Supplementary Table S1. 782 genes identified by piggyBac transposon mutagensis in HG3 cell line after fludarabine selection Supplementary Table S2. Gene Symbol/Gene Description Supplementary Table S3A. Molecular characteristics of BRAF mutant cases in the Spanish cohort. Supplementary Table S3B. Molecular characteristics of BRAF mutant cases in the UK LRF CLL4 cohort. Supplementary TableS 4. Genes differentially regulated in HG3 cells after fludarabine selection. Supplementary Table S5. Gene Set Name [# Genes (K)] Supplementary Table S6. Distribution of mutations in the BRAF gene in CLL.

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.

Supplementary Material and Methods in Word format

Supplementary Figure S1. schematic illustration of piggyBac transposon and transposase vectors used. PB IR refers to piggyBac inverted repeats. FRT is flippase recognition target. Supplementary Figure S2. Real-time quantitative PCR analysis confirms efficient depletion of BMP2K and DCK. Supplementary Figure S3. TP53 deficient HCT116 cell line exhibits reduced sensitivity to F-ara-A. Supplementary Figure S4. Protein expression following transient transfection of the putative fludarabine resistance genes DCK, NUDCD3, ARID5B and LARS in HCT116 cell line. Supplementary Figure S5. Vemurafinib normalizes the response to F-ara-A in cells expressing V600E mutant BRAF. Supplementary Figure S6. Hierarchical clustering of differentially expressed genes in pools of HG3 cells resistant to fludarabine. Supplementary Figure S7. Increased phosphorylation of ERK in HG3 pool resistant to fludarabine. Supplementary Figure S8. Distribution of mutations in the BRAF gene in CLL.

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Abstract Purpose: To identify resistance mechanisms for the chemotherapeutic drug fludarabine in chronic lymphocytic leukemia (CLL), as innate and acquired resistance to fludarabine-based chemotherapy represents a major challenge for long-term disease control. Experimental Design: We used piggyBac transposon-mediated mutagenesis, combined with next-generation sequencing, to identify genes that confer resistance to fludarabine in a human CLL cell line. Results: In total, this screen identified 782 genes with transposon integrations in fludarabine-resistant pools of cells. One of the identified genes is a known resistance mediator DCK (deoxycytidine kinase), which encodes an enzyme that is essential for the phosphorylation of the prodrug to the active metabolite. BMP2K, a gene not previously linked to CLL, was also identified as a modulator of response to fludarabine. In addition, 10 of 782 transposon-targeted genes had previously been implicated in treatment resistance based on somatic mutations seen in patients refractory to fludarabine-based therapy. Functional characterization of these genes supported a significant role for ARID5B and BRAF in fludarabine sensitivity. Finally, pathway analysis of transposon-targeted genes and RNA-seq profiling of fludarabine-resistant cells suggested deregulated MAPK signaling as involved in mediating drug resistance in CLL. Conclusions: To our knowledge, this is the first forward genetic screen for chemotherapy resistance in CLL. The screen pinpointed novel genes and pathways involved in fludarabine resistance along with previously known resistance mechanisms. Transposon screens can therefore aid interpretation of cancer genome sequencing data in the identification of genes modifying sensitivity to chemotherapy. Clin Cancer Res; 22(24); 6217–27. ©2016 AACR.

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel⁻/⁻). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel⁻/⁻ melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.

A major challenge in the post-genomic era is to understand how genome sequence variants (genotype) give rise to the enormous diversity observed in terms of morphology, physiology and behavior (phenotype) among living organisms. Domestic animals—with their tremendous phenotypic variation—are excellent model organisms for determining the relationships between genotype and phenotype. In this thesis, I describe the utilization of the chicken, in combination with modern genetic and genomic approaches, in developing our understanding of the genetic mechanisms underlying phenotypic variation. These studies provide novel information on the genetics behind variation in carotenoid- and melanin-based pigmentation—observed in many organisms—and also cast light on the genetic basis of chicken domestication. In paper I, we report that the yellow skin phenotype—observed in most commercial chickens—is caused by one or several tissue-specific mutations altering the expression of beta-carotene oxygenase 2 (BCO2 or BCDO2) in skin. In addition, we present the first conclusive evidence of a hybrid origin of the domestic chicken, since the allele causing yellow skin most likely originates from the grey jungle fowl (Gallus sonneratii) and not from the previously described sole ancestor, the red jungle fowl (Gallus gallus). In paper II, we detect a number of loci that were likely important during the domestication process of chicken and the later specialization into meat (broiler) and egg (layer) producing lines. One of the major findings was that worldwide, almost all domestic chickens carry a missense mutation in TSHR (thyroid stimulating hormone receptor) in a position that is completely conserved amongst vertebrates. We speculate that this “domestication-mutation” has played an important role in the transformation of the wild red jungle fowl ancestor into the modern domestic chicken. In paper III, we demonstrate that the dilution of red (pheomelanin) pigmentation—observed in the plumage of the Inhibitor of Gold chicken—is caused by a frame-shift mutation in the catechol-O-methyltransferase domain containing 1 (COMTD1) gene. The production and regulation of pheomelanin is poorly understood and this discovery advances our current knowledge of this pathway.

