A deletion at the X-linkedARHGAP36gene locus is associated with the orange coloration of tortoiseshell and calico cats

A deletion at the X-linked ARHGAP36 gene locus is associated with the orange coloration of tortoiseshell and calico cats | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A deletion at the X-linked ARHGAP36 gene locus is associated with the orange coloration of tortoiseshell and calico cats View ORCID Profile Hidehiro Toh , View ORCID Profile Wan Kin Au Yeung , View ORCID Profile Motoko Unoki , View ORCID Profile Yuki Matsumoto , Yuka Miki , Yumiko Matsumura , View ORCID Profile Yoshihiro Baba , View ORCID Profile Takashi Sado , View ORCID Profile Yasukazu Nakamura , View ORCID Profile Miho Matsuda , View ORCID Profile Hiroyuki Sasaki doi: https://doi.org/10.1101/2024.11.19.624036 Hidehiro Toh 1 Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan 2 National Institute of Genetics, Research Organization of Information and Systems , Mishima, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hidehiro Toh Wan Kin Au Yeung 1 Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan 3 Colledge of Liberal Arts, International Christian University , Mitaka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wan Kin Au Yeung Motoko Unoki 1 Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan 4 School of International Health, Graduate School of Medicine, The University of Tokyo , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Motoko Unoki Yuki Matsumoto 5 Data Science Center, Azabu University , Sagamihara, Japan 6 Research and Development Section, Anicom Specialty Medical Institute Inc. , Yokohama, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yuki Matsumoto Yuka Miki 1 Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan 7 School of Medicine, Nagoya University , Nagoya, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yumiko Matsumura 8 Inakazu Dog & Cat Kashii Hospital , Fukuoka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yoshihiro Baba 1 Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yoshihiro Baba Takashi Sado 9 Graduate School of Agriculture, and Agricultural Technology and Innovation Research Institute, Kindai University , Nara, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Takashi Sado Yasukazu Nakamura 2 National Institute of Genetics, Research Organization of Information and Systems , Mishima, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yasukazu Nakamura Miho Matsuda 10 Faculty of Dental Science, Kyushu University , Fukuoka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Miho Matsuda Hiroyuki Sasaki 1 Medical Institute of Bioregulation, Kyushu University , Fukuoka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hiroyuki Sasaki For correspondence: hsasaki{at}bioreg.kyushu-u.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The X-linked orange ( O ) locus in domestic cats controls an unknown molecular mechanism that causes the suppression of black-brownish pigmentation in favor of orange coloration. The alternating black-brownish and orange patches seen in tortoiseshell and calico cats are considered as classic examples of the phenotypic expression of random X-chromosome inactivation (XCI) occurring in female mammals. However, the O gene in the cat genome has not been identified, and the genetic variation responsible for the orange coloration remains unknown. We report here that a 5.1-kilobase (kb) deletion within an intron of the X-linked ARHGAP36 gene, encoding a Rho GTPase activating protein, is closely and exclusively associated with orange coloration. The deleted region contains a highly conserved putative regulatory element, whose removal presumably cause altered ARHGAP36 expression. Notably, ARHGAP36 expression in cat skin tissues is linked to the suppression of many melanogenesis genes, potentially shifting pigment synthesis from eumelanin to pheomelanin. Furthermore, we find evidence that the gene undergoes XCI in female human and mouse cells, and XCI-dependent CpG island methylation consistent with random XCI in female domestic cats. The 5.1-kb deletion seems widespread in domestic cats with orange coat coloration, suggesting a single origin of this coat color phenotype. Introduction The domestic cat ( Felis silvestris catus ) is a valued companion animal, a vermin-control agent, and a provider of important biomedical models, which most likely descended from a wildcat progenitor subspecies, Felis silvestris lybica , around 10,000 years ago 1 , 2 , 3 . Domestic cats display a diversity of phenotypic variation, including a range of coat coloration patterns resulting from interactions between multiple genetic loci, making them excellent models for studying gene function and regulation. The X-linked orange ( O ) locus is one of the coat coloration loci, which controls an unknown molecular mechanism that causes the suppression of black-brownish pigmentation (eumelanin) and promotion of orange coloration (pheomelanin) 4 . Phenotypic variants of this locus can be seen in the tortoiseshell (mottled orange and black-brownish pattern) and the calico cats (mosaic pattern of large orange, black-brownish, and white patches), which are almost exclusively female. These coloration patterns arise as a result of X-chromosome inactivation (XCI), which is an important epigenetic mechanism that equalizes X-linked gene dosage between females and males. More than 60 years ago, it was suggested that one of the two X chromosomes in a female