Auswahl der wissenschaftlichen Literatur zum Thema „Chromosome de levure“

Depuis les dix dernières années, les techniques de synthèse et d’assemblage d’ADN se sont grandement améliorées. La construction de molécules d’ADN synthétiques devient maintenant beaucoup plus simple et abordable de sorte qu’il est possible de reconstruire des chromosomes synthétiques complets. Nous assistons donc aux débuts de la génomique synthétique, qui vise la construction de génomes conçus sur mesure pour l’étude et l’utilisation de systèmes biologiques. De la synthèse des premiers génomes viraux jusqu’à la reconstruction des seize chromosomes de la levure, en passant par la première cellule bactérienne contrôlée par un génome entièrement synthétique, nous discutons des découvertes majeures, des aspects réglementaires et éthiques ainsi que du potentiel de cette nouvelle discipline pour le futur.

Les banques de grands fragments d’ADN constituent depuis une quinzaine d’années une avancée technologique remarquable pour l’étude des génomes complexes. En effet, chaque fragment cloné, jusqu’à 2 mégabases dans des chromosomes artificiels de levures (YAC) et 200 kilobases dans des chromosomes artificiels de bactéries (BAC), représente une petite région chromosomique qui contient un ou plusieurs gènes avec des éléments de régulation proximaux et distaux, ainsi que des marqueurs associés. Des collections de clones représentatives d’un génome entier ont été produites. Ces banques de grands fragments d’ADN sont intensivement utilisées pour la cartographie intégrée des génomes et sont les éléments fondateurs des programmes de grand séquençage, dont celui de l’Homme. Les clones de grands fragments d’ADN constituent, de plus, un matériel puissant pour élaborer les outils et concepts de la génomique fonctionnelle de demain.

Le développement de la génomique synthétique (GS) a permis l’élaboration d’outils et de méthodes innovantes permettant la synthèse, l’assemblage et la modification génétique précise de chromosomes bactériens complets. La raison principale de ce succès, ayant abouti à la création de la première cellule synthétique quasi-minimale JCVI-syn3.0, est l’utilisation de la levure Saccharomyces cerevisiae comme hôte temporaire d’accueil et de modification de ces génomes. Cependant, une autre technique a joué un rôle considérable dans le succès retentissant de ces travaux : la transplantation de génomes bactériens (TG). Cette technique, encore mal comprise, permet d’installer des génomes complets naturels ou synthétiques dans un contexte cellulaire favorable à leur expression et donner la vie. Une meilleure compréhension du processus de TG permettrait d’élargir l’ensemble des techniques de GS, appliquées actuellement quasi exclusivement à l’étude des mycoplasmes, à de nombreuses autres bactéries d’intérêt, y compris des bactéries génétiquement non-modifiables à ce jour.

Abstract Abstract 4798 Background: Acute monocytic leukemia (M5) is one kind of acute leukemia with poor prognosis, which has the characterization of high number of white blood cells, high risk of extramedullary metastasis and abnormal chromosome karyotype. Objective: To analyze clinical characterization and prognostic factors of adult acute monocytic leukemia. Methods: Clinical data of 144 patients diagnosed between Jan 1st 2006 and Dec 31 2010 were collected. Clinical manifestation, cytogenetics, immunophenotyping and minimal residual disease (MRD) were analyzed respectively. The numeration data was tested by χ2 test, while the measurement data by Levene test to variance homogeneity and by T test to the mean difference. The data were analyzed by SPSS 17.0. Results: 81.9% of 144 patients reached complete remission. 27.5% patients were abnormal in cytogenetics, including +8, −5/del (5q), −7/del (7q). Patients were divided into three groups according to chromosome karyotype. Intermediate prognostic group has better OS and DFS than poor prognostic group (P<0.05). 26 patients treated with SCT.These patients have higher OS rate for one year and low relapse rate than those who did not receive SCT.CR rate for old patients(≥55) and young patients (<55) were 69.4% and 86.1% respectively. Old patients had shorter OS and DFS than young patients, but no singnificant difference in FAB type, number of white blood cell and immunophenotyping. Old patients occur chromosome abnormalities more frequently than young patients. Relapse rate is 20.7% in MRD+ group versus 11.5% in MRD- group. Multivariate COX analysis showed that chromosome karyotype and MRD had most significant correlation with relapse. Conclusion: Chromosome karyotype, age, therapy, MRD were all prognostic factors of M5. Chromosome karyotype and MRD had most important significance in prognosis. Disclosures: No relevant conflicts of interest to declare.

Abstract Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones. In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and >60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance. 893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects’ CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause. We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms’ tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs. Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures: No relevant conflicts of interest to declare.

