Genome-Wide siRNA-Based Functional Genomics of Pigmentation Identifies Novel Genes and Pathways That Impact Melanogenesis in Human Cells

Abstract

Melanin 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 opthalmologic disorders (age related macular degeneration). Much of the core synthetic machinery driving melanin production has been identified; however, the spectrum of gene products participating in melanogenesis in different physiological niches is poorly understood. Functional genomics based on RNA-mediated interference (RNAi) provides the opportunity to derive unbiased comprehensive collections of pharmaceutically tractable single gene targets supporting melanin production. In this study, we have combined a high-throughput, cell-based, one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized, small interfering RNAs to identify novel biological pathways that govern melanin biogenesis in human melanocytes. Ninety-two novel genes that support pigment production were identified with a low false discovery rate. Secondary validation and preliminary mechanistic studies identified a large panel of targets that converge on tyrosinase expression and stability. Small molecule inhibition of a family of gene products in this class was sufficient to impair chronic tyrosinase expression in pigmented melanoma cells and UV-induced tyrosinase expression in primary melanocytes. Isolation of molecular machinery known to support autophagosome biosynthesis from this screen, together with in vitro and in vivo validation, exposed a close functional relationship between melanogenesis and autophagy. In summary, these studies illustrate the power of RNAi-based functional genomics to identify novel genes, pathways, and pharmacologic agents that impact a biological phenotype and operate outside of preconceived mechanistic relationships.

Autophagy is essential for mouse sense of balance

“…Thus, a reasonable hypothesis for the observed otoconial abnormalities in Atg4b- and Atg5-null mice is that autophagy inhibition results in a transcriptional downregulation and thus instability of sorting complexes, which in turn leads to an impairment in otoconial matrix assembly and secretion and finally to otoconial abnormalities and balance disorders (Figure 8). Consistent with this model, an essential role for autophagy has been recently proposed in melanogenesis, another process for which the activity of Ap-3 and Bloc-1 sorting complexes is essential (51). The mechanism connecting autophagy disruption with the observed transcriptional alterations in sorting complexes is not clear.…”

Atg7 is required for acrosome biogenesis during spermatogenesis in mice

“…Atg5 and Atg4B are involved in the secretion of otoconins by vestibular sensory cells of the inner ear [42]. Beclin1 and LC3-A have been implicated in the maturation of a specialized LRO, the melanosome [43]. As a novel LRO, the acrosome is regarded as a secretory lysosome not only because of its internal acid pH but also because it contains many enzymes associated with lysosomes in somatic cells [22].…”

[6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

“…It has been reported that skin pigmentation is regulated by a variety of intrinsic and extrinsic factors [7,8] , as well as diverse signaling pathways including protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinases (MAPKs) [9][10][11][12][13] . In particular, the MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) and the PI3K/Akt pathways have been shown to negatively regulate melanin synthesis in melanocytes and melanoma cells [14][15][16][17][18] .…”