunit of PP2A, to which IER5 directly binds. Our studies provide the first genome-wide view of the effects of Notch on gene expression in cutaneous squamous carcinoma cells, highlights previously unrecognized crosstalk between Notch and DNA response genes, and point to the existence of a Notch-IER5-PP2A signaling axis that coordinates keratinocyte differentiation.

Establishment of a Conditional Notch-on SCC Model
Timed, regulated ligand-mediated Notch activation is difficult to achieve because soluble Notch ligands do not induce signaling (Sun & Artavanis-Tsakonas, 1997). To bypass this limitation, we previously developed a simple method that relies on g-secretase inhibitor (GSI) washout ( Figure   1A), which delivers ICN to the nuclei of cells expressing mutated or truncated forms of membrane-tethered Notch in 15-30 minutes (Petrovic et al., 2019;Ryan et al., 2017;H. Wang et al., 2014;Weng et al., 2006). To create a SCC model in which GSI washout activates NOTCH1, we engineered a cDNA encoding a mutated truncated form of NOTCH1, DEGF-L1596H, that cannot respond to ligand and that has a point substitution in its negative regulatory region that produces ligand-independent, g-secretase-dependent Notch activation (Gordon et al., 2009;Malecki et al., 2006). DEGF-L1596H was transduced into two human SCC cell lines, IC8 and SCCT2, that have biallelic inactivating mutations in NOTCH1 and TP53 (Inman et al., 2018), lesions that were confirmed by resequencing on a clinical-grade targeted exome NGS platform (summarized in Tables 1 and 2). In pilot studies, we observed that the growth of IC8 and SCCT2 cells transduced with empty virus was unaffected by the presence or absence of GSI, whereas the growth of lines transduced with DEGF-L1596H was reduced by GSI washout (Figure 1, Supplemental Figure 1A, B). As anticipated, GSI washout was accompanied by rapid activation of NOTCH1 (ICN1), which was followed by delayed upregulation of markers of differentiation, such as involucrin (Figure 1, Supplemental Figure 1C, D).
To further characterize and optimize our system, we performed single cell-cloning of IC8-DEGF-L1596H cells and observed that growth arrest following GSI washout correlated with ICN1 accumulation (Figure 1, Supplemental Figure 2A, B). The subclone SC2, which showed moderate accumulation of ICN1 and sharply reduced growth following GSI washout, was selected for further study. We also observed that SC2 cells formed "skin-like" epithelia when seeded onto organotypic 3D cultures (Figure 1, Supplemental Figure 2C), whereas SCCT2-DEGF-L1596H cells did not (not shown); therefore, additional studies focused on IC8 cells and derivatives thereof. The ability of SC2 cells to form a multilayered epithelium in organotypic cultures in the Notch-on, growth suppressive state appears to stem from an emergent property, the Notch-dependent organization of these cells into a proliferating, ICN1-low basal layer in contact with matrix and a non-proliferating, ICN1-high suprabasal layer (Figure 1, Supplemental Figure 2C). Notably, growth arrest induced by GSI washout in SC2 cells was blocked by dominant-negative MAML1 (DN-MAML), a specific inhibitor of Notch-dependent transcription ( Figure 1B) (Nam, Sliz, Pear, Aster, & Blacklow, 2007;Weng et al., 2003), and was accompanied by increases in multiple markers of squamous differentiation, such as involucrin, keratin1, and plakophilin1, in both 2D ( Figure 1C-E) and in 3D cultures ( Figure 1F).

Identification of a Notch-induced program of gene expression in SCC
To determine early and delayed effects of Notch on gene expression, we performed RNA-seq on SC2 cells in 2D cultures in the Notch-off state and following Notch activation ( Figure 2A).
To confirm that these changes in gene expression are general features of Notch activation in IC8 cells (and not merely of the SC2 subclone) and to determine the kinetics of response, we performed RT-PCR analyses on a number of known and novel targets in pooled IC8-DEGF-L1596H transductants. This confirmed the Notch responsiveness of all genes tested, and also revealed variation in the kinetics of response, even among "canonical" Notch target genes. For example, HES1 showed fast induction followed by rapid down-regulation, consistent with autoinhibition (Hirata et al., 2002), whereas HES4, HES5, and NRARP (a feedback inhibitor of NTC function (Jarrett et al., 2019)) showed more sustained increases in expression ( Figure 2C).
Genes encoding non-structural proteins known to be linked to squamous differentiation also were "early" responders ( Figure 2D), as were genes linked to DNA damage/cell stress response ( Figure 2E). In the case of the latter novel rapidly responding genes, we used DN-MAML blockade to confirm that protein levels also rose in a Notch-dependent fashion ( Figure 2F). By contrast, increased expression of genes encoding structural proteins associated with keratinocyte differentiation (e.g., IVL, KRT1,KRT13,KRT14,KRT16,KRT17,LOR,FLG) was delayed, only emerging at 24-72h (Tables S1-S3). These findings suggest that Notch activation induces the expression of a core group of early direct target genes, setting in motion downstream events that lead to differentiation.

Notch target genes are associated with lineage-specific NTC-binding enhancer elements
To identify sites of NTC-binding to Notch-responsive regulatory elements in IC8-DEGF-L1596H cells, we performed ChIP-seq for endogenous Notch transcription complex components (RBPJ and MAML1) 4h after Notch activation, as well as for RBPJ prior to Notch activation. In the Notch-on state, most MAML1 binding sites also bound RBPJ (8533/9,187 sites, 93%; Figure   3A), in line with studies showing that MAML1 association with DNA requires both RBPJ and NICD (Nam, Sliz, Song, Aster, & Blacklow, 2006). Approximately 92% of RBPJ/MAML1 cobinding sites (hereafter designated NTC binding sites) are in intergenic or intronic regions consistent with enhancers ( Figure 3B). As predicted by past studies (Castel et al., 2013;Krejci & Bray, 2007;Ryan et al., 2017;H. Wang et al., 2014), NTC binding was associated with increases in RBPJ ChIP-Seq signals and H3K27ac signals at promoter and enhancer sites ( Figure 3C).
Motif analysis revealed that the most common motif lying within 300 bp of NTC ChIP-Seq signals is that of RBPJ ( Figure 3D), and that the motif for AP1, a factor not associated with NTC binding sites in other cell types (Chatr-Aryamontri et al., 2017;Drier et al., 2016;Petrovic et al., 2019;Ryan et al., 2017;H. Wang et al., 2014), is also highly enriched in this 600 bp window.
Based on the method of Severson et al. (Severson et al., 2017), approximately 13% of NTC binding sites in IC8 cells are predicted to be sequence paired sites ( Figure 3E), a specialized type of response element that binds NTC dimers (Arnett et al., 2010). Finally, particularly at early time points, NTC binding sites were spatially associated with genes that are upregulated by Notch, whereas genes that decreased in expression were no more likely to be associated with NTC binding sites than genes that did not change in expression ( Figure 3F). Taken together, these studies show that NTCs mainly bind lineage-specific enhancers in IC8 cells and that their loading leads to rapid "activation" of Notch responsive elements and upregulation of adjacent genes.
We also performed motif analysis on sites producing significant signals for only RBPJ or only MAML1. RBPJ "only" sites also were enriched for RBPJ (E value 1.3 e-124 ) and AP1 (E value 2.8 e-71 ) motifs but had lower average ChIP-Seq signals, suggesting these may be weak RBPJ binding sites. MAML1 "only" sites also were enriched for AP1 motifs (E value 2.9 e-181 ) but were not associated with RBPJ motifs. These sites were relatively few in number (N=654) and the associated AP1 motifs were distributed broadly around MAML1 signal peaks, arguing against direct physical interaction between MAML1 and AP1 family members on chromatin.
Thus, the significance of these "MAML1-only" peaks is uncertain, and it is possible that the observed ChIP-seq signals are non-specific, stemming from over-representation of "open" chromatin in ChIPs.

