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Contains fulltext : 251162.pdf (Publisher’s version ) (Open Access) The mitochondrial respiratory chain (MRC) is composed of four multiheteromeric enzyme complexes. According to the endosymbiotic origin of mitochondria, eukaryotic MRC derives from ancestral proteobacterial respiratory structures consisting of a minimal set of complexes formed by a few subunits associated with redox prosthetic groups. These enzymes, which are the "core" redox centers of respiration, acquired additional subunits, and increased their complexity throughout evolution. Cytochrome c oxidase (COX), the terminal component of MRC, has a highly interspecific heterogeneous composition. Mammalian COX consists of 14 different polypeptides, of which COX7B is considered the evolutionarily youngest subunit. We applied proteomic, biochemical, and genetic approaches to investigate the COX composition in the invertebrate model Drosophila melanogaster. We identified and characterized a novel subunit which is widely different in amino acid sequence, but similar in secondary and tertiary structures to COX7B, and provided evidence that this object is in fact replacing the latter subunit in virtually all protostome invertebrates. These results demonstrate that although individual structures may differ the composition of COX is functionally conserved between vertebrate and invertebrate species.

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs., Applying complexome profiling, Evers et al. unravel the composition of mitochondrial oxidative phosphorylation complexes in P. falciparum asexual and sexual blood stages. Abundance of these complexes differs between both stages, supporting the hypothesis that a mitochondrial metabolic switch is central to gametocyte development and functioning.

Protein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexome profiling with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.

Polycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model for comprehensively studying Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for proper recruitment of Polycomb group protein Rnf2. However, despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occur. These changes were mainly due to local dysregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important immediately after the body plan is formed to maintain spatially restricted expression profiles of transcription factors, and we highlight the differences that exist in the timing of PcG protein action between vertebrate species., Summary: Our unique zebrafish model of a maternal and zygotic mutant for the Polycomb group gene ezh2 reveals major conserved and divergent mechanisms in epigenetic gene repression during vertebrate development.

BackgroundGenome duplication has played a pivotal role in the evolution of many eukaryotic lineages, including the vertebrates. The most recent vertebrate genome duplication is that in Xenopus laevis, resulting from the hybridization of two closely related species about 17 million years ago [1]. However, little is known about the consequences of this duplication at the level of the genome, the epigenome and gene expression.ResultsOf the parental subgenomes, S chromosomes have degraded faster than L chromosomes ever since the genome duplication and until the present day. Deletions appear to have the largest effect on pseudogene formation and loss of regulatory regions. Deleted regions are enriched for long DNA repeats and the flanking regions have high alignment scores, suggesting that non-allelic homologous recombination (NAHR) has played a significant role in the loss of DNA. To assess innovations in the X. laevis subgenomes we examined p300 (Ep300)-bound enhancer peaks that are unique to one subgenome and absent from X. tropicalis. A large majority of new enhancers are comprised of transposable elements. Finally, to dissect early and late events following interspecific hybridization, we examined the epigenome and the enhancer landscape in X. tropicalis × X. laevis hybrid embryos. Strikingly, young X. tropicalis DNA transposons are derepressed and recruit p300 in hybrid embryos.ConclusionsThe results show that erosion of X. laevis genes and functional regulatory elements is associated with repeats and NAHR, and furthermore that young repeats have also contributed to the p300-bound regulatory landscape following hybridization and whole genome duplication.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens. This manuscript describes the "Drosophila Island Assay" and "Island Assay Analysis" algorithms for high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The "Drosophila Island Assay" algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The "Island Assay Analysis" script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

Fibromyalgia is a chronic condition characterized by widespread pain, fatigue, non-restorative sleep and cognitive difficulties that affects 2–4% of the general population. Recently a possible relationship between the FMR1 premutation and fibromyalgia has been pointed out. In attempt to gather more data we screened for the FMR1 CGG expansion 700 DNA samples from unrelated fibromyalgia patients. This data might be useful for evaluating the incorporation of this test in rheumatologic procedures for women with fibromyalgia. The observed frequency of FMR1 premutation carriers (3 of 700, 0.4%) is not significantly different from the estimated rate in the general female population (1/250–1/400) (P = 0.539, P = 0.716). Clinical examination of the FMR1 premutation carriers identified revealed that all of them had important neurological symptoms with regard to muscular symptoms, neurocognitive alterations and neurovegetative impairments. With regard to other clinical aspects of the disease the cases apparently did not differ from the average fibromyalgia patients. On the basis of our results an FMR1 screening among fibromyalgia female patients would not be recommended. However it would be worthwhile to further evaluate the different clinical presentations that fibromyalgia patients might present based on their FMR1 premutation carrier status.

