Biology subjects

Lazar-Stefanita, L.

Publications and source records attributed to Lazar-Stefanita, L..

6 recordsLinked to original sources

bioRxiv2025

Ancient co-option of LTR retrotransposons as yeast centromeres

The evolutionary origins of the genetic point centromere in the brewers yeast Saccharomyces cerevisiae, a member of the order Saccharomycetales, are still unknown. Competing hypotheses suggest that the point centromere tripartite genetic centromere DNA elements (CDEs) either evolved from ancestral epigenetic centromeres by descent with modification or were gained through horizontal transfer from selfish DNA plasmids.1,2 Here, we identified centromeres in the sister order Saccharomycodales and termed them "proto-point centromeres" due to sequence features that bridge the evolutionary gap between point centromeres and ancestral centromeres types. Comparative genomic analyses across multiple yeast orders showed an unexpected evolutionary link between point and proto-point centromeres to the long terminal repeats (LTRs) of Ty5 retrotransposons. Strikingly, one Saccharomycodales species, Saccharomycodes ludwigii, harbors compact Ty5-based centromeres, where its CDEII elements are divergent AT-rich Ty5 LTRs. These living fossil centromeres show how retrotransposon cis-regulation was likely co-opted for genetic centromere specification. These insights show that point centromeres are direct descendants of retrotransposons and have evolved by descent with modification. Ultimately, the many diverse centromere types across the yeast subphylum may share a common ancestry rooted in retrotransposon activity.

evolutionary biology↗

bioRxiv2022

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

The Sc2.0 project is building a eukaryotic synthetic genome from scratch, incorporating thousands of designer features. A major milestone has been achieved with the assembly of all individual Sc2.0 chromosomes. Here, we describe the consolidation of multiple synthetic chromosomes using endoreduplication intercross to generate a strain with 6.5 synthetic chromosomes. Genome-wide chromosome conformation capture and long-read direct RNA sequencing were performed on this strain to evaluate the effects of designer modifications, such as loxPsym site insertion, tRNA relocation, and intron deletion, on 3D chromosome organization and transcript isoform profiles. To precisely map "bugs", we developed a method, CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS", exploiting directed mitotic recombination in heterozygous diploids. Using this method, we first fine-mapped a synII defect resulting from two loxPsym sites in the 3 UTR of SHM1. This approach was also used to map a combinatorial bug associated with synIII and synX, revealing a highly unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. "Starvation" for tRNASerCGA leads to insufficient levels of the key positive inositol biosynthesis regulator, Swi3, which contains tandem UCG codons. Finally, to expedite consolidation, we employed a new method, chromosome swapping, to incorporate the largest chromosome (synIV), thereby consolidating more than half of the Sc2.0 genome in a single strain.

genomics↗

bioRxiv2020

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

Physical 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.

cell biology↗

bioRxiv2019

FACT mediates cohesin function on chromatin

Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion 1,2 and the organisation of higher-order structures during interphase 3 and mitosis 4,5. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes 6-8. Here we show that cohesin role in chromosome organisation requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin-dependent compartmentalization of chromosomes into loop domains.

molecular biology↗

bioRxiv2019

A major role for Eco1 in regulating cohesin-mediated mitotic chromosome folding

Understanding how chromatin organizes spatially into chromatid and how sister chromatids are maintained together during mitosis is of fundamental importance in chromosome biology. Cohesin, a member of the Structural Maintenance of Chromosomes (SMC) complex family, holds sister chromatids together 1-3 and promotes long-range intra-chromatid DNA looping 4,5. These cohesin-mediated DNA loops are important for both higher-order mitotic chromatin compaction6,7 and, in some organisms, compartmentalization of chromosomes during interphase into topologically associating domains (TADs) 8,9. Our understanding of the mechanism(s) by which cohesin generates large DNA loops remains incomplete. It involves a combination of molecular partners and active expansion/extrusion of DNA loops. Here we dissect the roles on loop formation of three partners of the cohesin complex: Pds5 10, Wpl1 11 and Eco1 acetylase 12, during yeast mitosis. We identify a new function for Eco1 in negatively regulating cohesin translocase activity, which powers loop extrusion. In the absence of negative regulation, the main barrier to DNA loop expansion appears to be the centromere. Those results provide new insights on the mechanisms regulating cohesin dependent DNA looping.

genetics↗

bioRxiv2016

Choreography of budding yeast chromosomes during the cell cycle

To ensure the proper transmission of the genetic information, DNA molecules must be faithfully duplicated and segregated. These processes involve dynamic modifications of chromosomes internal structure to promote their individualization, as well as their global repositioning into daughter cells (Guacci et al., 1994; Kleckner et al., 2014; Mizuguchi et al., 2014). In eukaryotes, these events are regulated by conserved architectural proteins, such as structural maintenance of chromosomes (SMC i.e. cohesin and condensin) complexes (Aragon et al., 2013a; Uhlmann, 2016). Although the roles of these factors have been actively investigated, the genome-wide chromosomal architecture and dynamics both at small and large-scales during cell division remains elusive. Here we report a comprehensive Hi-C (Dekker et al., 2002; Lieberman-Aiden et al., 2009) analysis of the dynamic changes of chromosomes structure over the Saccharomyces cerevisiae cell cycle. We uncover specific SMC-dependent structural transitions between the different phases of the mitotic cycle. During replication, cohesion establishment promotes the increase of long-range intra-chromosomal contacts. This process correlates with the individualization of chromosomes, which culminates at metaphase. Mitotic chromosomes are then abruptly reorganized in anaphase by the mechanical forces exerted by the mitotic spindle on the centromere cluster. The formation of a condensin-dependent loop, that bridges centromere cluster with the cenproximal flanking region of the rDNA, suggests that these forces may directly facilitate nucleolus segregation. This work provides a comprehensive overview of chromosome dynamics during the cell cycle of a unicellular eukaryote that recapitulates and unveils new features of highly conserved stages of the cell division.

genetics↗