IER5 is a direct Notch target gene
We were intrigued by the convergence of Notch target genes and genes linked to DNA damage/cell stress responses. To test the idea that DNA damage/cell stress response genes contribute to Notch-induced differentiation of squamous cells, we elected to study the gene IER5 in detail. IER5 is a member of the immediate early response gene family that encodes a 327 amino acid protein with a ~50 amino acid N-terminal IER domain and an C-terminal domain predicted to be unstructured. Previous studies have implicated IER5 in cellular responses to DNA damaging agents and heat shock (Ding et al., 2009;Kis et al., 2006), but a role in regulation of keratinocyte growth and differentiation has not been described.  Figure 4B and 4C, respectively) confirmed that the Notch responsiveness of these elements depend on RBPJ binding sites and also showed, in the case of enhancer D, that a flanking AP1 consensus site is also required. To determine the contributions of enhancers D and E within the genomic IER5 locus, we used CRISPR/Cas9 targeting to delete the regions containing RBPJ binding sites in these two enhancers in SC2 cells ( Figure 4D).
These deletions partially abrogated the Notch-dependent increase IER5 transcription ( Figure 4E) and suppressed the accumulation of IER5 protein following Notch activation ( Figure 4F), confirming that IER5 is directly regulated by Notch through these elements.

IER5 is required for "late" Notch-dependent differentiation events in squamous cells
To systematically determine the contribution of IER5 to Notch-dependent changes in gene expression, we compared the transcriptional response to Notch activation in SC2 cells, SC2 cells in which IER5 was knocked out (I5 cells), and I5 cells to which IER5 expression was restored (I5AB cells, Figure 5A). Different doses of IER5 had no effect on gene expression in the absence of Notch signaling, or on the expression of genes that are induced by Notch within 4h; however, by 24h and 72h of Notch activation, I5 cells failed to upregulate a large group of Notch responsive genes that were rescued by add-back of IER5 ( Figure 5B, denoted with red box). GO analysis revealed that IER5-dependent genes were associated with various aspects of keratinocyte differentiation and biology ( Figure 5C; summarized in Tables S6 and S7). An example of a differentiation-associated gene impacted by loss of IER5 is KRT1, a marker of spinous differentiation, expression of which is markedly impaired by IER5 knockout and restored by IER5 add-back ( Figure 5D). Similarly, IER5 was required for Notch-dependent expression of the late marker involucrin in 3D cultures ( Figure 5G). Thus, IER5 is necessary but not sufficient for expression of a group of genes that respond to Notch with delayed kinetics.
To determine if IER5 has similar effects in non-transformed keratinocytes, we performed studies in a TERT-immortalized oral keratinocyte cell line (NOK1) that differentiates following transfer to high Ca 2+ medium. We observed that IER5 protein levels increased in differentiation medium in a GSI-sensitive fashion ( Figure Figure 1A), as well as increased expression of NOTCH3, a known target of activated Notch. Suppression of IER5 transcript levels by GSI was partial, suggesting that additional pathways influence IER5 expression, consistent with the complex enhancer landscape around this gene. To study the effect of IER5 on NOK1 cell differentiation, we studied the effects of targeting IER5 with CRISPR/Cas9 and enforced expression of IER5 through retroviral transduction. IER5 knockout suppressed expression of multiple differentiation-associated genes ( Figure 5F), whereas overexpression of IER5 increased expression of each of these markers ( Figure 5G). Thus, IER5 is required for Notch-dependent differentiation of malignant and non-transformed keratinocytes.

IER5 binds B55a/PP2A complexes
The structure of IER5 suggests that it functions through protein-protein interactions. To identify interacting proteins in an unbiased way, we expressed a tagged form of IER5 in IER5 null I5 cells and performed tandem affinity purification followed by mass spectrometry (Adelmant, Garg, Tavares, Card, & Marto, 2019), which identified the B55a regulatory subunit of PP2A and PP2A scaffolding and catalytic subunits as potential interactors ( Figure 6A; summarized in Table   S8). We confirmed these associations by expressing tagged IER5 in I5 cells and tagged B55a in SC2 cells ( Figure 6B). Full-length IER5 and the N-terminal IER domain of IER5 co-precipitated endogenous B55a in Notch-independent fashion, whereas the C-terminal portion of IER5 did not ( Figure 6C). Similarly, tagged B55a co-precipitated endogenous IER5 in a fashion that was augmented by Notch activation ( Figure 6D), consistent with increased recovery of IER5 due to induction of IER5 expression by Notch. To confirm that IER5 binds B55a directly, we studied the interaction of purified recombinant proteins. IER5 exhibited saturable binding to B55acoated beads ( Figure 6E), and additional microscale thermophoresis studies showed that IER5 binds B55a with a Kd of approximately 100nM ( Figure 6F).