Sex-linked barring, a common plumage colour found in chickens, is characterized by black and white barred feathers. Previous studies have indicated that the white bands are caused by an absence of melanocytes in the feather follicle during the growth of this region. Here, we show that Sex-linked barring is controlled by the CDKN2A/B locus, which encodes the INK4b and ARF transcripts. We identified two non-coding mutations in CDKN2A that showed near complete association with the phenotype. In addition, two missense mutations were identified at highly conserved sites, V9D and R10C, and every bird tested with a confirmed Sex-linked barring phenotype carried one of these missense mutations. Further work is required to determine if one of these or a combined effect of two or more CDKN2A mutations is causing Sex-linked barring. This novel finding provides the first evidence that the tumour suppressor locus CDKN2A/B can affect pigmentation phenotypes and sheds new light on the functional significance of this gene.

Coat color and patterning phenotypes have been extensively studied as a model for advancing our understanding of the relationship between genetic and phenotypic variation. In this thesis, genes of relevance for pigment cell biology were investigated. The dissertation is divided in two parts. Forward genetics was used in the first part (Paper I and II) to identify the genes controlling the Silver and Sex-linked barring loci in chicken. In the second part, reverse genetics was employed to create a mouse line in which the PMEL17 protein is inactivated (Paper III). In Paper I, we report five mutations in SLC45A2 causing plumage color variants in both chicken and Japanese quail. Normal function of the SLC45A2 gene has previously been shown to be essential for the synthesis of both red/yellow pigment (pheomelanin) and brown/black pigment (eumelanin) in numerous species, including humans. The major discovery in this paper is the specific inhibition of pheomelanin in Silver chickens, whilst null mutations at this locus cause an almost complete absence of both pheomelanin and eumelanin. In Paper II, we report that Sex-linked barring in chickens is controlled by the CDKN2A/B tumor suppressor locus. The locus encodes two proteins, INK4B and ARF. The genetic analysis indicates that missense mutations in ARF or mutations in the promoter region of the ARF transcript are causing Sex-linked barring. In previous studies, mutations inactivating the CDKN2A/B tumor suppressor locus, have been shown to be responsible for familiar forms of human melanoma. Here we propose that these mutations in chicken CDKN2A/B cause the premature cell death of melanocytes as opposed to the cell proliferation and tumor growth associated with loss-of-function alleles in humans. In Paper III, we created a mouse line in which the PMEL17 protein is inactivated. Missense mutations in the gene encoding PMEL17 have previously been shown to be associated with reduced levels of eumelanin in epidermal tissues in several vertebrate species. The knockout mice are viable, fertile, and display no obvious developmental defects. The eumelanosomes within the melanocytes of these mice are spherical in contrast to the cigar-like shaped eumelanosomes present in wild-type animals. PMEL17 protein inactivation has only a subtle diluting effect on the coat color phenotype in four different genetic backgrounds. This suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations.

The Adhesion family of G protein-coupled receptors (GPCRs) includes 33 receptors and is the second largest GPCR family. Most of these proteins are still orphans and fairly little is known of their tissue distribution and evolutionary context. We report the evolutionary history of the Adhesion family protein GPR123 as well as mapping of GPR123 mRNA expression in mouse and rat using in situ hybridization and real-time PCR, respectively. GPR123 was found to be well conserved within the vertebrate lineage, especially within the transmembrane regions and in the distal part of the cytoplasmic tail, containing a potential PDZ binding domain. The real-time PCR data indicates that GPR123 is predominantly expressed in CNS. The in situ data show high expression in thalamic nuclei and regions containing large pyramidal cells like cortex layers 5 and 6 and subiculum. Moreover, we found distinct expression in amygdala, hypothalamus, inferior olive and spinal cord. The CNS specific expression, together with the high sequence conservation between the vertebrate sequences investigated, indicate that GPR123 may have an important role in the regulation of neuronal signal transduction.

