Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.

Objective The transcription factor PU.1 (encoded by Sfpi1) promotes myeloid differentiation but it is unclear what downstream genes are involved. MiRNAs are a class of small RNAs that regulate many cellular pathways including proliferation, survival and differentiation. The objective of this study was to identify miRNAs downstream of PU.1 that regulate hematopoietic development. Materials and Methods MiRNAs that change expression in a PU.1-inducible cell line were identified with microarrays. The promoter for a miRNA cluster upregulated by PU.1 induction was analyzed for PU.1 binding by electrophoretic mobility shift and chromatin immunoprecipitation assays. Retroviral transduction of hematopoietic progenitors was performed to evaluate the effect of miRNA expression on hematopoietic development in vitro and in vivo. Results We identified a miRNA cluster whose pri-transcript is regulated by PU.1. The pri-miRNA encodes three mature miRNAs: miR-23a, miR-27a, and miR-24-2. Each miRNA is more abundant in myeloid cells compared to lymphoid cells. When hematopoietic progenitors expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed a dramatic decrease in B lymphopoiesis and an increase in myelopoiesis compared to control cultures. In vivo, hematopoietic progenitors expressing the miR-23a cluster generate reduced numbers of B cells compared to control cells. Conclusions The miR-23a cluster is a downstream target of PU.1 involved in antagonizing lymphoid cell fate acquisition. Although miRNAs have been identified downstream of PU.1 in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in regulating the development of myeloid versus lymphoid cells.

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.

BackgroundProper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5′ endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism.MethodsWe hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells.ResultsOur data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival.ConclusionThis study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0912-8) contains supplementary material, which is available to authorized users.

Rodent incisors provide a classic model for studying epithelial-mesenchymal interactions in development. However, postnatal stem/progenitor cells in rodent incisors have not been exploited for tooth regeneration. Here, we characterized postnatal rat incisor epithelium and mesenchyme stem/progenitor cells and found that they formed enamel- and dentin-like tissues in vivo. Epithelium and mesenchyme cells were harvested separately from the apical region of postnatal 4–5 day rat incisors. Epithelial and mesenchymal phenotypes were confirmed by immunocytochemistry, CFU assay and/or multi-lineage differentiation. CK14+, Sox2+ and Lgr5+ epithelium stem cells from the cervical loop significantly enhanced amelogenin and ameloblastin expression upon BMP4 or FGF3 stimulation, signifying their differentiation towards ameloblast-like cells, whereas mesenchyme stem/progenitor cells upon BMP4, BMP7 and Wnt3a treatment robustly expressed Dspp, a hallmark of odontoblastic differentiation. We then control-released microencapsulated BMP4, BMP7 and Wnt3a in transplants of epithelium and mesenchyme stem/progenitor cells in the renal capsule of athymic mice in vivo. Enamel and dentin-like tissues were generated in two integrated layers with specific expression of amelogenin and ameloblastin in the newly formed, de novo enamel-like tissue, and DSP in dentin-like tissue. These findings suggest that postnatal epithelium and mesenchyme stem/progenitor cells can be primed by pivotal signals towards bioengineered tooth regeneration.