Summary One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3′-OH molecules that occur during double-strand break (DSB) repair at broken or stalled replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal new functions for breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase eta (Polη) to initiate DNA synthesis. PALB2, BRCA2 and Polη co-localize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη, and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D-loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D-loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair.

Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vasculature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogenesis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelial growth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulating angiogenesis, and have been the targets for the development of drugs. However, most experimental assays assessing angiogenesis rely on animal models. We report an in vitro model using a microvessel-on-a-chip. It mimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a single angiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observed in vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, as demonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2 pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstrates that sorafenib impairs the endothelial barrier function, while sunitinib does not. Such in vitro human model provides a significant complimentary approach to animal models for the development of effective therapies.

APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions.

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-β (TGF-β), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-β2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-β2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-β2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-β and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.

One typical mechanism to promote genomic instability, a hallmark of cancer, is to inactivate tumor suppressors, such as PALB2. It has recently been reported that mutations in PALB2 increase the risk of breast cancer by 8–9-fold by age 40 and the life time risk is ∼3–4-fold. To date, predicting the functional consequences of PALB2 mutations has been challenging as they lead to different cancer risks. Here, we performed a structure–function analysis of PALB2, using PALB2 truncated mutants (R170fs, L531fs, Q775X and W1038X), and uncovered a new mechanism by which cancer cells could drive genomic instability. Remarkably, the PALB2 W1038X mutant, harboring a mutation in its C-terminal domain, is still proficient in stimulating RAD51-mediated recombination in vitro, although it is unusually localized to the cytoplasm. After further investigation, we identified a hidden NES within the WD40 domain of PALB2 and found that the W1038X truncation leads to the exposure of this NES to CRM1, an export protein. This concept was also confirmed with another WD40-containing protein, RBBP4. Consequently, our studies reveal an unreported mechanism linking the nucleocytoplasmic translocation of PALB2 mutants to cancer formation.

The vascular barrier is an important function of the endothelium and its dysfunction is involved in several diseases. The barrier function of the endothelial cell monolayer is governed by cell-cell, cell-extracellular matrix (cell-ECM) contacts, and inflammatory factors such as thrombin, histamine or vascular endothelial growth factor. Several in vivo and in vitro assays that measure the vascular permeability induced by these factors have been developed. However, they suffer limitations such as being challenging for assessing details of biological processes at a cellular level or lacking the architecture of a vessel, that raise the need for new methods. In vitro 3D model-based assays have thus been developed but assays for investigating compounds that protects the barrier function are lacking. Here we describe the development of an in vitro three-dimensional (3D) vascular endothelium model in which we can manipulate the endothelial barrier function and permeability to molecules, which have a molecular weight similar to human serum albumin, allowing to assess the protective effect of compounds. A microvessel was prepared by culturing human umbilical vein endothelial cells (HUVECs) within a collagen gel on a polydimethylsiloxane (PDMS) chip. Using fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa, FITC-dextran) and confocal fluorescence microscopy, we showed that the microvessel presented an effective barrier function. We were then able to induce the loss of this barrier function by treatment with the inflammatory factor thrombin. The loss of barrier function was quantified by the extravasation of FITC-dextran into collagen matrix. Furthermore, we were able to analyze the protective effect on the endothelial barrier function of the cyclic adenosine monophosphate (cAMP) analog, 8-pCPT-2'-O-Me-cAMP (also called 007). In an attempt to understand the effects of thrombin and 007 in our model, we analyzed the adherens junctions and cytoskeleton through immunostaining of the vascular endothelial cadherin and actin, respectively. Our assay method could be used to screen for compounds modulating the barrier function of endothelial cells, as well as investigating mechanistic aspects of barrier dysfunction.

Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci −/− mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci−/− Fancd2−/− also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

A 3D in vitro microvessel model consisting of pericytes and endothelial cells which enables visualization of the multistep process of angiogenesis induced by VEGF.