The wind rose of human keratinocyte cell fate

Extensive efforts have been made to understand the molecular actors that control epithelial cell fate. Although pieces of information have been obtained from single-gene function investigations, the entire picture of the molecular mechanisms involved in the regulation of epithelial homeostasis is still mysterious. Growing data indicate that gene networks rather than single “master” genes dictate cell fate. In an attempt to characterize such gene networks, we have been investigating the human keratinocyte proliferation and differentiation genes that act downstream of the transcription factor p63, a major regulator of epidermal homeostasis. We identified two networks: the cell cycle network that controls cell proliferation and the keratinocyte cell fate network. Through further analysis of the existing data on epithelial tumorigenesis and induced pluripotent stem cells, we propose a wind rose model of cell fate that is based on a balance between these two different networks that ultimately control human keratinocyte fate and epidermal homeostasis. Electronic supplementary material The online version of this article (doi:10.1007/s00018-014-1758-1) contains supplementary material, which is available to authorized users.

Although p63 and MYC are important in the control of epidermal homeostasis, the underlying molecular mechanisms governing keratinocyte proliferation or differentiation, downstream of these two genes are not completely understood. By analyzing the transcriptional changes and phenotypic consequences of the loss of either p63 or MYC in human developmentally mature keratinocytes, we have characterized the networks acting downstream of these two genes to control epidermis homeostasis. We show that p63 is required to maintain growth and to commit to differentiation by two distinct mechanisms. Knockdown of p63 led to downregulation of MYC via the Wnt/-catenin and Notch signaling pathways and in turn reduced keratinocyte proliferation. We demonstrate that a p63-controlled keratinocyte cell fate (KCF) network is essential to induce the onset of keratinocyte differentiation. This network contains several secreted proteins involved in cell migration/adhesion, including fibronectin 1 (FN1), interleukin 1 beta (IL1B), cysteine-rich protein 61 (CYR61), and jagged-1 (JAG1), that act downstream of p63 as key effectors to trigger differentiation. Our results characterized for the first time a connection between p63 and MYC and a cell adhesion-related network that control differentiation. Furthermore, we show that the balance between the MYCcontrolled cell-cycle progression network and the p63-controlled cell adhesionrelated network could dictate skin cell fate.
The outer layer of human skin, the epidermis, is a self-renewing, stratified squamous epithelial tissue. In adult tissue, the maintenance of epidermal homeostasis depends on an exquisite regulation of the balance between keratinocyte proliferation and differentiation (1,2). However, the specific molecular mechanisms governing each of these processes are not completely understood.
p63, a member of the p53 tumor suppressor gene family, is known as a key regulator of epidermis development and keratinocyte differentiation. Striking developmental defects have been discovered during embryonic development in p63knockout mice (3,4). In addition, p63 is required for the maintenance of proliferative potential in epithelial stem cells (3,5,6), epithelial lineage commitment (4), differentiation of keratinocytes (7), and epithelial cell adhesion and survival (8). Dual roles of p63 in the initiation of epithelial stratification and maintenance of stem cell proliferative potential have been established (6). As simultaneous p63 and p53 knockdown rescued cell proliferation defect of p63 knockdown alone, but failed to restore differentiation, the roles of p63 in proliferation and differentiation of developmentally mature keratinocytes appear to be distinct (7).
In vivo, as keratinocytes commit to differentiation, they detach from basal layer and migrate outward into spinous layer, accompanied with the expression of early differentiation markers K1 and K10 (9). The effects of p63 in keratinocyte differentiation have been investigated by gain and loss of function experiments. Ectopic expression of p63 induces the early markers K1, but suppresses the expression of late differentiation markers such as loricrin and filagrin (10). Loss of p63 inhibited both stratification and differentiation in skin organotypic culture (7). However little is known on key genes acting downstream of p63 to regulate the dynamic equilibrium of keratinocytes differentiation and proliferation as well as epidermal homeostasis (11).
Similar to p63, the MYC oncogene is predominantly expressed in the basal cell layers of epidermis and is absent in suprabasal layers (12,13). MYC plays a vital role not only in keratinocyte proliferation (14)(15)(16) but also in accelerating the differentiation of epidermal stem cells (17)(18)(19)(20). Specifically, MYC activation leads to epidermal stem cell proliferation, and the sustained elevated expression of MYC in turn stimulates the proliferating stem cells to enter the transit amplifying compartment, thereby initiating terminal differentiation (21). Although p63 and MYC have been independently demonstrated to be key regulators in the dynamic equilibrium between proliferation and differentiation in epidermal stem cells, no connection between p63 and MYC has been reported so far. The genetic basis underlying the processes regulated by p63 and MYC in mature keratinocytes, as well as the nature of their downstream effectors, remains mostly unknown.