S*S (Silver), S*N (wild type/gold), and S*AL (sex-linked imperfect albinism) form a series of alleles at the S (Silver) locus on chicken (Gallus gallus) chromosome Z. Similarly, sex-linked imperfect albinism (AL*A) is the bottom recessive allele at the orthologous AL locus in Japanese quail (Coturnix japonica). The solute carrier family 45, member 2, protein (SLC45A2), previously denoted membrane-associated transporter protein (MATP), has an important role in vesicle sorting in the melanocytes. Here we report five SLC45A2 mutations. The 106delT mutation in the chicken S*AL allele results in a frameshift and a premature stop codon and the corresponding mRNA appears to be degraded by nonsense-mediated mRNA decay. A splice-site mutation in the Japanese quail AL*A allele causes in-frame skipping of exon 4. Two independent missense mutations (Tyr277Cys and Leu347Met) were associated with the Silver allele in chicken. The functional significance of the former mutation, associated only with Silver in White Leghorn, is unclear. Ala72Asp was associated with the cinnamon allele (AL*C) in the Japanese quail. The most interesting feature concerning the SLC45A2 variants documented in this study is the specific inhibition of expression of red pheomelanin in Silver chickens. This phenotypic effect cannot be explained on the basis of the current, incomplete, understanding of SLC45A2 function. It is an enigma why recessive null mutations at this locus cause an almost complete absence of both eumelanin and pheomelanin whereas some missense mutations are dominant and cause a specific inhibition of pheomelanin production.

G protein-coupled receptors (GPCRs) are one of the largest families of proteins, and here we scan the recently sequenced chicken genome for GPCRs. We use a homology-based approach, utilizing comparisons with all human GPCRs, to detect and verify chicken GPCRs from translated genomic alignments and Genscan predictions. We present 557 manually curated sequences for GPCRs from the chicken genome, of which 455 were previously not annotated. More than 60% of the chicken Genscan gene predictions with a human ortholog needed curation, which drastically changed the average percentage identity between the human-chicken orthologous pairs (from 56.3% to 72.9%). Of the non-olfactory chicken GPCRs, 79% had a one-to-one orthologous relationship to a human GPCR. The Frizzled, Secretin, and subgroups of the Rhodopsin families have high proportions of orthologous pairs, although the percentage of amino acid identity varies. Other groups show large differences, such as the Adhesion family and GPCRs that bind exogenous ligands. The chicken has only three bitter Taste 2 receptors, and it also lacks an ortholog to human TAS1R2 (one of three GPCRs in the human genome in the Taste 1 receptor family [TAS1R]), implying that the chicken's ability and mode of detecting both bitter and sweet taste may differ from the human's. The chicken genome contains at least 229 olfactory receptors, and the majority of these (218) originate from a chicken-specific expansion. To our knowledge, this dataset of chicken GPCRs is the largest curated dataset from a single gene family from a non-mammalian vertebrate. Both the updated human GPCR dataset, as well the chicken GPCR dataset, are available for download.

G protein-coupled receptors (GPCR) are integral membrane proteins with seven α-helices that translate a remarkable diversity of signals into cellular responses. The superfamily of GPCRs is among the largest and most diverse protein families in vertebrates.We have searched the human genome for GPCRs and show that the family includes approximately 800 proteins, which can divided into five main families; Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin. This study represents one of the first overall road maps of the GPCR family in a mammalian genome. Moreover, we identified eight novel members of the human Adhesion family which are characterized by long N-termini with various domains. We also investigated the GPCR repertoire of the chicken genome, where we manually verified a total of 557 chicken GPCRs. We detected several specific expansions and deletions that may reflect some of the functional differences between human and chicken.Substantial effort has been made over the years to find compounds that can bind and activate the melanocortin 4 receptor (MC4R). This receptor is involved in food intake and is thus an important target for antiobesity drugs. We used site-directed mutagenesis to insert micromolar affinity binding sites for zinc between transmembrane (TM) regions 2 and 3. We generated a molecular model of the human MC4R which suggests that a rotation of TM3 is important for activation of the MC4R.Furthermore, we present seven new vertebrate prolactin releasing hormone receptors (PRLHRs) and show that they form two separate subtypes, PRLHR1 and PRLHR2. We performed a pharmacological characterization of the human PRLHR which showed that the receptor can bind neuropeptide Y (NPY) related ligands. We propose that an ancestral PRLH peptide has coevolved with a redundant NPY binding receptor, which then became PRLHR. This suggests how gene duplication events can lead to novel peptide ligand/receptor interactions and hence spur the evolution of new physiological functions.