ts, and pathways that regulate differentiated cellular phenotypes. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19861958 play a role in the etiology of age related macular degeneration and Parkinson’s disease. Additionally, melanin is aberrantly regulated in human skin disorders such as vitiligo and melasma. Harnessing the molecular mechanisms that regulate melanogenesis to selectively modulate melanin production in the skin, eye, or brain could lead to novel treatments for multiple human pathologies. Pharmacologic modulation of melanin production has primarily focused on identifying inhibitors of tyrosinase, the rate limiting step in pigment production.These results GW 501516 site validate that the siRNAs selectively impact the expression of the cognate target gene, although this may not conceivably hold true for all of the siRNAs used in our screen. To eliminate siRNA pools with off-target effects on melanogenesis, the four siRNAs comprising each siRNA pool were retested individually. We found that at least two independent siRNAs against each target gene significantly inhibited pigment production, suggesting that pigmentation phenotypes are not a common consequence of siRNA off-target phenomena. Together, these studies demonstrate that the genome wide siRNA screening platform accurately identified gene targets that specifically impact pigment production. Initial examination of existing GO annotation data for our pigment regulators exposed a wide variety of cellular processes represented by the validated and candidate hits. Therefore, we employed a focused unbiased approach to identify regulators of tyrosinase, the rate limiting enzyme specifying melanogenesis among novel validated genes supporting MNT-1 pigmentation. Relative accumulation of tyrosinase, the melanogenesis transcription factor MITF, and the melanosomal marker protein Melan-A were examined 96 hours post siRNA transfection. Remarkably, over half of the validated pigment genes appear to be required for tyrosinase protein accumulation. This defect did not appear to be a gross inhibition of cell fate specification, as Melan-A expression was mostly unaffected. In addition, the sub cellular morphology of PMEL17, a melanosome structural protein, was normal at the level of immunofluorescence detection. Of those pigment genes impacting tyrosinase accumulation, approximately half appear to act at the level of tyrosinase mRNA accumulation, and most of these also impaired MITF mRNA accumulation. Given that tyrosinase is an MITF target gene, the pigmentation genes in this later class may represent action at the level of MITF mRNA. A caveat to this interpretation is our observation that siRNA-mediated turnover of tyrosinase mRNA can also lead to inhibition of MITF gene expression through a relationship that remains to be defined. Preliminary studies indicated that this phenotype was not a consequence of siRNA off-target phenomenon. While pigmentation in humans is a complex multigenic trait, the degree of genetic variation that contributes to melanocyte autonomous pigment production is unknown. To examine the phenotypic penetrance of novel pigmentation genes, identified in MNT-1 cells, in diverse genetic backgrounds, we employed primary human melanocyte cultures isolated from two different individuals. Remarkably, the majority of targets that 
regulated tyrosinase expression in MNT-1 cells also impacted tyrosinase expression when depleted from darkly pigmented primary melanocytes. Approximately half of these targets also inhibited tyrosinase expression w