Tetrallogy of Fallot (ToF) is the most widespread congenital heart defect characterized by a wide anatomic spectrum and clinical manifestations, which depend on the degree of stenosis of the pulmonary artery, and can be associated with chromosomal abnormalities. Hypertrophic cardiomyopathy (HCM) is a rare genetic disorder that often occurs in the autosomal-dominant type and has a high risk of cardiac death and is associated with abnormalities in certain gene loci. Clinical case. We present this rare association in a 9-month-old girl, without previous history of heart defect, who was admitted to intensive care unit with a clinical presentation of severe hypoxic spell. A rare case of the combination of ToF and HCM has been reported in a 9-month-old child admitted to the reanimation department of the Lviv Regional Children’s Clinical Hospital «OKHMATDYT» for malignant and cyanotic attacks. The girl was hospitalized for reanimation in a jaundice-cyanotic crisis with a saturation rate of 44%. On examination, a pronounced sciatica, pallor and cyanosis of the lips were detected. Auscultatively, the heart tones were rhythmic, muffled, the heart rate - 202 beats per second, systolic murmur was detected on the left side of the chest 5/6 on the Levine scale. Electrocardiogram showed signs of systolic hypertension and hypertrophy of the right ventricle. 2D-echocardiographic examination revealed hypertrophy of the left and right ventricular walls and interventricular septum as well as signs of ToF. On the magnetic resonance imaging the diagnosis was confirmed. After the stabilization of the general condition the child was transported to the Center of Pediatric Cardiology and Cardiac Surgery in Kyiv for surgical treatment, where it was recommended to continue the medical treatment with beta-blockers. After 2 months, the 1-year-old child was operated on routinely - radical correction of ToF, the post-operative state was good. Conclusions. Association of ToF and HCM is extremely rare. ToF is often associated with chromosomal aberration, while hypertrophic cardiomyopathy associates with certain gene loci. Surgical treatment of ToF associated with HCM differs greatly from surgical treatment of usual ToF and physiology of both conditions have to be considered prior to the surgery, as combination of ToF and HCM is associated with high postoperative mortality, as the LVOT progresses and increased risk of development of ventricular arrhythmias and heart failure develops. The association between ToF and HCM is extremely rare. ToF is associated with chromosomal abnormalities, whereas HCM is associated with abnormalities in certain gene loci. The prognosis for patients with TF and HCM is associated with high postoperative mortality due to progression of obstruction of the left ventricular tract, development of ventricular arrhythmias and cardiovascular failure during the postoperative period. The research was carried out in accordance with the principles of the Helsinki Declaration. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors. Key words: Tetrallogy of Fallot, hypertrophic cardiomyopathy, cyanotic spell, echocardiography, children.

Abstract Chromosome 8q24 is of particular importance for cancer susceptibility. Located in this region is the Plasmacytoma Variant Translocation 1 (PVT1) gene, a long noncoding RNA that has been implicated in multiple cancers including prostate cancer. Amplification of the PVT1 gene locus is a common event in many malignant diseases and is associated with poor clinical outcomes. The pioneering role of PVT1, and its alternatively spliced transcripts, as a cancer biomarker is progressively becoming established. We have demonstrated that copy numbers of PVT1 exons 4A, 4B and 9 is quantifiable in cancer cells, tissue, and serum from cancer patients. In this study, we assessed clinically annotated serum samples from 40 prostate cancer patients to investigate the clinical relevance of PVT1 exons 4A, 4B, and 9 as a biomarker across cancer risk levels and ethnicity/race. Explorative data analysis for the development of composite score for prostate cancer was performed using Kruskal-Wallis Rank Sum Test. We observed significantly higher copy numbers of PVT1 exons 4B and 9 across all races (White, Black and Hispanic) and Blacks and Hispanics when compared to the control. Additionally, using a 3-level cancer risk rating assessment in which 0 = healthy, 1 = low risk and 2 = high risk, we observed that PVT1 exon 9 may distinguish between cancerous and noncancerous cases across all races, but may not help distinguish between indolent and aggressive cancer cases. Notably, PVT1 exon 4B may help distinguish between indolent and aggressive cancer cases for Blacks and Hispanics. The results of this study suggest that using PVT1 exon 4B or 9 may identify cancer regardless of ethnicity/race, and that utilization of serum PVT1 exon 4B copy number may help distinguish between indolent and aggressive prostate cancer in Blacks and Hispanics. Citation Format: Fayola Levine, Emmanuel Asante-Asamani, Gargi Pal, Michael Liss, Olorunseun Ogunwobi. Investigating the clinical relevance in prostate cancer of the serum biomarkers PVT1 exons 4A, 4B and 9 across risk levels and ethnicity/race [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C060.