IER5 is epistatic to PPP2R2A in SCC cells
To gain insight into the role of B55a in regulation of Notch-and IER5-sensitive genes, we isolated SC2 cell clones that were knocked out for IER5, PPP2R2A, or both genes ( Figure 7A).
Knockout of PPP2R2A did not affect the levels of ICN1 following GSI washout ( Figure 7A), but increased the expression of KRT1 in 2D cultures ( Figure 7B) and the accumulation of involucrin in 3D cultures ( Figure 7C), effects that were suppressed by add-back of B55a, suggesting a model in which IER5 suppresses a B55a-dependent activity. This was supported by assays performed with IER5/PPP2R2A double knockout cells ( Figure 7A), in which expression of the late genes such as KRT1 was restored in the absence of IER5 ( Figure 7D). These results suggest that Notch-mediated suppression of B55a-PP2A activity via IER5 is important in regulating the complex series of events downstream of Notch that lead to squamous cell differentiation.

Discussion
Our work provides a genome-wide view of the direct effects of Notch in squamous cells, in which Notch activation induces growth arrest and differentiation. The phenotypic changes induced by Notch are mediated by a largely squamous cell-specific transcriptional program that includes genes linked to keratinocyte differentiation and DNA damage responses, including IER5, which modulates the activity of B55a-containing PP2A complexes. Upregulation of these Notch-responsive genes are associated with binding of NTCs to RBPJ sites within lineagespecific enhancers, including a minority of sequence-paired sites, a specialized dimeric NTC binding element recently implicated in anti-parasite immune responses in mice (Kobia et al., 2020). The Notch target genes and elements identified here in malignant squamous cells are likely to be relevant for understanding Notch function in non-transformed squamous cells, as many of the NTC binding enhancers found near Notch target genes in SCC cells are also active in normal human keratinocytes (based on review of ENCODE data for non-transformed human keratinocytes). These observations have a number of implications for understanding how Notch regulates the growth and differentiation of squamous cells and highlight the potential for Notch to influence the activity of diverse signaling pathways in keratinocytes through modulation of PP2A.
Prior work has suggested that p53-mediated upregulation of Notch expression and activity is a component of the DNA damage response in keratinocytes (Mandinova et al., 2008).
Conversely, our work shows that Notch activation, even in a TP53 mutant background, induces the expression of genes that are components of the keratinocyte DNA damage/cell stress response. In cells of most lineages, p53 responses serve to suppress cell growth and, if DNA damage is severe or cannot be repaired, to induce apoptosis. However, our work suggests that in cells of squamous lineage these two pathways share a core set of genes that induce differentiation. In the case of p53, this function may act to help to eliminate damaged cells from the epidermal stem cell pool without inducing apoptosis, which could comprise the barrier function of squamous epithelial surfaces. Consistent with this idea, recent work has shown that low-dose radiation induces the differentiation of esophageal squamous cells at the expense of self-renewal in vivo (Fernandez-Antoran et al., 2019). TP53 and Notch genes are frequently comutated in squamous carcinomas of the skin (as in our model system) and other sites; although co-mutation of tumor suppressor genes in a particular cancer is typically taken as evidence of complementary anti-oncogenic activities, our work suggests that in the cases of squamous carcinoma it may reflect, at least in part, redundant Notch and p53 functions. Further work delineating the crosstalk between p53 and Notch signaling in well controlled model systems will be needed to further test this idea.
Among the genes linked to Notch and p53 is IER5, an immediate early response gene that is a component of the DNA-damage response in a number of cell types (Ding et al., 2009;Kis et al., 2006;Kumar et al., 1998;Yang et al., 2016). Several lines of investigation suggest that IER5 modulates the function of PP2A complexes containing B55 (Asano et al., 2016; and here we demonstrate that IER5 directly binds B55a protein in a purified system. However, the exact effect of IER5 on PP2A function is uncertain. One model suggests that IER5 augments the ability of B55/PP2A complexes to recognize and dephosphorylate specific substrates such as S6 kinase and HSF1 Kawabata et al., 2015), the latter leading to HSF1 activation as part of the heat shock response. However, our work with double knockout cells suggests IER5 inhibits at least some activities that are attributable to B55a-containing PP2A complexes. Further work in purified systems may be helpful in clarifying how IER5 influences B55/PP2A function. We also note that while IER5 is necessary for upregulation of genes that are induced by Notch with delayed kinetics, it is not sufficient; therefore, it is likely that other PP2Aregulated factors that work in concert with Notch to induce expression of this class of genes await discovery.
Finally, we note that our small screen of SCC cell lines suggests that squamous cell carcinomas retain the capacity to respond to Notch signals by undergoing growth arrest and differentiation. Although originally identified as an oncogene, sequencing of cancer genomes has revealed that Notch most commonly acts as a tumor suppressor, particularly in squamous cell carcinoma, which is difficult to treat when advanced in stage. While restoring the expression of defective Notch receptors is challenging, detailed analysis of crosstalk between Notch and other pathways may reveal druggable targets leading to reactivation of tumor suppressive signaling nodes downstream of Notch, which would constitute a new therapeutic strategy for squamous cancers.

