H3K4 methylation by SETD1A/BOD1L facilitates RIF1-dependent NHEJ.

The 53BP1-RIF1-shieldin pathway maintains genome stability by suppressing nucleolytic degradation of DNA ends at double-strand breaks (DSBs). Although RIF1 interacts with damaged chromatin via phospho-53BP1 and facilitates recruitment of the shieldin complex to DSBs, it is unclear whether other regulatory cues contribute to this response. Here, we implicate methylation of histone H3 at lysine 4 by SETD1A-BOD1L in the recruitment of RIF1 to DSBs. Compromising SETD1A or BOD1L expression or deregulating H3K4 methylation allows uncontrolled resection of DNA ends, impairs end-joining of dysfunctional telomeres, and abrogates class switch recombination. Moreover, defects in RIF1 localization to DSBs are evident in patient cells bearing loss-of-function mutations in SETD1A. Loss of SETD1A-dependent RIF1 recruitment in BRCA1-deficient cells restores homologous recombination and leads to resistance to poly(ADP-ribose)polymerase inhibition, reinforcing the clinical relevance of these observations. Mechanistically, RIF1 binds directly to methylated H3K4, facilitating its recruitment to, or stabilization at, DSBs.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Notch signaling induces apoptosis in primary human CD34+ hematopoietic progenitor cells.

Notch signaling regulates diverse cell fate decisions during development and is reported to promote murine hematopoietic stem cell (HSC) self-renewal. The purpose of this study was to define the functional consequences of activating the Notch signaling pathway on self-renewal in human HSCs. Subsets of human umbilical cord blood CD34(+) cells were retrovirally transduced with the constitutively active human Notch 1 intracellular domain (N1ICD). N1ICD-transduced cells proliferated to a lesser extent in vitro than cells transduced with vector alone, and this was accompanied by a reduction in the percentage and absolute number of CD34(+) cell populations, including CD34(+)Thy(+)Lin(-) HSCs. Ectopic N1ICD expression inhibited cell cycle kinetics concurrent with an upregulation of p21 mRNA expression and induced apoptosis. Transduction of cells with HES-1, a known transcriptional target of Notch signaling and a mediator of Notch function, had no effect on HSC proliferation, indicating that the mechanism of the Notch-induced effect is HES-1-independent. The results of this study show that activation of the Notch signaling pathway has an inhibitory effect on the proliferation and survival of human hematopoietic CD34(+) cells populations. These findings have important implications for strategies aimed at promoting self-renewal of human HSCs.

Generation of a conditionally immortalized myeloid progenitor cell line requiring the presence of both interleukin-3 and stem cell factor to survive and proliferate.

The H-2Kappab temperature-sensitive (ts) A58 transgenic (Immorto) mouse has been used previously to generate conditionally immortalized cells from a number of tissues. The present study aimed to investigate characteristics of primitive myeloid precursor cells derived from H-2Kappab-tsA58 bone marrow. Cell populations were enriched for granulocyte/macrophage progenitors by centrifugal elutriation, and were cultured in the presence and absence of cytokines at the permissive and restrictive temperatures for the A58 oncogene. Cells derived from H-2Kappab-tsA58 mice required both A58 activation and the growth factors, stem cell factor (SCF) and interleukin-3 (IL-3), for long-term cell survival and growth; cells were maintained for > 300 d in culture under these conditions. IL-3- and SCF-dependent clonal cell lines were derived with a phenotype (lin-, Sca-1+, CD34+, ER-MP 58+, ER-MP 12+, ER-MP 20-) characteristic of primitive myeloid progenitors. These cells differentiated on addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) and acquired mature cell morphology with some upregulation of differentiation markers. In conclusion, the A58 oncogene can immortalize haemopoietic progenitor cells. These cells require two cytokines for growth, IL-3 and SCF; as such, they constitute a useful resource for the study of synergistic interactions between growth factors. The ability to develop monocytic cell characteristics also permits the investigation of cytokine-mediated early haemopoietic progenitor cell development.