De novo variants in ATXN7L3 lead to developmental delay, hypotonia and distinctive facial features.

Deubiquitination is critical for the proper functioning of numerous biological pathways such as DNA repair, cell cycle progression, transcription, signal transduction, and autophagy. Accordingly, pathogenic variants in deubiquitinating enzymes (DUBs) have been implicated in neurodevelopmental disorders (ND) and congenital abnormalities. ATXN7L3 is a component of the DUB module of the SAGA complex, and two other related DUB modules, and serves as an obligate adaptor protein of 3 ubiquitin-specific proteases (USP22, USP27X or USP51). Through exome sequencing and GeneMatching, we identified nine individuals with heterozygous variants in ATXN7L3. The core phenotype included global motor and language developmental delay, hypotonia, and distinctive facial characteristics including hypertelorism, epicanthal folds, blepharoptosis, a small nose and mouth, and low-set posteriorly rotated ears. In order to assess pathogenicity, we investigated the effects of a recurrent nonsense variant [c.340C>T; p.(Arg114Ter)] in fibroblasts of an affected individual. ATXN7L3 protein levels were reduced, and deubiquitylation was impaired, as indicated by an increase in histone H2Bub1 levels. This is consistent with the previous observation of increased H2Bub1 levels in Atxn7l3-null mouse embryos, which have developmental delay and embryonic lethality. In conclusion, we present clinical information and biochemical characterization supporting ATXN7L3 variants in the pathogenesis of a rare syndromic ND.

Assembly of the threespine stickleback Y chromosome reveals convergent signatures of sex chromosome evolution.

BACKGROUND: Heteromorphic sex chromosomes have evolved repeatedly across diverse species. Suppression of recombination between X and Y chromosomes leads to degeneration of the Y chromosome. The progression of degeneration is not well understood, as complete sequence assemblies of heteromorphic Y chromosomes have only been generated across a handful of taxa with highly degenerate sex chromosomes. Here, we describe the assembly of the threespine stickleback (Gasterosteus aculeatus) Y chromosome, which is less than 26 million years old and at an intermediate stage of degeneration. Our previous work identified that the non-recombining region between the X and the Y spans approximately 17.5 Mb on the X chromosome. RESULTS: We combine long-read sequencing with a Hi-C-based proximity guided assembly to generate a 15.87 Mb assembly of the Y chromosome. Our assembly is concordant with cytogenetic maps and Sanger sequences of over 90 Y chromosome BAC clones. We find three evolutionary strata on the Y chromosome, consistent with the three inversions identified by our previous cytogenetic analyses. The threespine stickleback Y shows convergence with more degenerate sex chromosomes in the retention of haploinsufficient genes and the accumulation of genes with testis-biased expression, many of which are recent duplicates. However, we find no evidence for large amplicons identified in other sex chromosome systems. We also report an excellent candidate for the master sex-determination gene: a translocated copy of Amh (Amhy). CONCLUSIONS: Together, our work shows that the evolutionary forces shaping sex chromosomes can cause relatively rapid changes in the overall genetic architecture of Y chromosomes.

A key metabolic gene for recurrent freshwater colonization and radiation in fishes.

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential ω-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization.

Centromere inactivation on a neo-Y fusion chromosome in threespine stickleback fish.

Having one and only one centromere per chromosome is essential for proper chromosome segregation during both mitosis and meiosis. Chromosomes containing two centromeres are known as dicentric and often mis-segregate during cell division, resulting in aneuploidy or chromosome breakage. Dicentric chromosome can be stabilized by centromere inactivation, a process which reestablishes monocentric chromosomes. However, little is known about this process in naturally occurring dicentric chromosomes. Using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on metaphase chromosome spreads, we demonstrate that centromere inactivation has evolved on a neo-Y chromosome fusion in the Japan Sea threespine stickleback fish (Gasterosteus nipponicus). We found that the centromere derived from the ancestral Y chromosome has been inactivated. Our data further suggest that there have been genetic changes to this centromere in the two million years since the formation of the neo-Y chromosome, but it remains unclear whether these genetic changes are a cause or consequence of centromere inactivation.

Identification of the centromeric repeat in the threespine stickleback fish (Gasterosteus aculeatus).

Centromere sequences exist as gaps in many genome assemblies due to their repetitive nature. Here we take an unbiased approach utilizing centromere protein A (CENP-A) chomatin immunoprecipitation followed by high-throughput sequencing to identify the centromeric repeat sequence in the threespine stickleback fish (Gasterosteus aculeatus). A 186-bp, AT-rich repeat was validated as centromeric using both fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on interphase nuclei and metaphase spreads. This repeat hybridizes strongly to the centromere on all chromosomes, with the exception of weak hybridization to the Y chromosome. Together, our work provides the first validated sequence information for the threespine stickleback centromere.

Molecular and developmental contributions to divergent pigment patterns in marine and freshwater sticklebacks.

Pigment pattern variation across species or populations offers a tractable framework in which to investigate the evolution of development. Juvenile threespine sticklebacks (Gasterosteus aculeatus) from marine and freshwater environments exhibit divergent pigment patterns that are associated with ecological differences. Juvenile marine sticklebacks have a silvery appearance, whereas sticklebacks from freshwater environments exhibit a pattern of vertical bars. We investigated both the developmental and molecular basis of this population-level variation in pigment pattern. Time course imaging during the transition from larval to juvenile stages revealed differences between marine and freshwater fish in spatial patterns of chromatophore differentiation as well as in pigment amount and dispersal. In freshwater fish, melanophores appear primarily within dark bars whereas iridophores appear within light bars. By contrast, in marine fish, these chromatophores are interspersed across the flank. In addition to spatially segregated chromatophore differentiation, pigment amount and dispersal within melanophores varies spatially across the flank of freshwater, but not marine fish. To gain insight into the molecular pathways that underlie the differences in pigment pattern development, we evaluated differential gene expression in the flanks of developing fish using high-throughput cDNA sequencing (RNA-seq) and quantitative PCR. We identified several genes that were differentially expressed across dark and light bars of freshwater fish, and between freshwater and marine fish. Together, these experiments begin to shed light on the process of pigment pattern evolution in sticklebacks.

