Global pigmentation variability in the hair, eyes, and skin is among the most striking phenotypic human traits. Differences in genomic regions associated with these traits show some of the strongest signals of selection in the human genome, indicating the importance of pigmentation throughout human evolution. Hair and eye color are especially variable across Europe, and several previous studies have queried the genome to determine where the mutations causing blond hair are located. In a recent paper by HHMI research specialist Catherine Guenther (David Kingsley’s lab) and colleagues, the team explored why blond hair occurs when an allele strongly associated with blond hair in Europeans is found (1).
The impact of mutations near the gene KITLG on skin pigmentation has been debated in previous studies in humans and sticklebacks (2, 3), but one regulatory region has an undeniably strong association with European blond hair (4). The allele most strongly associated with blond hair is 350 kb upstream of KITLG in a highly conserved region. As a starting point to characterize the molecular basis of the blond mutation, the authors looked at fur pigmentation in a Kitl mutant mouse strain (Steel panda or Slpan) with a 65 Mb inversion that includes sequences orthologous to the putatively causal blond hair allele. Compared to the background strain, heterozygotes and homozygotes had increasingly lighter hair. They also had reduced Kitl expression.
Lightening the candidate load
The authors next wanted to narrow down the region that causally regulates blond hair pigmentation in Kitl. The authors narrowed down the potential blond target to a 17 kb window bounding the human association signal (see Figure 1 below).
The most strongly associated SNP in humans was in the center of the window and in the only highly conserved region. They cloned 3 reporter constructs, H1, H2, and H3, into mice. Each clone contained a candidate regulatory region subsetting the 17 kb window driving lacZ expression. Only one of the clones, H2, drove consistent expression of lacZ. The authors repeated their cloning process by further subdividing the region spanned by H2 into two candidate regulatory peaks, HFE and H2b, both spanning conservation peaks. The HFE peak, which contains the most strongly associated blond hair SNP, drove consistent expression in hair follicles and epithelial cells of developing hair and skin, while the H2b peak drove consistent expression in kidneys. Their results indicate that the H2 region is an important gene enhancer, that the HFE region drives expression in the hair follicles, while the H2b region drives expression in kidneys.
The authors next made clones with the H2 region containing the exact ancestral (H2ANC) and derived (H2DER) mutations putatively causing blond hair in Europeans. The H2ANC and H2DER embryos qualitatively looked similar, and further quantification of lacZ expression in keratinocyte cell lines using the smaller 1.9 kb HFE clones showed significantly less expression in HFE-DER compared to HFE-ANC.
When the authors wanted to know why the blond allele is regulating expression in hair follicles, they turned to ENCODE. Previous ChIP-seq studies showed that the transcription factor family TCF/LEF strongly bound the region of interest in a colorectal epithelial cell line. The authors discovered a well-conserved LEF binding motif disrupted by the blond allele. LEF1 is a transcription factor expressed during hair follicle development, and Lef1 knockout mice are light-furred, providing a potential mechanism for upstream regulation of Kitl. Consistent with previous LEF1 binding site analyses, the authors showed reduced LEF1 responsiveness from the derived blond allele experimentally.
Finally, the authors integrated a single copy of the hair follicle enhancer into the same genomic location including either the blond allele (BLD-Kitl) or the ancestral allele (ANC-Kitl) into mice. The extra Kitl enhancer darkened both mice, but the mice with the BLD-Kitl insertion had only 79% of Kitl expression as the ANC-Kitl mice, and BLD-Kitl mice were noticeably lighter.
The molecular explanation behind the European blond hair allele identified previously in a GWAS is intrinsically very biologically interesting. Many pigmentation associations are in non-coding regions far from canonical pigmentation genes. Strong signals of positive selection have been identified upstream of KITLG as well as near many other pigmentation genes. Some other pigmentation associations also come from highly conserved regions, and this work has provided a framework for dissecting the regulatory function of pigmentation variants.
The implications of this study also extend beyond pigmentation. The time and cost to disentangle the molecular basis of a single GWAS likely means that most GWAS variants or regions will likely not be biologically characterized as carefully any time soon. The driver of blond hair was in a distal regulatory region 350 kb from the gene in an important transcription factor binding site where the consensus sequence does not perfectly match. Additionally, the expression change of the Kitl gene resulting from the causal mutation was by only a fraction of the ancestral case. Diseases without such an obvious phenotypic readout will likely be harder to dissect.
On the other hand, the abundance of publicly available data enabled much of the work presented here. Multiple studies have implicated the GWAS SNP in pigmentation phenotypes, increasing its likelihood of causality. Additionally, the authors were able to identify a potential upstream mechanism of Kitl regulation via the GWAS region with ENCODE ChIP-seq data alone. As larger genomic, interaction, regulatory datasets, etc. become available, our ability to explain phenotypic variation identified in humans with less costly and time-consuming in vivo models increases.
1. C. A. Guenther, B. Tasic, L. Luo, M. A. Bedell, D. M. Kingsley, A molecular basis for classic blond hair color in Europeans., Nat. Genet. 46, 748–52 (2014).
2. S. Beleza et al., R. A. Spritz, Ed. Genetic Architecture of Skin and Eye Color in an African-European Admixed Population, PLoS Genet. 9, e1003372 (2013).
3. C. T. Miller et al., cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans., Cell 131, 1179–89 (2007).
4. P. Sulem et al., Genetic determinants of hair , eye and skin pigmentation in Europeans, 39, 1443–1452 (2007).