Thierry Gostan

Cells preserve genome stability while occasionally generating genetic diversity through mutations and chromosomal rearrangements, but how these opposing outcomes are balanced remains unclear. Because late-replicating regions evolve more rapidly, we investigated the consequences of reduced origin licensing and lengthened S phase in yeast. Under these conditions, cells progress slowly through S phase without checkpoint activation, and enter mitosis with under-replicated DNA. We show that mitotic cyclin-dependent kinase (M-CDK) phosphorylates DNA polymerase α (Pol α), displacing it from replication forks and triggering fork collapse at mitotic entry. The resulting DNA damage response provides a window for mitotic DNA synthesis (MiDAS), enabling completion of replication through error-prone, recombination-dependent mechanisms resembling break-induced replication (BIR) and Fork Stalling & Template Switching (FoSTeS). These findings identify Pol α as a critical M-CDK target that promotes the transition from canonical replication to recombination-driven DNA synthesis when cells enter mitosis with incompletely-duplicated chromosomes. This mechanism offers a potential explanation for the accelerated evolution of late-replicating genomic regions.

RNA dysregulation is a recognized contributor to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the most common motor neuron (MN) disease. However, the molecular mechanisms linking defects in RNA metabolism to selective neuronal vulnerability remain poorly understood. Alterations in the cellular levels of R-loops –structures forming by reannealing of the nascent RNA with the template DNA during transcription- have been observed in neurodegeneration, but it is unclear how perturbations in R-loop homeostasis contribute to neuronal dysfunction. Here we investigate the molecular basis of a juvenile form of ALS dubbed ALS4 that is caused by mutations in the helicase SETX, which plays important roles in the resolution of R-loops and transcription termination. Using isogenic human induced pluripotent stem cell-derived MNs, we show that ALS4-associated SETX mutations induce progressive axonal defects and widespread transcriptomic alterations, including reduced expression or altered splicing of transcripts critical for neuronal function. ALS4 MNs exhibit a transcriptional signature marked by cellular stress, aberrant cell cycle re-entry, and compromised neuronal identity that is partially shared by other forms of ALS. Mechanistically, these defects are partly driven by downstream aberrant activation of the TGF-β signaling pathway, whose pharmacological inhibition ameliorates axonal defects. Finally, our analyses support a link between mutant SETX ectopic activity at R-loops and the observed alterations in RNA expression and splicing, providing new insights into how RNA dysregulation can drive neuronal dysfunction Altogether, our work reveals how perturbations at the interface of transcription and R-loop metabolism can reshape neuronal identity and drive disease. Teaser: Deregulation of TGF-β signaling drives axonal defects and compromised motor neuron identity in senataxin-mediated ALS

Despite advances in the identification of chromatin regulators and genome interactions, the principles of higher‐order chromatin structure have remained elusive. Here, we applied FLIM‐FRET microscopy to analyse, in living cells, the spatial organisation of nanometre range proximity between nucleosomes, which we called “nanocompaction.” Both in naive embryonic stem cells (ESCs) and in ESC‐derived epiblast‐like cells (EpiLCs), we find that, contrary to expectations, constitutive heterochromatin is much less compacted than bulk chromatin. The opposite was observed in fixed cells. HP1α knockdown increased nanocompaction in living ESCs, but this was overridden by loss of HP1β, indicating the existence of a dynamic HP1‐dependent low compaction state in pluripotent cells. Depletion of H4K20me2/3 abrogated nanocompaction, while increased H4K20me3 levels accompanied the nuclear reorganisation during EpiLCs induction. Finally, the knockout of the nuclear cellular‐proliferation marker Ki‐67 strongly reduced both interphase and mitotic heterochromatin nanocompaction in ESCs. Our data indicate that, contrary to prevailing models, heterochromatin is not highly compacted at the nanoscale but resides in a dynamic low nanocompaction state that depends on H4K20me2/3, the balance between HP1 isoforms, and Ki‐67.