Biology subjects

Fischer, G.

Publications and source records attributed to Fischer, G..

32 records · Page 2Linked to original sources

bioRxiv2023

Telomerase-independent survival leads to a mosaic of complex subtelomere rearrangements in Chlamydomonas reinhardtii

Telomeres and subtelomeres, the genomic regions located at chromosome extremities, are essential for genome stability in eukaryotes. In the absence of the canonical maintenance mechanism provided by telomerase, telomere shortening induces genome instability. The landscape of the ensuing genome rearrangements is not accessible by short-read sequencing. Here, we leverage Oxford Nanopore Technologies long-read sequencing to survey the extensive repertoire of genome rearrangements in telomerase mutants of the model green microalga Chlamydomonas reinhardtii. In telomerase mutant strains grown for [~]700 generations, most chromosome extremities were capped by short telomere sequences that were either recruited de novo from other loci or maintained in a telomerase-independent manner. Other extremities did not end with telomeres but only with repeated subtelomeric sequences. The subtelomeric elements, including rDNA, were massively rearranged and involved in breakage-fusion-bridge cycles, translocations, recombinations and chromosome circularization. These events were established progressively over time and displayed heterogeneity at the subpopulation level. New telomere-capped extremities composed of sequences originating from more internal genomic regions were associated with high DNA methylation, suggesting that de novo heterochromatin formation contributes to restore chromosome end stability in C. reinhardtii. The diversity of alternative strategies to maintain chromosome integrity and the variety of rearrangements found in telomerase mutants are remarkable and illustrate genome plasticity at short timescales.

genomics↗

bioRxiv2022

142 telomere-to-telomere assemblies reveal the genome structural landscape in Saccharomyces cerevisiae

As population genomics is transitioning from single reference genomes to pangenomes, major improvements in terms of genome contiguity, phylogenetic sampling, haplotype phasing and structural variant (SV) calling are required. Here, we generated the Saccharomyces cerevisiae Reference Assembly Panel (ScRAP) comprising 142 reference-quality genomes from strains of various geographic and ecological origins that faithfully represent the genomic diversity and complexity of the species. The ca. 4,800 non-redundant SVs we identified impact the expression of genes near the breakpoints and contribute to gene repertoire evolution through disruptions, duplications, fusions and horizontal transfers. We discovered frequent cases of complex aneuploidies, preferentially involving large chromosomes that underwent large SVs. We also characterized the evolutionary dynamics of complex genomic regions that classically remain unassembled in short read-based projects, including the 5 Ty families and the 32 individual telomeres. Overall, the ScRAP represents a crucial step towards establishing a high-quality, unified and complete S. cerevisiae pangenome.

genomics↗

bioRxiv2021

Spectral Separation of Evoked and Background Cortical Activity - Application to Short Latency Median Nerve Evoked Potentials

BackgroundSpectral analysis of repeatedly evoked potentials (EPs) is challenging since recordings contain a superposition of evoked signals and background activity. We developed a novel approach, N-interval Fourier Transform Analysis (N-FTA), which allows for reliable separation and accurate assessment of evoked and background spectral components. We applied this approach to spectral analysis of median nerve short latency EPs for identifying spectral bands of clinical relevance. MethodsWe performed right median nerve stimulation in two volunteers at 2.46 Hz and 3.95 Hz stimulation rate (600 and 1000 repetitions respectively). We applied N-FTA for splitting the periodically repeated evoked components from irregular background activity and investigated spectral components in the low, medium and high frequency (LF-, MF-, HF-) bands. We present a signal processing approach, which allows for accurately extracting diagnostically relevant features (signal morphology, latency and amplitude) using an 18 Hz to 240 Hz bandwidth. ResultsIn the LF-band < 10 Hz, evoked-to-background ratio (EBR) was below -15 dB due to the high level near DC background activity (1/f-activity, eye blinks,{theta} -band). Highest EBR was near -5 dB and obtained at a few tens of Hz. Relatively broad continuous segments of evoked activity were detected in the MF band. These frequencies of a few hundred Hz were linked to the signal segment between the N20 and P25 peaks in the time domain. High frequency oscillations (HFOs) near 600 Hz were approximately -25 dB below the background level and near the limit of the sensitivity of N-FTA. By subtracting stimulation artifacts and applying zero-phase filters it was possible to extract diagnostically relevant short latency EP features (signal morphology, latency and amplitude) only from the MF-band with a similar accuracy as a routinely used broad-band setting. HFOs displayed amplitudes of a few tenths of {micro}V. SummaryN-FTA allows for accurate, simultaneous spectral analysis of evoked and background activity in individual trials. The approach allowed for identifying target frequency bands of high evoked activity and for tailoring signal processing such, that morphology and latency can be obtained at a significantly reduced bandwidth. This should allow for development of more robust and faster recording techniques in the near future.