Abstract Objective: Outcomes for patients with metastatic or relapsed osteosarcoma (OS) remain poor. Many OS samples have demonstrated a high proportion of mutational signature 3 (ms3), suggestive of homologous recombination DNA repair deficiency (HRD) or a BRCAness molecular phenotype. The HRD score, which quantifies chromosomal structural rearrangements by combining loss of heterozygosity (LOH), large state transitions (LST), and telomere allelic imbalances (TAI), has been used to assess HRD status in patients with breast and ovarian cancer, with high scores found to be predictive of PARP inhibitor sensitivity. While high HRD scores have been described in OS, little is known about how HRD scores vary by stage, site, pre vs post-therapy, presence of homologous recombination pathway or TP53 alterations, and their correlation to ms3 contribution. Methods: To address this issue, we evaluated 48 OS samples with tumor purity ≥ 20% from 46 patients who had 80x whole genome sequencing (WGS) performed through our pediatric expanded genomics platform. HRD scores were then estimated using scarHRD, a computational tool, which quantifies LOH, LST, and TAI events from WGS data and has shown a good correlation to SNP-array based methods. Results: Of the 48 samples analyzed, 25 samples were from primary sites and 23 from metastatic sites of disease. The median HRD score was 45 with a minimum score of 6 and a maximum score of 79. The 25th, 75th, and 90th percentiles of values were 35.7, 57, and 66.3 respectively. There was a significant difference in HRD score in patients with localized disease (n=22) vs metastatic disease (n=26) at time of diagnosis with median HRD (mHRD) scores of 41 and 51.5 (p = 0.027). The presence of a TP53 structural variant or single nucleotide variant (mHRD=47) was associated with higher HRD scores than TP53 wild-type samples (mHRD=29.5, p=0.04). There was no statistically significant difference in HRD scores in pre vs post-treated samples or primary site vs metastatic site samples. Samples with homologous recombination pathway alterations (n=10) were not associated with increased HRD score. There was a positive correlation (Spearman’s rho=0.425, p=0.001) between the number of mutations attributed to ms3 and HRD score. Conclusions: The range and distribution of HRD scores in our OS cohort closely resemble BRCA1/2 deficient breast cancer. Patients with metastatic disease at diagnosis were found to have higher HRD scores, representing a higher degree of genomic instability and increasing potential to accumulate resistance mutations. Higher HRD scores in TP53 mutated samples may be due to chromosomal instability caused by mechanisms other than HRD. The BRCAness phenotype in OS is now being targeted in a phase II clinical trial, combining the PARP inhibitor olaparib with the ATR inhibitor ceralasertib (NCT0441706), which includes integrated correlative biology to prospectively assess biomarkers of response such as HRD score. Citation Format: Michael David Kinnaman, Julia Pena, Max Levine, Nancy Bouvier, Elli Papaemmanuil, Suzanne Forrest, Katherine Janeway, Paul Meyers, Julia Glade Bender. Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2590.

Abstract Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability suggesting an alternate role in malignant transformation. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic stem/progenitor cells. We therefore investigated the impact of both complete loss and haploinsufficiency of Smc3, an obligate member of the cohesin complex, in normal hematopoiesis and in myeloid transformation by developing a conditional Smc3 knockout allele. Somatic loss of Smc3 in hematopoietic cells induced lethal bone marrow aplasia (median survival 11 days; p<0.001), with premature sister chromatid separation and abnormal nucleolar organization. Competitive transplant assays showed that Smc3 loss completely abrogated stem cell self-renewal in vivo. These data are consistent with an absolute requirement for the cohesin complex in hematopoietic stem/progenitor cells. By contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, with increased serial replating, expanded hematopoietic stem/progenitor cells, and a self-renewal/engraftment advantage in competitive transplantation assays in vivo (Figure a). Smc3 haploinsufficiency altered coordinated transcriptional output, including reduced expression of master regulatory transcription factors governing lineage commitment. Consistent with these data, Smc3 loss resulted in expanded Cd150+ Cd48+ ST-HSC (p=0.008), reduction in Cd150+ Cd48- LT-HSC (p=0.001), and altered chromatin architecture with dysregulated expression of genes with specific chromatin architecture footprints. Smc3 haploinsufficiency cooperated with Flt3ITD to induce acute leukemia in vivo (Figure b), with dysregulated expression of hematopoietic master regulators and altered nucleolar topology similar to that observed in germline cohesinopathy syndromes and in AML patients with cohesin mutations (Figure c). To further explore the mechanism by which Smc3 loss cooperates with Flt3ITD to induce leukemia, we investigated chromatin cis-regulatory architecture with transposase hypersensitivity assays (ATAC-seq). We hypothesized that increased accessibility at cis-regulatory elements and the alterations in gene expression seen in cells with combined Smc3 haploinsufficiency and Flt3ITD may be in a large part driven by potentiated Stat signaling at chromatin. We analyzed 146 transcription factor recognition motifs within the THS differentially observed in Smc3Δ/+Flt3ITD and wild-type cells. Chromatin accessibility gained in Smc3Δ/+Flt3ITD cells are enriched in Stat family transcription factor binding sites, including Stat5. We also observed enrichment of the Stat5 gene expression signature in the Smc3Δ/+Flt3ITD cells compared to Smc3Δ/+, Flt3ITD and wild-type cells, suggesting the divergent mutations cooperate to potentiate oncogenic Stat5 signaling in HSPCs. Our results demonstrate a key dose-dependent role for the cohesin complex in hematopoiesis, and show that reduced cohesin functions to alter enhancer-mediated transcription and contribute to aberrant self-renewal and myeloid transformation. Figure 1. Figure 1. Disclosures Levine: Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Les télomères sont les complexes nucléoprotéiques des extrémités des chromosomes linéaires. Ils ont notamment pour fonction de maintenir l'intégrité du génome en protégeant les extrémités chromosomiques contre les fusions. Chez la levure Saccharomyces cerevisiae, la protéine Rap1 est le facteur liant à haute densité les séquences télomériques. Dans cette étude, nous utilisons un nouvel allèle nul conditionnel de RAP1 et nous montrons que la perte de Rap1 provoque des fusions télomère-télomère, détectées par PCR. Un grand nombre de ces fusions a été cloné et le point de fusion a été analysé par restriction. Cette analyse montre que les fusions impliquent des télomères de taille sauvage. Ces fusions ne sont pas détectées dans les cellules déficientes pour l'un des facteurs du " Non-Homologous End-Joining " (NHEJ), incluant les protéines des complexes de la ligase IV (Lig4, Lif1, Lif2), KU (Yku70, Yku80) et Mre11 (Mre11, Rad50, Xrs2). Tous ces résultats tendent à démontrer que Rap1 est essentiel pour bloquer les événements de NHEJ entre télomères. Rap1 étant un facteur télomérique conservé à travers l'évolution, il est fort probable que le rôle de Rap1 dans la protection des télomères contre le NHEJ soit universel
Fusions between chromosomes would compromise genome integrity. In particular, Non-Homologous End Joining (NHEJ) must be excluded from telomeres. Rap1p is the prominent telomere binding-factor in Saccharomyces cerevisiae and homologues are found in human cells and Schyzosaccharomyces pombe. Instead of binding directly the telomeric repeats, spRap1 and hRap1 are recruited by the major telomere binding-factor in these organisms, Taz1 and TRF2, respectively. It has been demonstrated that Taz1 and TRF2 protect chromosomes against end fusions by NHEJ. We took advantage in this study of a new conditional allele of RAP1, rap1-(∆), to test a role for Rap1p in telomere protection. In this allele, Rap1p is lost when cells progress toward stationary phase. This loss correlated with the appearance of end-to-end fusions detected by PCR. Telomere fusions were cloned. The fusion point seems difficult to sequence. However, the presence of a restriction site at the junction of some cloned fusions allowed us to determine that fusions occurred between telomeres of near wild-type length. Furthermore, we observed that the sequence at the fusion point seems random. Telomere fusions were not observed in rap1-(∆) cells defective for each factor required for NHEJ in budding yeast: Lig4p, Lif1p, Lif2p, Yku70p, Yku80p, Mre11p, Rad50p, and Xrs2p. The NHEJ-DNA polymerase Pol4p is also required. Sae2p and Tel1p, two known regulators of the Mre11p-Rad50p-Xrs2p complex not required for NHEJ, did not seem to be involved in telomere fusions. Thus, Rap1p protects telomeres from NHEJ. This newly described role is likely to be conserved