Cell lines and 2D cultures
Cells were grown under 5% C02 at 37 o C in media supplemented with glutamine and streptomycin/penicillin. IC8 cells (N. J. Wang et al., 2011) were cultured in Keratinocyte medium as described (Purdie, Pourreyron, & South, 2011). EGF-L1596H cDNA cloned into pBABE-puro was packaged into pseudotyped retrovirus and used to transduce IC8 and SCCT2 cells, which were selected with puromycin (1 ug/ml). In some instances, cells were also transduced with pseudotyped MigRI retrovirus encoding dominant negative MAML1 fused to GFP (Weng et al., 2003).

Cell growth assays
Cell numbers were estimated using CellTiter Blue (Promega) per the manufacturer's recommendations. Fluorescence was measured using a SpectraMax M3 microplate reader (Molecular Devices).

Organotypic 3D cultures
3D raft cultures were performed on a matrix containing 5x10 5 J2 3T3 fibroblast cells and rat collagen as described (Arnette, Koetsier, Hoover, Getsios, & Green, 2016). Briefly, rafts were allowed to mature for 6-7 days and then were seeded with 5x10 5 SCC cells in E-medium with or without GSI. After 2 days, rafts were raised to the fluid-air interface, and medium was refreshed every 2 days for a total of 12 additional days.

Targeted exon sequencing
NGS was performed on IC8 and SCCT2 cell genomic DNA using the "oncopanel" assay (Abo et al., 2015;Wagle et al., 2012), which covers 447 cancer genes. Briefly, DNA (200 ng) was enriched with the Agilent SureSelect hybrid capture kit and used for library preparation.

Preparation of ChIP-Seq and RNA-seq Libraries
Chromatin was prepared as described (H. Wang et al., 2014) and was immunoprecipitated with antibodies against MAML1 (clone D3K7B) or RBPJ (clone D10A11, both from Cell Signaling Technology) and Dynabeads bearing sheep anti-rabbit Ig (Thermo Fisher Scientific). H3K27ac ChIPs were prepared using the ChIP assay kit (Millipore) and H2K27ac antibody (ab4729, Abcam). ChIP-seq libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit (New England BioLabs). Total RNA was prepared with Trizol (Life Technology) and RNeasy Mini columns (Qiagen). RNA libraries were constructed using the NEBNext Ultra II RNA Library Prep kit (New England BioLab). ChIP-seq and RNA-seq libraries were sequenced on an Illumina NextSeq 500 instrument. ChIP-seq and RNA-seq data sets will be deposited in GEO.
After first-pass DE analysis, a control set of 733 genes with FDR>0.5 in all pairwise comparisons was used with RUVg (k=1) to identify unwanted variation. Second-pass edgeR analysis included the RUVg weights in the model matrix. Genes with FDR<5% and absolute logFC >1 were retained for further analysis. DAVID v.6.8 (Huang da, Sherman, & Lempicki, 2009) was used for gene ontology (GO) enrichment analysis of the DE gene lists. EdgeR cpm function with library size normalization and log2 conversion was used to generate expression values, which were displayed using pheatmap (R package version 1.0.8). Other plots were made using in-house R scripts (available upon request).

