University of Oklahoma researcher publishes a study identifying 135 previously unknown pigmentation genes with broad implications for human diversity, cell biology and medicine.
The skin, hair and eye color of more than eight billion humans is determined by the light-absorbing pigment known as melanin. An article recently published in the journal Science features research from Vivek Bajpai, Ph.D., lead author and an assistant professor in the School of Sustainable Chemical, Biological and Materials Engineering at the University of Oklahoma, and collaborators from Stanford University. Their research has identified 135 new genes associated with pigmentation.
Melanin is produced within special structures called melanosomes. Melanosomes are found inside melanin-producing pigment cells called melanocytes. Although all humans have the same number of melanocytes, the amount of melanin they produce differs and gives rise to the variation in human skin color.
“To understand what actually causes different amounts of melanin to be produced, we used a technology called CRISPR-Cas9 to genetically engineer cells,” Bajpai said. “Using CRISPR, we systematically removed more than 20,000 genes from hundreds of millions of melanocytes and observed the impact on melanin production.”
To identify which genes influence melanin production, cells that lost melanin during the gene removal process needed to be separated from millions of other cells that did not. Using in vitro cell cultures, Bajpai developed a novel method to achieve this goal that detects and quantifies the melanin-producing activity of melanocytes. By passing light through the melanocytes, he could record if the light was either absorbed or scattered by the melanin inside.
“If there are a lot of melanin-producing melanosomes, the light will scatter much more than in cells with little melanin,” Bajpai said. “Using a process called side-scatter of flow cytometry, we were able to separate cells with more or less melanin. These separated cells were then analyzed to determine the identity of melanin-modifying genes. We identified both new and previously known genes that play important roles in regulating melanin production in humans.”
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.
The researchers found 169 functionally diverse genes that impacted melanin production. Of those, 135 were not previously associated with pigmentation. They further identified the function of two newly discovered genes: KLF6 and COMMD3. The DNA-binding protein KLF6 led to a loss of melanin production in humans and animals, confirming the role KLF6 plays in melanin production in other species as well. The COMMD3 protein regulated melanin synthesis by controlling the acidity of melanosomes.
Historically, darker pigmentation has been needed to protect against ultraviolet radiation in areas closer to the equator and for people who spend hours in direct sunlight. As humans moved into areas with less direct sunlight or fewer hours of daylight overall, less melanin was needed. Over time, this resulted in melanosomes that produced less melanin, thus absorbing more sunlight.
“By understanding what regulates melanin, we can help protect lighter-skinned people from melanoma, or skin cancer,” Bajpai said. “By targeting these new melanin genes, we could also develop melanin-modifying drugs for vitiligo and other pigmentation diseases.”
The technological processes developed and used by the research team could also be applied to identify genes that regulate melanin production in fungi and bacteria. Melanin production in fungi and bacteria enables them to be more pathogenic to humans or crops. Researchers could develop effective interventions against these microbes and their diseases by discovering and targeting such melanin-producing genes.
Bajpai’s role in the study was completed during his professorship at the University of Oklahoma. However, a portion of this research took place during his postdoctoral research fellowship at Stanford University. A grant from the Oklahoma Center for Adult Stem Cell Research supported the study. Additional funding was provided by the U.S. Department of Defense, CA160997; Howard Hughes Medical Institute; National Institute of General Medical Sciences, NIH R35 GM131757; Stinehart-Reed Award; and the Ludwig Center for Cancer Stem Cell Research and Medicine.
Story source: University of Oklahoma
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.
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.
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.
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.