Le projet international de synthèse des chromosomes de S. cerevisiae (projet Sc2.0) a débuté il y a une dizaine d'années en suivant des principes établis par le Pr. Jef Boeke. Les chromosomes synthétiques ont été conçus pour augmenter la stabilité du génome en supprimant toutes les séquences répétées (ARNt, éléments transposables...), tout en y ajoutant un système d'évolution inductible dépendant du système Cré/LoxP (système SCRaMbLE), permettant de générer rapidement des réarrangements chromosomiques. Bien que le design du projet Sc2.0 soit très conservateur en ce qui concerne le contenu des gènes, la suppression de plusieurs classes de séquences répétées peut affecter l'organisation du génome et potentiellement altérer les fonctions cellulaires. En utilisant la méthode de capture de conformation de chromosome couplée au séquençage de seconde génération (Hi-C), mon objectif a été de caractériser l'organisation 3D des génomes des souches synthétiques et évoluées. À ce jour, huit chromosomes (syn I, II, III, V, VI, IX-R, X et XII) ont été entièrement assemblés séparément. En utilisant les souches contenant un ou plusieurs de ces chromosomes, nous avons pu montrer que leur organisation génomique n'est globalement pas affectée par leur présence. Quelques exceptions subsistent, avec synIII dont les cassettes HML et HMR ont été retirées, et synXII d'où l'ADNr a été déplacé sur un autre chromosome. À ce stade, nous concluons que l'ADN répétitif dispersé ne conduit pas la conformation moyenne globale du génome de S. cerevisiae. Nous avons aussi exploité les cartes de contacts pour identifier les réarrangements dans les souches SCRaMbLE
The international project Sc2.0 started 10 years ago by the Pr. Jef Boeke aims to build a fully synthetic genome of S. cerevisiae which increases the genome stability by removing all repeated sequences (tRNA, transposable elements, etc.), and implements SCRaMbLE (for Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution), an inducible, high-throughput chromosome rearrangement system. This design is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes. However, it may affect genome organization and potentially alter cellular functions. To determine wether those modifications affected the three-dimensional conformation of synthetic chromosmes, we investigated it using chromosomes conformation capture coupled to second generation sequencing method (Hi-C). Currently, eight synthetic chromosomes (synI, synII, synIII, synV, synVI, synIX-R, synX et synXII) have been fully assembled. Using these strains we observed that the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploited the contact maps to detect rearrangements induced in these SCRaMbLE strains

