Mechanisms and Evolutionary Patterns of Mammalian and Avian Dosage Compensation

A large-scale comparative gene expression study reveals the different ways in which the chromosome-wide gene dosage reductions resulting from sex chromosome differentiation events were compensated during mammalian and avian evolution

Large scale assessment of regulatory evolution and transcriptome complexity in mammals

AbstractIn addition to genetic changes affecting the function of gene products, changes in gene expression have been suggested to underlie many or even most of the phenotypic differences among mammals. However, detailed gene expression comparisons were, until recently, restricted to closely related species, owing to technological limitations. Thus, we took advantage of the latest technologies (RNA-Seq) to generate extensive qualitative and quantitative transcriptome data for a unique collection of somatic and germline tissues from representatives of all major mammalian lineages (placental mammals, marsupials and monotremes) and birds, the evolutionary outgroup.In the first major project of my thesis, we performed global comparative analyses of gene expression levels based on these data. Our analyses provided fundamental insights into the dynamics of transcriptome change during mammalian evolution (e.g., the rate of expression change across species, tissues and chromosomes) and allowed the exploration of the functional relevance and phenotypic implications of transcription changes at a genome-wide scale (e.g., we identified numerous potentially selectively driven expression switches).In a second project of my thesis, which was also based on the unique transcriptome data generated in the context of the first project we focused on the evolution of alternative splicing in mammals. Alternative splicing contributes to transcriptome complexity by generating several transcript isoforms from a single gene, which can, thus, perform various functions. To complete the global comparative analysis of gene expression changes, we explored patterns of alternative splicing evolution. This work uncovered several general and unexpected patterns of alternative splicing evolution (e.g., we found that alternative splicing evolves extremely rapidly) as well as a large number of conserved alternative isoforms that may be crucial for the functioning of mammalian organs.Finally, the third and final project of my PhD consisted in analyzing in detail the unique functional and evolutionary properties of the testis by exploring the extent of its transcriptome complexity. This organ was previously shown to evolve rapidly both at the phenotypic and molecular level, apparently because of the specific pressures that act on this organ and are associated with its reproductive function. Moreover, my analyses of the amniote tissue transcriptome data described above, revealed strikingly widespread transcriptional activity of both functional and nonfunctional genomic elements in the testis compared to the other organs. To elucidate the cellular source and mechanisms underlying this promiscuous transcription in the testis, we generated deep coverage RNA-Seq data for all major testis cell types as well as epigenetic data (DNA and histone methylation) using the mouse as model system. The integration of these complete dataset revealed that meiotic and especially post-meiotic germ cells are the major contributors to the widespread functional and nonfunctional transcriptome complexity of the testis, and that this "promiscuous" spermatogenic transcription is resulting, at least partially, from an overall transcriptionally permissive chromatin state. We hypothesize that this particular open state of the chromatin results from the extensive chromatin remodeling that occurs during spermatogenesis which ultimately leads to the replacement of histones by protamines in the mature spermatozoa. Our results have important functional and evolutionary implications (e.g., regarding new gene birth and testicular gene expression evolution).Generally, these three large-scale projects of my thesis provide complete and massive datasets that constitute valuables resources for further functional and evolutionary analyses of mammalian genomes

Étude exploration des fonctions cognitives chez des individus de 21 à 40 ans ayant l'ARSCS