Quantitative RT-PCR
Total RNA was isolated using RNAeasy Mini Kit (Qiagen) and cDNA was prepared using an iScript cDNA synthesis kit (BioRad). qPCR was carried out using a CFX384 Real-Time PCR Detection System. Gene expression was normalized to GAPDH using the DD CT method. Primer sets used are available on request.

In Situ Hybridization
In situ hybridization (ISH) reagents were from Advanced Cell Diagnostics. Deidentified normal human skin was obtained from the paraffin archives of the Department of Pathology at Brigham and Women's Hospital under institutional review board protocol #2014P001256. Briefly, 4µ sections of skin were deparaffinized, processed using RNAscope 2.5 Universal Pretreatment Reagent, and hybridized to probes specific for human IER5, human PPIB (peptidylprolyl isomerase B), or bacterial DapB in a HybEZ II oven. ISH signal was developed using the RNAscope 2.5 HD Assay.
Luciferase assays were performed using Dual Luciferase Assay Kit (Promega) as described (Malecki et al., 2006) using lysates from cultured cells that were co-transfected with firefly luciferase and internal control Renilla luciferase plasmids using Lipofactamine 2000 (Thermo Fisher Scientific).

Tandem affinity purification of IER5 complexes and mass spectrometry
Mass spectroscopy was performed on tandem affinity purified IER5 complexes prepared from IE5 knockout (I5) cells expressing tandem tagged IER5 48h after GSI washout. Lysis of cells and subsequent tandem purification were as described . Tryptic peptides were analyzed by electrospray mass spectrometry (QExactive HF mass spectrometer, Thermo Fisher; Digital PicoView electrospray source platform, New Objective). Spectra were recalibrated using the background ion (Si(CH3)2O)6 at m/z 445.12 +/-0.03 and converted to a Mascot generic file format (.mgf) using multiplierz scripts Parikh et al., 2009). Spectra were searched using Mascot (v2.6) against three databases: i) human protein sequences (downloaded from RefSeq); ii) common lab contaminants; and iii) a decoy database generated by reversing the sequences from these two databases. Spectra matching peptides from the reverse database were used to calculate a global FDR and were discarded, as were matches to the forward database with FDR>1.0% and those present in >1% of 108 negative tandem affinity purification controls (Rozenblatt-Rosen et al., 2012).

Recombinant protein expression and purification
A cDNA encoding B55a with 6xHis-SUMO N-terminal tag was cloned into the baculovirus transfer vector pVL1392. High-titer baculovirus supernatants were used to infect insect Sf9 cells grown at a density of 4.0x10 6 cell/mL. After 72h of incubation at 27°C, conditioned media was isolated by centrifugation, supplemented with 20mM Tris buffer, pH 7.5, containing 150mM NaCl, 5mM CaCl2, 1mM NiCl2, and 0.01mM ZnCl2, re-centrifuged to remove residual debris, and applied to a Ni-NTA column. After washing with 20mM Tris buffer, pH 7.5, containing 150mM NaCl and 5 mMCaCl2, B55a was eluted in the same buffer supplemented with 500mM imidazole. This eluate was concentrated with a centrifugal filter and then subject to S200 size exclusion chromatography in 20mM Na cacodylate, pH 6.0, containing 150mM NaCl, and 5mM CaCl2. Fractions containing B55a were further purified by ion exchange chromatography on a MonoQ column in 20 mM Na cacodylate, pH 6.0, using a linear NaCl gradient. The purified protein was buffer-exchanged into 20mM HEPES buffer, pH 7.5, containing 150mM NaCl, prior to flash freezing and storage at −80°C. A cDNA encoding IER5 with a N-terminal 6xHis-SUMO tag was cloned into pTD6 and used to transform Rosetta E. coli cells. Expression of IER5 was induced at 37 o C for 4h with IPTG induction followed by inclusion body preparation. Lysates were centrifuged at 4 o C for 20min at 30,000 x g and pellets were resuspended in 10mL wash buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, containing 1% Triton X-100 and 1M urea) per gram cell weight, and incubated at 23 o C for 5min. Following multiple washes, inclusion bodies were resuspended in extraction buffer (50mM Tris-HCl, pH 7.5, 8M urea, 1mM bmercaptoethanol, 1mM PMSF) and incubated at room temperature for 1h. The solubilized proteins were then dialyzed overnight against a 100-fold volume of wash buffer and cleared by centrifugation. 6x-his-IER5 was then concentrated on Ni-NTA beads, eluted with 500mM imidazole cleaved using SUMO protease (SUMOpro, Lifesensors), and passed back over Ni-NTA beads. Untagged IER5 was then purified by chromatography on S200 and MonoQ columns as described for B55a.