Le projet international de synthèse des chromosomes de S. cerevisiae (projet Sc2.0) a débuté il y a une dizaine d'années en suivant des principes établis par le Pr. Jef Boeke. Les chromosomes synthétiques ont été conçus pour augmenter la stabilité du génome en supprimant toutes les séquences répétées (ARNt, éléments transposables...), tout en y ajoutant un système d'évolution inductible dépendant du système Cré/LoxP (système SCRaMbLE), permettant de générer rapidement des réarrangements chromosomiques. Bien que le design du projet Sc2.0 soit très conservateur en ce qui concerne le contenu des gènes, la suppression de plusieurs classes de séquences répétées peut affecter l'organisation du génome et potentiellement altérer les fonctions cellulaires. En utilisant la méthode de capture de conformation de chromosome couplée au séquençage de seconde génération (Hi-C), mon objectif a été de caractériser l'organisation 3D des génomes des souches synthétiques et évoluées. À ce jour, huit chromosomes (syn I, II, III, V, VI, IX-R, X et XII) ont été entièrement assemblés séparément. En utilisant les souches contenant un ou plusieurs de ces chromosomes, nous avons pu montrer que leur organisation génomique n'est globalement pas affectée par leur présence. Quelques exceptions subsistent, avec synIII dont les cassettes HML et HMR ont été retirées, et synXII d'où l'ADNr a été déplacé sur un autre chromosome. À ce stade, nous concluons que l'ADN répétitif dispersé ne conduit pas la conformation moyenne globale du génome de S. cerevisiae. Nous avons aussi exploité les cartes de contacts pour identifier les réarrangements dans les souches SCRaMbLE
The international project Sc2.0 started 10 years ago by the Pr. Jef Boeke aims to build a fully synthetic genome of S. cerevisiae which increases the genome stability by removing all repeated sequences (tRNA, transposable elements, etc.), and implements SCRaMbLE (for Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution), an inducible, high-throughput chromosome rearrangement system. This design is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes. However, it may affect genome organization and potentially alter cellular functions. To determine wether those modifications affected the three-dimensional conformation of synthetic chromosmes, we investigated it using chromosomes conformation capture coupled to second generation sequencing method (Hi-C). Currently, eight synthetic chromosomes (synI, synII, synIII, synV, synVI, synIX-R, synX et synXII) have been fully assembled. Using these strains we observed that the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploited the contact maps to detect rearrangements induced in these SCRaMbLE strains