embryo is randomly selected and inactivated in each cell during early development, and that descendant cells maintain the same inactivation pattern as the ancestral cell 5 . As a result, the alternative expression of the orange allele versus the wildtype allele in different skin patches creates a mosaic coloration pattern in female cats heterozygous at the O locus 5 . Rare occurrence of the mosaic phenotype in males can be explained by sex chromosome aneuploidy (XXY), chimerism, mosaicism, or somatic mutations 6 . While the coat color phenotypes of tortoiseshell and calico cats are often quoted as visually recognizable examples of XCI in female mammals, the gene and the genetic variation responsible for the orange coloration remain unknown. A previous genetic mapping study using microsatellite markers located the O locus within a 9.7-megabase (Mb) region of the cat X chromosome 7 . A more recent study using a 63K DNA array identified a 1.5-Mb haplotype block associated with orange coloration within this 9.7-Mb region 8 . The study also provided a list of twelve genes located within the haplotype region as the candidates for the O gene; however, none were known to regulate melanin synthesis. In this study, we searched for the gene and genetic variation associated with the orange coloration by comparing the genomic sequences of cats with orange coloration, including tortoiseshell and calico cats, and control cats. We identified a 5.1-kilobase (kb) deletion in one of the protein-coding genes located within the haplotype region, which was closely and exclusively associated with orange coloration. We further present evidence that this gene undergoes XCI, most likely random XCI, in multiple mammalian species. Results Systematic screening for genetic variations responsible for orange coloration using whole-genome sequencing (WGS) To identify the genetic variation responsible for the orange coloration and X-linked O gene in domestic cats, we sequenced the DNA samples from 18 cats, including eight calico cats, one tortoiseshell cat, one cat with both orange and white coat regions, and eight cats without orange coloration (Supplementary Tables 1 and 2, Extended Data Fig. 1A). We mapped the WGS reads to the AnAms1.0 cat genome, a high-quality chromosome-scale sequence assembly with only one short unmapped scaffold 9 . Thirteen putative protein-coding genes, overlapping with the twelve genes previously reported 8 , were identified in the 1.5-Mb haplotype region (Supplementary Table 3). The AnAms1.0 genome was derived from a silver tabby American Shorthair 9 (Extended Data Fig. 1A), and we used this sequence as a reference (wildtype) to identify genetic variations associated with the orange coloration. Within the 1.5-Mb haplotype region, we identified 2,569 single nucleotide polymorphisms (SNPs) in ten cats with the orange coloration, and 2,073 SNPs in eight cats without orange coloration. Removal of SNPs shared by the two groups identified 594 SNPs unique to the orange coloration group (Extended Data Fig. 2A). Using published WGS data from nine cats with color photographs, contributed by the 99 Lives Cat Genome Sequencing Initiative 10 (Supplementary Table 4) (photographs available in the paper or at https://cvm.missouri.edu/research/feline-genetics-and-comparative-medicine-laboratory/99-lives/successfully-sequenced-cats/ ), we identified 901 SNPs in seven non-orange cats. After removing the SNPs shared with this pool, a total of 450 SNPs unique to the orange group remained, none of which were frameshift, missense, or nonsense mutations in the protein-coding exons (Extended Data Fig. 2B). To search for other types of variations, we analyzed the sequence depth across the 1.5-Mb O haplotype region and identified a 5.1-kb deletion present only in the orange cat group ( Fig. 1A ). This deletion was located within the first intron of ARHGAP36 and was surprisingly present in all ten sequenced cats with the orange coloration, but in none of the eight cats without orange coloration ( Fig. 1A , Extended Data Fig. 3A). Notably, the eight calico cats and one tortoiseshell cat were heterozygous for the deletion, consistent with the mosaic coat color phenotype. Furthermore, analysis of the published data from the above-described nine cats with coat color information confirmed the presence of this deletion only in those with the orange coloration, namely a calico cat named Cali and her father, Orion 10 (Extended Data Fig. 3B, Supplementary Tables 4 and 5). Again, the calico cat was heterozygous for the deletion. While we could not exclude at this stage that one of the identified SNPs was responsible for the O allele, the consistent and exclusive presence of the deletion made it a strong candidate for the causative genetic variation. Interestingly, the SNP (position 108,984,866 in AnAms1.0, or 110,230,748 in felCat9) (hereafter we use AnAms1.0 coordinate) previously found to be most closely associated with the orange coloration 11 was located in an intron of ENOX2 , a protein-coding gene located closest to ARHGAP36 (Supplementary Table 3). Download figure Open in new tab Figure 1. Identification of the 5.1-kb deletion within the O haplotype region. (A) Location of the 5.1-kb deletion associated with the orange coloration in the cat X chromosome. An orange bar indicates the 9.7-Mb O region identified using microsatellite markers 7 and a red bar marks the 1.5-Mb O haplotype region identified using SNP microarray 8 . A pink box