Biology subjects

Beckouet, F.

Publications and source records attributed to Beckouet, F..

6 recordsLinked to original sources

bioRxiv2023

Transcription promotes discrete long-range chromatin loops besides organizing cohesin-mediated DNA folding

The multi-layered arrangement of eukaryotic genomes and chromosome spatial organization dynamics are of functional importance for gene expression, DNA replication and segregation. SMC complexes are essential instruments of chromosome folding by carrying out long range intra-chromatid DNA looping. Cohesin, in addition to tether sister chromatids, also ensures dynamic regulation of gene expression in mammals by promoting interaction between distal regulatory elements and promoters whereas transcription affects genome folding in numerous organisms and in multiple ways. Here, we comprehensively dissect the relative contributions of transcription and cohesin complexes, as well as their interplay, on the yeast S. cerevisiae genome organization through DNA borders and loops. Transcription activation specifically induces appearance of DNA borders and loops, independently of SMC complexes, while also directly interfering in addition with cohesin-mediated loop expansion.

genomics↗

bioRxiv2023

Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription.

The RNA polymerase I (Pol I) enzyme that synthesizes large rRNA precursors, exhibits high rate of pauses during elongation, indicative of a discontinuous process. We show here that Premature Termination of Transcription (PTT) by Pol I is a critical regulatory step limiting rRNA production in vivo. The Pol I mutant, SuperPol (RPA135-F301S), produces 1.5-fold more rRNA than the wild type (WT). Combined CRAC and rRNA analysis link increased rRNA production in SuperPol to reduced PTT, resulting in shifting polymerase distribution toward the 3 end of rDNA genes. In vitro, SuperPol shows reduced nascent transcript cleavage, associated with more efficient transcript elongation after pauses, to the detriment of transcriptional fidelity. Notably, SuperPol is resistant to BMH-21, a drug impairing Pol I elongation and inducing proteasome-mediated degradation of Pol I subunits. Compared to WT, SuperPol maintains subunit stability and sustains high transcription levels upon BMH-21 treatment. These comparative results show that PTT is alleviated in SuperPol while it is stimulated by BMH-21 in WT Pol I.

molecular biology↗

bioRxiv2023

Sister chromatid cohesion halts DNA loop expansion

Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin and Smc5/6. This organization regulates different DNA-related processes along the cell cycle such as transcription, recombination, segregation and DNA repair. During G2/M stages, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion in G2.

molecular biology↗

bioRxiv2022

Exogenous chromosomes reveal how sequence composition drives chromatin assembly, activity, folding and compartmentalization

Genomic sequences co-evolve with DNA-associated proteins to ensure the multiscale folding of long DNA molecules into functional chromosomes. In eukaryotes, different molecular complexes organize the chromosomes hierarchical structure, ranging from nucleosomes and cohesin- mediated DNA loops to large scale chromatin compartments. To explore the relationships between the DNA sequence composition and the spontaneous loading and activity of these DNA-associated complexes in the absence of co-evolution, we characterized chromatin assembly and activity in yeast strains carrying exogenous bacterial chromosomes that diverged from eukaryotic sequences over 1.5 billion years ago. We show that nucleosome assembly, transcriptional activity, cohesin-mediated looping, and chromatin compartmentalization can occur in a bacterial chromosome with a largely divergent sequence integrated in a eukaryotic host, and that the chromatinization of bacterial chromosomes is highly correlated with their sequence composition. These results are a step forward in understanding how foreign sequences are interpreted by a host nuclear machinery during natural horizontal gene transfers, as well as in synthetic genomics projects.

genetics↗

bioRxiv2021

Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin dependent chromatin loops

Chromosome spatial organization and dynamics influence DNA-related metabolic processes. SMC complexes like cohesin are essential instruments of chromosome folding. Cohesin-dependent chromatin loops bring together distal loci to regulate gene transcription, DNA repair and V(D)J recombination processes. Here we characterize further the roles of members of the cohesin holocomplex in regulating chromatin loop expansion, showing that Scc2, which stimulates cohesin ATPase activity, is essential for the translocation process required to extend DNA loop length. Eco1-dependent acetylation of Smc3 during S phase counteracts this activity through the stabilization of Pds5, to finely tune loop sizes and stability during G2. Inhibiting Pds5 in G2 leads to a strong enlargement of pre-established, stable DNA loops, in a Scc2-dependent manner. Altogether, the study strongly supports a Scc2-mediated translocation process driving expansion of DNA loops in living cells.

genetics↗

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↗