Neural crest cells are an embryonic structure unique to vertebrates that gives rise to multiple lineages including the pigment-producing melanocytes. Pigmentation, defined as the placement of pigment in skin, hair, and eyes for coloration, is distinctive because the location, amount, and type of pigmentation provides a visual manifestation of genetic heterogeneity in pathways regulating the pigment-producing cells. The scope of this genetic heterogeneity ranges from normal to pathological pigmentation phenotypes. Clinically, normal human pigmentation encompasses a variety of skin and hair color as well as punctate pigmentation such as melanocytic nevi (moles) or ephelides (freckles), while abnormal human pigmentation exhibits markedly reduced or increased pigment levels, known as hypopigmentation and hyperpigmentation, respectively. Elucidation of the molecular genetics underlying pigmentation has revealed genes important for melanocyte development and function. Furthermore, many pigmentation disorders show additional defects in cells other than melanocytes, and identification of the genetic insults in these disorders has revealed pleiotropism, where a single gene is required for various functions in different cell types. Thus, unraveling the genetics of easily visualized pigmentation disorders has identified molecular similarities between melanocytes and less visible cell types/tissues, arising from a common developmental origin and/or shared genetic regulatory pathways. We utilize a variety of genetic and genomic approaches to discover the etiology of human pigmentation disorders, often focusing on the fact that the developmental mutations disrupting pigmentation are instructive for understanding abnormal pathways governing related disorders. 1. Melanoma, skin cancer of melanocytes. We have previously shown that the transcription factor SOX10 is essential for survival and proper differentiation of neural crest cell lineages, where it plays an important role in the generation and maintenance of melanocytes. SOX10 is also highly expressed in melanoma tumors, but a role in disease progression has not been established. We have now shown that melanoma tumor cell lines require wild-type SOX10 expression for proliferation, and SOX10 haploinsufficiency reduces melanoma initiation in a transgenic mouse model of melanoma. Stable SOX10 knockdown in human melanoma cells arrested cell growth, altered cellular morphology, and induced senescence. Melanoma cells with stable loss of SOX10 were arrested in the G1 phase of the cell cycle. Since cell cycle dysregulation is a core event in neoplastic transformation, the role for SOX10 in maintaining cell cycle control in melanocytes suggests a rational new direction for targeted treatment or prevention of melanoma. We are currently pursuing approaches to modulate SOX10 function as a means of providing novel approaches for melanoma intervention. 2. Genomics of melanocyte gene regulation. Regulation of gene expression at the level of transcription is an essential process in all organisms. In eukaryotes, DNA sequence elements in combination with chromatin modification play a major role in regulating transcription during normal development and in oncogenic transformation. We have undertaken a multilevel approach to defining the genomic elements and chromatin modifications in the melanocyte lineage, with the goals of determining how they correlate with developmental processes and how they are altered in neural crest and pigmentation disorders such as melanoma. Using large-scale enhancer discovery facilitated by chromatin marks analysis, we have isolated a set of over 2400 melanocyte enhancer loci in the mammalian genome that are evolutionarily constrained, enriched for sequence motifs predicted to bind key melanocyte transcription factors, located near genes relevant to melanocyte biology, and capable of driving reporter gene expression in melanocytes in culture and in transgenic zebrafish. We are combining these results with chromatin isolated from melanocytes and melanoma lines at various cellular states, and also examining additional chromatin binding proteins and transcription factors. For example, comparison of SOX10 ChIP-seq in melanocytes with genomic profiles of EP300 and H3K4Me1 reveals SOX10 binding categories reflecting distinct regulatory components. SOX10-bound genomic segments include not only developmental and pigmentation genes, as expected, but also implicate genes involved in regulation of kinase activities, cellular macromolecular complex assembly and negative regulation of proliferation. Continuation of this genomic level approach will provide a valuable resource to the neural crest, pigment cell and melanoma communities.

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Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2013
Total Cost
$764,833
Indirect Cost
Name
National Human Genome Research Institute
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Type
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Hwang, Hun-Way; Baxter, Laura L; Loftus, Stacie K et al. (2014) Distinct microRNA expression signatures are associated with melanoma subtypes and are regulated by HIF1A. Pigment Cell Melanoma Res 27:777-87
Harris, Melissa L; Pavan, William J (2013) Postnatal lineage mapping of follicular melanocytes with the Tyr::CreER(T) (2) transgene. Pigment Cell Melanoma Res 26:269-74
Harris, Melissa L; Buac, Kristina; Shakhova, Olga et al. (2013) A Dual Role for SOX10 in the Maintenance of the Postnatal Melanocyte Lineage and the Differentiation of Melanocyte Stem Cell Progenitors. PLoS Genet 9:e1003644
Cronin, Julia C; Watkins-Chow, Dawn E; Incao, Art et al. (2013) SOX10 Ablation Arrests Cell Cycle, Induces Senescence, and Suppresses Melanomagenesis. Cancer Res 73:5709-18
Loftus, Stacie K; Antonellis, Anthony; Matera, Ivana et al. (2009) Gpnmb is a melanoblast-expressed, MITF-dependent gene. Pigment Cell Melanoma Res 22:99-110
Loftus, Stacie K; Baxter, Laura L; Buac, Kristina et al. (2009) Comparison of melanoblast expression patterns identifies distinct classes of genes. Pigment Cell Melanoma Res 22:611-22
Antonellis, Anthony; Huynh, Jimmy L; Lee-Lin, Shih-Queen et al. (2008) Identification of neural crest and glial enhancers at the mouse Sox10 locus through transgenesis in zebrafish. PLoS Genet 4:e1000174
Dutton, James R; Antonellis, Anthony; Carney, Thomas J et al. (2008) An evolutionarily conserved intronic region controls the spatiotemporal expression of the transcription factor Sox10. BMC Dev Biol 8:105