Functional Analysis of Red Hair Colour Using a Humanised Mouse Model

Although much of the normal variation between humans is due to genetics, very little is known about the genes that cause this variation. An exception is the genetics of hair colour for which, largely thanks to studies in mice, a number of genes are known that are associated with both blond hair and red hair. The genetics of red hair is the simplest and this study is addressing the genes and interactions between them which result in red hair.

We showed a number of years ago that people with red hair have changes in a gene known as MC1R, but not everyone with a variant MC1R gene has red hair; some variants frequently result in red hair whilst others more often than not do not. There must be other genes that determine whether or not MC1R affects hair colour. We have studied the DNA of tens of thousands of people in the UK Biobank study, and identified genes that appear to affect hair colour. There are many different genes associated with blond hair, but only a handful that result in red hair. To study these genes and their interactions in more detail we plan to study the effect of some of the variants in cells grown in culture. In order to fully understand the impact of these variants in the whole body, we will go on to make mice that carry these genetic changes, and look at the impact of the changes on mouse hair colour. This will act as a model system for the study of other traits controlled by more complex genetic interactions.

Mice which lack the MC1R gene have yellow fur rather than the normal black or "agouti" (a grey/brown colour). The human and mouse MC1R proteins have somewhat different properties, and the respective genes are regulated differently to compensate. If we engineer one of the "strong" red hair changes into the mouse gene it has surprisingly little impact on the colour of the mice; the differences in gene regulation mask the effect of the variant. We have produced a mouse strain that lacks its own MC1R gene but contains the human gene, along with all the DNA required for the normal function of the gene, and this gives the mice their normal coloured hair. This human gene will allow us to study changes seen in red haired people by engineering changes in the DNA. We will produce mice with a "strong" and a "weak" change in the coding part of the human MC1R gene and assess their impact on mouse hair colour. We have also found changes in the DNA that control the MC1R gene in red-haired people. We will engineer these changes into our "humanised" mouse model and assess it's impact on hair colour.

We have also found that DNA changes near a gene called MSX2 are associated with red hair. This gene codes for a protein that can control other genes, and indirect evidence suggests that it may control a gene called ASIP, which codes for an inhibitor of MC1R. We will investigate where MSX2 interacts with DNA around the ASIP gene, and we will ask what is the effect on hair colour of removing those sites in mice.

The genetic basis of most normal human variation is not well understood. One exception is hair colour, for which GWAS studies have identified genes which in many cases were already known from studies on model organisms and human disease. We have carried out GWAS on a cohort over 100,000 individuals for red hair and for blonde hair. Blonde hair is associated with many loci at genome-wide significance whereas analysis of red hair finds very few associated loci. Understanding the genetic interactions that result in human variation can be addressed in part using mouse models, and we propose to analyse candidate red hair variants in this way.

Almost all red hair individuals have variants in the GPCR, MC1R, but not all those with two variant alleles have red hair; there are modifier loci. Some MC1R variants have high penetrance, whilst other have low penetrance, indicating that some alleles are more dependent on modifiers than others. Mice lacking MC1R have yellow fur, but engineering a high penetrance variant into the mouse gene by CRISPR has little effect on pigmentation because of differences in pharmacology and regulation. We have a mouse line containing the human coding sequence and the necessary regulatory sequences, which rescues the yellow phenotype. We will use CRISPR to generate mouse lines with coding and non-coding variant alleles in the human transgene and assess the impact on transcription of MC1R and on the pigmentation. We will also model the interaction with an identified modifier gene, ASIP.

We find a novel association with a transcription factor, MSX2, which is a candidate regulator of ASIP. We will carry out ChIP-Seq of keratinocytes and fibroblasts to identify binding sites at ASIP and elsewhere and will edit these in cultured cells and in transgenic mice to analyse the effect on ASIP transcription, response to BMP4 and on pigmentation in mice.

This project will have two major impacts. Firstly, we anticipate significant impact in the academic community. The use of a humanised mouse model to test human variation in vivo is novel and the project will act as an exemplar for this approach. In order to communicate this impact to the community we will engage through scientific meetings and through the scientific literature.

Professor Jackson is currently President of the European Society for Pigment Cell Research, and will be participating in their meetings, in 2018 and 2019. In 2017 the world-wide pigment cell community will meet at the International Pigment Cell Conference in Denver, Colorado. Professor Jackson will attend this meeting as a member of the Scientific Program Committee. We anticipate that Professor Jackson or the postdoctoral researcher on this project will present at the European Pigment Cell Conferences in 2018 and 2019. Possibly more important to maximise impact is the presentation of these results to the genetics community as a demonstration of the power of this approach to interpreting SNP associations. We will present the work at the American Society of Human Genetics Conference in 2019 in Houston, Texas and at the European Society of Human Genetics conference in 2020, in Berlin. In addition there is a lot of effort in the animal breeding field which identifies DNA variant associations with traits of agricultural importance, and the approaches in this project will be relevant to translating these traits to functional consequence. This impact would be relevant to commercial organisations who are also involved in animal breeding.

The essential route to impact to the academic community will be via publication in high impact, open access journals. Professor Jackson has a long history of publication in top journals, including senior author papers recently in PLoS Genetics, Nature Communications, Human Molecular Genetics and the American Journal of Human Genetics.

The second important impact is on the public as a whole. Hair colour genetics is a topic that is particularly interesting to the public, and serves as a great way of communicating ideas in genetics. The notion that MC1R variants "cause" red hair is well understood as an example of recessive inheritance. However, this message can be nuanced with the work of this project to bring in the notion of modifier genes and oligogenic inheritance. Professor Jackson has an excellent track record in communicating his research to the general public. In the last few years he has presented his work on red hair at Cafes Scientifique in Edinburgh, Glasgow and Cockermouth and has presented a show; "Red Hair, Truth Myths and Lies" in the Cabaret of Dangerous Ideas at the Edinburgh Festival Fringe. In addition he has participated in both live and recorded radio interviews, and spoken to many journalists from the print and online media. He is one of the experts used by the Science Media Centre as a point of contact for journalists to comment on mouse genetics and transgenic animals, in particular.