Biology subjects

Buonomo, S.

Publications and source records attributed to Buonomo, S..

4 recordsLinked to original sources

bioRxiv2024

Supervised and unsupervised deep learning-based approaches for studying DNA replication spatiotemporal dynamics

In eukaryotic cells, DNA replication is organised both spatially and temporally, as evidenced by the stage-specific spatial distribution of replication foci in the nucleus. Despite the genetic association of aberrant DNA replication with numerous human diseases, the labour-intensive methods employed to study DNA replication have hindered large-scale analyses of its roles in pathological processes. In this study, we first demonstrate that a convolutional neural network trained to classify S-phase stages based on DAPI and EdU patterns could identify altered replication dynamics in Rif1-deficient mouse embryonic stem cells (mESCs), revealing a skewed distribution across the various S-phase stages. Given the possible practical limitations associated with a supervised framework, we proceed to show that the abnormal replication profile of Rif1-deficient mESCs could further be detected by an unsupervised approach (based on self-supervised representation learning), which could additionally reconstruct progression through S-phase. Finally, we extend our approach to a well-characterised cellular model of inducible deregulated origin firing, involving cyclin E overexpression. Through parallel EdU- and PCNA-based analyses, we demonstrate the potential applicability of our method to patient samples, offering a means to identify the contribution of deregulated DNA replication to a plethora of pathogenic processes.

cell biology↗

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↗

bioRxiv2020

A RIF1 and KAP1-based, double-bookmarking system generates a toggle switch that stabilises the identities of the active and inactive X chromosomes during random X inactivation in mouse

The onset of random X inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. Here we show that RIF1 and KAP1 are two fundamental factors for the definition of the asymmetry. Our data show that at the onset of mESC differentiation, upregulation of the long non-coding RNA Tsix weakens the symmetric RIF1 association with the Xist promoter, and opens a window of opportunity for a more stable association of KAP1. KAP1 is required to sustain high levels of Tsix, thus reinforcing and propagating the asymmetry, and, as a result, marking the future active X chromosome. Furthermore, we show that RIF1 association with the future inactive X chromosome is essential for Xist upregulation. This double-bookmarking system, based on the mutually exclusive relationships of Tsix and RIF1, and RIF1 and KAP1, thus coordinates the identification of the inactive and active X chromosomes and initiates a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X chromosome state.

molecular 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↗