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122580.Full.Pdf bioRxiv preprint doi: https://doi.org/10.1101/122580; this version posted March 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 A protein phosphatase network controls the temporal and spatial dynamics of 2 differentiation commitment in human epidermis 3 1 1 1 2 4 Ajay Mishra , Angela Oliveira Pisco *, Benedicte Oules *, Tony Ly *, Kifayathullah 1 1 1 1 5 Liakath-Ali *, Gernot Walko , Priyalakshmi Viswanathan , Jagdeesh Nijjhar , Sara-Jane 3 2 1† 6 Dunn , Angus I. Lamond and Fiona M. Watt 7 1 th 8 King’s College London, Centre for Stem Cells and Regenerative Medicine, 28 Floor, Tower 9 Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, UK. 2 10 Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, 11 Dow Street, Dundee, DD1 5EH, UK. 3 12 Computational Science Laboratory, Microsoft Research, Cambridge CB1 2FB, UK 13 14 *These authors contributed equally to the work 15 † 16 Correspondence to: [email protected] 17 18 Abstract 19 Epidermal homeostasis depends on a balance between stem cell renewal and terminal 1,2 20 differentiation . While progress has been made in characterising the stem and 3 21 differentiated cell compartments , the transition between the two cell states, termed 4 22 commitment , is poorly understood. Here we characterise commitment by integrating 23 transcriptomic and proteomic data from disaggregated primary human keratinocytes 24 held in suspension for up to 12h. We have previously shown that commitment begins at 5 25 approximately 4h and differentiation is initiated by 8h . We find that cell detachment 26 induces a network of protein phosphatases. The pro-commitment phosphatases – 27 including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote terminal 28 differentiation by negatively regulating ERK MAPK and positively regulating key AP1 29 transcription factors. Their activity is antagonised by concomitant upregulation of the 30 anti-commitment phosphatase DUSP10. The phosphatases form a dynamic network of 31 transient positive and negative interactions, with DUSP6 predominating at commitment. 32 Boolean network modelling identifies a mandatory switch between two stable states 33 (stem cell and differentiated cell) via an unstable (committed) state. In addition 1 bioRxiv preprint doi: https://doi.org/10.1101/122580; this version posted March 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 34 phosphatase expression is spatially regulated relative to the location of stem cells, both 35 in vivo and in response to topographical cues in vitro. We conclude that an auto- 36 regulatory phosphatase network maintains epidermal homeostasis by controlling the 37 onset and duration of commitment. 38 39 Introduction 40 Commitment is a transient state during which a cell becomes restricted to a particular 41 differentiated fate. Under physiological conditions commitment is typically irreversible and 6 42 involves selecting one differentiation pathway at the expense of others . While commitment is 43 a well-defined concept in developmental biology, it is still poorly understood in the context of 4,6 44 adult tissues . This is because end-point analysis fails to capture dynamic changes in cell 45 state, and rapid cell state transitions can depend on post-translational events, such as protein 7 46 phosphorylation and dephosphorylation . For these reasons a systems approach to analyse 47 commitment is required. 48 49 We set out to examine commitment in human interfollicular epidermis, which is a multi- 2 50 layered epithelium formed by keratinocytes and comprises the outer covering of the skin . 51 The stem cell compartment lies in the basal layer, attached to an underlying basement 52 membrane, and cells that leave the basal layer undergo a process of terminal differentiation as 53 they move through the suprabasal layers. In the final stage of terminal differentiation the cell 54 nucleus and cytoplasmic organelles are lost and cells assemble an insoluble barrier, called the 2 55 cornified envelope, which is formed of transglutaminase cross-linked proteins and lipids . 56 Cells can become committed to differentiate at any phase of the cell cycle, and upon 5 57 commitment they are refractory to ECM-mediated inhibition of differentiation . 