OCA2

肤色差异是不同人群最显著的表型特征之一。肤色是人类适应太阳的紫外线辐射而自然选择形成的，也是人类在不同纬度长期生活适应性进化的一个重要标志。现代人类起源于非洲，目前非洲人群具有世界上最丰富的遗传多样性和表型多样性。不同地区的非洲人群，肤色差异很大。特别是居住在非洲南部的桑人（KhoeSan）,他们具有人类最古老的遗传成分，而且肤色最浅，但其浅肤色的遗传学基础以及适应性进化的机制尚缺乏研究。研究人类肤色多样性的遗传机制及其生物学基础，不仅可以增进对人类表型进化的理解，还可以为皮肤相关疾病的诊治提供指导。2024年1月10日，宾夕法尼亚大学Sarah A. Tishkoff教授团队（冯园庆博士为第一作者，谢宁博士为第二作者）与合作者在Nature Genetics在线发表题为“Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans”的研究论文，该研究运用整合基因组学和功能基因组学对非洲人群肤色进行研究，揭示了人类肤色多样性和适应性进化的遗传机制。肤色是具有高度遗传性的复杂性状，受到数百个基因位点的影响。全基因组关联分析表明人类肤色相关突变中，90%以上都是位于非编码区，如何研究非编码区突变的功能是一个挑战。研究者运用了两种方法寻找与皮肤色素相关的候选单核苷酸突变（SNP），一种是通过对多个种族的非洲人群进行肤色相关的全基因组关联分析（GWAS）获得GWAS-SNP，一种是通过自然选择扫描（natural selection scan）对浅肤色的桑人群与其他深肤色的非洲人群进行比较获得显著差异的Di-SNP。大规模平行报告基因检测技术(massively parallel reporter assays, MPRA)可以同时检测基因组数千个调控元件及突变的调控活性。研究者挑选出位于黑色素细胞的开放染色质区域中的候选SNP，使用MPRA检测了这些候选SNP的调控活性。为了识别这些SNP的潜在靶基因，使用 Hi-C和H3K27ac HiChIP技术，构建了高分辨率的染色质相互作用图谱。最后，结合荧光素酶报告基因检测、CRISPR介导的基因组编辑、转录组分析和黑色素丰度检测，发现了多个显著影响黑素细胞中黑色素水平的新突变、增强子和新基因，揭示了调控人类皮肤色素的遗传机制。从20,000多个通过全基因组关联分析和自然选择扫描得到的候选SNP中，研究者选择了1,157个 SNP用于MPRA，鉴定出165个可以改变调控活性的功能性SNP(MFV)（图1）。进一步验证了其中16个MFV可以调控增强子活性，并使用功能基因组方法鉴定了它们的靶基因。图1. 大规模筛选与非洲人肤色相关的遗传突变。左图，筛选候选SNP示意图。右图，MPRA流程图。与其他非洲人群相比，非洲南部的桑人群肤色最浅，并且拥有人类最古老的遗传谱系。研究发现MITF附近的rs111969762、LEF1附近的rs17038630和TRPS1附近的 rs11985280，这三个Di-SNP均能显著影响增强子活性及其靶基因的表达，可能对桑人皮肤色素的适应性进化发挥了作用。MITF、LEF1和TRPS1参与调节黑色素细胞的分化和毛发的发育，这表明在桑人中观察到的影响肤色的突变也可能影响其独特的头发形态。值得注意的是，TRPS1附近的突变rs11985280，与浅肤色相关的突变频率在桑人和大多数非洲以外人群中接近100%，而与深肤色相关的突变频率在大多数深肤色的非洲人群和深肤色的美拉尼西亚人群中很高，这是一个全球对紫外线照射的适应的典型例子。研究发现OCA2（一种与白化病相关的基因）增强子的突变rs6497271可导致黑色素细胞里的黑色素水平降低75%。并且，在同一个OCA2增强子中，有两个位置相邻很近的突变均可显著影响调控活性，这两个突变分别有120万年和5.7万年的历史，后者与人类迁出非洲的时期很巧合的一致。这个研究说明人类的肤色在不断演变，并且这样的演变在不同的时期发生在同一个重要基因的同一个增强子上。研究还发现GWAS-SNP rs7948623的主要靶基因是CYB561A3和TMEM138（图2）。重要的是，通过功能实验，进一步发现了CYB561A3是一个影响人类皮肤色素的新基因，它能够调控线粒体呼吸和黑素生成相关的基因表达。图2. GWAS-SNP rs7948623 的主要靶基因是CYB561A3和TMEM138。左图，MPRA 在 DDB1 基因座附近鉴定出的3个调控突变。右图，Hi-C 和 H3K27ac HiChIP 鉴定 DDB1基因座附近的染色质相互作用。本研究综合群体遗传学和功能基因组学，GWAS和进化分析，MPRA技术，结合Hi-C、CRISPR基因编辑等实验技术，一次性检测成千上万个非编码突变的功能，鉴定了功能突变的靶基因，发现了调控色素的新基因。这种整合基因组学方法，可广泛推广应用于高通量地研究人类疾病或复杂性状的遗传因素，鉴定功能性突变以及新的致病基因，为精准医疗和药物靶点鉴定提供指导。人类肤色的差异变化是一种复杂的适应性特征，受到众多遗传位点的影响。本研究发现了非编码突变在决定人类肤色上发挥着重要作用，强调了影响肤色的遗传因素的复杂性，也凸显出研究具有高度的遗传多样性和表型多样性的人群，对确定复杂性状的遗传基础十分重要。同时，研究提供了肤色多样性的历史进化的新信息，有助于更清晰地描述人类进化。参考文献：Feng Y, Xie N, Inoue F, Fan S, Saskin J, et al. Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans. Nat Genet. 2024 Jan 10. doi: 10.1038/s41588-023-01626-1. Epub ahead of print. PMID: 38200130.                 责编|探索君排版|探索君文章来源|“iNature”End往期精选围观王晓红教授团队：绘制单次冷冻囊胚移植成功的女性植入期外周血microRNA动态表达谱，开拓领域创新研究热文维生素B12在细胞重编程与组织再生中发挥关键作用！热文不孕症治疗新方向！又一导致女性不孕的关键因素被发现热文Let-7家族miRNA或为癌症潜在免疫治疗靶点热文一项最新发现或可用于阿尔茨海默病药物开发点击“阅读原文”，了解更多~

Researchers have discovered key insights into the molecular basis of skin color variations among African populations. Their findings broaden the understanding of human evolution and the genetics underpinning contemporary human skin color diversity. Researchers from the University of Pennsylvania have discovered key insights into the molecular basis of skin color variations among African populations. Their findings, published in Nature Genetics, broaden the understanding of human evolution and the genetics underpinning contemporary human skin color diversity. "Despite the abundant genetic diversity within African populations, they have been historically underrepresented in genetic studies," says senior author Sarah Tishkoff, a