We hypothesized that the regulation of the equilibrium between proliferation and differentiation of keratinocytes could rely on gene networks acting downstream of p63 and MYC, rather than on a single gene.  (25). RNA was isolated, amplified, labeled, and hybridized following a published protocol (25). For each condition, three independent biological replicates were obtained and the knockdown of corresponding genes was verified before RNA extraction ( Figure S2). Each biological replicate was hybridized to the arrays in 4 replicates using a dye-swap strategy. Slides were scanned with an Agilent G2565AA Microarray Scanner (Agilent Technologies). Data analyses, including intensity-dependent Lowess normalization of raw data and differential analysis, were performed with GeneSpring 7.0 software (Silicon Genetics). Differentially expressed genes were identified using ANOVA (p-value corrected with Benjamini and Hoehberg False Discovery Rate) and only genes with a fold change ≥1.2 and p<0.01 were used for further analysis. Gene pathway and genetic network analyses were performed by Ingenuity Pathway Analysis (IPA) (Ingenuity Systems) http://www.ingenuity.com.
Ontologies attached to each gene were used to classify altered genes according to main biological themes. The following criteria enter into generating the networks in the Ingenuity Pathways Analysis application: 1. We have designated the molecules of interest in the Analysis Parameters before running the analysis. Molecules that a) meet the cut-off and/or filter criteria and which b) interact with other molecules in Ingenuity's Knowledge Base are identified as focus molecules (also called Network Eligible molecules). Focus molecules serve as the "seeds", or focal points, for generating networks. 2. Networks are preferentially enriched for focus molecules with the most extensive interactions, and for which interactions are specific with the other molecules in the network (rather than molecules that are promiscuous, those that interact with a broad selection of molecules throughout Ingenuity's knowledge base). 3. Additional non-focus molecules from the dataset and from Ingenuity's knowledge base are then recruited and added to the growing networks. 4. Networks are scored for the likelihood of finding the focus molecule(s) in that given network. The higher the score, the lower the probability that you would find the focus molecules(s) you see in a given network by random chance. 5. In the current version of the application, there is a cut-off of 35 molecules per network to keep networks to a usable size. All the microarrays data used in this study have been deposited into the NCBI Gene Expression Omnibus under accession number GSE17394. mRNA expression analysis. RNA was extracted with an RNeasy Mini Kit (QIAGEN). For real-time quantitative PCR, 2 μg RNA was reverse-transcribed in a total volume of 20 μl with a SuperScript II RNase H reverse transcriptase system (Invitrogen) and random primers according the manufacturer's instructions. Reverse transcription reactions were diluted to 500 μl of water, and 5 μl of the diluted cDNA was used for each quantitative PCR. Quantitative PCR was carried out with a Platinum Quantitative PCR SuperMix-UDG Kit (Invitrogen) using an ABI 7500 Fast Real-Time PCR system (Applied Biosystems). All experiments were run in triplicate, and the results were normalized to 18S rRNA expression. Primer sequences are listed in Table S1.
Cell cycle analysis. Cell cycle phase was determined by analyzing total DNA content using flow cytometry. HaCaT cells were synchronized by culturing in serum-free DMEM medium 24 hours before transfection. Cells were transfected using INTERFERin with 10 nM of siRNA. 48 hours post-transfection, cells were collected, fixed, and stained with a Cell Cycle Phase Determination Kit (Cayman chemical). Flow cytometry analysis was performed in a MoFlo cell sorter (DakoCytomation).
Luciferase reporter assay. HaCaT cells were cotransfected with plasmid and siRNA in 24-well plates using Amaxa Nucleofector II (Amaxa Biosystems) according to the manufacturer's recommendations. The human MYC promoter cloned into a luciferase plasmid was described previously (26). A TK-Renilla reporter was used as an internal normalization control. The ratio of firefly luciferase plasmid to Renilla luciferase was 10:1 in each nucleofection (2 µg: 200 ng). At 48 hours post-transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega).

