14 research outputs found
SOX10 post-translational modifications identified in MG132-treated 501mel cells.
<p>This schematic representing the SOX10 protein indicates known domains, SOXE conserved regions, and phosphorylated residues, as follows: black bars show known phosphorylation sites, green bars show known sites that were confirmed in this study, and red bars show novel sites from this study. The phosphorylated residues S224, S232, T240 and T244 were observed on numerous peptide fragments, and one or all four are plausible; their close proximity and the limited fragmentation capability in the digest restrict more precise determination among these residues. The nuclear localization and nuclear export signal regions are unaffected by the phosphorylation sites.</p
Identification and functional analysis of SOX10 phosphorylation sites in melanoma
<div><p>The transcription factor SOX10 plays an important role in vertebrate neural crest development, including the establishment and maintenance of the melanocyte lineage. SOX10 is also highly expressed in melanoma tumors, and SOX10 expression increases with tumor progression. The suppression of SOX10 in melanoma cells activates TGF-β signaling and can promote resistance to BRAF and MEK inhibitors. Since resistance to BRAF/MEK inhibitors is seen in the majority of melanoma patients, there is an immediate need to assess the underlying biology that mediates resistance and to identify new targets for combinatorial therapeutic approaches. Previously, we demonstrated that SOX10 protein is required for tumor initiation, maintenance and survival. Here, we present data that support phosphorylation as a mechanism employed by melanoma cells to tightly regulate SOX10 expression. Mass spectrometry identified eight phosphorylation sites contained within SOX10, three of which (S24, S45 and T240) were selected for further analysis based on their location within predicted MAPK/CDK binding motifs. SOX10 mutations were generated at these phosphorylation sites to assess their impact on SOX10 protein function in melanoma cells, including transcriptional activation on target promoters, subcellular localization, and stability. These data further our understanding of SOX10 protein regulation and provide critical information for identification of molecular pathways that modulate SOX10 protein levels in melanoma, with the ultimate goal of discovering novel targets for more effective combinatorial therapeutic approaches for melanoma patients.</p></div
Mutation of SOX10 phosphorylation sites causes distinct changes in protein stability.
<p>Cycloheximide pulse-chase assays in 501mel (A-C) and MeWo (D-F) cells revealed altered stability of SOX10 phospho-mutants compared to WT SOX10 protein. A. WT SOX10 showed a half-life of 8.3 hours in 501mel cells. B,C. Stability of SOX10 phospho-mutants S24A and T240A is not significantly different from WT SOX10 in 501mel cells (two-way ANOVA, p = 0.25). D. WT SOX10 stabilty in MeWo cells exhibited a half-life of 19.5 hours. E. S24A SOX10 mutant protein showed reduced stability in MeWo cells with a half-life of 4.7 hours. F. T240A SOX10 mutant protein showed reduced stability in MeWo cells with a half-life of 11.7 hours. Both the S24A and the T240A mutant proteins exhibited significant differences relative to WT SOX10 protein in MeWo cells (two-way ANOVA, p = 0.0057 for protein type, p<0.0001 for time and interaction); by Bonferroni’s multiple comparisons post-test, these differences were significant for SOX10 S24A from 4 hours through 10 hours, and were significant for SOX10 T240A from 4 hours through 16 hours (P-values: *≤0.05, **≤0.01, ***≤0.001, ***≤0.0001). Data are compiled from 3 independent assays, with standard deviations plotted.</p
Mass spectrometry analysis identifies SOX10 phosphorylation sites.
<p>A. Workflow schematic of SOX10 protein analysis by mass spectrometry. 501mel cells were treated with MG132 proteasomal inhibitor before scraping cells and performing immunoprecipitation (IP) using SOX10 antibody to isolate protein. The eluted proteins were separated by SDS-page, followed by staining and removal of bands corresponding to 55kD, 75kD and 100kD. All three gel bands were subjected to destaining, in-gel digestion and extraction before running LC-MS/MS. B. Portions of IP samples were separated on SDS-page gel, followed by transfer onto PVDF membrane and Western blotting to confirm SOX10 isolation in the eluted samples being used for mass spectrometry. C. The three phosphorylation sites selected for mutation and characterization are shown in the context of full length SOX10 (Genbank ID NM_006941).</p
SOX10 phospho-mutants exhibit cell-specific differences in activation of the MITF promoter.
<p>A. Over-expression of WT and phospho-mutant SOX10 yields similar protein levels in HeLa cells at 48 hours on Western blot. SOX10 phospho-mutants show bands running at slightly different sizes; the SOX10 Sumo3x mutant is included as a control for protein band shifting that results from altering amino acid residues at sites of post-translational modifications. B,C. Representative luciferase data showing activation of p<i>MITF</i> from WT and SOX10 phospho-mutants in HeLa (B) and NIH3T3 cells (C); the S24A and T240A constructs showed significantly greater promoter activation in HeLa cells, while the S24A, S45A construct showed significantly greater promoter activation in NIH3T3 cells. Replicate data sets for p<i>MITF</i> can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190834#pone.0190834.s002" target="_blank">S2 Fig</a>. D. p<i>TYR</i> promoter luciferase data showed no significant differences between phospho-mutants and WT SOX10 in HeLa cells; representative dataset is shown. E. p<i>DCT</i> promoter luciferase data showed no significant differences between phospho-mutants and WT SOX10 in HeLa cells; representative dataset is shown. Statistics were calculated using one-way ANOVA with Bonferroni’s multiple comparison test, three independent assays per promoter construct.</p
SOX10 phosphorylation mutants retain nuclear localization.