marks the 5.1-kb region deleted in cats with the orange coloration. Exon-intron structure of ARHGAP36 is shown with exon usage generating three isoforms. Representative WGS read-depth profiles across the 5.1-kb region are shown at the bottom for three cats with indicated coat color and sex. (B) Sequence similarity profiles of the region containing the 5.1-kb deletion. Sequence similarity between domestic cats and indicated species is shown. A pink box highlights the deleted region, while a blue box marks the UCE-like sequence. (C) ENCODE annotations for the UCE-like sequence on human chromosome X. Shown are features of the hg38 131,041,688-131,042,122 region corresponding to the cat UCE-like sequence (boxed by blue lines) and adjacent regions, retrieved from the UCSC Genome Browser. Purple bars indicate regions showing distal enhancer-like signatures and a green bar highlights the DNase I hypersensitive site cluster identified in neuroblastoma. Gray bars represent ChIP-seq peak clusters for indicated transcription factors in neuroblastoma. Strong association between the deletion and orange coloration To confirm the association between the 5.1-kb deletion within an intron of ARHGAP36 and the orange coloration, additional DNA samples (n = 40) were analyzed by polymerase chain reaction (PCR). The study showed that all six calico cats, including one rare male calico cat (#24) (Extended Data Fig. 1B), and all five tortoiseshell cats had the deletion in one of the two X-chromosomes (Extended Data Fig. 3C, Supplementary Table 1). The fact that the male calico was heterozygous for the deletion is consistent with the presence of sex chromosome aneuploidy (presumably XXY) 6 . Three additional cats with the orange coloration but no black-brownish coloration (#30, #31, and #32) had this deletion in a hemizygous (male) or homozygous manner (female), while none of the non-orange cats did (n = 26) (Supplementary Table 1). Combining our WGS and PCR data, a total of 58 cats (24 orange and 34 non-orange) demonstrated a 100% link between the deletion and the orange coloration (Fisher’s exact test: P = 2.2e-16) (Table, Supplementary Table 1). Addition of the information from the publicly available WGS data of the above-described nine photographed cats further supported this link in a total of 67 cats (26 orange and 41 non-orange) ( P = 2.2e-16). Next, we sought to determine whether any of the 450 SNPs found only in the orange cat group were linked to the 5.1-kb deletion because such a SNP could also contribute to the phenotype. We found that only one SNP (C->T, at position 109,185,709), located 0.5-kb upstream of the deletion within an intron of ARHGAP36 ( Fig. 1A ), was present in all ten sequenced cats of our orange group (Extended Data Fig. 2C,D). We then used 258 WGS datasets, mostly from the 99 Lives Cat Genome Sequencing Initiative 10 (including those from the nine cats with color images), and found that, while all 61 cats carrying the 5.1-kb deletion (21 hemizygous males, 12 homozygous females, and 28 heterozygous females) had the SNP, none of the 197 cats without it did (Supplementary Tables 4 and 5). All 28 females heterozygous for the deletion were also heterozygous for the SNP. These results indicate that the deletion and a linked SNP are present within an intron of ARHGAP36 in cats with the orange coat coloration. Ultraconserved element (UCE)-like sequence in the deleted region We then performed a more detailed analysis of the deletion and SNP. Sanger sequencing of the PCR products confirmed a 5,076-nucleotide (nt) deletion spanning from position 109,186,183 to 109,191,258 (Extended Data Fig. 3D). The terminal ‘AA’ dinucleotide likely served as an acceptor template. Importantly, the deleted region contained a 436-nt sequence showing strong evolutionary conservation reminiscent of UCE (ref. 12) ( Fig. 1B , Extended Data Fig. 3E). According to the data from the human ENCODE project 13 , this UCE-like sequence contained a DNase I hypersensitive site cluster in human neuroblastoma cells and chromatin immunoprecipitation-sequencing (ChIP-seq) clusters for transcription factors including GATA2/3, MXI1, and RCOR1 ( Fig. 1C ). Interestingly, while GATA family proteins are transcriptional activators 14 , MXI1 (Max interactor 1, dimerization protein) and RCOR1 (REST corepressor 1 or CoREST) are transcriptional repressors 15 , 16 . Therefore, this putative cis -regulatory element could function as a transcriptional enhancer or a repressor. In contrast, the SNP located about 0.5-kb upstream of the deletion (position 109,185,709) was not in a region conserved among species and therefore unlikely to have a role in gene regulation. It is likely that the 5.1-kb deletion removed a critical regulatory region, leading to altered expression of a nearby gene(s) such as ARHGAP36 . Negative correlation between ARHGAP36 and melanogenesis gene expression We hypothesized that the O gene would show differential expression depending on the presence/absence of the deletion in skin tissues, especially in orange and black-brownish coat regions. We first performed RNA sequencing (RNA-seq) on skin tissues obtained from the auricles of different adult calico cats (#19, #20, and #22, females, precise ages unknown) (Supplementary Table 2). Of the thirteen protein-coding genes found in the O haplotype region, only ARHGAP36 showed differential expression in different coat color regions, with its expression detected only in the orange region (Extended