Penn Integrates Knowledge University Professor with appointments in the Perelman School of Medicine and School of Arts & Sciences. "Our findings offer novel information about the genetic basis and evolutionary history of skin color diversity, contributing to a clearer depiction of human evolution." The story of human evolution is as rich and diverse as the adaptations found across the world's populations, Tishkoff says. She notes that, among many adaptive traits, skin color stands out as one of the most well-known. Darker skin tones, prevalent in equatorial regions, serve as nature's very own sunblock, evolving over millennia to shield these populations from the sun's intense ultraviolet radiation. Conversely, lighter pigmentation, as seen in populations closer to the poles, is an adaptation to mitigate the risks of insufficient sun exposure by maximizing vitamin D production, which is triggered by UV exposure. "Our approach involved genome-wide association studies of skin color from more than 1,500 eastern and southern African individuals as well as scanning the genome to identify genetic variants that are highly differentiated between lightly-pigmented Khoesan-speaking San population and other darkly pigmented Africans and may play a role in local adaptation in that population," says Yuanqing Feng, first author of the paper and a postdoctoral researcher in the Tishkoff Lab. The researchers note that pigmentation is a complex trait influenced by hundreds of variants scattered across the genome, with the majority situated in noncoding regions. These noncoding variants may affect the expression of genes located up to one million bases away. The vast number of mutations associated with skin color and the uncertainty surrounding the target genes regulated by these mutations make it particularly arduous for researchers to find the precise genetic mechanisms governing this trait. Feng and collaborators used massively parallel reporter assays to discern the regulatory activities of thousands of variants. This high-throughput technique narrowed down the thousands of candidates to 165 functional variants. To identify the target genes of these functional variants, Feng further constructed high-resolution chromatin interaction maps in melanocytic cells using chromatin conformation capture assays. "This is a high-resolution 3D genome map in melanoma cells that will be valuable for gene regulation studies in pigmentation and melanoma biology," Feng says. Using CRISPR/Cas9-based genome editing, the researchers discovered that mutations in an enhancer of OCA2, a gene associated with albinism, could lead to a 75% reduction in melanin levels when compared to control cells. Within the same OCA2 enhancer, the researchers identified two closely located regulatory variants, estimated to be 1.2 million years old and 57 thousand years old, with the latter coinciding with the period of human migration from Africa. "This case illustrates the continuous evolution of human skin color, and it's remarkable to observe the significant effects on skin pigmentation attributed to a single enhancer," Feng says. San people have relatively lighter pigmentation compared to other African populations and possess the oldest genetic lineages in humans. While it is hypothesized that the light skin color of the San may result from adaptation to a southern African environment, the genetic underpinnings