<p>A,B. HeLa cells (A) and 501mel melanoma cells (B) were transfected with WT and phospho-mutant SOX10 constructs, and after 48 hours were fixed and stained to visualize subcellular localization of WT SOX10 and SOX10 phosphoryation mutant proteins. The Sumo3x SOX10 mutant was used as a post-translational modification control, as it is known to express in the nucleus despite mutations in all 3 sumoylation sites. No differences in localization are seen in the SOX10 phosphorylation mutants relative to WT SOX10. The V5 antibody (V5-488) stains exogenous SOX10 in both cell lines, while the SOX10 antibody (SOX10-568) stains both exogenous and endogenous SOX10 in 501mel cells (HeLa cells do not express endogenous SOX10).</p
TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF
<div><p>Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why <i>TFAP2A</i> expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in <i>Tfap2a</i>, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including <i>Dct</i>, <i>Mc1r</i>, <i>Mlph</i>, and <i>Pmel</i>. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the “master regulator” of melanocyte development. For example, the promoter of <i>TRPM1</i> is bound by both TFAP2A and MITF, and we show that the activity of a minimal <i>TRPM1</i> promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of <i>Trpm1</i> is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs <i>Tfap2a</i> and <i>Tfap2b</i> are both expressed in mouse melanocytes, and we show that mouse embryos with <i>Wnt1-Cre</i>-mediated deletion of <i>Tfap2a</i> and <i>Tfap2b</i> in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between <i>tfap2a</i> and <i>mitfa</i> in zebrafish, but find that artificially elevating expression of <i>tfap2a</i> does not increase levels of melanin in <i>mitfa</i> hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.</p></div
TFAP2A binds active enhancers and promoters in mouse melanocytes.
<p>(A) Pie chart showing distribution of mouse TFAP2A peaks with respect to genomic features. TSS, transcription start site; TTS, transcription termination site. (B) Distance from TSS to the nearest TFAP2A peak for genes in three expression categories: highest 1000, median 1000, or lowest 1000. Promoter-proximal TFAP2A peaks are enriched at highly expressed genes (RNA-seq on mouse melan-a cells at GSE87051). (C) Examples of active enhancer signatures defined by H3K4me1 peaks flanking a p300 peak [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref065" target="_blank">65</a>], and partial enhancer signatures, overlapping TFAP2A peaks upstream of the melanocyte differentiation gene <i>Slc45a2</i>. (D) Distance from TSS to the nearest TFAP2A peaks that overlap the active enhancer signature for genes in three expression categories: highest 1000, median 1000, or lowest 1000. (E) Overlap of TFAP2A ChIP-seq peaks in mouse melanocytes with published TFAP2A ChIP-seq peaks in mouse kidney and epididymis cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref068" target="_blank">68</a>]. 34% of melanocyte peaks were shared with one or both of the other cell types. (F) MEME-ChIP analysis of unique peaks from each cell type. Melanocyte-unique peaks are significantly enriched for SOX10 and M-Box MITF binding motifs, while kidney-unique and epididymis-unique peaks are not. *All motifs shown are a result of <i>de novo</i> MEME-ChIP enrichment analysis except the M-Box, which we specifically searched using the Analysis of Motif Enrichment (AME) tool [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref109" target="_blank">109</a>].</p
TFAP2A peaks are associated with genes involved in pigmentation.
<p>(A-C) Density-based clustering of H3K27ac signal at (A) TFAP2A peaks, (B) MITF peaks, and (C) TFAP2A peaks that overlap MITF peaks in human melanocytes (H3K27ac data from GSM1127072 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref064" target="_blank">64</a>]), MITF peaks from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref018" target="_blank">18</a>]). (D) Overlap between genes associated with active TFAP2A peaks and genes associated with active MITF peaks in human melanocytes. (E) Typical enhancers (gray) and super-enhancers (colored) in human melanocytes that overlap neither TFAP2A nor MITF peaks, TFAP2A peaks only, MITF peaks only, or both TFAP2A and MITF peaks. Labels identify melanocyte genes of interest. (F) Diagram of the <i>TRPM1</i> -700 bp promoter element depicting the positions of four TFAP2A binding sites (A1–A4) and the previously reported E-box 1 MITF binding site (E1). (G) Luciferase assays in M21 melanoma cells. Deletion of all four TFAP2A binding sites (ΔAP2) significantly reduced reporter activity compared to the intact <i>TRPM1</i> -700bp element (Student’s t-test, **p = 0.01).</p
Forced expression of <i>tfap2a</i> does not rescue melanocytes in <i>mitfa</i> mutant zebrafish.
<p>(A-D) Nuclei of melanocytes expressing mosaic <i>Tg(mitfa</i>:<i>tfap2a-Myc)</i> are brightly labeled after anti-Myc immunostaining. At 48 hpf, labeled melanocytes in <i>mitfa</i><sup><i>z25/z25</i></sup> mutant embryos (A, B) and <i>mitfa</i><sup><i>w2/z25</i></sup> mutant embryos (C, D) display no apparent improvement in pigmentation or dendricity compared to adjacent unlabeled cells (B and D, white arrowheads). (E, F) In <i>mitfa</i><sup><i>w2/w2</i></sup> mutants, all labeled cells are unpigmented and resemble other cell types including xanthophores (F, white arrowhead).</p