Mistimed origin licensing and activation stabilize common fragile sites under tight DNA-replication checkpoint activation.

Genome integrity requires replication to be completed before chromosome segregation. The DNA-replication checkpoint (DRC) contributes to this coordination by inhibiting CDK1, which delays mitotic onset. Under-replication of common fragile sites (CFSs), however, escapes surveillance, resulting in mitotic chromosome breaks. Here we asked whether loose DRC activation induced by modest stresses commonly used to destabilize CFSs could explain this leakage. We found that tightening DRC activation or CDK1 inhibition stabilizes CFSs in human cells. Repli-Seq and molecular combing analyses showed a burst of replication initiations implemented in mid S-phase across a subset of late-replicating sequences, including CFSs, while the bulk genome was unaffected. CFS rescue and extra-initiations required CDC6 and CDT1 availability in S-phase, implying that CDK1 inhibition permits mistimed origin licensing and firing. In addition to delaying mitotic onset, tight DRC activation therefore supports replication completion of late origin-poor domains at risk of under-replication, two complementary roles preserving genome stability.

A non-transcriptional function of Yap regulates the DNA replication program in Xenopus laevis.

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death.

The identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca(2+) rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr(-/-)) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

Vasoinhibins regulate the inner and outer blood-retinal barrier and limit retinal oxidative stress.

Vasoinhibins are prolactin fragments present in the retina, where they have been shown to prevent the hypervasopermeability associated with diabetes. Enhanced bradykinin (BK) production contributes to the increased transport through the blood-retina barrier (BRB) in diabetes. Here, we studied if vasoinhibins regulate BRB permeability by targeting the vascular endothelium and retinal pigment epithelium (RPE) components of this barrier. Intravitreal injection of BK in male rats increased BRB permeability. Vasoinhibins prevented this effect, as did the B2 receptor antagonist Hoe-140. BK induced a transient decrease in mouse retinal and brain capillary endothelial monolayer resistance that was blocked by vasoinhibins. Both vasoinhibins and the nitric oxide (NO) synthase inhibitor L-NAME, but not the antioxidant N-acetyl cysteine (NAC), blocked the transient decrease in bovine umbilical vein endothelial cell (BUVEC) monolayer resistance induced by BK; this block was reversed by the NO donor DETANONOate. Vasoinhibins also prevented the BK-induced actin cytoskeleton redistribution, as did L-NAME. BK transiently decreased human RPE (ARPE-19) cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC. DETANONOate reverted the blocking effect of vasoinhibins. Similar to BK, the radical initiator Luperox induced a reduction in ARPE-19 cell monolayer resistance, which was prevented by vasoinhibins. These effects on RPE resistance coincided with actin cytoskeleton redistribution. Intravitreal injection of vasoinhibins reduced the levels of reactive oxygen species (ROS) in retinas of streptozotocin-induced diabetic rats, particularly in the RPE and capillary-containing layers. Thus, vasoinhibins reduce BRB permeability by targeting both its main inner and outer components through NO- and ROS-dependent pathways, offering potential treatment strategies against diabetic retinopathies.