58 59 Results 60 Although there are currently no markers of commitment, we have previously used 61 suspension-induced differentiation of disaggregated human keratinocytes in methylcellulose- 5 62 containing medium to define its timing (Fig. 1a). Since keratinocytes increase in size as they 5 63 differentiate , we enriched for undifferentiated cells in the starting population by a filtration 64 step. By determining when cells recovered from suspension could no longer resume clonal 65 growth on replating (Fig. 1b; Extended Data Fig. 1a), we confirmed that there is a marked 66 drop in colony forming ability between 4 and 8 hours. This correlates with an increase in the 2 bioRxiv preprint doi: https://doi.org/10.1101/122580; this version posted March 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 67 proportion of cells expressing the terminal differentiation markers involucrin (IVL) and 68 transglutaminase 1 (TGM1) (Fig. 1c, d; Extended Data Fig. 1b) and downregulation of genes 69 that are expressed in the basal layer of the epidermis, including α6 integrin (ITGα6) and TP63 70 (Fig. 1d). 71 72 We next collected keratinocytes after 4, 8 and 12h in suspension and performed genome-wide 73 transcript profiling using Illumina based microarrays and proteome-wide peptide analysis by 74 SILAC-Mass-Spectrometry (MS) (Fig.1e, f; Extended Data Figs. 1c-f; Extended Data Tables 75 1, 2). Keratinocytes collected immediately after trypsinization served as the 0h control. When 76 comparing the starting (0h) cell population with cells suspended for 4, 8 or 12h, t-SNE 77 analysis of the gene expression data indicated that the 4h cell state was distinct from the 8 and 78 12h cell states (Fig. 1e). Unsupervised hierarchical clustering of differentially expressed 79 genes (Extended Data Fig. 1c) or proteins (Fig. 1f) also indicated that the 4h sample clustered 80 separately from the 8 and 12h samples. GO term enrichment analysis of differentially 81 expressed transcripts or proteins showed enrichment of terms associated with epidermal 8,9 82 differentiation at 8h and 12h (Extended Data Figs. 1e, f) , which is consistent with the drop 83 in clonogenicity seen at these time points. 84 85 When we compared the significantly differentially expressed proteins (p-value <0.05) that 86 changed ≥2 fold at one or more time points with their corresponding transcripts 87 (Fig. 1g-i), there was a moderately positive correlation at 8h and 12 h (Pearson correlations of 88 0.51 and 0.68, respectively), consistent with the correlation between bulk mRNA and protein 10,11 89 levels seen in previous studies of mammalian cells . However, at 4h transcripts and 90 proteins were only weakly correlated (Pearson correlation 0.19, Fig. 1g). 91 92 The poor correlation between protein and transcript levels at 4h suggested a potential role for 93 post-transcriptional mechanisms in regulating commitment. To investigate this we performed 94 unbiased SILAC-MS based phospho-proteomic analysis. SILAC-labelled peptides isolated 95 from cells at 0, 4 and 8h time points were enriched for phosphopeptides using HILIC pre- 96 fractionation and titanium dioxide affinity chromatography (Extended Data Fig. 1d; Extended 97 Data Tables 3, 4). Over 3,500 high confidence phosphorylation sites were identified with an 98 Andromeda search score >= 30 and quantified at both the 4 and 8h timepoints. At 4h, 3 bioRxiv preprint doi: https://doi.org/10.1101/122580; this version posted March 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 99 approximately two thirds of the changes involved dephosphorylation (Fig. 1j). These 100 dephosphorylation events could not be attributed to decreases in protein abundance, as shown 101 by the discordance between total protein abundance and changes in protein phosphorylation 102 (Fig. 1k). 103 104 Analysis of the integrated proteomic and genomic datasets revealed 45 phosphatases that were 105 differentially expressed between 0 and 4h, 20 of which were upregulated (Fig. 2a, b). 106 Interrogation of published datasets revealed that these phosphatases were also dynamically 12 107 expressed during Calcium-induced stratification of human keratinocytes and differentiation 13 108 of reconstituted human epidermis (Extended Data Fig. 2a, b). 109 110 To examine the effects on keratinocyte self-renewal of knocking down each of the 20 111 phosphatases that were upregulated on commitment in suspension, we transfected primary 112 human keratinocytes with SMART pool siRNAs and measured colony formation in culture 113 (Fig. 2c; Extended Data Fig. 3a, b). As a control we also included INPP5J, which did not 114 change in expression during suspension-induced differentiation. Live cell imaging was used 115 to monitor cell growth for three days post-transfection (Extended Data Fig. 3b). Knocking 116 down seven of the phosphatases significantly increased clonal growth (Fig 2c; Extended Data 117 Fig. 3d), with five phosphatases – DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA - having 118 the most pronounced effect (p-value <0.001).
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