of this adaptation remain elusive. The researchers pinpointed several crucial regulatory variants near MITF, LEF1, and TRPS1 that contribute to the skin color adaptation observed in the San. "MITF, LEF1, and TRPS1 are involved in signaling pathways regulating both melanocyte differentiation and hair development," Tishkoff says. "This suggests that the variants influencing the lighter skin pigmentation observed in the San people may also contribute to their distinctive hair morphology." Notably, the variant near TRPS1 associated with lighter skin color is at nearly 100% frequency in the San and in most non-Africans, whereas the variant associated with darker skin color is common in most other African populations and in the darkly pigmented Melanesian population, a striking example of global adaptations to UV exposure." Additionally, the researchers found a novel gene impacting human skin pigmentation, CYB561A3, which regulates iron homeostasis and influences melanin levels in melanocytic cells. "To our knowledge, the role of CYB561A3 in skin pigmentation has not been reported before. Intriguingly, there have been reports linking intravenous iron infusion to skin hyperpigmentation. Given that CYB561A3 encodes an iron reductase, I am curious about the role of this protein in this process," Tishkoff says. "Our findings underscore the complexity of genetic factors influencing skin color and the benefits of including ethnically diverse and underrepresented populations in genetic studies," she says. "Conducting functional studies on the impact of noncoding variants will enhance our comprehension of the genetics underlying complex human traits and disease risk." "The populations included in this study are from remote regions of Africa and required the use of a mobile lab set up in the field sites," Tishkoff says. "The collaboration with our partners in Africa was key to the success of this research project." In future research, the Tishkoff lab would like to use its innovative functional genomics approach to identify more genetic variants contributing to human pigmentation and other adaptive traits in a larger sample of ethnically diverse Africans. Sarah Tishkoff is the David and Lyn Silfen University Professor in Genetics and Biology and a Penn Integrates Knowledge University Professor with appointments in the Perelman School of Medicine's Department of Genetics and Department of Medicine and in the School of Arts & Sciences' Department of Biology at the University of Pennsylvania. Yuanqing Feng is a postdoctoral fellow in the Tishkoff lab at Penn. Other authors include Ning Xie, Chao Zhang, Fang Zhang, and Matthew E.B. Hansen of Penn; Fumitaka Inoue of Kyoto University; Shaohua Fan of Fudan University; Thomas Nyambo of Hubert Kairuki Memorial University; Sununguko Wata Mpoloka and Gaonyadiwe George Mokone of the University of Botswana; Charles Fokunang and Alfred K. Njamnshi of the University of Yaoundé; Gurja Belay of Addis Ababa University; Michael S. Marks of the Children's Hospital of Philadelphia Research Institute; Elena Oancea of Brown University; and Nadav Ahituv of the University of California, San Francisco. This research was supported by the National Institutes of Health (grants R35GM134957-01, 3UM1HG009408-02S1, 1R01GM113657-01, 5R01AR076241-02, and 1S10OD010786-01) and the Penn Skin Biology and Disease Resource-based Center (Grant NIH P30-AR069589).