IER5/B55a binding assays
For bead pulldown assays, 5µM Purified His-SUMO-B55a bait was bound to 100ul Ni-NTA beads, which were was and mixed with different concentrations of purified IER5 in 20mM HEPES buffer, pH 7.5, containing 150mM NaCl for 1h. Control binding assays were conducted with purified His-SUMO bond to Ni-NTA beads. Following extensive washing, proteins were eluted by boiling in SDS-PAGE loading buffer and analyzed on SDS-PAGE gels followed by Western blotting. Microscale thermophoresis assays were performed on a NanoTemper ® Monolith NT.115 instrument with blue/red filters (NanoTemper Technologies GmbH, Munich, Germany). Samples were prepared in 20mM Tris-HCl, pH 7.5, containing 150mM NaCl, 1mM b-mercaptoethanol, 1% glycerol, 0.05% Tween-20, and loaded into premium treated capillaries.
Measurements were performed at 22° C using 20% MST power with laser off/on times of 5s and 30s, respectively. His-SUMO-B55a target labeled with red fluorescent detector as per the NanoTemper His-labeling kit was used at a concentration of 10nM and mixed with 16 serial dilutions of purified IER5 ligand from 5µM to 0.000153µM. All experiments were repeated three times. Data analyses were performed using NanoTemper® analysis software.

CRISPR/Cas9 targeting and site directed mutagenesis
Guide RNAs (gRNAs) were designed using software available at http://crispr.mit.edu. To score the effects of gene editing in bulk cell populations, gRNAs were cloned into pL-        transcript levels in SC2 cells targeted with control AAVS1 CRISPR/Cas9 plasmids (SC2/con) or with CRISPR/Cas9 plasmids that remove the RPBJ sites in enhancers D and E (SC2/DD+E).

Figure Legends
Cells were either maintained in GSI or were harvested 2h following GSI washout (WO).
Transcript abundance was measured in experimental triplicates by RT-PCR and normalized against GAPDH. Error bars represent standard errors of the mean. F) Western blots showing IER5 protein levels in SC2/con cells and SC2/DD+E cells that were either maintained in GSI or harvested 1, 2, or 4 h following GSI washout (WO).  on differentiation-associated transcripts in NOK1 cells. In F, NOK1 cells were transfected with CRISPR/Cas9 and IER5 (I5KO) gRNA or AAVS1 control (con) gRNA. In G, NOK1 cells were transduced with empty retrovirus (con) or IER5 cDNA. In F and G, analyses were done on pooled transfectants and transductants, respectively, which were maintained in low Ca2+ medium or moved to high Ca2+ medium for 3 days (F) or 5 days (G) prior to harvest. Inset Western blots show the extent of IER5 loss (F) and IER5 overexpression (G) relative to control cells. In D, F, and G, transcript abundance was measured in experimental triplicates by RT-PCR and normalized against GAPDH. Error bars represent standard deviations of the mean.   clones. Cells were maintained in GSI or harvested 72h following GSI washout (WO). KRT1 transcript abundance was measured in experimental triplicates by RT-PCR and normalized against GAPDH. Error bars represent standard errors of the mean.  Data and materials availability: All genome-wide data sets will be made available through GEO following publication. Cell lines, primer sequences, gRNA sequences, and other reagents/methodology will be made available on request.

Supplemental Material
Supplemental Table 1. Differentially expressed genes after 4h of Notch activation, SC2 cells.
Supplemental Table 4. Unclustered GO annotations of Notch-sensitive genes, SC2 cells, at 72h of Notch activation.
Supplemental Table 5. Clustered GO annotations of Notch-sensitive genes, SC2 cells, at 72h of Notch activation.