Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes.

Summary Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes we present a proof-of-principle approach that addresses these limitations. Combining ChIP-Seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. As Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.

Methylation of the regulatory region of the elongation of very‐long‐chain fatty acids‐like 2 (ELOVL2) gene, an enzyme involved in elongation of long‐chain polyunsaturated fatty acids, is one of the most robust biomarkers of human age, but the critical question of whether ELOVL2 plays a functional role in molecular aging has not been resolved. Here, we report that Elovl2 regulates age‐associated functional and anatomical aging in vivo, focusing on mouse retina, with direct relevance to age‐related eye diseases. We show that an age‐related decrease in Elovl2 expression is associated with increased DNA methylation of its promoter. Reversal of Elovl2 promoter hypermethylation in vivo through intravitreal injection of 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dc) leads to increased Elovl2 expression and rescue of age‐related decline in visual function. Mice carrying a point mutation C234W that disrupts Elovl2‐specific enzymatic activity show electrophysiological characteristics of premature visual decline, as well as early appearance of autofluorescent deposits, well‐established markers of aging in the mouse retina. Finally, we find deposits underneath the retinal pigment epithelium in Elovl2 mutant mice, containing components found in human drusen, a pathologic hallmark of age related macular degeneration. These findings indicate that ELOVL2 activity regulates aging in mouse retina, provide a molecular link between polyunsaturated fatty acids elongation and visual function, and suggest novel therapeutic strategies for the treatment of age‐related eye diseases.

Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, Nmf9, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with Nmf9 expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian Nmf9. Intriguingly, homozygotes for null mutations in the Drosophila homolog, CG45058, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes.

Advances in DNA sequencing technologies have greatly facilitated the discovery of rare genetic variants in the human genome, many of which may contribute to common disease risk. However, evaluating their individual or even collective effects on disease risk requires very large sample sizes, which involves study designs that are often prohibitively expensive. We present an alternative approach for determining genotypes in large numbers of individuals for all variants discovered in the sequence of relatively few individuals. Specifically, we developed a new imputation algorithm that utilizes whole-exome sequencing data from 25 members of the South Dakota Hutterite population, and genome-wide SNP genotypes from >1400 individuals from the same founder population. The algorithm relies on identity-by-descent sharing of phased haplotypes, a different strategy than the linkage disequilibrium methods found in most imputation methods. We imputed genotypes discovered in the sequence data to on average ~77% of chromosomes among the 1400 individuals. Median R2 between imputed and directly genotyped data was > 0.99. As expected, many variants that are vanishingly rare in European populations have risen to larger frequencies in the founder population, and would be amenable to single-SNP analyses.

Methylation of the regulatory region of the Elongation Of Very Long Chain Fatty Acids-Like 2 (ELOVL2) gene, an enzyme involved in elongation of long-chain polyunsaturated fatty acids, is one of the most robust biomarkers of human age, but the critical question of whether ELOVL2 plays a functional role in molecular aging has not been resolved.Here, we report that Elovl2 regulates age-associated functional and anatomical aging in vivo, focusing on mouse retina, with direct relevance to age-related eye diseases. We show that an age-related decrease in Elovl2 expression is associated with increased DNA methylation of its promoter. Reversal of Elovl2 promoter hypermethylation in vivo through intravitreal injection of 5-Aza-2'-deoxycytidine (5-aza-dc) leads to increased Elovl2 expression and rescue of age-related decline in visual function. Mice carrying a point mutation C234W that disrupts Elovl2-specific enzymatic activity show electrophysiological characteristics of premature visual decline, as well as early appearance of autofluorescent deposits, well-established markers of aging in the mouse retina. Finally, we find deposits underneath the retinal pigment epithelium in Elovl2 mutant mice, containing components of complement system and lipid metabolism. These findings indicate that ELOVL2 activity regulates aging in mouse retina, provide a molecular link between polyunsaturated fatty acids elongation and visual functions, and suggest novel therapeutic strategies for treatment of age-related eye diseases.

Endogenous retroviruses and retrotransposons contribute functional genetic variation in animal genomes. In mice, Intracisternal A Particles (IAPs) are a frequent source of both new mutations and polymorphism across laboratory strains. Intronic IAPs can induce alternative RNA processing choices, including alternative splicing. We previously showed IAP I∆1 subfamily insertional mutations are suppressed by a wild-derived allele of the major mRNA export factor, Nxf1. Here we show that a wider diversity of IAP insertions present in the mouse reference sequence induce insertion-dependent alternative processing that is suppressed by Nxf1CAST alleles. These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation. Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles. Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.

How and to what degree gene duplication events create regulatory innovation, redundancy, or neofunctionalization remain important questions in animal evolution and comparative genetics. Ankfn1 genes are single copy in most invertebrates, partially duplicated in jawed vertebrates, and only the derived copy retained in most mammals. Null mutations in the single mouse homolog have vestibular and neurological abnormalities. Null mutation of the single Drosophila homolog is typically lethal with severe sensorimotor deficits in rare survivors. The functions and potential redundancy of paralogs in species with two copies is not known. Here we define a vestibular role for Ankfn1 homologs in zebrafish based on simultaneous disruption of each locus. Zebrafish with both paralogs disrupted showed vestibular defects and early lethality from swim bladder inflation failure. One intact copy at either locus was sufficient to prevent major phenotypes. Our results show that vertebrate Ankfn1 genes are required for vestibular-related functions, with at least partial redundancy between ancestral and derived paralogs.