Biology subjects

Gnan, S.

Publications and source records attributed to Gnan, S..

6 recordsLinked to original sources

bioRxiv2023

The balance between ATR and DDK activities controls TopBP1-mediated locking of dormant origins at the pre-IC stage

Replication stress, a major hallmark of cancers, and ensuing genome instability source from impaired progression of replication forks. The first line of defense against fork slowing is compensation, a long-described process that elicits firing of otherwise dormant origins. It remains unclear whether compensation requires activation of the DNA replication checkpoint or passively results from lengthening of the window of time during which dormant origins can fire when fork progression slows, or both. Using molecular DNA combing we show here that a linear relationship ties inter-origin distances to fork speeds, independently of the checkpoint status. We called this line "stressline" and further show that its slope enables precise quantification of the compensation efficiency. Comparison of the slopes in different genetic backgrounds reveals that compensation requires ATR, not CHK1, while TopBP1 and CDC7/DBF4 repress dormant origin activation. These results strongly suggest that TopBP1 locks dormant origins at the pre-IC stage and that ATR and DDK oppose to control the conversion of dormant pre-ICs into functional salvage origins. Both passive and active processes thus contribute to compensation. Moreover, Repli-seq and OK-seq analyses confirm the activating role of ATR and permit development of ATRAP-seq, a new procedure allowing mapping of early constitutive origins.

genetics↗

bioRxiv2023

RIF1 regulates replication origin activity and early replication timing in B cells

The mammalian DNA replication timing (RT) program is crucial for the proper functioning and integrity of the genome. The best-known mechanism for controlling RT is the suppression of late origins of replication in heterochromatin by RIF1. Here, we report that in antigen-activated B lymphocytes, RIF1 binds predominantly to early-replicating active chromatin, regulates early origin firing and promotes early replication. RIF1 has a minor role in gene expression and genome organization in B cells. Furthermore, we find that RIF1 functions in a complementary and non-epistatic manner with minichromosome maintenance (MCM) proteins to establish early RT signatures genome-wide and, specifically, to ensure the early replication of highly transcribed genes. These findings reveal new layers of regulation within the B cell RT program, driven by the coordinated activity of RIF1 and MCM proteins.

molecular biology↗

bioRxiv2021

Kronos scRT: a uniform framework for single-cell replication timing analysis

Mammalian genomes are replicated in a cell-type specific order and in coordination with transcription and chromatin organization. Although the field of replication is also entering the single-cell era, current studies require cell sorting, individual cell processing and have yielded a limited number (<100) of cells. Here, we have developed Kronos scRT (https://github.com/CL-CHEN-Lab/Kronos_scRT), a software for single-cell Replication Timing (scRT) analysis. Kronos scRT does not require a specific platform nor cell sorting, allowing the investigation of large datasets obtained from asynchronous cells. Analysis of published available data and droplet-based scWGS data generated in the current study, allows exploitation of scRT data from thousands of cells for different mouse and human cell lines. Our results demonstrate that, although most cells replicate within a close timing range for a given genomic region, replication can also occur stochastically throughout S phase. Altogether, Kronos scRT allows investigating the RT program at a single-cell resolution for both homogeneous and heterogeneous cell populations in a fast and comprehensive manner.

genomics↗

bioRxiv2021

GC content but not nucleosome positioning directly contributes to intron-splicing efficiency in Paramecium

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the >90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur co-transcriptionally and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We showed that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of [~]151 bp, amongst the smallest periodicities reported. Our analysis revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint to locate introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for the nonsense-mediated decay effectors, we showed that introns located at the edge of nucleosomes display higher splicing efficiency than those at the centre. However, multiple regression analysis indicated that the GC content, rather than nucleosome positioning, directly contributes to intron splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning and intron evolution in Paramecium.

genomics↗

bioRxiv2020

Unscheduled origin building in S-phase upon tight CDK1 inhibition suppresses CFS instability

Genome integrity requires replication to be completed before chromosome segregation. This coordination essentially relies on replication-dependent activation of a dedicated checkpoint that inhibits CDK1, delaying mitotic onset. Under-replication of Common Fragile Sites (CFSs) however escapes surveillance, which triggers chromosome breakage. Using human cells, we asked here whether such leakage results from insufficient CDK1 inhibition under modest stresses used to destabilize CFSs. We found that tight CDK1 inhibition suppresses CFS instability. Repli-Seq and molecular combing analyses consistently showed a burst of replication initiations in mid S phase across large origin-poor domains shaped by transcription, including CFSs. Strikingly, CDC6 or CDT1 depletion or CDC7-DBF4 inhibition during the S phase prevented both extra-initiations and CFS rescue, showing that CDK1 inhibition promotes targeted and mistimed building of functional extra-origins. In addition to delay mitotic onset, checkpoint activation therefore advances replication completion of chromosome domains at risk of under-replication, two complementary roles preserving genome stability.

cell biology↗

bioRxiv2019

Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing program and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. RIF1s role in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing program and nuclear architecture.

genomics↗