La mitose est une étape clé du cycle cellulaire, très préservée chez toutes les cellules eucaryotes, durant laquelle le matériel génétique de la cellule (les chromosomes) réparti de manière égale dans les deux cellules filles. Cette équipartition du matériel génétique est cruciale pour le maintien de la stabilité génétique. Durant ce processus, les chromosomes, composés des chromatides soeurs, établissent une plaque métaphasique au centre du fuseau mitotique. Chaque chromatide est attachée à un pôle du fuseau mitotique respectif (on parle d'attachement bipolaire) vers lequel elle se dirigera durant l'anaphase. Les chromatides sont l'unité indivisible du matériel génétique durant la mitose, à l'image des atomes dans une molécule. Initialement, une fois la chromatine condensée en chromosomes, chacun de ces " objets " est détaché et réparti suivant une position précise appellée territoires chromosomiques. Toute la complexité de la mitose est de capturer chacune des chromatides et de les positionner sur la plaque métaphasique avant leur séparation et migration vers leur pôle respectif durant l'anaphase. Cette étape de la division cellulaire requiert donc non seulement un réseau complexe d'interaction et de signalisation biochimique comme dans beaucoup d'autres processus biologiques mais aussi un fin contrôle spatio-temporel du mouvement et du positionnement de ces objets de grande taille à l'échelle de la cellule. Il semblerait que l'origine du mouvement des chromosomes provienne pour une grande part de la dynamique des microtubules. Ce qui est moins certain est la part relative accordée aux différents processus régulant cette dynamique; que ce soit la dynamique intrinsèque (appelée instabilité dynamique des microtubules) ou l'effet de différentes protéines sur les microtubules comme les MAPs (Microtubule Associated Proteins) et les kinésines (protéines motrices). On notera par ailleurs que le mécanisme de transfert d'énergie entre la dynamique des microtubules et le mouvement des chromosomes est encore très largement hypothétique. La dynamique des chromosomes durant la mitose est aussi largement contrôlée par un grand nombre d'acteurs autres que les microtubules. Certains d'entre eux étant responsables de l'attachement MTs-kinétochore comme les complexes NDC80 et DAM1, tandis que d'autres sont impliqués dans la régulation de la dynamique des microtubules comme la kinésine-8 et la kinésine-13. Durant mon travail de thèse, j'ai étudié la dynamique des chromosomes en mitose chez la levure à fission, modèle celulaire dont les mécanismes primordiaux qui contrôlent la mitose sont conservés avec les eucaryotes supérieurs. En effet, j'ai caractérisé deux de ces mécanismes conservés au cours de l'évolution: l'alignement des chromosomes durant la métaphase ainsi qu'un mouvement de va et vient plus ou moins régulier le long du fuseau aussi appelé oscillation des chromosomes. J'ai montré, en analysant les trajectoires des chromosomes que ces deux processus sont pour une large part indépendants [@Mary2015]. De plus, le processus d'alignement des chromosomes, encore mal compris, est en partie contrôlé par la kinésine-8 via une activité dépendante de la longueur des microtubules. Il semblerait donc que cette kinésine soit capable de fournir une information spatiale le long du fuseau mitotique afin de positionner correctement les chromosomes. Enfin, j'ai utilisé un modèle mathématique de la ségrégation des chromosomes précédemment développé dans l'équipe afin de tester de manière quantitative les hypothèses de mécanisme du centrage des chromosomes par la kinésine-8. L'ensemble de mon travail porte donc sur le contrôle du mouvement, de l'attachement et du positionnement des chromosomes durant la mitose afin de mieux comprendre les processus biophysiques associés à la mitose
Mitosis is a highly preserved process in all eukaryotic cells during which the genetic material (chromosomes) is divided in two parts which spread in both daughter cells. This equipartition is crucial for maintaining genetic stability. During this process, chromosomes form a metaphasic plate at the center of the mitotic spindle. Each chromatid is attached to its respective spindle pole (called bipolar attachment) toward which it will move during anaphase. Chromatids are the indivisible units of genetic material during mitosis just like atoms in a molecule. Originally each of these "\ objects\ " are detached and organized in chromosomes territories. All the complexity of mitosis resides in the capture of each chromatid by the spindle pole to exert forces to position them on the metaphase plate before their separation and migration towards their respective poles in anaphase. This step of cell division not only requires complex interaction networks and metabolic signaling pathways just like many other biological processes but also a fine spatio-temporal control of movement and positioning of these big objects relative to cell size. It is usually accepted that the origin of chromosome movement arises from microtubule dynamics. However, what is less clear is the relative importance of each of these processes regulating chromosome movement: the intrinsic dynamic instability of microtubules or the effect of their associated proteins such as MAPs and kinesins. It is also important to note that the mechanism controlling the transfer of energy between microtubule dynamics and chromosome movement is still largely hypothetical. Moreover, chromosome dynamics during mitosis is regulated by a large number of actors apart from microtubules. Some of them being responsible for MT-kinetochore attachment such as NDC80 and DAM1 complex. While others are involved in the regulation of MT dynamics such as Kinesin-8 and Kinesin-13. During my PhD, I studied fission yest chromosome dynamic during mitosis. This cellular model has the advantage of sharing many fundamental mechanisms of symmetrically dividing higher eukaryotic cells. I characterized two of these conserved mechanisms: chromosome alignment during metaphase and back and forth movement along the spindle, called chromosome oscillation. By analyzing chromosome trajectories, I showed that both processes are performed through independent mechanisms [@Mary2015]. Moreover, chromosome alignment process, which is still poorly understood, is regulated by Kinesin-8 via a length dependent activity on microtubules. This suggests that Kinesin-8 is able to provide spatial information along the mitotic spindle to properly position chromosomes. Finally, I used a mathematical model of chromosome segregation in order to test quantitatively different hypotheses of chromosome centering process. This work is thus deciphering the control of movement, attachment and positioning of chromosomes during mitosis and seeks to better understand the biophysical processes controlling mitosis

Ce travail a ete effectue dans le cadre d'un programme de la communaute europeenne. Il comprend trois parties: 1) construction d'un contig du chromosome x de s. Cerevisiae, 2) sequencage du fragment de 41 kb, 3) analyse de la sequence de ce fragment. Un contig de 250 clones recombinants contenant l'adn du chromosome x a ete assemble en progressant a l'aide de ribosondes t3 et t7. Sa validite a ete confirmee par diverses approches. Une carte de restriction ecori de l'ensemble du contig a ete etablie. Un fragment de 41 kb a ete sequence en utilisant une methode aleatoire de fractionnement a l'aide d'un sequenceur abi 373a. 16 phases de lecture ouverte (plo) nouvelles ont ete identifiees. 13 plo ne montrent pas ou peu similitude avec d'autres proteines et 3 plo indiquent des similitudes significatives. Parmi ces dernieres plo, l'une presente une forte similitude avec les proteines des helicases, une seconde represente vraisemblablement la premiere proteine du canal cl# identifiee chez la levure. Par ailleurs, 2 nouveaux loci d'arnt ont ete identifies