RESULTS
siRNA-mediated depletion of either p63 or MYC decreased mature keratinocyte proliferation, but only the loss of p63 inhibited differentiation. To investigate the specific roles of MYC and p63 in human keratinocyte differentiation, we knocked down each gene in HaCaT cells using specific small interfering RNA (siRNA) and analyzed the phenotypic consequences. Because of the existence of six different isoforms of p63, we used a siRNA targeting the conserved DNA binding domain in all genes to achieve ablation of all p63 isoforms. We first verified that the expression of either MYC or p63 in siRNAtransfected keratinocytes was specifically knocked down at both the transcript ( Figure  1A) and protein levels ( Figure 1B) 48 hours post-transfection. Compared to cells treated with control siRNA, we observed a 70% and 90% downregulation of MYC and p63 expression, respectively. It is noteworthy that a significant loss of both genes was still observable after 10 days of culture ( Figure  S1). Two days after siRNA transfection, we observed a reduced ATP content ( Figure  1C), while both EdU ( Figure 1D) and Ki67 staining ( Figure 1E) significantly decreased, both in p63-or MYC-knockdown HaCaT cells. Together these data demonstrate a defect in keratinocyte proliferation upon ablation of either p63 or MYC. We next monitored the capacity of these cells to differentiate in vitro during 10 days of culture in the appropriate medium. As we focused on the commitment to differentiation, we chose to monitor the expression of two early differentiation markers, keratin 1 (K1) and keratin 10 (K10), rather than later ones, such as involucrin or filagrin. Indeed, K1 and K10 are markers of the basal-spinous layers transition in epidermis. Reduction of p63 levels in keratinocytes significantly inhibited the expression of K1 and K10 at the transcript level ( Figure 1F). In contrast, cells lacking MYC still expressed high levels of both markers, suggesting that these cells were still able to differentiate and did so even faster than the control ( Figure 1F). On the protein level, K1 was continuously induced except in cells lacking p63, which exhibited upregulation of K1 until day 8, followed by a weak decrease in expression ( Figure 1G). Expression of the K10 protein started late in all conditions but became significant in MYC-depleted cells after 8 days of culture ( Figure 1G). In addition, we observed that loss of p63 in cultures of primary human keratinocytes (PHK) inhibited expression of both K1 and K10 compared to control or MYC-depleted cells ( Figure 1H). These results demonstrate that the ablation of either MYC or p63 significantly reduced human mature keratinocyte proliferation, while only the siRNA-mediated loss of p63 inhibited differentiation.
Knockdown of either MYC or p63 leads to cell cycle arrest. To determine the potential origin of the proliferation defect in keratinocytes lacking either p63 or MYC, we analyzed cell death and the cell cycle. Knockdown of either p63 or MYC triggered a significant arrest in G0/G1 (Figure 2A), without affecting cell death ( Figure 2B). To identify the molecular mechanisms controlling this cell cycle arrest, we analyzed the gene expression profiles of keratinocytes depleted of either MYC or p63 after transfection (Table S2). For these experiments we have particularly insisted on robustness of the data. We performed three independent siRNA transfections, three independent RNA extractions ( Figure S2), along with several technical replicates, to generated three independent expression profiles that we then averaged for either myc-or p63-depleted keratinocytes. This transcriptome analysis showed that a small network of genes strongly associated with cell cycle regulation (p<6.2x10 -35 ) was significantly downregulated in keratinocytes lacking either MYC or p63 ( Figure S3A and Figure 2C). The majority of genes in this network were similarly regulated in both p63-depleted and MYC-depleted cells, except CSK2, GADD45A, and CCND2 ( Figure S3A). Analysis by qRT-PCR of the major cell cycle inhibitors confirmed that p15 and p21 were both significantly upregulated in response to the knockdown of either MYC or p63 in HaCaT cells ( Figure  2D, E). Interestingly the same trend was observed in primary keratinocytes ( Figure  2F, G). We also observed that MYC, a major hub (highly connected node) in this cell cycle-controlling network, was downregulated in both HaCaT and primary keratinocyte cells lacking p63 ( Figure 2C, D and F). This result suggests that p63 is necessary for the proper expression of MYC in human keratinocytes.  Figure S4) (27). Using specific siRNA targeting either Np63 or TAp63, we were able to demonstrate that only the knockdown of Np63 isoforms triggered downregulation of MYC expression ( Figure  3C). This was consistent with the fact that Np63 is the isoform predominantly expressed in adult keratinocytes. Conversely overexpression of either Np63 or TAp63 in HaCaT cells had no effect on MYC expression ( Figure S5). Together these results demonstrated that p63 is necessary for the proper expression of MYC.