Data Fig. 4A). We then analyzed multiple skin tissues obtained from a young calico cat that died of unknown causes (#01, female, three weeks old) (Extended Data Fig. 1C, Supplementary Table 2). Of the genes in the O haplotype region, ARHGAP36 and IGSF1 showed differential expression ( Fig. 2A ). ARHGAP36 was persistently expressed in the orange regions but variably expressed in the black-brownish regions ( Fig. 2A , Extended Data Fig. 4A). One of the two black-brownish regions showed virtually no ARHGAP36 expression. IGSF1 , a gene located next to ARHGAP36 , behaved similarly, suggesting that the two genes might share a regulatory mechanism, although the expression level of IGSF1 was extremely low (< 0.6 fragments per kilobase of exon per million reads mapped [FPKM]) ( Fig. 2A ). Notably, ARHGAP36-203 was the only mRNA isoform produced in the skin samples (Extended Data Fig. 4B). Since the expression pattern observed in the black-brownish regions should be the wildtype pattern, the more persistent ARHGAP36 , and possibly IGSF1 , expression induced by the deletion could shift the pigment synthesis from eumelanin to pheomelanin in the orange regions. Download figure Open in new tab Figure 2. Negative correlation between ARHGAP36 and melanogenesis gene expression. (A) Expression levels of the thirteen protein-coding genes of the O haplotype region in cat skin tissues. RNA-seq data were obtained in skin regions of different coat colors from a calico cat (#01). Expression levels of key melanocyte genes, KIT and MC1R , are also shown. (B) Top ten KEGG pathways in which genes negatively and positively correlated with ARHGAP36 are enriched. The pathways are ranked by significance. (C) KEGG diagram of the melanogenesis pathway highlighting genes negatively correlated with ARHGAP36 expression (red boxes). The original KEGG pathway diagram was slightly modified for clarity. Based on the publicly available gene expression databases, such as the Genotype-Tissue Expression (GTEx) 17 , both ARHGAP36 and IGSF1 are highly expressed in neuro-endocrinological tissues, such as the hypothalamus, pituitary gland, and adrenal gland. Notably, these tissues are rich in neural crest-derived cells, from which melanocytes also originate 18 . IGSF1 encodes a member of the immunoglobulin superfamily, and loss-of-function mutations in this gene cause X-linked central hypothyroidism and testicular enlargement (OMIM300888) 19 . There is currently no known role for IGSF1 in hair follicle development or melanogenesis. In addition, its expression was very low in cat skin tissues as described above ( Fig. 2A , Extended Data Fig. 4A). ARHGAP36 encodes a member of the Rho GTPase-activating protein family, but a systematic study has shown that this protein lacks the GTPase-activating activity 20 . Rather, it has been reported that ARHGAP36 mediates and activates Hedgehog (HH) signaling and suppresses protein kinase A (PKA) signaling 21 – 25 . Importantly, the HH and PKA signaling pathways mediate hair follicle development and melanogenesis, respectively, and dysregulation of human ARHGAP36 is implicated in X-linked Bazex-Dupré-Christol syndrome (BDCS, OMIM301845) 26 , characterized by follicular atrophoderma, congenital hypotrichosis, and multiple basal cell naevi and carcinomas. Hence, its known biological roles make ARHGAP36 an excellent candidate for the O gene. We then sought genes that were positively and negatively correlated with ARHGAP36 expression in the skin samples from orange and black-brownish regions, excluding white regions where no melanogenesis is expected. We found 341 genes showing positive correlation with ARHGAP36 and 517 genes showing negative correlation (Supplementary Table 6). Strikingly, an analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) in the David system 27 , 28 revealed that the negatively correlated genes were enriched in the melanogenesis pathway ( Fig. 2B ). Examples of the melanogenesis genes negatively correlated with ARHGAP36 included ADCY7 , DCT , LEF1 , TCF7 , TYR , TYRP1 , WNT10B , WNT3A , WNT5A , and WNT6 . The roles of the protein products were distributed in the whole melanogenesis pathway, from ligands activating the signaling pathways to enzymes directly involved in the melanin synthesis ( Fig. 2C ). Notably, among the melanin synthesis enzymes, TYRP1 and DCT are involved in eumelanin synthesis but not pheomelanin synthesis. Therefore, their downregulation may attenuate eumelanin synthesis and cause a shift towards pheomelanin synthesis 29 . The negative correlation between ARHGAP36 and melanin synthesis enzyme genes was also seen in the auricle skin samples from different calico cats (Extended Data Fig. 4C). ARHGAP36 is subject to XCI in female mammalian cells The mosaic coat color of tortoiseshell and calico cats is attributed to the random inactivation of the X-linked O gene in O / o female cells 5 . However, a proportion of X-linked genes is known to escape XCI to varying degrees 30 . We therefore wished to examine whether ARHGAP36 of the domestic cat is subject to random XCI in female cells and whether melanocytes in black-brownish and orange regions express the different alleles. To do this, we would need tissue samples from female cats (preferably from tortoiseshell or calico cats) carrying informative SNPs within the ARHGAP36 transcripts. However, we have not yet had a chance to access such samples. The XCI is investigated in detail in humans and mice. One of the authors (T.S.) previously investigated the inactivation of the X-linked genes using fibroblast cells derived from female mouse embryos obtained by crossing a JF1 female and a C57Bl/6J male 31 , 32 . In the study, the X chromosome from C57Bl/6J was fixed to be active by an internal deletion of Xist (X B6-ΔA ), a gene essential for XCI 31 . We reprocessed the RNA-seq data and found that all 172 SNP-containing Arhgap36 reads were from the X B6-ΔA , indicating that the gene is subject to XCI in mice. We also reprocessed the chromatin RNA-seq data from a similar study using embryonic fibroblast cells where the X chromosome from Mus musculus castaneus was genetically fixed to be inactive 33 and found that all Arhgap36 transcripts were from the other X (27 reads containing informative SNPs). Regarding the XCI of human ARHGAP36 , a SNP-based allelic imbalance analysis was performed for a total of 409 X-linked genes in multiple lymphoblastoid and fibroblast cells 34 . The data indicated that human ARHGAP36 (designated FLJ30058 in that study) was subject to XCI in all ten female cell-lines possessing informative SNPs. These results show that, while direct evidence is lacking for domestic cats, ARHGAP36 is subject to XCI in multiple mammalian species. DNA methylation status of cat ARHGAP3 6 is consistent with random XCI DNA methylation can serve as a hallmark for genes that are subject to XCI, as X-linked CpG islands (CGIs), especially promoter-associated ones, are highly methylated on the inactive X but almost unmethylated on the active X (ref. 35). Using published whole-genome bisulfite sequencing (WGBS) datasets from human and mouse tissues 36 , 37 , we first compared the methylation levels of the ARHGAP36 CGIs between the sexes, which showed significantly higher methylation levels in females of both species (Extended Data Fig. 5A,B), consistent with their XCI-dependent methylation. We then investigated the published allelic methylation data from hybrid mouse embryonic fibroblast cells carrying a Mus musculus castaneus -derived inactive X (ref. 33) and found that the Arhgap36 CGI is more methylated on the inactive X (Extended Data Fig. 5A). To determine whether the CGIs of cat ARHGAP36 undergo XCI-dependent methylation, we performed WGBS on skin tissues of different colors from a female calico cat (#01) and peripheral blood cells from two other cats (female #04 and male #10) (Supplementary Table 2). We also obtained publicly available WGBS data from a male cat named Boris 38 . Globally, X-linked promoter CGIs (n = 362) were more methylated in females, ranging from 20% to 40%, than in males, ranging from 0% to 5% ( Fig. 3A ), consistent with the XCI-dependent methylation. We then looked at the ARHGAP36 CGIs and found higher methylation in females than in males, just as the most other X-linked CGIs ( Fig. 3B ). In contrast, the CGIs of known XCI-escaper genes, such as KDM6A, MED14, and DDX3X (ref. 39), were unmethylated in both sexes (Extended Data Fig. 5C). We then analyzed the allelic methylation status of one of the ARHGAP36 CGIs where two SNPs were available (A/G and C/A substitutions at positions 109,204,156 and 109,204,409, respectively). The result showed that both alleles are moderately methylated ( Fig. 3C ), consistent with the random XCI in tissues consisting of a heterogeneous cell population, rather than parental-origin-dependent (imprinted) or other types of skewed XCI (ref. 40). Altogether, our results corroborate Lyon’s hypothesis that the color patterns of tortoiseshell and calico cats are the results of random XCI of the O gene 5 . Download figure Open in new tab Figure 3. DNA methylation profiles of the X chromosome and ARHGAP36 in female and male cats. (A) Scatter plots and violin plots comparing CpG methylation levels of 362 X-linked promoter CGIs between female and male cats. The WGBS data were from skin (cat #01, orange patch 1) and peripheral blood cells (cats #04 and #10). (B) CpG methylation profiles of the cat ARHGAP36 region in female (red) and male cats (blue). The WGBS data were obtained from skin regions (cat #01) and peripheral blood cells (cats #04 and #10). The WGBS data of peripheral blood cells from a male cat named Boris was obtained from a publicly available dataset (SRX548709). A pink box indicates the region corresponding to the 5.1-kb deletion, and blue boxes indicate the CGIs. (C) Allelic CpG methylation of CGI #1 of ARHGAP36 . The CpG methylation levels (left) and the number of CpGs analyzed using SNP-containing WGBS reads (right) are shown. The data were from different skin regions of a calico cat (#01). Two SNPs were used to analyze allele-specific reads: cis association of the SNPs with the deletion (Δ) was determined using the data from cats hemizygous (males) and homozygous (females) for the deletion. Note that CGI #1 overlaps with the most active promoter in the skin (Extended Data Fig. 4B). Origins of the genetic variations responsible for the coat colors As described above, all 24 cats with orange coloration that we analyzed had the 5.1-kb deletion (Table, Supplementary Table 1). They were either owned or feral cats, which were mostly from the Fukuoka City area of Japan. Also, sixty-one of the 258 cats contributed by the 99 Lives Cat Genome Sequencing Initiative 10 (Supplementary Table 4), which had exactly the same deletion, included those from the United States, Europe, and the Middle East 41 . Thus, it is likely that the 5.1-kb deletion is widely distributed in domestic cats