Rodriguez Colman, M. J. | bioRxiv Science

bioRxiv2020

A FOXO-dependent replication checkpoint restricts proliferation of damaged cells

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Stalling or slowing of the replication fork as a result of replication stress leads to formation of aberrant single-stranded DNA (ssDNA) stretches and potentially DNA double-stranded-breaks (DSBs). Accumulation of ssDNA activates the ATR-dependent DNA replication stress checkpoint response that slows progression from S/G2- to M-phase to protect genomic integrity (1). However, whether mild replication stress restricts proliferation remains controversial (2-6). Here we identify a novel cell cycle exit mechanism, that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the CDH1 bound Anaphase Promoting Complex / Cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead Box O (FOXOs) mediated repression of its transcriptional regulator E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible, and thereby play an important role in maintaining genomic stability.

molecular biology↗

bioRxiv2019

Mitochondria define intestinal stem cell differentiation downstream of a FOXO/Notch axis

Differential signalling of the WNT and Notch pathways regulates proliferation and differentiation of Lgr5+ crypt-based columnar cells (CBCs) into all cell lineages of the intestine. We have recently shown that high mitochondrial activity in CBCs is key in maintaining stem cell function. Interestingly, while high mitochondrial activity drives CBCs, it is reduced in the adjacent secretory Paneth cells (PCs). This observation implies that during differentiation towards PCs, CBCs undergo a metabolic rewiring involving downregulation of mitochondrial number and activity, through a hitherto unknown mechanism. Here we demonstrate, using intestinal organoids that FoxO transcription factors and Notch signalling functionally interact in determining CBC cell fate. In agreement with the organoid data, combined Foxo1 and 3 deletion in mice increases PC number in the intestine. Importantly, we show that FOXO and Notch signalling converge onto regulation of mitochondrial fission, which in turn provokes stem cell differentiation into the secretory types; Goblet cells and PCs. Finally, mapping intestinal stem cell differentiation based on pseudotime computation of scRNA-seq data further supports the role of FOXO, Notch and mitochondria in determining secretory differentiation. This shows that mitochondria is not only a discriminatory hallmark of CBCs and PCs, but that its status actively determines lineage commitment during differentiation. Together, our work describes a new signalling-metabolic axis in stem cell differentiation and highlights the importance of mitochondria in determining cell fate.

cell biology↗