Le projet de sequencage systematique du genome de la levure saccharomyces cerevisiae a demarre en 1989. C'est dans le cadre de ce projet europeen que notre laboratoire a participe au sequencage d'un fragment de 43 kb du chromosome ii. Pour ma part, j'ai contribue au sequencage de 2 fragments contigus de 6 kb et 12,5 kb (faisant partie du fragment de 43 kb). Au cours de ces 2 projets, des strategies differentes ont ete employees. Nous avons montre qu'il est preferable d'utiliser une methode de digestion avec la dnasei pour generer des fragments aleatoires, et de sequencer a partir des deux extremites des inserts plasmidiques plutot que de realiser des digestions partielles avec des enzymes de restriction et de cloner les fragments aleatoires obtenus dans des vecteurs simple-brin. Dans un deuxieme temps, la sequence du fragment de 43 kb du chromosome ii a ete analysee. Celle-ci presente 23 orfs parmi lesquelles, 10 correspondent a des genes connus, 8 presentent des similitudes avec des proteines des banques de donnees et 5 correspondent a de nouvelles orfs putatives. Nous avons detecte plusieurs genes connus, sup46 et urp1 (proteines ribosomales), rim2 (replication in mitochondria), msi1 (multicopy suppressor of ira1), pgi1 (phosphoglucoisomerase), bem1 (bud emergency), dur1,2 (uree amidolyase), ybr1307 (suppresseur d'afg3), ytaf#i#i90 (tata-binding protein associating factor) et cdc47 (implique dans l'initiation de la replication d'adn). Parmi les autres orfs, certaines presentent des similitudes avec des proteines connues, ce qui permet d'emettre des hypotheses sur leurs fonctions: ybr1445 et ybr14411 (mannosyltransferases putatives), ybr1245 (facteur de transcription putatif), ybr1444 (lipase peroxysomale putative). Parmi toutes ces orfs, 6 d'entre d'elles ont ete deletees. Pour ybr1403 et ybr1410, nous avons montre que leurs deletions entrainent un phenotype de letalite. Pour la souche deletee pour ybr1405 (msi1), nous avons analyse les arn#ms des genes impliques dans la cascade ampc. Nous avons ainsi montre que msi1 n'est pas implique dans la regulation transcriptionnelle de ces genes

La condensation mitotique des chromosomes est l'un des mécanismes assurant la transmission fidèle de l'information génétique. Les complexes condensines et leur association à la chromatine sont nécessaires à cette condensation. Cependant, les mécanismes par lesquels ces complexes s'associent aux chromosomes et contribuent à leur condensation sont mal compris. L'objectif de ma thèse était d'identifier et de caractériser des facteurs de condensation encore inconnus collaborant avec le complexe condensine présent chez S. pombe. Par un crible génétique, nous avons recherché des mutants viables lorsque le complexe condensine est complètement fonctionnel mais morts lorsque ce complexe est partiellement défectif. Nous avons ainsi identifié 7 protéines jusqu'alors jamais impliquées dans la condensation mitotique. Parmi ces dernières, nous avons identifié des protéines impliquées dans le remodelage de la chromatine et des facteurs de transcription comme Gcn5, une HAT très conservée, connue pour son rôle de coactivateur de la transcription ; suggérant un lien entre la condensation et la machinerie transcriptionnelle. Gcn5 s'associe à la chromatine au niveau des promoteurs des gènes où elle acétyle principalement H3K9, H3K14 et H3K18. Sa présence au niveau des promoteurs est directement corrélée avec le niveau de transcription des gènes correspondants. Bien que la majorité de la chromatine soit dé-acétylée et que la présence de Gcn5 soit réduite au niveau des chromosomes en mitose, des traces de H3K9 acétylée persistent au niveau de certains promoteurs. Nos résultats suggèrent que cette acétylation persistante pourrait être liée au recrutement du complexe condensine à la chromatine
From yeasts to human, Condensin is essential for mitotic chromosome condensation. However, how Condensin binds to chromatin and, in this context, shapes mitotic chromosome remain poorly understood. Mappings performed from yeasts to mouse have revealed that condensin is enriched near highly expressed genes along chromosome arms, suggesting that as yet identified features associated with transcription take part in condensin binding to chromatin. To identify factors that collaborate with Condensin we performed a synthetically lethal genetic screen in fission yeast. We searched for mutants that are alive when Condensin is fully functional but dead when Condensin is partly defective. We identified 7 proteins never known for their roles in the mitotic condensation, such as some chromatin remodelling and some transcription factors. All these results were consistent with a link between condensation and transcription. Among theses 7 proteins, we found Gcn5, which encodes a conserved HAT, well known for the role it plays as a transcriptional co-activator. Gcn5 binds to gene promoters where it acetylates mainly H3K9, K14 and K18, and its occupancy correlates with transcription rates. Remarkably, although the bulk of chromatin is de-acetylated and Gcn5 reduced from chromatin upon mitosis entry, traces of Gcn5 dependant H3K9 acetylated persist at condensin binding sites. Here, we provide evidence that Gcn5-mediated histone H3 K9 acetylation could assist the binding of Condensin to chromatin