worldwide, suggesting the single origin of the orange coat color phenotype. Calico cats have white coat regions in addition to orange and black-brownish patches. A feline endogenous retrovirus insertion within the KIT gene on chromosome B1 has been identified as the causative genetic variation for white spotting 42 . To detect the white spotting allele ( w s ) in our cat samples, we performed a PCR assay. All cats with white coat regions (n = 25), including all fourteen of the calico cats, carried at least one w s allele (Supplementary Table 1), consistent with a previous report 42 . In contrast, cats without white coat regions (n = 33), including all six tortoiseshell cats, did not carry this allele (Supplementary Table 1). Altogether, the genetic basis of the coat color patterns of calico and tortoiseshell cats is likely to be common worldwide. Lastly, the golden hamster ( Mesocricetus auratus ) exhibits orange coloration due to the activity of an X-linked gene designated Sex-linked yellow ( Sly ) 43 . Using a high-quality chromosome-scale sequence assembly of the golden hamster genome 44 , we examined whether the genomic position of Arhgap36 corresponds to the genetic location of Sly . This analysis showed that the two loci are located in different regions of the X chromosome (Extended Data Fig. 6), supporting the previous conclusion reached by others that Sly and O are distinct 43 . Together with the knowledge obtained so far in humans and mice, this result suggests that the genetic contribution of ARHGAP36 and the UCE-like sequence to orange coloration may be unique to certain mammalian species. Discussion More than 60 years ago, Lyon suggested that the alternative black-brownish and orange patches of tortoiseshell cats are consistent with the random inactivation of the heretofore unidentified X-linked O gene in heterozygous females 5 . Our systematic screening now identifies a 5.1-kb deletion within an intron of ARHGAP36 , which is among the previously listed candidate genes in the O haplotype region 8 , as the genetic variation responsible for the orange coloration. Although no non-invasive method is available at present for direct demonstration of the causal relationship in this companion animal species, a perfect association between the existence of the identified genetic variation deleting a putative regulatory element and the orange coat coloration strongly supports our conclusion. While ARHGAP36 and its adjacent gene, IGSF1 , appear to share some regulatory mechanisms, the observed higher expression of the former in the skin, its known roles in signaling pathways related to hair follicle development and melanogenesis (the HH and PKA pathways), and its implications in human skin diseases involving hair follicles and naevi 26 all point to it as an excellent candidate for the long-sought O gene. Furthermore, we provide evidence that ARHGAP36 is subject to XCI in female human and mouse cells. Our results on the allelic DNA methylation status of the ARHGAP36 CGIs are also consistent with the random inactivation of this gene in female cats. ARHGAP36 encodes a member of the Rho GTPase activating protein family and is implicated in neuronal development, bone formation, hair follicle development, and cancer development or progression through positive and negative regulation of the HH and PKA signaling pathways, respectively 21 – 26 , but no role has been reported in melanogenesis. In fact, no locus affecting hair or coat color has been mapped at the orthologous location in humans or mice. Additionally, the location of the X-linked Sly, which is responsible for the orange coat coloration of the golden hamster, is distinct from both the O locus 43 and the ARHGAP36 gene (this study). Thus, ARHGAP36 protein is a novel factor regulating the melanogenesis pathway, and this role may be specific to the domestic cat or the feline family. Consistent with this, our attempts to generate mouse models by introducing the genetic variation have not been successful: neither a deletion corresponding to the one found in domestic cats nor a more targeted deletion restricted to the UCE-like sequence caused no discernible coat color change in several engineered mouse lines. How does ARHGAP36 regulate melanogenesis? According to the public databases, ARHGAP36 is highly expressed in tissues containing neural crest derivatives, but it is unknown whether the melanocyte lineage, which is also a neural crest derivative, are the primary site of ARHGAP36 expression in the skin. A previous immunostaining study however reported the detection of ARHGAP36 expression in a small number of hair follicle cells in the outer root sheath of healthy human skin 26 . Since our study using commercially available antibodies did not work for cat skin samples, the precise site of ARHGAP36 expression remains an open question. In any case, our RNA-seq data shows that ARHGAP36 expression in melanocytes, or cells communicating with them, negatively regulates a variety of melanogenesis pathway genes including those encoding signaling molecules (ligands) and enzymes directly involved in melanin synthesis. It is interesting that ARHGAP36 protein appears to lack the GTPase activating activity 20 but has the potential to inhibit PKA signaling directly by blocking the catalytic activity of PKA or by accelerating ubiquitin-mediated degradation of the catalytic subunit of PKA 22 . The PKA signaling elicited by MC1R bound to melanocyte stimulating hormone is a