Variation in epidermal pigmentation is one of the most striking features of human populations and is critical for protection against the harmful effects of ultraviolet radiation. Many genes that control pigmentation have been identified in animal models, but little is known about the genetic basis of normal human skin pigment variation. Filling this knowledge gap will likely reveal novel molecular and cellular mechanisms underlying melanocyte biology, melanoma risk and rare genetic diseases. Because of their high genetic diversity and wide spectrum of skin tone, African populations are particularly informative for discovering genes that regulate pigmentation. Accordingly, our team recently used a genome wide association study and scans of natural selection in a unique dataset from 1600 ethnically diverse Africans to identify genetic variants that affect skin tone in Africans. These included genes with previously unknown functions in pigmentation such as MFSD12, encoding a predicted transmembrane transporter that localizes to lysosomes but not to melanosomes and thus regulates pigmentation by a novel mechanism. Preliminary analyses also identified additional candidate transporters and regulators of receptor-mediated signaling that were not known to function in pigmentation and that may regulate melanogenesis in novel ways. Here, employing a multi-PI team headed by experts in human genetics, membrane trafficking/ organelle biology, and membrane signaling/ ion transport in collaboration with three additional experts, we propose to identify (i) new pigmentation genes in a larger set of 3,000 Africans for which we have whole genome sequence data, and (ii) the cellular and molecular mechanisms by which the products of these genes control human skin pigmentation. We will accomplish our goal by integrating human genomic and pigmentation data in our unique African cohort, genetic analyses of the impact of non-coding sequence variants on gene expression, and cell culture studies of melanocyte cell biology and physiology. The three specific aims will test our hypotheses that variation in pigmentation results from genetic variations in the regulatory elements of genes encoding proteins that alter either the melanosomal milieu directly or indirectly via signaling mediated by receptors such as the melanocortin-1 receptor (MC1R). Specifically, we will employ the following three specific aims: 1. Identify novel genetic variants influencing skin pigmentation and test whether the variants influence gene expression from nearby genes; 2. Define novel mechanisms by which transporters in lysosomes or melanosomes influence pigmentation by defining how MFSD12 and other newly identified transporters impact melanogenesis; 3. Determine the function of newly identified receptor-mediated signaling proteins that regulate pigmentation resulting from our new analyses and test whether and how they influence MC1R signaling.