We next investigated the molecular mechanisms underlying the p63 knockdowntriggered downregulation of MYC expression. Using luciferase reporter constructs fused to truncated MYC promoter regions ( Figure 3D), we characterized the sequence upstream of MYC and identified a putative p63-controlled region. As demonstrated in Figure 3E, this region extended from -349 to -607 bp upstream of the transcription start site. A close-up view of this region revealed the absence of the p63 consensus binding site, in agreement with our previous published results, which showed that MYC was not a direct target of p63 in human keratinocytes (28,29). These results suggested that the expression of MYC is under the indirect control of p63. The region upstream of the MYC gene also contains several other binding sites for transcription factors (TFs), including TCF-4 (TF responding to the Wnt/-catenin signaling pathway), YY-1 (TF responding to the Notch signaling pathway), c-FOS, and c-JUN (AP1 TFs) ( Figure 3F). Because these TF are known to play important roles in the control of keratinocyte proliferation and differentiation (30-32), we investigated whether or not they are involved in the p63 knockdown-triggered downregulation of MYC promoter activity. In keratinocytes lacking p63, we observed a moderate inhibition of both TCF4 or YY1-dependent luciferase activities, but no change or a slight activation in JUN and FOS-driven luciferase expression ( Figure 3G). These results suggest that TCF4 and YY1, the TFs responding to the Wnt/-catenin and Notch signaling pathways, respectively, were partially responsible for the p63-dependent downregulation of MYC expression.
The p63 knockdown-triggered downregulation of MYC expression is mediated by the Notch and Wnt/-catenin signaling pathways. To determine whether the signaling cascades leading to the activation of YY1 and TCF4 are affected by p63 levels, we scrutinized the expression profiles of keratincoytes lacking either p63 or MYC. Interestingly we found that the Wnt/-catenin and Notch signaling cascades were indeed potently inhibited in keratinocytes lacking p63. JAG1 and DLL1, ligands of the Notch signaling pathway, were both downregulated in p63-knockdown cells ( Figure 4A). In addition, several inhibitors of the Wnt/-catenin pathway were upregulated, while several activators were downregulated, likely resulting in the inhibition of this signaling pathway ( Figure  4A). At the protein level, the ablation of p63 resulted in the downregulation of both cleaved Notch1 (NICD) and total -catenin ( Figure 4B). Taken together, these results suggest that both pathways were turned down in p63-knockdown cells. Inhibition of the Notch signaling pathway in presence of p63-targeting siRNA was also indicated by the reduced expression of two reporter genes of Notch-signaling cascade, HES1 and HEY1 ( Figure 4C). To further confirm the potential involvement of the Wnt and Notch pathways in down-regulation of MYC expression, we restored downstream signaling by transfecting HaCaT cells with either WNT3 or NICD expression vectors, together with p63-targeting siRNA or control siRNA. We observed that the over expression of either WNT3 or NCID induced the upregulation of MYC expression both at the transcript ( Figure 4D) and protein levels ( Figure 4E). This would partially restore keratinocyte proliferation. However, overexpression of those two genes was not sufficient to restore proper differentiation of keratinocytes lacking p63 (data not shown). These results suggest that the molecular role of p63 in differentiation of human mature keratinocytes is likely different from the ones that dictate their proliferation.
A cell migration/adhesion-related network acts downstream of p63 to induce the onset of keratinocyte differentiation. Our results demonstrated that MYC is downregulated in cells lacking p63 (Figure 2 and 3), thus functionally corresponding, at least partially, to a siRNA-mediated knockdown of MYC. However, these cells exhibited completely opposite differentiation outcomes ( Figure  1E). To investigate the molecular mechanisms enabling keratinocyte differentiation downstream of p63, we compared the expression profiles of p63depleted and MYC-depleted cells.
As demonstrated in Figure 5A, 546 genes were common to both expression profiles. It is noteworthy that there were more genes common to both profiles than specific to the p63-depleted cells. This again suggests that part of the transcriptional response to p63 ablation in human keratinocytes was also due to the downregulation of MYC. The downregulated genes in keratinocytes lacking either MYC ( Figure S6A) or p63 ( Figure S6B) shared similar Gene Ontology (GO) terms: e.g., cell cycle, DNA replication, and DNA repair. Genes upregulated in either MYC-( Figure  S6C) or p63-depleted keratinocytes ( Figure  S6D) also shared some GO terms, such as cellular movement or cell death.
Among the 546 genes common to both expression profiles, we found 71 genes that were antagonistically regulated (Table  S3).
