Harnessing RNAi-Based Functional Genomics to Unravel the Molecular Complexity Underlying Skin Pigment Variation

Abstract

Melanin, the primary chromophore in human skin, protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson’s disease), auditory disorders (Waardenburg’s syndrome), and ophthalmologic disorders (age-related macular degeneration). Extensive studies have identified over 150 genes that regulate the production of melanin pigment in human cells. Despite this extensive investigation, the phenotypic variation in human skin color cannot simply be explained by the aberrant regulation of these 150 genes. We recently utilized RNAi-based functional genomics to unravel the molecular complexity underlying skin pigment variation. These studies identified 92 novel genes that regulate melanogenesis in human cells, novel pharmacologic inhibitors of melanin production, and novel pathways that regulate melanogenesis. In this chapter, we will discuss an integrative approach to identify novel genes and pathways that regulate melanogenesis from our dataset. First, we will discuss strategies to integrate protein–protein interaction network algorithms with our dataset to better define pathways that regulate melanogenesis in human cells. Two aspects of our RNAi screen dataset were unique: our screen failed to identify many of the known regulators of melanogenesis among our top tier of hits, indicating that the approach had an appreciable false negative rate. Instead, our approach identified a large number of genes that regulate melanogenesis by uncharacterized mechanisms. Therefore, we sought to define our targets by measuring their impact on known melanogenesis regulatory machinery. Initial studies segregated these genes into functional classes by examining their impact on known regulators of melanogenesis, tyrosinase and MITF. An annotation-based approach was used to identify targets that potentially regulate melanosome trafficking, uncovering an unexpected link between autophagy and melanogenesis. In vivo studies validated that autophagy impacts melanogenesis. Recent studies have developed an integrative functional genomics strategy utilizing both siRNA and shRNA platforms to define the impact of individual genes on melanogenesis. In this chapter, we present a model to integrate protein–protein interaction network datasets, functional genomics data, siRNA and shRNA loss of function platforms, and pharmacologic databases to identify functional gene networks that govern human phenotypes and novel treatments for clinical disorders.