fundamental pathway regulating melanin synthesis 45 and, in fact, genetic variations in MC1R are associated with red hair and fair skin of humans (increased pheomelanin and decreased eumelanin) 46 . So, if ARHGAP36 is expressed in melanocytes, it could directly modulate the PKA pathway to alter the balance between eumelanin and pheomelanin. The genomic region of the 5.1-kb deletion contains a UCE-like sequence containing putative binding sites for transcriptional activators and repressors. Based on the expression patterns of cat ARHGAP36 in colored coat regions, we speculate that the deletion disrupts its regulated expression and causes inappropriate activation. However, we do not know the precise change caused by the deletion since the follicle melanogenesis is a complex process regulated by a variety of intrinsic and extrinsic factors. For example, it is restricted to the anagen stage of the hair cycle and coupled to the life cycle of melanocytes with changes in their distribution and differentiation 45 . Therefore, we would first need to know the precise site and timing of ARHGAP36 expression in healthy cat follicles. Since samples required for such a systematic study are not easily accessible in this non-experimental animal species, we are hoping that recently derived feline induced pluripotent stem (iPS) cells 47 and their use in constructing in vitro systems including organoids would resolve some of the problems. In particular, derivation of naïve female iPS cells carrying two active X chromosomes and their differentiation into melanocytes and other cells would recapitulate the random XCI and production of different pigment species in different cells. Further investigation warrants detailed understanding of how the deletion eventually regulates pheomelanin synthesis. In conclusion, we have identified the X-linked ARHGAP36 gene as the strong candidate as the long-sought O gene of the domestic cat. Although it is still unclear how the identified deletion switches eumelanin to pheomelanin, the variation likely dominates the cat population with orange coloration. We also provide evidence that ARHGAP36 is most likely subject to random XCI in the domestic cat as previously suggested by Lyon 5 . How this gene became employed in melanogenesis in certain species and how it exerts pleiotropic biological effects in mammals are interesting future questions. View this table: View inline View popup Download powerpoint Table Association between orange coloration and a 5.1-kb deletion within ARHGAP36 Methods Ethics statement Sampling of domestic cats was carried out in accordance with the ethical guidelines of Kyushu University using the protocols approved by the Animal Experiment Committee (#A21-433-1). Blood samples and small tissue pieces were acquired by veterinarians during routine veterinary practice or spaying/neutering surgery. Skin tissues from an animal that died of unknown causes were voluntarily donated by a breeder. DNA and RNA extraction Genomic DNA was extracted from whole blood cells using the NucleoSpin Blood kit (Macherey-Nagel) or from tissue samples by a standard method involving proteinase K digestion and isopropanol precipitation. Total RNA was extracted from tissue samples using TRIzol Reagent (Thermo Fisher Scientific). WGS and data analysis Libraries were prepared from genomic DNA using the TruSeq DNA PCR-Free LT Library Prep Kit (Illumina) and sequenced to generate 53-nt paired-end reads on an Illumina NovaSeq 6000 equipped with NVCS v1.6.0 and RTA v3.4.4. The reference genome of the domestic cat (AnAms1.0) was obtained from the Cats-I database 9 . All paired-end reads were aligned to the reference genome using the Burrows-Wheeler Aligner (BWA v0.7.17) 48 . After removing duplicate reads, single nucleotide substitutions and small insertion-deletion variants (indels) were identified. Variant Call Format files were generated using the Genome Analysis Toolkit (GATK v4.1.3) 49 . These files were annotated using SnpEff v4.3 (ref. 50) to assess the effect of variants on gene function. Raw fastq files from published WGS datasets of 258 cats, including nine with clear color images, contributed primarily by the 99 Lives Cat Genome Sequencing Initiative 10 , 41 , were obtained from the Sequence Read Archive. The datasets were analyzed using our proprietary pipeline. Human (hg38 and hg19) and mouse (mm10) X chromosome sequences and ENCODE data were downloaded from the UCSC Genome Browser. Sequence alignment between multiple mammalian species was performed using mVISTA (ref. 51) and ClustalW v2.1 (ref. 52). The golden hamster genome sequence was obtained from the Sequence Read Archive (DRA010879) 44 . Genotyping The cats were genotyped for alleles of ARHGAP36 (ϑ′/+) and KIT ( w + / w s ) by PCR using KOD One PCR Master Mix (TOYOBO) with primers listed in Supplementary Table 7. Some animals were also examined for the presence of SRY on the Y chromosome. Sanger sequencing Sanger sequencing of PCR products was performed to determine the exact position and size of the deletion using BigDye Terminator v3.1 (Thermo Fisher Scientific) and primers listed in Supplementary Table 7. RNA-seq and data analysis Libraries were prepared from total RNA using NEBNext rRNA Depletion Kit, NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB). Sequencing was performed using a SP Reagent Kit (Illumina) on an Illumina NovaSeq 6000 equipped with NVCS v1.6.0 and RTA v3.4.4 to generate 53-nt paired-end reads. Adapter