BarkBase: Epigenomic Annotation of Canine Genomes

Dogs are an unparalleled natural model for investigating the genetics of health and disease, particularly for complex diseases like cancer. Comprehensive genomic annotation of regulatory elements active in healthy canine tissues is crucial both for identifying candidate causal variants and for designing functional studies needed to translate genetic associations into disease insight. Currently, canine geneticists rely primarily on annotations of the human or mouse genome that have been remapped to dog, an approach that misses dog-specific features. Here, we describe BarkBase, a canine epigenomic resource available at barkbase.org. BarkBase hosts data for 27 adult tissue types, with biological replicates, and for one sample of up to five tissues sampled at each of four carefully staged embryonic time points. RNA sequencing is complemented with whole genome sequencing and with assay for transposase-accessible chromatin using sequencing (ATAC-seq), which identifies open chromatin regions. By including replicates, we can more confidently discern tissue-specific transcripts and assess differential gene expression between tissues and timepoints. By offering data in easy-to-use file formats, through a visual browser modeled on similar genomic resources for human, BarkBase introduces a powerful new resource to support comparative studies in dogs and humans

Supplementary Materials for Comparative genomics of Balto, a famous historic dog, captures lost diversity of 1920s sled dogs

The PDF file includes: Supplementary Text; Materials and Methods; Figs. S1 to S10; Tables S1 to S15; References.-- Other Supplementary Material for this manuscript includes the following: MDAR Reproducibility Checklist; Data S1 to S5; Model and ScriptsN

Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements.

Vocal production learning (vocal learning) is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution

​Comparative genomics of Balto, a famous historic dog, captures lost diversity of 1920s sled dogs

Tomàs Marquès-Bonet forma part del Zoonomia ConsortiumWe reconstruct the phenotype of Balto, the heroic sled dog renowned for transporting diphtheria antitoxin to Nome, Alaska in 1925, using evolutionary constraint estimates from the Zoonomia alignment of 240 mammals and 682 genomes from dogs and wolves of the 21st century. Balto shares just part of his diverse ancestry with the eponymous Siberian husky breed. Balto's genotype predicts a combination of coat features atypical for modern sled dog breeds, and a slightly smaller stature. He had enhanced starch digestion compared with Greenland sled dogs and a compendium of derived homozygous coding variants at constrained positions in genes connected to bone and skin development. We propose that Balto's population of origin, which was less inbred and genetically healthier than modern breeds, was adapted to the extreme environment of 1920s Alaska

The genomic landscape of canine osteosarcoma cell lines reveals conserved structural complexity and pathway alterations.

The characterization of immortalized canine osteosarcoma (OS) cell lines used for research has historically been based on phenotypic features such as cellular morphology and expression of bone specific markers. With the increasing use of these cell lines to investigate novel therapeutic approaches prior to in vivo translation, a much more detailed understanding regarding the genomic landscape of these lines is required to ensure accurate interpretation of findings. Here we report the first whole genome characterization of eight canine OS cell lines, including single nucleotide variants, copy number variants and other structural variants. Many alterations previously characterized in primary canine OS tissue were observed in these cell lines, including TP53 mutations, MYC copy number gains, loss of CDKN2A, PTEN, DLG2, MAGI2, and RB1 and structural variants involving SETD2, DLG2 and DMD. These data provide a new framework for understanding how best to incorporate in vitro findings generated using these cell lines into the design of future clinical studies involving dogs with spontaneous OS

​Comparative genomics of Balto, a famous historic dog, captures lost diversity of 1920s sled dogs

We reconstruct the phenotype of Balto, the heroic sled dog renowned for transporting diphtheria antitoxin to Nome, Alaska, in 1925, using evolutionary constraint estimates from the Zoonomia alignment of 240 mammals and 682 genomes from dogs and wolves of the 21st century. Balto shares just part of his diverse ancestry with the eponymous Siberian husky breed. Balto's genotype predicts a combination of coat features atypical for modern sled dog breeds, and a slightly smaller stature. He had enhanced starch digestion compared with Greenland sled dogs and a compendium of derived homozygous coding variants at constrained positions in genes connected to bone and skin development. We propose that Balto's population of origin, which was less inbred and genetically healthier than that of modern breeds, was adapted to the extreme environment of 1920s Alaska

DNA damage-induced cell death and <i>gypsy</i> ERV expression contribute hTDP-43 mediated toxicity.

<p>(A) Lifespan analysis shows that co-expression of <i>loki</i>(IR) (<i>Repo</i> > <i>loki</i>(IR) + hTDP-43) fully rescues the lifespan deficit exhibited by flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (B) Co-expression of <i>loki</i>(IR) (<i>ELAV</i> > <i>loki</i>(IR) + hTDP-43) likewise fully rescues the lifespan deficit exhibited by flies expressing neuronal hTDP-43 (<i>ELAV</i> > hTDP-43). (C) Central projections of whole-mount TUNEL stained brains reveal a noticeable reduction in the apoptotic activity induced by glial hTDP-43 expression (<i>Repo</i> > hTDP-43 + GFP(IR)) when <i>gypsy</i> expression is knocked down (<i>Repo</i> > hTDP-43 + <i>gypsy</i>(IR)), while knocking down <i>loki</i> completely alleviates the apoptosis induced by glial hTDP-43 expression (<i>Repo</i> > hTDP-43 + <i>loki</i>(IR)). (D) Quantification of (H), normalized to the positive control (<i>Repo</i> > hTDP-43 + GFP(IR)). <i>N</i> = 12 for <i>Repo</i> / +; <i>N</i> = 9 for <i>Repo</i> > hTDP-43 + GFP(IR); <i>N</i> = 7 for <i>Repo</i> > hTDP-43 + <i>gypsy</i>(IR); and <i>N</i> = 7 for <i>Repo</i> > hTDP-43 + <i>loki</i>(IR). <b>*</b>All of the lifespans with the exception of the NRTI feeding experiments shown in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.g004" target="_blank">4</a> and 5, were performed concurrently in order to ensure comparability across groups. Therefore, appropriate controls are shared across panels.</p

Glial and neuronal hTDP-43 expression erodes siRNA-mediated silencing.