We hypothesized that these antagonistically regulated genes could mechanistically explain, at least partially, the oppose differentiation outcomes between p63-and MYC-lacking keratinocytes. We used the Ingenuity knowledge base using IPA software to analyze the networks and functions associated with these 71 genes. Strikingly, a network of 41 nodes was extracted and significantly associated with a single function, cell migration/adhesion (p<310 -14 ). In cells lacking p63, this network was strongly downregulated ( Figure  5B), while in MYC-depleted keratinocytes, this same network was upregulated ( Figure  S3B). We further validated the expression of some genes in the network by qRT-PCR in HaCaT cells ( Figure 5C) and in primary keratinocytes ( Figure 5D). As expected, the expression of these genes was upregulated in MYC-depleted cells and downregulated in keratinocytes lacking p63. These results suggest that this migration/adhesion-related gene network could contain potential effectors acting downstream of p63 to induce the onset of terminal differentiation in human keratinocytes.
To validate our hypothesis, we used several functional approaches. First, we searched for known phenotypes associated with these 41 genes network in the Mouse Genome Informatics database. There are 19 knockout mice with abnormal skin phenotypes reported in that database. Strikingly, 15 genes out of these 19 KO mice were present in this network (Table 1). These data suggest that near 80% of all known skin dysfunction-related genes belongs to the network we have characterized and functionally validate it. We also monitored in vitro differentiation of keratinocyte lacking different genes belonging to this migration/adhesion network: FN1, MMP13, JAG1, IL1B and CYR61 ( Figure 6A). Except for MMP13, we observed a strong inhibition of differentiation in cells lacking any of these genes, as demonstrated by the delayed expression of K1 ( Figure 6B) and K10 ( Figure 6C) transcripts. It is noteworthy that although the ablation of IL1B was only partial (40%), yet it significantly inhibited differentiation. Furthermore ablation of all these gene together (siCocktail) strongly inhibited keratinocyte differentiation. Finally, if the genes belonging to this network promote commitment to differentiation we postulated that their expression should be down-regulated in non-differentiated and/or pluripotent cells. We data-mined the NCBI Gene Expression Omnibus database and interestingly, we found that 7 hubs in this network, PLAU, FN1, IL1B, ADM, DUSP10, GADD45A, RAC2, were significantly down-regulated in induced pluripotent stem cells (iPS) and are even part of the iPS transcriptomic signature ( Figure 6D) (33). Together, these results show that a p63-controlled migration/adhesion-related network plays a key role in the onset of human mature keratinocyte differentiation. As a consequence we named this network, the Keratinocyte Cell Fate (KCF) network.

DISCUSSION
Our findings confirm that sustained expression of both p63 and MYC, two major regulators of epidermal homeostasis (5)(6)(7)18,21,34,35), is required to maintain growth and differentiation of human developmentally mature keratinocytes. However, we demonstrate that their respective roles are very different, as already suggested by some authors (36). We propose a model to illustrate the distinct mechanisms of action of p63 on human developmentally mature keratinocyte proliferation and differentiation (Figure 7). P63 is required fro the proper expression of of MYC expression through the combined regulation of the Wnt/-catenin and Notch signaling pathways, leading in turn to cell cycle regulation and cell proliferation. P63 also regulates a KCF network that contains several potential "differentiation effectors", some located in the extracellular space. The upregulation of KCF network would promote the onset of terminal differentiation of keratinocyte (Figure 7). In this study, we show that the siRNA-mediated loss of MYC triggers downregulation of the "proliferation network" and upregulation of the KCF network, promoting human keratinocyte differentiation; in contrast, p63 knockdown downregulates both cell proliferation and mobility/adhesion-related networks, thus inhibiting differentiation. These results were observed both in HaCaT cell line (mutated p53) and in normal human primary keratinocytes (wild-type p53), suggesting that the model we propose is independent of the p53 status of skin cells.
As MYC is a transcriptional repressor of p15 and p21, two cyclindependant kinase inhibitors (37)(38)(39), and p63 was reported to repress p21 and p16 (40)(41)(42), it was not a surprise to observe reduced proliferation in cells lacking either gene. More surprising was the p63 knockdowntriggered downregulation of MYC expression that we report here for the first time. This result suggests that although MYC is not a direct target of p63 (28,29), the expression of this transcription factor is necessary for the proper expression of MYC both in HaCaT cell line and primary human keratinocytes culture. Our results establish that MYC expression is mediated, at least in part, by the Wnt/-catenin and Notch signaling pathways. Interestingly, both pathways are important regulators of proliferation and differentiation in epidermal stem cell maintenance and wound healing (1,(43)(44)(45)(46). The interplay between the Wnt/catenin and Notch pathways has been reported in epidermal homeostasis and differentiation as well (47,48). We show that both pathways act in concert downstream of p63 to control the proper expression of MYC and, in turn, regulate keratinocyte cell cycle.