sequences and low-quality bases were removed from the 5’ and 3’ ends, respectively. Reads were aligned to the AnAms1.0 genome by HISAT2 v2.0.5 (ref. 53). Transcripts were assembled using StringTie v2.1.4 (ref. 54). To characterize the gene regulatory network involving ARHGAP36 , we identified genes that showed positive and negative correlation with ARHGAP36 expression in skin samples from a calico cat (#01). Only the orange and black-brownish regions (n = 2 each) were used for the analysis because we did not expect melanogenesis in the white regions. (Indeed, we observed no ARHGAP36 expression in the white region.) We then grouped the four datasets into two, ARHGAP36 positive (two orange and one black-brownish samples) and ARHGAP36 negative (one black-brownish sample). Then, a gene was considered to be positively correlated if its FPKM value was greater than 1 and at least five times higher in all three ARHGAP36 -positive samples compared with the ARHGAP36 -negative sample. Conversely, a gene was considered to be negatively correlated if its FPKM was greater than 1 and at least five times higher in the ARHGAP36 -negtive sample compared to the ARHGAP36 -positive samples. Then, genes positively and negatively correlated with ARHGAP36 were subjected to the KEGG pathway analysis using DAVID 27 , 28 . RNA-seq data from mouse embryonic fibroblasts (GSE112097 and GSE121184) 31 , 33 were reprocessed as described in the original manuscript for SNP-based allelic expression analysis of mouse Arhgap36 . RNA-seq data from various cat organs and tissues were acquired from the publicly available dataset (PRJNA312519) and processed using the aforementioned pipeline. WGBS and data analysis Genomic DNA spiked with 2 ng lambda phage DNA (Promega) was subjected to bisulfite conversion. WGBS libraries were prepared using the post-bisulfite adapter tagging method 55 . Sequencing was performed using a S1 Reagent Kit (Illumina) on an Illumina NovaSeq 6000 equipped with NVCS v1.6.0 and RTA v3.4.4 to generate 108-nt single-end reads. Adapter sequences and low-quality bases were removed from the 5’ and 3’ ends, respectively. Reads were aligned to the AnAms1.0 genome using Bismark v0.10.0 (ref. 56) with a seed length of 28, allowing a maximum of one mismatch in the seed and enabling the “--pbat” option. Only uniquely aligned reads were used in the subsequent analysis. Data from both DNA strands were combined. Bisulfite conversion rate was estimated using reads uniquely aligned to the lambda phage genome. Allele-specific CpG methylation levels were calculated by using SNPsplit v0.3.2 (ref. 57), based on a list of SNPs identified in ARHGAP36 . Publicly available WGBS datasets were obtained from the Sequence Read Archive for a male cat named Boris (SRX548709) 38 , human cytotrophoblasts (NBDC No. hum0086) 37 , and mouse liver (GSE106379) 36 . These data were processed using the same pipeline as the cat data analysis. In addition, pre-calculated bedGraph files on DNA methylation of mouse embryonic fibroblasts were obtained from the NCBI Gene Expression Omnibus (GSE122094) 33 . CGIs were predicted in the AnAms1.0 genome sequence using the method described by Gardiner-Garden and Frommer 58 . Data availability All sequencing data related to this study are publicly available in the DDBJ/ENA/NCBI databases under BioProject No. PRJDB18200. Author contributions M.U., M.M., and H.S. conceived the study and planned the experiments. M.U., Y. Matsumoto, Y. Matsumura, M.M. collected the cat samples. W.K.A.U., M.U., Y. Miki, and M.M. performed the experiments and H.T., W.K.A.U., Y.N., and H.S. analyzed the data. Y.B. and T.S. analyzed the data from mouse studies. Y.B., Y.N., M.M., and H.S. supervised the entire project. H.T., W.K.A.U., M.U., M.M., and H.S. interpreted the results and wrote the paper with input from all authors. Competing interests The authors declare no competing interests. Additional information Extended data is available for this paper. Supplementary information The online version contains supplementary material. 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Share A deletion at the X-linked ARHGAP36 gene locus is associated with the orange coloration of tortoiseshell and calico cats Hidehiro Toh , Wan Kin Au Yeung , Motoko Unoki , Yuki Matsumoto , Yuka Miki , Yumiko Matsumura , Yoshihiro Baba , Takashi Sado , Yasukazu Nakamura , Miho Matsuda , Hiroyuki Sasaki bioRxiv 2024.11.19.624036; doi: https://doi.org/10.1101/2024.11.19.624036 Share This Article: Copy Citation Tools A deletion at the X-linked ARHGAP36 gene locus is associated with the orange coloration of tortoiseshell and calico cats Hidehiro Toh , Wan Kin Au Yeung , Motoko Unoki , Yuki Matsumoto , Yuka Miki , Yumiko Matsumura , Yoshihiro Baba , Takashi Sado , Yasukazu Nakamura , Miho Matsuda , Hiroyuki Sasaki bioRxiv 2024.11.19.624036; doi: https://doi.org/10.1101/2024.11.19.624036 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetics Subject Areas All Articles Animal Behavior and Cognition (7643) Biochemistry (17717) Bioengineering (13910) Bioinformatics (42017) Biophysics (21480) Cancer Biology (18628) Cell Biology (25537) Clinical Trials (138) Developmental Biology (13392) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22530) Immunology (17755) Microbiology (40438) Molecular Biology (17200) Neuroscience (88705) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4832) Physiology (7657) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9828) Zoology (2272)