君不见，高堂明镜悲白发，朝如青丝暮成雪……不论头发是在不知不觉间由黑转白，还是因为遭遇重大变故而“一夜白头”，都是令人伤感遗憾的事。头发的变白似乎总和衰老脱不开关系，既是由于人们总因衰老而白头，也是因为白发会让人看起来苍老更多。然而现在，除了染发、戴假发之外，人们有希望真正做到将头发由白变黑！4月19日，纽约大学格罗斯曼医学院研究团队在Nature上发表最新篇论文“Dedifferentiation maintains melanocyte stem cells in a dynamic niche”，揭秘了头发变白的机制，并指出这一机制可以被逆转。图1 研究成果（图源：[1]）一般情况下，头发呈现出黑色，是由于黑色素细胞合成的真黑色素（Eumelanin）将头发“染”成了黑色。黑色素细胞由黑色素干细胞（melanocyte stem cell，McSC）分化成熟而来。McSC是成体干细胞的一种。一般而言，成体干细胞会有两种截然不同的命运走向：一种通过自我更新维持自身，另一种则先进入名为“过渡扩增”（transit-amplifying，TA）的中间状态，可以快速增殖，然后分化成熟为具体的细胞类型。成体干细胞能否维持这两种走向，决定了身体组织器官能否正常地更新和修复，比如伤口愈合和毛发生长，都有赖于此。然而，格罗斯曼医学院的研究人员通过谱系追踪（可以跟踪一种细胞及其后代）和3D活体成像的方法对小鼠体内的McSC进行持续的跟踪。结果发现，McSC的行为与其他的成体干细胞并不完全相同，甚至颠覆了以往的教条。毛发的生长是由毛囊（hair follicles，HF）周期性地生长、退化、再生所驱动的。研究人员发现，在毛囊的休止期（telogen），大多数McSC位于毛囊的毛基质（hair germ，HG）区，少数情况下，McSC会出现在毛囊的隆起（bulge）区。这表明，对于头发着色和McSC的自我维持，HG区的McSC是主力军。图2 毛囊结构（图源：[2]）随后，研究人员追踪了HG McSC在毛囊周期其余时间的命运走向。结果发现，在毛囊生长初期（anagen），在毛球（hair bulb）区，出现了HG McSC生成的成熟黑色素细胞，这些成熟的黑色素细胞会在初期结束时死亡。同时，HG McSC还出现在隆起和上部外根鞘（upper outer root sheath，ORS up）的干细胞生态位，并表现出自我更新能力。在毛囊的生长中晚期（catagen），隆起/ORS up区的大多数McSC出现在毛囊下部。到下一个休止期，它们大多数回到了HG区域。图3 HG McSC在毛囊生长周期中的两种命运走向，上图为自我维持走向，下图为分化成熟走向（图源：[1]）研究人员进一步通过形态学观察和单细胞RNA测序对生长期早期的McSC的行为进行了考察，结果发现不论是形态上还是基因表达上，生长初期早期的McSC及其子细胞都变得更接近分化成熟的黑色素细胞，然而这种变化会在McSC迁移到隆起/ORS up区后退回。McSC的这种循环往复甚至可以维持两年之久——接近小鼠成年后的寿命。鉴于此，研究人员提出大胆假设，猜想大部分HG McSC会随着HG的生长而分化为“过渡扩增”状态，McSC的自我维持依赖于它们的去分化。这有别于以往对成体干细胞的认知，即分化程度只会越来越深，不会出现逆转的情况。研究人员接着着眼于一种晚期色素沉着基因Oca2，来追踪HG McSC中分化程度最高的那部分细胞的后续命运。结果发现，这群高度分化、甚至是位于HG最下部（称为“过渡扩增”区）的Oca2+ McSC都可以退回成未分化的McSCs；而且，没有McSC天生免于分化，所有的McSC都会经历“过渡扩增”状态；此外，即使长期处在促进分化的环境中，如紫外线照射，McSC依然可以恢复到未分化状态。