Dear Editor, Expression of MITF is often regarded as the key event in melanocyte specification from neural crest cells (Sommer, 2011). The central role of MITF in melanocyte biology and ‘phenotype-switching’ in melanoma depending on the levels of MITF has been documented (Hoek and Goding, 2010), and the importance of the fine-tuning of MITF levels in melanoma was proposed by several groups (Pinner et al., 2009; Shah et al., 2010). Factors such as SOX10, PAX3, CBP, and b-catenin / LEF1 control MITF expression in melanocyte development in mouse model (Hou and Pavan, 2008); however, the mechanisms regulating MITF expression in human melanocyte and melanomas remains obscure. SOX2 is expressed in 45% primary melanomas and 40% metastasis, and SOX2-knockdown inhibited A2058 melanoma growth upon transplantation (Laga et al., 2010). Recent studies suggested SOX2 involvement in melanocyte development in mice (Adameyko et al., 2012) and in human melanoma progression (Girouard et al., 2012). Here, we prvide evidence that SOX2 and MITF expression is correlated with normal human melanocytes and three human melanoma cell lines at a single-cell level. We found that SOX2 regulates the levels of endogenous MITF in these cell lines, suggesting a role for SOX2-MITF axis in biology of normal human melanocyte and human melanomas. We established a human embryonic stem cell (hESC) model of neural crest stem cells and neural crest lineages (Curchoe et al., 2010) and demonstrated a key role of SOX2 in peripheral neurogenesis (Cimadamore et al., 2011). Because MITF marks melanocyte progenitors shortly after the emigration of neural crest cells (Sommer, 2011), we investigated the expression of MITF in hESC-derived neural crest lineage (hESC-NC). MITF is expressed in _25% of hESC-NC (Figure S1A), which are uniformly positive for SOX2 under these culture conditions (Cimadamore et al., 2011). We have previously validated hESC-NC cells engineered with SOX2-specific / scrambled shRNA under the control of Tet-inducible promoter (Cimadamore et al., 2011). Knockdown of SOX2 transcripts decreased MITF mRNA and MITF protein, suggesting that SOX2 is required for endogenous MITF levels in hESC-NC (Figure S1B, C). We investigated the relationship between SOX2 and MITF in normal human melanocytes, which express various levels of both transcription factors at a single-cell level (Figures 1A, S2A). We used immunofluorescence to colocalize both transcription factors and then quantified the coexpression of these factors in the same cell. We found that SOX2 and MITF expression is well correlated at the single-cell level (r = 0.65) (Figure 1B), and the knockdown of SOX2 (using lentiviral delivery of SOX2-specific shRNA) reduced the levels of MITF in these cells (Figure 1C). These results suggest that SOX2 is required for the endogenous levels of MITF in hESC-NC and normal human melanocytes in vitro. Figure 1 SOX2 regulates MITF expression in primary melanocytes and melanoma cell lines. (A, D, G, and J) Costaining for SOX2 and MITF in primary melanocytes (A), MEL501 (D), MeWo (G), and Lu1205 (J) melanoma cell lines. Scale bars = 50lm. (B, E, H, and K) Single-cell ... Next, we analyzed the expression of SOX2 and MITF in three human melanoma cell lines (i.e., MEL501, MeWo, and Lu1205). Although several MITF isoforms have been described (Amae et al., 1998; Hershey and Fisher, 2005), melanoma cells predo- minantly express MITF-M isoform (Amae et al., 1998). Western blot analysis confirmed that MITF-M is a predominant isoform in all three melanoma lines analyzed in the current study (Figure S3). At a single-cell level, the expression of both SOX2 and MITF is highly variable in melanoma lines (Figures 1D, G, J, S2B, C, D). Therefore, we used immunofluorescence to colocalize both transcription factors and then quantified the coexpression of these factors in the same cells. SOX2 and MITF expression was well correlated with MEL501 and MeWo melanoma lines (r = 0.9 and 0.56, respectively) (Figure 1E, H). In Lu1205 melanoma cells, which express lower levels of MITF compared with MEL501 and MeWo lines (Figure S3), SOX2 and MITF expression was moderately correlated (r = 0.52) (Figure 1K). Taken together, these results demonstrate significant correlation of the levels of SOX2 and MITF in normal human melanocytes and melanoma lines, consistent with the role of SOX2 as a modulator of MITF expression. Indeed, we observed no MITF-expressing cells that were negative for SOX2 in these cell lines by immunofluorescence. Next, we investigated functional requirement of SOX2 for MITF expression in three human melanoma cell lines MEL501, MeWo, and Lu1205 expressing high, intermediate, and low levels of MITF as demonstrated by Western blot analysis (Figure S3). All melanoma lines were engineered with lentiviral vectors expressing SOX2-specific shRNA or scrambled control shRNA. The knockdown of SOX2 induced pronounced decrease in the levels of MITF protein as assayed by quantitative immunofluorescence (Figures 1F, I, L, S4). These results suggest that SOX2 function is required for the endogenous levels of MITF in MEL501, MeWo, and Lu1205 melanoma cells expressing very different levels of MITF. Finally, we investigated the effect of SOX2 overexpression (using SOX2-IRES-GFP lentivirus) in MeWo and Lu1205 melanoma cells expressing MITF at variable high and low levels, respectively (Figures 2A, C, S3). The SOX2 overexpression in MeWo cells significantly decreased MITF levels (Figure 2A, B), but not completely eliminated MITF protein as compared to SOX2 knockdown (Figures 2A, B, versus S4B). The SOX2 overexpression in Lu1205 line had no significant effect on MITF levels (Figure 2C, D). These contradictory nonlinear effects of SOX2 on MITF expression would suggest that SOX2 can function as an activator or as a repressor under different conditions of signaling, in vivo versus in vitro, and / or in the context of additional modifiers (e.g., BRAF). Figure 2 Effect of SOX2 overexpression in meWo and Lu1205 cell lines. (A, C) Immunostaining for MITF in MeWo (A) and Lu1205 (C) melanoma cell lines overexpressing GFP (control) or SOX2- IRES-GFP. (B, D) Quantification of MITF expression from A and C. Only GFP-positive ... Recent studies in mice proposed cross-regulatory interactions between SOX2 and MITF (Adameyko et al., 2012). The SOX2 overexpression results in human MeWo melanoma line are consistent with SOX2 suppression of MITF in B16–F10 mouse melanoma line (Adameyko et al., 2012). However, our study also suggests that SOX2 is required to maintain the endogenous levels of MITF in normal human melanocytes and melanoma cell lines. The effect of SOX2 could be indirect, although SOX2 binding to the MITF proximal promoter was observed in mouse model (Adameyko et al., 2012), raising the possibility that SOX2 may interact with other factors to regulate MITF expression (e.g., PAX3, CBP, and b-catenin / LEF1).