Item does not contain fulltext We review and document the evolutionary origin of all complex I assembly factors and nine supernumerary subunits from protein families. Based on experimental data and the conservation of critical residues we identify a spectrum of protein function conservation between the complex I representatives and their non-complex I homologs. This spectrum ranges from proteins that have retained their molecular function but in which the substrate specificity may have changed or have become more specific, like NDUFAF5, to proteins that have lost their original molecular function and critical catalytic residues like NDUFAF6. In between are proteins that have retained their molecular function, which however appears unrelated to complex I, like ACAD9, or proteins in which amino acids of the active site are conserved but for which no enzymatic activity has been reported, like NDUFA10. We interpret complex I evolution against the background of molecular evolution theory. Complex I supernumerary subunits and assembly factors appear to have been recruited from proteins that are mitochondrial and/or that are expressed when complex I is active. Within the evolution of complex I and its assembly there are many cases of neofunctionalization after gene duplication, like ACAD9 and TMEM126B, one case of subfunctionalization: ACPM1 and ACPM2 in Yarrowia lipolytica, and one case in which a complex I protein itself appears to have been the source of a new protein from another complex: NDUFS6 gave rise to cytochrome c oxidase subunit COX4/COX5b. Complex I and its assembly can therewith be regarded as a treasure trove for pathway evolution. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Contains fulltext : 174204.pdf (Publisher’s version ) (Closed access) Transposable elements are parasitic genomic elements that can be deleterious for host gene function and genome integrity. Heterochromatic histone modifications are involved in the repression of transposons. However, it remains unknown how these histone modifications mark different types of transposons during embryonic development. Here we document the variety of heterochromatic epigenetic signatures at parasitic elements during development in Xenopus tropicalis, using genome-wide ChIP-sequencing data and ChIP-qPCR analysis. We show that specific subsets of transposons in various families and subfamilies are marked by different combinations of the heterochromatic histone modifications H4K20me3, H3K9me2/3 and H3K27me3. Many DNA transposons are marked at the blastula stage already, whereas at retrotransposons the histone modifications generally accumulate at the gastrula stage or later. Furthermore, transposons marked by H3K9me3 and H4K20me3 are more prominent in gene deserts. Using intra-subfamily divergence as a proxy for age, we show that relatively young DNA transposons are preferentially marked by early embryonic H4K20me3 and H3K27me3. In contrast, relatively young retrotransposons are marked by increasing H3K9me3 and H4K20me3 during development, and are also linked to piRNA-sized small non-coding RNAs. Our results implicate distinct repression mechanisms that operate in a transposon-selective and developmental stage-specific fashion.

AimsThe causes of intellectual disability, which affects 1%–3% of the general population, are highly heterogeneous and the genetic defect remains unknown in around 40% of patients. The application of next-generation sequencing is changing the nature of biomedical diagnosis. This technology has quickly become the method of choice for searching for pathogenic mutations in rare uncharacterised genetic diseases.MethodsWhole-exome sequencing was applied to a series of families affected with intellectual disability in order to identify variants underlying disease phenotypes.ResultsWe present data of three families in which we identified the disease-causing mutations and which benefited from receiving a clinical diagnosis: Cornelia de Lange, Cohen syndrome and Dent-2 disease. The genetic heterogeneity and the variability in clinical presentation of these disorders could explain why these patients are difficult to diagnose.ConclusionsThe accessibility to next-generation sequencing allows clinicians to save much time and cost in identifying the aetiology of rare diseases. The presented cases are excellent examples that demonstrate the efficacy of next-generation sequencing in rare disease diagnosis.