While the inhibition of proliferation of cells lacking p63 was partially MYCdependent, the differentiation defects appeared to be independent of MYC. Indeed, the siRNA-mediated loss of MYC did not impair keratinocyte commitment to terminal differentiation. The differentiation was even slightly accelerated in MYC-depleted cells. By comparing expression profiles from mature keratinocytes lacking either MYC or p63, we found 546 common genes, of which 71 were antagonistically regulated. Strikingly, from that list of 71 genes we extracted a gene network containing 41 genes, that were significantly associated (p<310 -14 ) with a single function -cell migration/adhesion. These results are consistent with recent reports showing that p63 functions as an inhibitor of cell migration (27) and that a p53 mutant forms a complex with p63 to antagonize its cell migration-inhibitory function, leading to TGF-dependent metastasis (49). Similarly, it was shown that p63 regulates a cell adhesion program, including integrins, in epithelial cells (8). Although we cannot exclude the possibility that other genes among the 546 common genes, but not found in our network, might regulate early differentiation, we have clearly demonstrated that this p63-controlled cell migration/adhesion network contains several effectors acting downstream of p63 to trigger differentiation of mature keratinocytes.
Some of the differentiation effectors we have identified (such as integrins, FN1, PLAU, JAG1, IL1, and CYR61) are also involved in cancer progression in inducible human tissue neoplasia (50). Integrin signaling plays an important role in epidermal adhesion, growth, and differentiation (51,52). JAG1 is a ligand of the Notch signaling pathway and acts on keratinocyte differentiation (32,53). JAG1 is also transcriptional target of p63 (54). Interleukin 1 (IL1) is implicated in human epidermal keratinocyte proliferation (55) and even in the regeneration of epidermal tissue in vitro (56). IL1 is the active form of interleukin-1 in human epidermis. IL1 was considered non-functional in keratinocytes, but our data suggest that IL1 is necessary to induce keratinocyte differentiation.
Although, most of these "differentiation effectors" do not seem to be direct transcriptional targets of p63, the control exerted by p63 on this network was dominant. Indeed we were unable to rescue differentiation of p63-depleted keratinocytes by ectopic expression of NICD or JAG1, with the use of JAG1 or IL1 recombinant proteins, or even with an acellular matrix obtained from normal fully differentiated keratinocytes (data not shown).
We report for the first time the role of this p63-regulated cell migration/adhesion network in the commitment of developmentally mature keratinocytes to differentiation. A normal expression of this network seems to be required to trigger differentiation, while its downregulation prevents it. Furthermore, we believe that misregulation of this gene network may play a major role in tumorigenesis. Indeed, Khavari's group recently reported a core tumor progression signature (CTPS) network in keratinocytes, which contained 282 nodes and was involved in carcinogenesis (50). This CTPS network contained several oncogene hubs, and 8 of the top 10 nodes were extracellular or cell surface proteins. It is noteworthy that 4 out of these 8 extracellular oncogene hubs, PLAU, CYR61, FN1, and IL1, also belong to the p63-regulated cell migration/adhesion network we describe in this study. Other oncogene hubs reported in the CTPS network (50), such as SERPINE1 and ITGA6, were also downregulated in human keratinocytes depleted of p63 (Table S2).
In conclusion, the siRNA-mediated loss of MYC triggers downregulation of the "proliferation network" and upregulation of the "KCF migration/adhesion-related network", promoting human keratinocyte differentiation; in contrast, p63 knockdown downregulates both cell proliferation and KCF networks, thus inhibiting differentiation. We believe that the balance between levels of expression of both cell proliferation and KCF networks, could dictate keratinocyte cell fate. Furthermore, we think that this network approach would reconcile much of the existing data on the regulation of the balance between proliferation and differentiation in skin.                Table S1. List of all PCR primers used in this study. Table S2. List of genes differentially expressed in siMYC or siP63 transcriptome analysis. Table S3. List of common genes modulated inversely in the siMYC and siP63 transcriptomes.    Figure S6 A B C D 8   Table S3 List of common genes modulated oppositely in siTP63 and siMYC transcriptome. Fold changes were given.