Nous avons cherché à évaluer l’impact des réarrangements chromosomiques sur l’évolution des génomes de levures selon deux approches. La première approche a consisté à retracer les réarrangements chromosomiques au cours de l’évolution des Saccharomycotina. Nous avons construit un arbre phylogénétique à partir de 66 génomes issus de bases de données publiques et reconstruit la structure des génomes ancestraux des 66 espèces. La comparaison des génomes ancestraux a permi d’inférer 5150 réarrangements chromosomiques passés. Nous avons montré que selon les clades considérés, les génomes évoluent plutôt par inversion ou par translocation et que les réarrangements chromosomiques et les mutations non-synonymes s’accumulent à un rythme coordonné au cours de l’évolution. La seconde approche a consisté à quantifier l’impact phénotypique des variations structurelles (SV) du génome en termes de taux de croissance végétative et de viabilité méiotique chez Saccharomyces cerevisiae. Nous avons développé une technique pour induire à façon des SV ciblés dans le génome de S. cerevisiae, en induisant deux coupures simultanées dans le génome de S. cerevisiae avec CRISPR/Cas9 et à guider la réparation des cassures par recombinaison homologue avec des oligonucléotides chimériques. Nous avons alors adapté cette technique pour induire en une étape un grand nombre de SV aléatoires. L’impact phénotypique des SV obtenus a été quantifié en méiose et en croissance végétative. Ces travaux montrent que même des réarrangements chromosomiques balancés n’affectant aucune phase codante génèrent une grande diversité phénotypique qui participe à l’adaptation des organismes à leur environnement
The aim of this work was to assess the impact of chromosomal rearrangements on the evolution of yeast genomes with two approaches. The first approach consisted in retracing past rearrangements during the evolution of Saccharomycotina yeast genomes. We have built a phylogenetic tree of 66 genomes gathered from public databases, then reconstructed the structure of all ancestral genomes of these species. By comparing the structure of reconstructed ancestral genomes, we have inferred 5150 past rearrangements. We showed that depending on the clades, genomes tend to evolve mostly by inversion or by translocation. In addition, we showed that chromosomal rearrangements and non-synonymous mutations tend to accumulate at a coordinated pace during evolution. The second approach aimed at quantifying the phenotypic impact of structural variations of chromosomes (SVs) in terms of vegetative growth and meiotic viability in Saccharomyces cerevisiae. We developed a technique to induce easily targeted SVs in the genome of S. cerevisiae by inducing two chromosomal breaks with CRISPR/Cas9 and providing the cells with chimerical donor oligonucleotides to repair the split chromosomes by homologous recombination. We have then adapted this technique to induce multiple random SVs in a single step. The phenotypic impact of obtained variants on vegetative growth and on spore viability was quantified. These results show that even balanced chromosomal rearrangements that do not affect coding sequence generate a wide phenotypic diversity that contributes to the adaptation of organisms to their environment

Les génomes sont des structures hautement dynamiques et les grandes variations structurelles (SVs) des chromosomes, telles que les inversions, contribuent à l'évolution des génomes et à l'adaptation des espèces. Il est essentiel de comprendre l'impact fonctionnel des inversions sur la diversité phénotypique, car il existe de plus en plus de preuves que les inversions joueraient un rôle important dans les variations phénotypiques. Afin d'expliquer l'impact phénotypique des inversions, nous avons choisi la levure de boulangerie comme modèle eucaryote unicellulaire dans notre travail. Sur la base d'un catalogue de 104 événements d'inversion caractérisés parmi un panel de 142 assemblages de génomes complets, nous nous sommes concentrés sur une inversion spéciale de 32kb localisée sur le chromosome XIV et qui est trouvée de manière récurrente dans diverses souches de Saccharomyces cerevisiae et S. paradoxus. Nous avons utilisé la méthodologie CRISPR/Cas9 d'édition du génome pour générer des bibliothèques de souches de S. cerevisiae contenant cette région dans les deux orientations par l'introduction de 2 cassures double-brin ((DSB pour Double-Strand Break) de l'ADN aux extrémités de cette région. Nous avons construit ce type d'inversions dans 3 souches hôtes présentant des fonds génétiques différents, S288C, YPS128 et Y12. Afin de tester les relations entre ce type de variation génétique et les traits phénotypiques, nous avons étudié l'impact fonctionnel des inversions pendant les cycles cellulaires sexuels et végétatifs, y compris le taux de croissance dans différentes conditions de culture, l'efficacité de la sporulation, l'efficacité des croisements et la viabilité des spores. Ce travail nous a permis de déterminer la contribution de cette inversion aux variations phénotypiques et son rôle adaptatif au cours de l'évolution
Genomes are highly dynamic structures and large-scale Structural Variations (SVs) of chromosomes such as inversions contribute to genome evolution and species adaptation. Understanding the functional impact of inversion on phenotypic diversity is essential because there are growing evidence that inversions play an important role in phenotypic variation. For the purpose of explaining the phenotypic impact of inversions, we choose yeast as single cell eukaryotic model in our work. Based on a catalogue of 104 inversion events characterized among a panel of 142 complete genome assemblies, we focused on a special 32kb inversion on chromosome XIV that is recurrently found in various strains of Saccharomyces cerevisiae and S. paradoxus. CRISPR/Cas9 methodology of genome editing is applied to generate strain libraries in S. cerevisiae containing this region in both orientations through the introduction of DNA double-strand breaks (DSBs) at the inversion boundaries. We constructed such inversion models in 3 different host strains with different genetic background, S288C, YPS128 and Y12. In order to test the relationships between this type of genetic variation and phenotypic traits, we investigated the functional impact of the inversions during both sexual and asexual cell cycles, including growth ratio in different culture conditions, sporulation efficiency, mating efficiency and spore viability. This work allows us to determine the contribution of inversions to phenotypic variations and their adaptive role during evolution