L’Ataxie récessive spastique de Charlevoix-Saguenay (ARSCS) est une maladie héréditaire présente dans plusieurs pays (Masciullo et al., 2012; Pedroso et al., 2011; Pimenta et al., 2017; Anheim et al., 2008), mais présentant une prévalence plus élevée au Québec (Bouchard, Barbeau, Bouchard, & Bouchard, 1978 ; Bouchard, Bouchard, Bouchard & Barbeau., 1979 ; Giasson, 1992). Cette ataxie cérébelleuse est dégénérative et se manifeste par des atteintes pyramidales, cérébelleuses et neuropathiques (Bouchard et al., 1978). L’implication du cervelet dans les fonctions cognitives, notamment les fonctions exécutives, langagières et visuospatiales, est de plus en plus documentée (Schmahmann & Sherman, 1998; De Smet, Paquier, Verhoeven, & Mariën, 2013). Étant donné l’atrophie du vermis cérébelleux supérieur observée chez les individus ayant l’ARSCS (Gazulla et al., 2011; Martin, Sylvain, Bouchard, St-Onge, & Truchon, 2007; Pedroso et al., 2011; Prodi et al., 2013; Shimazaki, Sakoe, Niijima, Nakano, & Takiyama, 2007; Synofzik et al., 2013), il est important d’investiguer les fonctions cognitives. Peu d’études ont été faites à ce niveau chez des adultes ayant l’ARSCS (Bouchard et al., 1978; Boucher, 2017; Brassard, 2020; Verhoeven et al., 2012) et leurs résultats tendent à démontrer une vulnérabilité cognitive sans, toutefois, présenter de portrait homogène. Cette étude vise donc l’exploration des portraits cognitifs de quatre adultes, âgés entre 21 et 40 ans, ayant l’ARSCS. Afin d’avoir des portraits complets, plusieurs instruments neuropsychologiques ont été utilisés. Le fonctionnement intellectuel, les habiletés visuoperceptuelles, ainsi que les fonctions mnésiques, attentionnelles et langagières ont été ciblés pour l’évaluation. L’étude de cas multiples a permis de dresser un portrait cognitif des quatre participants. Ces résultats ont été comparés entre eux de façon à faire ressortir les similarités. Une grande variabilité interpersonnelle a été observée dans les résultats. Néanmoins, les résultats démontrent deux fonctions présentant une altération pour l’ensemble des participants, soit la mémoire de travail et la vitesse du traitement de l’information. Les résultats de cette étude suggèrent, aux cliniciens qui travaillent avec cette clientèle, de porter une attention particulière à ces fonctions cognitives, afin d’offrir des interventions plus adaptées en cas de problématiques

Birth and expression evolution of mammalian microRNA genes.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression, yet their origins and functional evolution in mammals remain little understood due to the lack of appropriate comparative data. Using RNA sequencing, we have generated extensive and comparable miRNA data for five organs in six species that represent all main mammalian lineages and birds (the evolutionary outgroup) with the aim to unravel the evolution of mammalian miRNAs. Our analyses reveal an overall expansion of miRNA repertoires in mammals, with threefold accelerated birth rates of miRNA families in placentals and marsupials, facilitated by the de novo emergence of miRNAs in host gene introns. Generally, our analyses suggest a high rate of miRNA family turnover in mammals with many newly emerged miRNA families being lost soon after their formation. Selectively preserved mammalian miRNA families gradually evolved higher expression levels, as well as altered mature sequences and target gene repertoires, and were apparently mainly recruited to exert regulatory functions in nervous tissues. However, miRNAs that originated on the X chromosome evolved high expression levels and potentially diverse functions during spermatogenesis, including meiosis, through selectively driven duplication-divergence processes. Overall, our study thus provides detailed insights into the birth and evolution of mammalian miRNA genes and the associated selective forces

Light-activated cell identification and sorting (LACIS) for selection of edited clones on a nanofluidic device.

Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4-targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, individual colonies are identified based on their specific genotype. Each colony is split and sequentially exported for on-target sequencing and further off-chip clonal expansion of the validated clones. Using this method, single-clone editing efficiencies, including the rate of mono- and bi-allelic indels or precise nucleotide replacements, can be assessed within 10 days from Cas9 ribonucleoprotein introduction in cells

A Comparison of mRNA Sequencing with Random Primed and 3'-Directed Libraries.

Creating a cDNA library for deep mRNA sequencing (mRNAseq) is generally done by random priming, creating multiple sequencing fragments along each transcript. A 3'-end-focused library approach cannot detect differential splicing, but has potentially higher throughput at a lower cost, along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) that correct PCR bias. Here, we compare an implementation of such a 3'-digital gene expression (3'-DGE) approach with "conventional" random primed mRNAseq. Given our particular datasets on cultured human cardiomyocyte cell lines, we find that, while conventional mRNAseq detects ~15% more genes and needs ~500,000 fewer reads per sample for equivalent statistical power, the resulting differentially expressed genes, biological conclusions, and gene signatures are highly concordant between two techniques. We also find good quantitative agreement at the level of individual genes between two techniques for both read counts and fold changes between given conditions. We conclude that, for high-throughput applications, the potential cost savings associated with 3'-DGE approach are likely a reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable many larger scale studies focusing on not only differential expression analysis, but also quantitative transcriptome profiling