neuroscience↗

bioRxiv2021

Consensus tandem tetratricopeptide repeat proteins are complex superhelical nanosprings

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs) -- superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between folded and unfolded conformations. We rationalise the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the centre towards both termini simultaneously. Strikingly, we also directly observe the proteins superhelical tertiary structure in the force signal. Using protein engineering, crystallography and single-molecule experiments, we show how the superhelical geometry can be altered by carefully placed amino-acid substitutions and examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications. Significance statementRepetition of biological building blocks is crucial to modulating and diversifying structure and function of biomolecules across all organisms. In tandem-repeat proteins, the linear arrangement of small structural motifs leads to the formation of striking supramolecular shapes. Using a combination of single-molecule biophysical techniques and modelling approaches, we dissect the spring-like nature of a designed repeat protein and demonstrate how its shape and mechanics can be manipulated by design. These novel insights into the biomechanical and biochemical characteristics of this protein class give us a methodological basis from which to understand the biological functions of repeat proteins and to exploit them in nanotechnology and biomedicine.

biophysics↗

bioRxiv2020

SARS-CoV-2 escapes CD8 T cell surveillance via mutations in MHC-I restricted epitopes

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control, though direct evidence has been lacking so far. Here, we identified non-synonymous mutations in MHC-I restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV- 2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding, which was associated with a loss of recognition and functional responses by CD8+ T cells isolated from HLA-matched COVID-19 patients. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through escape mutations in MHCI-restricted viral epitopes. This provides evolutionary evidence for CD8+ T cell immunity controlling SARS-CoV-2 with consequences for COVID-19 vaccine design.

immunology↗

bioRxiv2020

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily-conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted -methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionising radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

cancer biology↗

bioRxiv2019

Phylogenetic reconstruction based on synteny block and gene adjacencies

Gene order can be used as an informative character to reconstruct phylogenetic relationships-between species independently from the local information present in gene/protein sequences. PhyChro is a reconstruction method based on chromosomal rearrangements, applicable to a wide range of eukaryotic genomes with different gene contents and levels of synteny conservation. For each synteny breakpoint issued from pairwise genome comparisons, the algorithm defines two disjoint sets of genomes, named partial splits, respectively supporting the two block adjacencies defining the breakpoint. Considering all partial splits issued from all pairwise comparisons, a distance between two genomes is computed from the number of partial splits separating them. Tree reconstruction is achieved through a bottom-up approach by iteratively grouping sister genomes minimizing genome distances. PhyChro estimates branch lengths based on the number of synteny breakpoints and provides confidence scores for the branches. PhyChro performance isevaluatedon two datasets of 13 vertebrates and 21 yeast genomes by using up to 130 000 and 179 000 breakpoints respectively, a scale of genomic markers that has been out of reach until now. PhyChro reconstructs very accurate tree topologies even at known problematic branching positions. Its robustness has been benchmarked for different synteny block reconstruction methods. On simulated data PhyChro reconstructs phylogenies perfectly in almost all cases, and shows the highest accuracy compared to other existing tools. PhyChro is very fast, reconstructing the vertebrate and yeast phylogenies in less than 15 min. AvailabilityPhyChro will be freely available under the BSD license after publication Contactalessandra.carbone@lip6.fr