那么，McSC是如何完成去分化的呢？过去的研究表明，WNT信号是McSC成熟分化不可或缺的，缺乏WNT信号将使得黑色素细胞再生不足，但是持续的、过多的WNT信号则会耗尽McSC。这次，研究人员观察到，随着毛囊向下生长伸长，WNT活性区域也随之向下移动，而在隆起/ORS up区，WNT信号遭到抑制。因此，研究人员猜测，WNT信号的下调与否，决定了McSC能否恢复到未分化状态。随后的实验证实了这一猜测，即McSC在干细胞生态位（隆起/ORS up区）和过渡扩增区（HG/毛球区）的移动能力，使它们能够面对不同的WNT信号水平，从而可逆地走向分化或去分化。过去，科学家们发现，McSC比其他成体干细胞群失效更早，导致了人和小鼠出现白发。现在，研究人员所发现的这一机制的脆弱性说明了背后的原因——McSC在两个区域之间的移动容易遭到损害。比如，当人或小鼠进入老年，毛囊在经历了多轮生长周期之后，越来越多的McSC会“卡在”隆起/ORS up区域而无法回到HG区域，虽然它们仍能在隆起/ORS up区域再生，但无法在HG区域进入“过渡扩增”状态并最终分化为黑色素细胞，也就无法为头发着色。对小鼠进行重复拔毛和强制再生加速毛囊老化的实验也证实了这一点，在隆起区域中含有McSC的毛囊比例从原来的10%上升到了50%。看来，白头发越拔越多，还是有一定道理的。图4 反复拔毛强制再生使得小鼠毛发变白（图源：[1]）研究的第一作者、纽约大学朗格健康中心的博士后Qi Sun表示，发现的这一机制可能同样存在与人类身上，因此，通过帮助“卡在”隆起区的McSC重新回到HG区域，或许是预防或扭转头发变白的一种可行方案。研究的资深作者、纽约大学朗格尼健康中心Ronald O. Perelman皮肤病学系和细胞生物学系教授Mayumi Ito表示，团队正在计划研究恢复McSC移动的方法，或者直接在物理上将它们移回HG区域，使之恢复产生色素的能力。另外，值得注意的是，McSC还是致命癌症黑色素瘤的关键参与者。这个研究中所发现的McSC发育可塑性或许解释了为什么黑色素瘤如此难以治疗。另一方面，McSC的过早老化或许是预防黑色素瘤形成的进化策略——如果细胞停止复制，也就无法产生导致癌症的突变。 责编|风立宵校对|风立宵End参考资料：[1]Sun, Q., Lee, W., Hu, H. et al. Dedifferentiation maintains melanocyte stem cells in a dynamic niche. Nature (2023). https://doi.org/10.1038/s41586-023-05960-6[2]https://www.skin168.net/2013/10/hair-anatomy.html[3]https://www.eurekalert.org/news-releases/986164 [4]https://www.nature.com/articles/d41586-023-00918-0 本文系生物探索原创，欢迎个人转发分享。其他任何媒体、网站如需转载，须在正文前注明来源生物探索。 往期精选 围观 ‍张锋团队再发Science：又一种新的基因编辑工具要来了？ 热文教科书级新发现：调控基因表达的“交通灯”，将为癌症治疗提供新靶点 热文发现“渐冻症”用药潜在新靶点，可遏制蛋白质错误折叠！ 热文纳米粒子加速蛋白质药物纯化，降低生产成本，让更多的地区用得起！ 热文 全天化无人值守！百迈客发布全自动化实验平台百灵2000，加速拉开全民测序序幕点击“阅读原文”，了解更多~