Context-dependent neocentromere activity in synthetic yeast chromosome VIII

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae. With the completion of synthetic chromosome VIII (synVIII) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII, we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (~500 bp) of the CDEIII-proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality </p

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or ‘‘CRISPR D-BUGS,’’ to map phenotypic variants caused by specific designer modifications, known as ‘‘bugs.’’ We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASer CGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating &gt;50% of the Sc2.0 genome in one strain </p

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes. </p

Réorganisation fonctionnelle du génome de la levure durant le cycle cellulaire

Decades of studies showed that chromatin structure is tightly linked to DNA related metabolic processes, through the dynamic regulation of a myriad of molecular factors. The proper organization of chromosomes is notably important to ensure the maintenance of DNA integrity during cell cycle progression. Using the model S. cerevisiae, the aim of my PhD project was to characterize to which extent chromatin reorganization during the cell cycle may influence chromosome stability. To do so, we first generated a comprehensive genome-wide study of the reorganization of yeast’s chromosomes during an entire cell cycle. This work, besides recapitulating expected chromosomal features of the replication and mitotic stages, led to the characterization of peculiar chromosome structures such as a DNA loop bridging the rDNA and the centromeres. The role of structural maintenance of chromosomes (SMC) complexes and of microtubules were thoroughly investigated. A second part of my work focused on describing features of the chromatin organization of cells that exited the proliferative cell cycle and entered into quiescence. We characterized the dense status of silenced heterochromatin at specific loci, such as telomeres, in relation to the silent information regulators (SIRs). Finally, we tried to achieve a better understanding of the functional interplay between chromosome stability and the 3D genome architecture during replication, by investigating the genomic stability at replication pausing sites. Overall, our results point at a striking plasticity of replication structures to different stresses. Future work aims to map replication-dependent chromosomal rearrangements on the genomic maps.Des décennies d'études ont montré que la structure de la chromatine est étroitement liée aux processus métaboliques de l'ADN. Une bonne organisation des chromosomes tout au long du cycle cellulaire est particulièrement importante pour assurer le maintien de l'intégrité de l'ADN. Le but de mon projet de doctorat était de caractériser dans quelle mesure la réorganisation de la chromatine pendant le cycle cellulaire pourrait influencer la stabilité des chromosomes. Pour ce faire, nous avons d'abord effectué une étude complète de la réorganisation des chromosomes de la levure modèle Saccharomyces cerevisiae pendant tout un cycle cellulaire. Ce travail, en plus de récapituler les caractéristiques chromosomiques attendues, a conduit à la caractérisation de structures chromosomiques particulières, telle qu'une boucle d'ADN reliant l'ADNr et les centromères. Le rôle des complexes SMC et des microtubules a été étudié en profondeur. Une deuxième partie de mon travail a porté sur la description de l'organisation de la chromatine de cellules qui ont quitté le cycle cellulaire prolifératif et sont entrées en quiescence. Nous avons ainsi caractérisé le statut dense de l'hétérochromatine silencieuse dans des loci spécifiques tels que les télomères. Enfin, nous avons essayé de mieux comprendre l'interaction fonctionnelle entre la stabilité chromosomique et l'architecture 3D du génome durant la réplication en étudiant la stabilité génomique à des sites de pause de réplication. Nos résultats indiquent une adaptabilité frappante des structures de réplication sous différentes contraintes. Le travail futur vise à cartographier les réarrangements chromosomiques dépendants de la réplication

Sir3 mediates long-range chromosome interactions in budding yeast

International audiencePhysical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here, we investigate the well-conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing SIR complex composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long-range contacts between distant regions, including the rDNA, telomeres, and internal Sir3-bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach, we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, Sir4), Sir2 activity, or chromosome context. Altogether, these data suggest that Sir3 acts as a molecular bridge that stabilizes long-range interactions

The silencing factor Sir3 is a molecular bridge that sticks together distant loci

Posté dans BioRxiv le 29 juin 2020Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here we investigate the well conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing complex SIR composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3 , we show that the binding of Sir3 directly promotes long range contacts between distant regions, including the rDNA, telomeres, and internal Sir3 bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, and Sir4), Sir2 activity or chromosome context. Altogether these data suggest that Sir3 represents an uncommon example of protein able to bridge directly distant loci

Karyotype engineering reveals spatio-temporal control of replication firing and gene contacts

International audienceEukaryotic genomes vary in terms of size, chromosome number, and genetic complexity. Their temporal organization is complex, reflecting coordination between DNA folding and function. Here, we used fused karyotypes of budding yeast to characterize the effects of chromosome length on nuclear architecture. We found that size-matched megachromosomes expand to occupy a larger fraction of the enlarged nucleus. Hi-C maps reveal changes in the three-dimensional structure corresponding to inactivated centromeres and telomeres. De-clustering of inactive centromeres results in their loss of early replication, highlighting a functional correlation between genome organization and replication timing. Repositioning of former telomere-proximal regions on chromosome arms exposed a subset of contacts between flocculin genes. Chromatin reorganization of megachromosomes during cell division remained unperturbed, and it revealed that centromere-rDNA contacts in anaphase, extending over 0.3 Mb on wild-type chromosome, cannot exceed ∼1.7 Mb. Our results highlight the relevance of engineered karyotypes to unveiling relationships between genome organization and functio

Karyotype engineering reveals spatio-temporal control of replication firing and gene contacts

International audienceEukaryotic genomes vary in terms of size, chromosome number, and genetic complexity. Their temporal organization is complex, reflecting coordination between DNA folding and function. Here, we used fused karyotypes of budding yeast to characterize the effects of chromosome length on nuclear architecture. We found that size-matched megachromosomes expand to occupy a larger fraction of the enlarged nucleus. Hi-C maps reveal changes in the three-dimensional structure corresponding to inactivated centromeres and telomeres. De-clustering of inactive centromeres results in their loss of early replication, highlighting a functional correlation between genome organization and replication timing. Repositioning of former telomere-proximal regions on chromosome arms exposed a subset of contacts between flocculin genes. Chromatin reorganization of megachromosomes during cell division remained unperturbed, and it revealed that centromere-rDNA contacts in anaphase, extending over 0.3 Mb on wild-type chromosome, cannot exceed ∼1.7 Mb. Our results highlight the relevance of engineered karyotypes to unveiling relationships between genome organization and functio

Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners

International audienceCohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions