Rationale Inactivating mutations in the FOXF1 gene locus are frequently found in patients with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACD/MPV), a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardio-vascular and gastro-intestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal and gall bladder morphogenesis. Objective While FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and ACD/MPV patients remain uncharacterized due to lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. Methods and Results A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia and vascular abnormalities in the lung, placenta, yolk sac and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited VEGF signaling and decreased expression of endothelial genes critical for vascular development, including VEGF receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2 and the non-coding RNA Fendrr. ChIP assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1 and Tie2 genes are direct transcriptional targets of FOXF1. Conclusions FOXF1 is required for formation of embryonic vasculature by regulating endothelial genes critical for vascular development and VEGF signaling.

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. IntroductionFanconi anemia (FA) is characterized by developmental defects, bone marrow (BM) failure, and higher predisposition to both hematologic and nonhematologic cancers. 1 The primary reason for morbidity and mortality in FA patients is progressive BM failure because of the depletion of hematopoietic stem cells. 1,2 Although BM transplant significantly reduces hematologic deficiencies and improves outcomes, FA patients still have a greater risk of developing myelodysplastic syndrome, acute myeloid leukemia, and other solid tumors, such as squamous cell carcinomas. [1][2][3] Diagnostic features of the disease are increased chromosomal breaks and hypersensitivity of FA cells to DNA interstrand cross-linking (ICL) agents. 1 FAis a genetically heterogeneous disease, comprising 15 complementation groups; the genes mutated in these groups have been identified. 3 Eight of the FA proteins (FANCA, -B,-C, -E, -F, -G, -L, and -M) and 5 associated factors (FAAP100, FAAP24, HES1, MHF1, and MHF2) form the FA nuclear core complex. The core complex is required for mono-ubiquitination of FANCD2-FANCI dimer on DNA damage, which results in activation of downstream DNA repair and tolerance reactions. 3,4 The downstream FA proteins include FANCD1/BRCA2, FANCJ/BACH1, FANCN/PALB2, FANCO/RAD51C, and FANCP/ SLX4, along with FA-associated proteins FAN1, RAD18, and RAD51. 3 Together, these proteins function in the "FA-BRCA" pathway, which facilitates DNA cross-link repair and coordinates other DNA damageresponsive events, thereby stabilizing stalled replication forks, conveying signals to DNA checkpoint pathways, and facilitating recovery of replication forks. 3,4 Discovery of several new members of the core complex in the past decade contributed much to the understanding of this pathway. Despite the isolation and characterization of several core complex members, a clear understanding of the core complex is far from clear. To better understand the functions of the core complex, it is necessary to isolate and characterize all core-complex proteins and associated subcomplexes. Our previous attempt to better define the composition of the core complex led to the discovery of FANCB, FANCL, FANCM, FAAP100, MHF1, and MHF2. [5][6][7][8][9] In this study, we report the isolation and characterization of a novel core complex protein, FAAP20. Methods Cloning and constructsThe pMIEG3 retroviral vector was used for protein expression in mammalian cells. 9 The pMYFP retroviral...

SummaryHelicases are involved in the metabolism of nucleic acid; this is very sensitive to the abiotic stresses that reduce plant growth and productivity. However, the molecular targets responsible for this sensitivity have not been well studied. Here we report on the isolation and characterization of cold-and salinity stress-induced pea DNA helicase 47 (PDH47). The transcript of PDH47 was induced in both shoots and roots under cold (4°C) and salinity (300 mM NaCl) stress, but there was no change in response to drought stress. Tissue-specific differential regulation was observed under heat (37°C) stress. ABA treatment did not alter expression of PDH47 in shoots but induced its mRNA in roots, indicating a role for PDH47 in both the ABA-independent and ABA-dependent pathways in abiotic stress. The purified recombinant protein (47 kDa) contains ATP-dependent DNA and RNA helicase and DNA-dependent ATPase activities. With the help of photoaffinity labeling, PDH47 was labeled by [a-32 P]-ATP. PDH47 is a unique bipolar helicase that contains both 3¢ to 5¢ and 5¢ to 3¢ directional helicase activities. Anti-PDH47 antibodies immunodeplete the activities of PDH47 and inhibit in vitro translation of protein. Furthermore, the PDH47 protein showed upregulation of protein synthesis. The activities of PDH47 are stimulated after phosphorylation by protein kinase C at Ser and Thr residues. Western blot analysis and in vivo immunostaining, followed by confocal microscopy, showed PDH47 to be localized in both the nucleus and cytosol. The discovery of cold-and salinity stress-induced DNA helicase should make an important contribution to a better understanding of DNA metabolism and stress signaling in plants. Its bipolar helicase activities may also be involved in distinct cellular processes in stressed conditions.

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Fanconi anemia (FA) is a genome-instability syndrome that has been associated with both cancer predisposition and bone-marrow failure. FA proteins are involved in cellular response to replication stress in which they coordinate DNA repair with DNA replication and cell cycle progression. One regulator of the replication stress response is the ATP-dependent DNA translocase FANCM which we have shown to be hyperphosphorylated in response to various genotoxic agents. However, the significance of this phosphorylation remained unclear. Here we show that genotoxic stress-induced FANCM phosphorylation is ATR dependent and that this modification is highly significant for the cellular response to replication stress. We identified serine (S1045) residue of FANCM that is phosphorylated in response to genotoxic stress and this effect is ATR dependent. We show that S1045 is required for FANCM functions including its role in FA pathway integrity, recruiting FANCM to the site of interstrand cross links, preventing the cells from entering mitosis prematurely, and efficient activation of the CHK1 and G2/M checkpoints. Overall, our data suggest that an ATR-FANCM feedback loop is present in the FA and replication-stress-response pathways, and that it is required for both efficient ATR/CHK1-checkpoint activation and FANCM function.