Melanin is a complex polymer present across all life forms ranging from unicellular to multicellular organisms. In humans, melanin is typically found in skin, eye and hair and plays physiological role in maintaining homeostasis. Melanin is synthesized inside specialized organelles called melanosomes found in melanocytes. Melanocytes produce and transport melanin filled melanosomes to neighboring keratinocytes resulting in skin pigmentation. Irrespective of skin color, all humans have same number of melanocytes and similar anatomical distribution. Therefore, only variable melanin content within melanosomes give rise to variation in skin color in humans. Melanin synthesis dysregulation leads to various pigmentation diseases like melasma, vitiligo, albinism, etc. Additionally, differences in melanin levels lead to differential disease susceptibility. For example, melanoma more frequently occurs in light colored individuals. Although biochemical pathway of melanin synthesis has been known, the genes and pathways regulating melanogenesis have not been completely understood. Understanding gene regulation of skin pigmentation can help us develop therapies for pigmentation associated diseases. Additionally, during engineered skin graft transplantations, matching skin color and tone of the graft to the patient is a challenge due to lack of knowledge about gene regulatory pathways associated with melanogenesis. To fill this knowledge gap, our lab recently conducted a genome-wide CRISPR screen to identify regulators of melanogenesis. We demonstrated that cellular melanin concentration determines light scattering (measured as side scatter or SSC through flow cytometry) properties of melanocytes. Making use of this unique feature and using SSC as a proxy for melanin levels, we performed a CRISPR-Cas9 based genetic screen that led to the identification of novel gene regulators of human skin pigmentation (Bajpai, et.al, Science 2023). Our CRISPR screen identified several members of ‘copper metabolism protein family MURR1 domain-containing (COMMD)’ gene family (e.g., COMMD 2,3,4,5,8 and 10) as regulators of melanin synthesis. COMMD family of genes play important roles in human health and disease by participating in a variety of cellular functions, including transcription factor regulation, endosomal sorting, ion transport, and copper metabolism. In this work, we investigated the role of COMMD factors in human melanogenesis.
To examine the gene expression levels of COMMD genes in human melanocytes of different levels of melanin, we performed RNA-seq studies using melanocytes isolated from differently colored human skin. We found among COMMD genes, COMMD2 and COMMD3 were highly expressed, COMMD8 and COMMD10 were moderately expressed in dark skin melanocytes indicating their prominent role in melanogenesis. To gain insights into COMMD3 function, we deleted the COMMD3 gene using CRISPR and conducted RNA-seq studies (Figure) using MNT-1 cells, which is a melanin producing melanoma cell line that recapitulates melanin synthesis process like primary human melanocytes. The gene ontology analysis enriched for pathways involved in ‘organelle organization’ and ‘protein targeting’ suggesting possible role of COMMD3 in protein trafficking to the melanosomes. We next, genetically tagged COMMD3 deleted and control cells with a GPR143-HA-mScarlet reporter construct that selectively expresses on melanosomes. Upon performing immunoblotting after immunoprecipitation with anti-HA antibody coated magnetic beads, we found that COMMD3 is physically present in melanosome fraction of the cells further supporting its role in melanosomes function. In summary, our studies identify COMMD family of proteins as novel regulators of human melanogenesis which are differently expressed in light and dark color human skin melanocytes. COMMD3 is physically present on melanosomes and functions as putative protein trafficking gene. Taken together, our studies shed new insights into melanocyte biology, which have implication for pathophysiological human pigmentation and melanized engineered skin grafts.
