In:
Science, American Association for the Advancement of Science (AAAS), Vol. 381, No. 6658 ( 2023-08-11)
Abstract:
Melanin is a heterogenous polymer found across life forms and has roles in pigmentation, environmental adaptation, and species survival. In humans, melanin synthesis is compartmentalized within a subcellular organelle called the melanosome, which is present in specialized pigment cells called melanocytes. Variable melanin synthesis within melanosomes results in variation in skin, hair, and eye color in human populations, whereas perturbations in melanogenesis are associated with diseases. RATIONALE Melanin’s particular physicochemical properties such as high refractive index determine its optical properties. We reasoned that an accumulation of melanin within melanosomes would change melanocytes’ light-scattering properties. Thus, measuring light-scatter index with flow cytometry could serve as a proxy for cellular melanin content of live pigment cells and could be used as an assay for a CRISPR-Cas9–based genetic screen to identify factors that govern melanin synthesis within melanosomes. RESULTS Using pigmented cell lines and human melanocytes at different stages of melanosomal maturation, we established a quantitative linear relationship between melanin levels and light scattering measured as side scatter (SSC) through flow cytometry. We further demonstrated that SSC changes capture dynamic shifts in melanin levels within melanosomes. Using SSC as a proxy for melanin content, we then conducted a genome-wide CRISPR-Cas9 genetic screen to systematically uncover regulators of melanogenesis. Our screen identified both previously known pigmentation genes and 135 previously unidentified hits that we refer to as melanin-promoting genes, and whose deletion resulted in decreased melanin production. The melanin-promoting genes are involved in diverse biological pathways such as transcription regulation, RNA processing, and endosomal transport, among others. Consistent with their melanin-promoting role, the expression of the majority of our screen hits is elevated in darkly pigmented, compared with lightly pigmented, human melanocytes. Our analyses revealed that select melanin-promoting genes are associated with skin color variation and show evidence of local adaptation in human populations. We further characterized the products of two genes, KLF6 and COMMD3 , for their role in melanogenesis. We showed that deletion of the transcription factor KLF6 inhibits melanosome maturation and reduces pigmentation in vitro and in vivo, whereas COMMD3, a protein involved in endosomal transport, regulates melanosomal pH. Loss of COMMD3 leads to a decreased melanosomal pH, which in turn perturbs melanosome maturation; this effect can be reversed by chemicals that raise the melanosomal pH. CONCLUSION Our work demonstrates that changes in melanin content can be robustly quantified by measuring the side scattering property of live pigment cells. By exploiting this relationship in the context of a genetic screen, we identified melanin-promoting genes with diverse biological functions. By focusing on specific previously unidentified candidates, we implicated a new cargo recycling pathway in melanosome function and identified a transcription factor involved in melanosome maturation. Our work provides a rich resource for further studies of melanogenesis and its relationship with skin color variation and human diseases. Genetic screen finds previously unidentified loci for human melanogenesis. By exploiting melanin’s light-scattering properties, we conducted a genome-wide screen and uncovered genes regulating melanin content and hence, melanogenesis, in human cells. These melanin-promoting genes are expressed at higher levels in darkly pigmented melanocytes and show association with pigmentation in human populations. KLF6 deletion reduces melanogenesis and pigmentation in vivo, whereas COMMD3 exerts melanogenic effect by modulating melanosomal pH and tyrosinase (TYR) activity.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.ade6289
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2023
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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