The ability to generate complete genomic sequences will provide unique opportunities to explore how variation impacts evolution and human health, to discover mechanisms regulating organism development, and to manage disease diagnosis and intervention. The effective utilization of this genomic information requires a detailed understanding of the function of encoded information. While tremendous progress has been made in defining individual components of genomic sequences, we still do not understand the function of most annotated genes, we have a limited understanding of the role of non-coding sequences in gene regulation, and we have just started to define the contribution of genomic alterations to human disease. Our section directly addresses these issues by using genomic tools and genetic manipulation of model organisms to unravel genome function and to dissect gene regulatory pathways in development and disease. We integrate data from basic science with clinical information to: 1) identify pathways that regulate mammalian development, 2) understand how alterations in these pathways lead to disease states, and 3) develop paradigms for therapeutic interventions. Our group has demonstrated that mice heterozygous for mutations in the transcription factor SOX10 exhibit multiple defects in neural crest development including reduced numbers of melanocytes in the skin and absence of myenteric ganglia in the colon. We have also shown that SOX10 homozygous mutants die in utero and also exhibit extensive defects in the entire peripheral nervous system. The human congenital disorder Hirschsprung disease can be caused by SOX10 mutations, and it also exhibits rectocolic aganglionosis and can be associated with hypopigmentation. Thus SOX10 mice serve as mouse models for human disease as well as broadly inform us about neural crest development and disease. Our goal is to understand the function of SOX10 in mammalian development and use this information to understand the pathology of and develop treatments for neural crest disorders. SOX10 and adult stem cell genetics. Hair graying in mouse is attributed to the loss of melanocyte stem cell function and the progressive depletion of the follicular melanocyte population. Single-gene, hair graying mouse models have pointed to a number of critical pathways involved in melanocyte stem cell biology; however, the broad range of phenotypic variation observed in human hair graying suggests that additional genetic variants involved in this process may yet be discovered. Using a sensitized approach, we ask here whether natural genetic variation influences a predominant cellular mechanism of hair graying in mouse, melanocyte stem cell differentiation. We developed an innovative method to quantify melanocyte stem cell differentiation by measuring ectopically pigmented melanocyte stem cells in response to the melanocyte-specific transgene Tg(Dct-Sox10). We make the novel observation that the production of ectopically pigmented melanocyte stem cells varies considerably across strains. The success of sensitizing for melanocyte stem cell differentiation by Tg(Dct-Sox10) sets the stage for future investigations into the genetic basis of strain-specific contributions to melanocyte stem cell biology. BRAF. Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors. BRG1 signaling. Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function. Genome editing. Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

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23
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2018
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Human Genome Research
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Cronin, Julia C; Loftus, Stacie K; Baxter, Laura L et al. (2018) Identification and functional analysis of SOX10 phosphorylation sites in melanoma. PLoS One 13:e0190834
Ruiz, Rolando; Jahid, Sohail; Harris, Melissa et al. (2017) The RhoJ-BAD signaling network: An Achilles' heel for BRAF mutant melanomas. PLoS Genet 13:e1006913
Watkins-Chow, Dawn E; Varshney, Gaurav K; Garrett, Lisa J et al. (2017) Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection. G3 (Bethesda) 7:719-722
Marathe, Himangi G; Watkins-Chow, Dawn E; Weider, Matthias et al. (2017) BRG1 interacts with SOX10 to establish the melanocyte lineage and to promote differentiation. Nucleic Acids Res 45:6442-6458
Ibiza, Sales; García-Cassani, Bethania; Ribeiro, Hélder et al. (2016) Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence. Nature 535:440-443
Harris, Melissa L; Levy, Denise J; Watkins-Chow, Dawn E et al. (2015) Ectopic differentiation of melanocyte stem cells is influenced by genetic background. Pigment Cell Melanoma Res 28:223-8
Watkins-Chow, Dawn E; Cooke, Joanna; Pidsley, Ruth et al. (2015) Correction: Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes. PLoS Genet 11:e1005682
Saldana-Caboverde, Amy; Perera, Erasmo M; Watkins-Chow, Dawn E et al. (2015) The transcription factors Ets1 and Sox10 interact during murine melanocyte development. Dev Biol 407:300-12
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
Harmacek, Laura; Watkins-Chow, Dawn E; Chen, Jianfu et al. (2014) A unique missense allele of BAF155, a core BAF chromatin remodeling complex protein, causes neural tube closure defects in mice. Dev Neurobiol 74:483-97

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