DUX4 activates germline genes, retroelements, and immune mediators: implications for facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression.

Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration.

BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

DHODH modulates transcriptional elongation in the neural crest and melanoma.

Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.

Endothelial progenitor cell function, apoptosis, and telomere length in overweight/obese humans.

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle-aged and older adults (42-67 years) were studied: 25 normal weight (normal weight; BMI: 18.5-24.9 kg/m(2)) and 42 overweight/obese (overweight/obese; BMI: 25.0-34.9 kg/m(2)). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) was determined in the absence and presence of phytohemagglutinin (10 microg/ml). Intracellular active caspase-3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell-derived factor-1alpha (SDF-1alpha; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin-stimulated release of VEGF (90.6 +/- 7.6 vs. 127.2 +/- 11.6 pg/ml) and G-CSF (896.1 +/- 77.4 vs. 1,176.3 +/- 126.3 pg/ml) was ~25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ~30% greater (P < 0.05) increase in active caspase-3 in EPCs from overweight/obese (2.8 +/- 0.2 ng/ml) compared with normal weight (2.2 +/- 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF-1alpha. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.

Aging and endothelial progenitor cell telomere length in healthy men.

BACKGROUND: Telomere length declines with age in mature endothelial cells and is thought to contribute to endothelial dysfunction and atherogenesis. Bone marrow-derived circulating endothelial progenitor cells (EPCs) are critical to vascular health as they contribute to both reendothelialization and neovascularization. We tested the hypothesis that EPC telomere length decreases with age in healthy adult humans. METHODS: Peripheral blood samples were collected from 40 healthy, non-obese, sedentary men: 12 young (age 21-34 years), 12 middle-aged (43-55 years) and 16 older (57-68 years). Putative EPCs were isolated from peripheral blood mononuclear cells and telomere length was determined using genomic DNA preparation and Southern hybridization techniques. RESULTS: EPC telomere length (base pairs) was approximately 20% (p=0.01) lower in the older (8492+523 bp) compared to the middle-aged (10,565+572 bp) and young (10,205+501 bp) men. Of note, there was no difference in EPC telomere length between the middle-aged and young men. CONCLUSIONS: These results demonstrate that EPC telomere length declines with age in healthy, sedentary men. Interestingly, telomere length was well preserved in the middle-aged compared to young men, suggesting that EPC telomere shortening occurs after the age of 55 years.

Gender and Endothelial Progenitor Cell Number in Middle-Aged Adults.

BACKGROUND: Between the ages of 45 and 65 years, the prevalence of cardiovascular disease is significantly lower in women compared with men. Circulating bone marrow-derived endothelial progenitor cells (EPCs) play an important role in vascular repair. Reduced EPC number is predictive of more cardiovascular events. It is currently unknown whether there is a sex-difference in EPC number in middle-aged adults. OBJECTIVE: We tested the hypothesis that circulating EPC number is higher in middle-aged women than men. METHODS: Peripheral blood samples were collected from 58 sedentary adults, 29 men (57 ± 1 yr) and 29 women (58 ± 1 yr). Mononuclear cells were isolated and fluorescence-activated cell sorting (FACS) analysis of cells negative for CD45 was performed for those positive for CD34, and triple positive for CD34, VEGFR-2, and CD133 according to the recommendations of the International Society for Hematotherapy and Graft Engineering. RESULTS: The number of CD45(-)/CD34(+) and CD45(-)/CD34(+)/ VEGFR-2(+)/CD133(+) were not significantly different between women and men (0.055 ± 0.006% vs 0.069 ± 0.008% and 0.0013 ± 0.0003% vs 0.0018 ± 0.0004%, respectively). CONCLUSIONS: These results demonstrate no sex-difference in EPC number in middle-age adults. Therefore, it is unlikely that differences in EPC number contribute to the gender-related differences in the prevalence of cardiovascular events in this population.

Reducing the lateral force acting on the patella does not consistently decrease patellofemoral pressures.

BACKGROUND: Extensor mechanism procedures that decrease the lateral component of the patellar tendon or quadriceps force acting on the patella do not consistently reduce pain. HYPOTHESIS: Patellofemoral treatments do not consistently decrease patellofemoral pressures because of variations in the moments acting on the patella. STUDY DESIGN: Computer simulation study. METHODS: Computational models of 4 knees were constructed to characterize the patellofemoral pressure distribution during simulated squatting from 40 degrees to 90 degrees. The knees were given an initial Q angle of 25 degrees. Patellofemoral treatments were simulated by increasing the percentage of the quadriceps force applied by the vastus medialis by 50% and by medializing the tibial tuberosity to decrease the Q angle to 15 degrees. RESULTS: Decreasing the Q angle caused a larger decrease in the lateral component of the force applied by the quadriceps and patellar tendon than did increasing the force applied by the vastus medialis and, therefore, was more effective at decreasing patellofemoral pressures and the force needed to resist lateral subluxation. Both treatments also decreased the moments acting to rotate the distal patella laterally and tilt the patella laterally during flexion. Variations in these moments increased patellofemoral pressures for some knees. CONCLUSIONS: Treatments that reduce patellofemoral subluxation can have an unexpected influence on patellofemoral pressures because of the moments acting on the patella. CLINICAL RELEVANCE: Extensor mechanism procedures that restore patellofemoral stability may not provide pain relief.