A molecular basis for classic blond hair color in Europeans

Abstract

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 unknown. Here we dissect a regulatory region of the KITLG gene (encoding KIT ligand) that is significantly associated with common blond hair color in northern Europeans. 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.

Figure 1. A distant regulatory region upstream of the KITLG gene controls hair pigmentation in humans and mice

(a) SNPs on human chromosome 12 are associated with blond hair in Europeans (modified from). The peak association is found at rs12821256 (red), which is 355 kb upstream of the KITLG transcription start site. (b) The frequency distribution of rs12821256 in different populations. The allele associated with blond hair, G (yellow), is most prevalent in northern Europe. (c) The Sl^pan allele at the mouse Kitlg locus consists of a 65.6 megabase chromosome inversion (GRCm38/mm10 chr10 breakpoints: 34.301 Mb - 99.916 Mb) that displaces upstream sequences orthologous to rs12821256 (red triangle). Heterozygous (Sl^pan/+) and homozygous (Sl^pan/Sl^pan) mice have lighter coats than control (+/+) animals, demonstrating that alteration of even a single copy of the region upstream of Kitlg can reduce hair pigmentation.

Figure 2. The human blond-associated region contains a functional hair enhancer

(a) A 17.1 kb region bounded by SNPs rs444647 and rs661114 defines the candidate interval for blondness. Within this region, a large block of mammalian sequence conservation overlaps peak marker rs12821256. Five human fragments were cloned upstream of a lacZ reporter gene and tested for in vivo enhancer activity in transgenic mice. (b–f) Representative transgenic embryos generated by pronuclear microinjection of different lacZ constructs, processed at E16.5 to reveal lacZ gene activity (blue staining). Scale bar, 1 mm. (b) H1. (c) H2. (d) H3. (e) HFE (for hair follicle enhancer). (f) H2b. Of the three clones spanning the entire interval, only the 6.7 kb clone, H2, produced consistent lacZ expression in skin and kidney (arrow). Analysis of two subclones from H2 separated HFE skin (e) and H2b kidney (f, arrow) enhancers. (g,h) 6 µm crosssections through E16.5 dorsal skin from (g) H2 and (h) HFE transgenic embryos counterstained with nuclear fast red. Strong lacZ expression is visible in the basal epithelium and developing hair follicles. Scale bar, 30 µm.

Figure 3. Variant hair follicle enhancers produce altered levels of gene expression

(a–c) Representative E16.5 transgenic embryos, generated by pronuclear injection of different 6.7 kb H2-lacZ constructs (shown below), processed for lacZ gene activity (blue). The full H2 region was used for these experiments, as expression in kidney provided a control for successful integration and expression of constructs even if expression in hair was disrupted. The clones tested were: (a) H2-ANC, "A" at rs12821256, (b) H2-BLD, "G" at rs12821256, or (c) H2-DEL, an 11 bp deletion that removes the rs12821256 position. lacZ gene activity was observed in developing hair follicles and kidneys (arrows) in all transgenic embryos. Although no consistent difference was noted between H2-ANC (N=15) and H2-BLD (N=9) embryos, H2-DEL embryos (N=8) (c) showed reduced activity in skin but normal kidney expression (arrow). Scale bar, 1 mm. (d) Expression analysis of different 1.9 kb HFE-luciferase reporters in the human HaCaT keratinocyte cell line. Bars represent the mean increase in luciferase gene activity over an empty vector control measured 48 hours after transfection from a typical experiment. The enhancers tested differed only by the following: HFEANC (A at rs12821256), HFE-BLD (G at rs12821256), and HFE-DEL (11 bp deletion removing rs12821256). Both the HFE-BLD and HFE-DEL constructs exhibited significantly reduced activity in HaCaT keratinocytes compared to the HFE-ANC plasmid. Error bars indicate s.e.m. Unpaired t test P-values; * P<0.05, *** P<5×10⁻⁴.

Figure 4. The blond allele at rs12821256 alters a TCF/LEF binding site and reduces LEF responsiveness in keratinocytes

(a) A 4 kb window centered on the blond SNP shows that TCF7L2 ChIP-seq reads from the ENCODE project accumulate over rs12821256 in the 1.9 kb HFE (NCBI36/hg18 chr12:87,852,100-87,853,992; HCT116 cells, TCF7L2 Sg data set). (b) The sequence surrounding rs12821256 resembles a consensus LEF binding motif. The blond-associated allele in humans changes a highly conserved, consensus-matching A nucleotide to a non-consensus G within the predicted LEF binding motif. (c) Response of mini-promoters to increasing levels of LEF protein 48 hours following co-transfection into HaCaT keratinocytes. The three luciferase reporter constructs tested contained 7 tandem copies of an artificial consensus LEF binding site (7X LEF) (SuperTOPFlash), seven copies of the human ancestral binding site (7X ANC), or seven copies of the blond-associated sequence variant (7X BLD). All three mini promoters demonstrated elevated activity in response to increased LEF protein. The magnitude of the response to moderate LEF levels corresponds with the predicted binding capabilities of the variant LEF sites, with 7X LEF >> 7X ANC >> 7X BLD. Note that the 7X BLD human variant shows significantly lower activation than the 7X ANC human sequence at every level of LEF1 tested (5 ng, P<0.0001; 10 ng, P<0.0001; 28ng P<0.0001; 50 ng, P<0.001; Mann-Whitney test). Representative experiment shown of N=2 replicates. Error bars indicate s.e.m.

Figure 5. Mouse lines differing at single base pair position in the KITLG hair follicle enhancer show obvious differences in hair color

(a) Schematic of the site-specific integration (SSI) strategy used to create matched BLD-Kitlg and ANC-Kitlg insertions in mice. Blond or ancestral hair enhancers (HE) were cloned upstream of an Hsp68 minimal promoter-Kitlg transgene (Methods). Blue arrows denote flanking attB sites that recombine with tandem attP sites (black arrows) in the murine chromosome 11 H11P3 locus upon pronuclear injection of a mix containing each SSI plasmid with ϕC31 mRNA. (b) Box plots representing quantitative RT-PCR analysis of Kitlg RNA expression in P8 dorsal skin. Both BLD-Kitlg/+ and ANC-Kitlg/+ heterozygotes exhibit significantly elevated levels of epidermal Kitlg compared to control animals. However, mice carrying the blond-variant transgene produce 21% less Kitlg than the matched ancestral transgene. Mann-Whitney P-values: BLD vs. +/+ = 5e-9; ANC vs. +/+ = 7e-10; BLD vs. ANC = 0.03146. (c) Representative 2-month old mice exhibiting the hair color phenotypes associated with a single copy of each SSI transgene. The mice pictured from left to right are: wild type (FVB/C57Bl/6J F1 hybrid), BLD-Kitlg/+, and ANC-Kitlg/+ heterozygotes. Mice carrying the blond-associated allele at rs12821256 are notably lighter than mice carrying the ancestral allele at the KITLG hair follicle enhancer (also see Supplementary Fig. 7).