Hair color differences are among the most obvious examples of phenotypic variation in humans. Although genome-wide association studies (GWAS) have implicated multiple loci in human pigment variation, the causative base-pair changes are still largely unknown1. Here we dissect a regulatory region of the KITLG gene (encoding KIT ligand) that is significantly associated with common blond hair color in northern Europeans2. Functional tests demonstrate that the region contains a regulatory enhancer that drives expression in developing hair follicles. This enhancer contains a common SNP (rs12821256) that alters a binding site for the lymphoid enhancer-binding factor 1 (LEF1) transcription factor, reducing LEF1 responsiveness and enhancer activity in cultured human keratinocytes. Mice carrying ancestral or derived variants of the human KITLG enhancer exhibit significant differences in hair pigmentation, confirming that altered regulation of an essential growth factor contributes to the classic blond hair phenotype found in northern Europeans.
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We thank R. Moon (University of Washington) for the XE237 LEF1 expression plasmid, R. Nusse (Stanford University) for the SuperTOPFlash plasmid, C. Lowe (Stanford University) for help with statistical and 1000 Genomes Project analysis and members of the Kingsley laboratory for useful comments on the manuscript. This work was supported in part by the University of Georgia Research Foundation (M.A.B.) and by US National Institutes of Health grants GM65393 (M.A.B.), R01-NS050835 (L.L.) and a Center of Excellence in Genomic Science award 5P50HG2568 (D.M.K.). L.L. and D.M.K. are investigators of the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Integrated supplementary information
Fifteen transgenic embryos produced by pronuclear injection with the 6.7-kb H2 plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. The skin (n = 13) and kidney (n = 14) were consistent sites of expression. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.
Thirteen transgenic embryos produced by pronuclear injection with the 1.5-kb H2b plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. The kidney (n = 12) was the only consistent site of expression. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.
Eleven transgenic embryos produced by pronuclear injection with the 1.9-kb HFE clone are pictured. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Hair/skin expression was visible in 8 of the 11 embryos. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.
Nine transgenic embryos produced by pronuclear injection with the 6.7-kb H2-BLD plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Hair/skin (n = 7) and kidney (n = 7) were consistent sites of expression. No clear difference in expression compared to the complete set of H2 (H2-ANC) transgenic embryos was evident (see Supplementary Fig. 1). The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.
Eight transgenic embryos produced by pronuclear injection with the 6.7-kb H2-DEL plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Seven of the embryos show hair/skin expression. However the strength of this staining appeared reduced compared to H2-ANC and H2-BLD embryos, particularly in embryos that showed comparably strong kidney expression (such as embryos 2 and 5). The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.
BLD-Kitl/+ and ANC-Kitl/+ heterozygotes exhibit several altered pigmentation patterns compared to wild-type (FVB/C57BL/6J F1 hybrid) littermates. At 2 months, ectopic pigmentation is seen on the muzzles (arrowheads in a) and the epithelium of the antitragus and ear canal (arrows in b–d). In contrast, BLD-Kitl/+ and ANC-Kitl/+ heterozygotes show reduced pigmentation in the whiskers (arrows in a) and the hair on the digits (arrows in e), perhaps because of competitive interactions between body sites for melanocyte colonization and development48 or premature differentiation of migrating melanocytes49. (f–h) Cross-sections (6 μm) through dorsal skin from 2-month-old (f) wild-type, (g) BLD-Kitl/+ and (h) ANC-Kitl/+ heterozygotes counterstained with nuclear fast red. Elevated Kitl expression controlled by both the BLD and ANC hair enhancers leads to ectopic pigmentation of the bulge region of hair follicles (arrows) and the basal epidermis (asterisks). DP, dermal papilla. Scale bars, 30 μm.
Supplementary Figure 7 Analysis of pigment levels in zigzag hairs from site-specific transgenic mice.
(a) Photographs of zigzag hairs from wild-type (+/+; FVB/C57BL/6J F1 hybrid), BLD (BLD-Kitl/+) line 2 and ANC (ANC-Kitl/+) line 2 heterozygotes at P21. Fifteen hairs per mouse were analyzed to determine the fraction of pigmented pixels per hair shaft. (b) Mean pigmentation density in different genotypes. Both BLD-Kitl/+ and ANC-Kitl/+ heterozygotes exhibit significantly higher levels of pigmentation than wild-type controls. Notably, the amount of pigment in BLD-Kitl/+ heterozygotes is also significantly less than is found in ANC-Kitl/+ heterozygotes (P = 0.0278). Error bars indicate s.e.m. Unpaired t-test values; *P < 0.05, **P < 5 ×10–3.
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Guenther, C., Tasic, B., Luo, L. et al. A molecular basis for classic blond hair color in Europeans. Nat Genet 46, 748–752 (2014). https://doi.org/10.1038/ng.2991
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