bioinformatics↗

bioRxiv2019

Macroevolutionary integration of phenotypes within and across ant worker castes

Phenotypic traits are often integrated into evolutionary modules: sets of organismal parts that evolve together. In social insect colonies the concepts of integration and modularity apply to sets of traits both within and among functionally and phenotypically differentiated castes. On macroevolutionary timescales, patterns of integration and modularity within and across castes can be clues to the selective and ecological factors shaping their evolution and diversification. We develop a set of hypotheses describing contrasting patterns of worker integration and apply this framework in a broad (246 species) comparative analysis of major and minor worker evolution in the hyperdiverse ant genus Pheidole. Using geometric morphometrics in a phylogenetic framework, we inferred fast and tightly integrated evolution of mesosoma shape between major and minor workers, but slower and more independent evolution of head shape between the two worker castes. Thus, Pheidole workers are evolving as a mixture of intra- and inter-caste integration and rate heterogeneity. The decoupling of homologous traits across worker castes may represent an important process facilitating the rise of social complexity.

evolutionary biology↗

bioRxiv2018

Reshuffling yeast chromosomes with CRISPR/Cas9

Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes including in some instances large segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations display large phenotypic diversity in a wide range of environmental conditions. No coding sequence or promoter region was altered by the multiple translocations showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.\n\nAUTHOR SUMMARYChromosomes are highly dynamic objects that often undergo large structural variations such as reciprocal translocations. Such rearrangements can have dramatic functional consequences, as they can disrupt genes, change their regulation or create novel fusion genes at their breakpoints. For instance, 90-95% of patients diagnosed with chronic myeloid leukemia carry the Philadelphia chromosome characterized by a reciprocal translocation between chromosomes 9 and 22. In addition, translocations reorganize the genetic information along chromosomes, which in turn can modify the 3D architecture of the genome and potentially affect its functioning. Quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. Here, we report a novel CRISPR/Cas9-based technology allowing to generate with high efficiency and at a base-pair precision either uniquely targeted or multiple reciprocal translocations in yeast, without leaving any marker or scar in the genome. Engineering targeted reciprocal translocations allowed us for the first time to untangle the phenotypic impacts of large chromosomal rearrangements from that of point mutations. In addition, the generation of multiple translocations led to a large reorganization of the genetic information along the chromosomes. Although no gene was disrupted, we showed that solely shuffling the genome resulted in the emergence of fitness advantage in stressful environmental conditions.

genomics↗

bioRxiv2018

Evolution of the latitudinal diversity gradient in the hyperdiverse ant genus Pheidole

AimThe latitudinal diversity gradient is the dominant pattern of life on Earth, but a consensus understanding of its origins has remained elusive. The analysis of recently diverged, hyper-rich invertebrate groups provides an opportunity to investigate latitudinal patterns with the statistical power of large trees while minimizing potentially confounding variation in ecology and history. Here, we synthesize global phylogenetic and macroecological data on a hyperdiverse (>1100 species) ant radiation, Pheidole, and evaluate the roles of three general explanations for the latitudinal gradient: variation in diversification rate, tropical conservatism, and ecological regulation.\n\nLocationGlobal.\n\nTime PeriodThe past 35 million years.\n\nMajor taxa studiedThe hyperdiverse ant genus Pheidole Westwood.\n\nMethodsWe assembled geographic data for 1499 species and morphospecies, and inferred a dated phylogeny of Pheidole of 449 species, including 150 species newly sequenced for this study. We tested correlations between diversification rate and latitude with BAMM, HiSSE, GeoSSE, and FiSSE, and examined patterns of diversification as Pheidole spread around the globe.\n\nResultsWe found that Pheidole diversification occurred in series of bursts when new continents were colonized, followed by a slowdown in each region. There was no evidence of systematic variation of net diversification rates with latitude across any of the methods. Additionally, we found latitudinal affinity is moderately conserved with a Neotropical ancestor and phylogenetic inertia alone is sufficient to produce the gradient pattern.\n\nMain ConclusionsOverall our results are consistent with tropical conservatism explaining the diversity gradient, while providing no evidence that diversification rate varies systematically with latitude. There is evidence of ecological regulation on continental scales through the pattern of diversification after colonization. These results shed light on the mechanisms underlying the diversity gradient, while contributing toward a much-needed invertebrate perspective on global biodiversity dynamics.

evolutionary biology↗

bioRxiv2017

The evolution of the temporal program of genome replication

Comparative analyses of temporal programs of genome replication revealed either a nearly complete conservation between closely related species or a comprehensive reprogramming between distantly related species. Therefore, many important questions on the evolutionary remodeling of replication timing programs remain unanswered. To address this issue, we generated genome-wide replication timing profiles for ten yeast species from the genus Lachancea, covering a continuous evolutionary range from closely related to more divergent species. The comparative analysis of these profiles revealed that the replication program linearly evolves with increasing evolutionary divergence between these species. We found that the evolution of the timing program mainly results from a high evolutionary turnover rate of the cohort of active replication origins. We detected about one thousand evolutionary events of losses of active replication origins and gains of newborn origins since the species diverged from their last common ancestor about 80 million years ago. We show that the relocation of active replication origins is independent from synteny breakpoints, suggesting that chromosome rearrangements did not drive the evolution of the replication programs. Rather, origin gains and losses are linked both in space, along chromosomes, and in time, along the same branches of the phylogenetic tree. New origins continuously arise with on average low to medium firing efficiencies and increase in efficiency and earliness as they evolutionarily age. Yet, a subset of newborn origins emerges with high firing efficiency and origin losses occur concomitantly to their emergence and preferentially in their direct chromosomal vicinity. These key findings on the evolutionary birth, death and conservation of active replication origins provide the first description of how the temporal program of genome replication has evolved in eukaryotes.

evolutionary biology↗

bioRxiv2017

Redesigning chromosomes to optimize conformation capture (Hi-C) assays

In all chromosome conformation capture based experiments the accuracy with which contacts are detected varies considerably because of the uneven distribution of restriction sites along genomes. In addition, repeated sequences as well as homologous, large identical regions remain invisible to the assay because of the ambiguities they introduce during the alignment of the sequencing reads along the genome. As a result, the investigation of homologs during meiosis prophase through 3C studies has been limited. Here, we redesigned and reassembled in yeast a 145kb region with regularly spaced restriction sites for various enzymes. Thanks to this Syn-3C design, we enhanced the signal to noise ratio and improved the visibility of the entire region. We also improved our understanding of Hi-C data and definition of resolution. The redesigned sequence is now distinguishable from its native homologous counterpart in an isogenic diploid strain. As a proof of principle, we track the establishment of homolog pairing during meiotic prophase in a synchronized population. This provides new insights on the individualization and pairing of homologs, as well as on their internal restructuration into arrays of loops during meiosis prophase. Overall, we show the interest of redesigned genomic regions to explore complex biological questions otherwise difficult to address.

synthetic biology↗

bioRxiv2017

Structure of the human pro-myostatin precursor and determinants of growth factor latency

Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have solved the structure of unprocessed promyostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated step-wise activation process, distinct from the prototypical pro-TGF-{beta}1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors.

biochemistry↗

bioRxiv2017

A Molecular Portrait Of De Novo Genes

New genes, with novel protein functions, can evolve \"from scratch\" out of intergenic sequences. These de novo genes can integrate the cells genetic network and drive important phenotypic innovations. Therefore, identifying de novo genes and understanding how the transition from noncoding to coding occurs are key problems in evolutionary biology. However, identifying de novo genes is a difficult task, hampered by the presence of remote homologs, fast evolving sequences and erroneously annotated protein coding genes. To overcome these limitations, we developed a procedure that handles the usual pitfalls in de novo gene identification and predicted the emergence of 703 de novo genes in 15 yeast species from two genera whose phylogeny spans at least 100 million years of evolution. We established that de novo gene origination is a widespread phenomenon in yeasts, only a few being ultimately maintained by selection. We validated 82 candidates, by providing new translation evidence for 25 of them through mass spectrometry experiments. We also unambiguously identified the mutations that enabled the transition from non-coding to coding for 30 Saccharomyces de novo genes. We found that de novo genes preferentially emerge next to divergent promoters in GC-rich intergenic regions where the probability of finding a fortuitous and transcribed ORF is the highest. We found a more than 3-fold enrichment of de novo genes at recombination hot spots, which are GC-rich and nucleosome-free regions, suggesting that meiotic recombination would be a major driving force of de novo gene emergence in yeasts.

evolutionary biology↗