From GWAS to Function: Transcriptional regulation of pigmentation genes in humans Transcriptional regulation of pigmentation genes in humans

Human pigmentation is one of the most explicit visual traits, which therefore has been subject of many research studies. With the emergence of large-scale genetic association studies like GWASs, numerous SNPs have been associated with a phenotype of interest, such as human eye, hair and skin color. Many of the identified pigmentation-associated SNPs have been implemented in forensic and/or anthropological applications that are developed to predict human pigmentation traits. The work described in this thesis aims to understand the functional biology underlying several of these highly associated pigmentation SNPs. This thesis starts with a general overview of the current knowledge on human pigmentation in Chapter 1, including its evolutionary history and biochemistry, the mechanisms of melanogenesis, and genetic variation of pigmentation genes. It also summarizes the essentials of transcriptional regulation and the key players involved in this complex process. Chapters 2-5 contain the experimental work performed during the course of this PhD study. Herein I focus on the biological function of SNPs that are strongly associated with human pigmentation phenotypes. In Chapter 2, I describe a detailed analysis of the regulatory function of an enhancer element that contains the intronic SNP rs12913832 which is strongly associated with human skin, eye and hair color, and controls expression of the pigmentation gene OCA2. Due to the original design of GWASs and the SNP arrays used, the genetic association signals prioritized in these studies are not necessarily the actual causal or functional SNPs. These causal SNPs need to be identified in order to study the functional biology underlying the detected genetic association signals. This is exemplified in Chapter 3, in which I describe the identification of the actual functional SNP (rs12350739) responsible for the detected skin pigmentation-associated signal in the BNC2 gene, followed by a detailed analysis of the transcriptional regulation of that gene and the involvement of rs12305739 therein. Regulatory elements, such as enhancers are typically located at large distances from their target genes, however this generally does not restrict the activity of these elements, as they are able to regulate transcription over large distances through long-range interactions. Chapter 4 focuses on chromatin structure to characterize the allele-specific regulatory mode-of-action of an intronic enhancer in which the pigmentation-associated SNP rs12203592 is located, and controls expression of the IRF4 gene. In Chapter 5 I investigate the genetic basis of human skin color by combining a series of GWASs. This is followed by functional analyses of one of the five genomic regions harboring skin-color associated SNPs detected in these GWASs. At the time the work on this thesis was done, this genomic region represented the least understood genetic association signal at the functional molecular level. Finally, Chapter 6 summarizes the results of the experimental research described in Chapters 2-5, and I discuss the in silico and experimental workflow as employed in Chapters 2-5 to unravel the functional biology underlying genetic association signals in a more general context.