In humans, epidermal melanocytes are responsible for skin pigmentation, defense against ultraviolet radiation, and the deadliest common skin cancer, melanoma. While there is substantial overlap in melanocyte development pathways between different model organisms, species dependent differences are frequent and the conservation of these processes in human skin remains unresolved 1-3 . Thus, the biology of developing and adult human melanocytes remains largely uncharacterized. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes derived directly from skin, capturing transcriptomes across different anatomic sites, developmental age, sexes, and multiple skin tones. Using donor-matched skin from distinct volar and non-volar anatomic locations, we uncovered subpopulations of melanocytes exhibiting site-specific enrichment that occurs during gestation and persists through adulthood. In addition, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we use these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis. Overall, the characterization of human melanocytes fresh from skin revealed new subpopulations, human-specific transcriptional programs, and valuable insights into melanoma dedifferentiation. INTRODUCTION:Epidermal melanocytes, the pigment producing cells of human skin, are responsible for skin tone and orchestrate the primary defense against damage from ultraviolet (UV) radiation. Some anatomic site-specific differences in pigmentation are due to environmental factors, such as the tanning response to UV exposure. Others, like the hypopigmentation at volar sites (such as palms and soles), are present at birth. In adult skin, mesenchymal -epithelial interactions are known to influence anatomic site-specific melanocyte survival and pigment production 4 but melanocyte intrinsic factors that contribute to site-specific specialization remain unclear.Model organisms are powerful tools for investigating melanocyte development. In chick and mouse, a transient, multipotent neural crest cell population gives rise to committed immature melanocyte precursors, called melanoblasts, via two spatially and temporally distinct pathways 2,3 . Such studies focus primarily on melanocytes in skin appendages (hair follicle, feather, and sweat gland). However, despite constituting the predominate subtype in human skin, resident epidermal melanocytes have not been the subject of analogous investigations into developmental trajectories and anatomic-specializations.Melanocytes can give rise to melanomas which present distinct phenotypic and genomic characteristics correlated with primary tumor location 5,6 . Like many cancers, melanoma progression is coupled to dedifferentiation of the cell of origin 7 . The aggressive nature of melanoma is proposed to be rooted in unique attributes of the melanocytic linage 8 . Decoding the transcriptome of epidermal mela...

BackgroundCancer/testis (CT) genes have expression normally restricted to the testis, but become activated during oncogenesis, so they have excellent potential as cancer-specific biomarkers. Evidence is starting to emerge to indicate that they also provide function(s) in the oncogenic programme. Human TEX19 is a recently identified CT gene, but a functional role for TEX19 in cancer has not yet been defined.MethodssiRNA was used to deplete TEX19 levels in various cancer cell lines. This was extended using shRNA to deplete TEX19 in vivo. Western blotting, fluorescence activated cell sorting and immunofluorescence were used to study the effect of TEX19 depletion in cancer cells and to localize TEX19 in normal testis and cancer cells/tissues. RT-qPCR and RNA sequencing were employed to determine the changes to the transcriptome of cancer cells depleted for TEX19 and Kaplan-Meier plots were generated to explore the relationship between TEX19 expression and prognosis for a range of cancer types.ResultsDepletion of TEX19 levels in a range of cancer cell lines in vitro and in vivo restricts cellular proliferation/self-renewal/reduces tumour volume, indicating TEX19 is required for cancer cell proliferative/self-renewal potential. Analysis of cells depleted for TEX19 indicates they enter a quiescent-like state and have subtle defects in S-phase progression. TEX19 is present in both the nucleus and cytoplasm in both cancerous cells and normal testis. In cancer cells, localization switches in a context-dependent fashion. Transcriptome analysis of TEX19 depleted cells reveals altered transcript levels of a number of cancer-/proliferation-associated genes, suggesting that TEX19 could control oncogenic proliferation via a transcript/transcription regulation pathway. Finally, overall survival analysis of high verses low TEX19 expressing tumours indicates that TEX19 expression is linked to prognostic outcomes in different tumour types.ConclusionsTEX19 is required to drive cell proliferation in a range of cancer cell types, possibly mediated via an oncogenic transcript regulation mechanism. TEX19 expression is linked to a poor prognosis for some cancers and collectively these findings indicate that not only can TEX19 expression serve as a novel cancer biomarker, but may also offer a cancer-specific therapeutic target with broad spectrum potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0653-4) contains supplementary material, which is available to authorized users.

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.

SUMMARYEpidermal melanocytes are present throughout the skin, one of the largest organs with distinct anatomical, morphological and functional characteristics. Clear differences in melanocytic disease manifestation and phenotype exist across different anatomical locations. Here, we investigate human melanocyte heterogeneity during development, homeostasis, and disease progression using single cell RNA sequencing of freshly isolated human fetal, neonatal, and adult skin from a demographically diverse cohort. Comparative analysis across developmental stages and between anatomical sites revealed distinct subclasses of melanocytes from lineages that diverge early in human development. Using differentiation programs delineated from healthy melanocytes, we identified melanoma gene expression signatures that are developmental in origin and are re-acquired during disease, signatures that are unique to melanoma progression, as well as signatures that offer prognostic value. This dataset provides a valuable resource for further investigations of the melanocytic lineage in health and disease.

Dozens of driver mutations and copy number changes have been implicated in human squamous cell carcinomas (SCCs), and each tumor harbors multiple putative driver mutations. However, the minimal set of mutations sufficient to transform a normal keratinocyte into squamous cell carcinoma is unknown. By analyzing TCGA data and previously published datasets, we have identified common combinations of driver mutations in SCCs. Among the most common driver events are mutations in TP53, CDKN2A, and NOTCH1. Using CRISPR editing at endogenous loci in primary human keratinocytes, we demonstrate that no single driver mutation is sufficient to transform keratinocytes, and in fact many single gene mutations appear toxic to keratinocytes. The combination of loss-of-function mutations in TP53 and CDKN2A is sufficient to immortalize both skin and oral primary human keratinocytes in 2D culture and confer a selective advantage in competition assays. Simultaneous mutation of TP53, CDKN2A, and NOTCH1 demonstrates a very similar phenotype in 2D culture as mutation of TP53 and CDKN2A alone. However, using 3D organotypic squamous epithelial cultures consisting of genome-edited keratinocytes grown on decellularized dermis impregnated with fibroblasts and grown at an air-liquid interface demonstrates that keratinocytes with TP53 and CDKN2A mutation alone are sufficient to induce dysplasia, whereas keratinocytes harboring TP53, CDKN2A, and NOTCH1 mutation exhibit an SCC-like phenotype, including invasion through the basement membrane. RNAseq analysis demonstrates that the transition to SCC by NOTCH1 loss involves a partial EMT-like program. Targeted hybrid capture sequencing of edited keratinocytes revealed no other driver mutations or copy number changes other than the experimentally induced ones, consistent with the notion that TP53, CDKN2A, and NOTCH1 mutations alone are sufficient to reprogram a normal keratinocyte into SCC. Thus, despite a high mutation burden and frequent chromosomal instability, as few as three mutational events appear to be sufficient to induce SCC. Citation Format: Vicente Planells-Palop, Angie Koo, Heydi Malave, Katharine Lee, Aaron Tward. Minimal mutational determinants of human squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B40.