<p>(A) Representative central projections show that co-expression of the hTDP-43 transgene, but not an unrelated tdTomato control transgene, interferes with the ability of a Dcr-2 processed IR (GFP(IR)) to silence a GFP transgenic reporter in glial cells using the <i>Repo-GAL4</i> driver. Quantification of GFP signal for each group is shown in the appropriate bar graph; values are represented as relative fold change over <i>Repo</i> > GFP + GFP(IR) (mean + SEM). A two-way ANOVA reveals significant effects of both genotype (p < 0.0001) and age (p < 0.0001), and a significant age x genotype interaction (p < 0.0001). <i>N</i> = 5 for <i>Repo</i> > GFP and <i>Repo</i> > GFP + GFP(IR); <i>N</i> = 10 for all other groups. (B) An equivalent analysis demonstrates that hTDP-43 has a similar effect in the neuronal cells of the <i>Drosophila</i> mushroom body using the <i>OK107-Gal4</i> driver, but with a later age of onset than hTDP-43 expression in glial cells. Quantification of GFP signal for each group is shown in the appropriate bar graph as in (A). A two-way ANOVA reveals significant effects of genotype (p = 0.0054) and age (p < 0.0001), as well as a significant age x genotype interaction (p = 0.0021). <i>N</i> = 5 for <i>OK107</i> > GFP and <i>OK107</i> > GFP + GFP(IR); <i>N</i> = 10 for all other groups. (C) Co-expression of hTDP-43, but not GFP, in the photoreceptor neurons of the fly eye under the <i>GMR-Gal4</i> driver interrupts the ability of a Dcr-2 processed IR to silence the endogenous <i>white</i>^<i>+</i> pigment gene with an age of onset similar to that observed with neuronal expression of hTDP-43 in the CNS under <i>OK107-Gal4</i>, resulting in characteristic clusters of red-pigmented ommatidia. <i>N</i> = 5 for <i>GMR</i> > <i>w</i>(IR) + Gal80^ts <i>OFF</i> and <i>GMR</i> > <i>w</i>(IR) + Gal80^ts <i>ON</i>; <i>N</i> = 20 for all other groups.</p

<i>gypsy</i> ERV expression contributes to hTDP-43 mediated toxicity.

<p>(A) Lifespan analysis shows that co-expression of <i>gypsy</i>(IR) (<i>Repo</i> > <i>gypsy</i>(IR) + hTDP-43) partially rescues the lifespan deficit exhibited by flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (B) Co-expression of an unrelated GFP(IR) control transgene (<i>Repo</i> > GFP(IR) + hTDP-43) does not effect the lifespan of flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (C) Co-expression of <i>gypsy</i>(IR) (<i>ELAV</i> > <i>gypsy</i>(IR) + hTDP-43) has no effect on lifespan in flies expressing neuronal hTDP-43 (<i>ELAV</i> > hTDP-43).</p

Glial hTDP-43 expression results in early and dramatic de-suppression of the <i>gypsy</i> ERV.

<p>(A) Transcript levels of <i>gypsy ORF2</i> (<i>Pol</i>) as detected by qPCR in whole head tissue of flies expressing hTDP-43 in neurons (<i>ELAV</i> > hTDP-43) versus glia (<i>Repo</i> > hTDP-43) at a young (2–4 Day) or aged (8–10 Day) time point. Transcript levels normalized to <i>Actin</i> and displayed as fold change relative to flies carrying the hTDP-43 transgene with no Gal4 driver (hTDP-43 / +) at 2–4 Days (means + SEM). A two-way ANOVA reveals a significant effect of genotype (p < 0.0001) but no effect of age (p = 0.5414). <i>N</i> = 8 for all groups. (B) An equivalent analysis shows that <i>gypsy ORF3</i> (<i>Env</i>) likewise displays a significant effect of genotype (p < 0.0001) and no effect of age (p = 0.6530). <i>N</i> = 4 for the 2–4 Day cohort and <i>N</i> = 5 for the 8–10 Day cohort. (C) Central projections of whole mount brains immunostained with a monoclonal antibody directed against <i>gypsy</i> ENV protein reveals dramatic, early accumulation of ENV immunoreactive puncta in brains expressing glial hTDP-43 (5–8 Days) in comparison to both age-matched genetic controls (<i>ELAV</i> / +; <i>Repo</i> / +; hTDP-43 / +) and flies expressing neuronal hTDP-43. This effect persists out to 19–25 Days post-eclosion. <i>ELAV</i> / +, 5–8 Day (<i>N</i> = 3), 19–25 Day (<i>N</i> = 4); <i>Repo</i> / +, 5–8 Day (<i>N</i> = 3), 19–25 Day (<i>N</i> = 3); hTDP-43 / +, 5–8 Day (<i>N</i> = 5), 19–25 Day (<i>N</i> = 2); <i>ELAV</i> > hTDP-43, 5–8 Day (<i>N</i> = 2), 19–25 Day (<i>N</i> = 4); <i>Repo</i> > hTDP-43, 5–8 Day (<i>N</i> = 7), 19–25 Day (<i>N</i> = 8).</p