Impaired Replication Stress Response in Cells from Immunodeficiency Patients Carrying Cernunnos/XLF Mutations

Non-Homologous End Joining (NHEJ) is one of the two major pathways of DNA Double Strand Breaks (DSBs) repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(D)J recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(D)J recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis

Mammalian Cdh1/Fzr mediates its own degradation

The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time

Topoisomerase 1-dependent R-loop deficiency drives accelerated replication and genomic instability.

DNA replication is a complex process tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here, we show that mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression, causing aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results dissect a mechanism of oncogene-induced DNA damage by aberrant replication fork acceleration

Selective Elimination of Human Pluripotent Stem Cells by an Oleate Synthesis Inhibitor Discovered in a High-Throughput Screen

SummaryThe use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns), nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn #1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs. PluriSIn #1 was also cytotoxic to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state. Finally, application of PluriSIn #1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments

LRRC6 mutation causes primary ciliary dyskinesia with dynein arm defects.

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects

Cernunnos/XLF mutation does not affect replication rate.

<p>A. Example of a DNA fiber showing the labeled IdU pulse (green signals and arrows) and the labeled CldU pulse (red signals and arrows). The estimated origin of replication is marked by a black arrow. B. Distribution of fork rate in untreated Cernunnos/XLF mutated and complemented cells. The data presented is based on two independent experiments. In each experiment at least 50 molecules each containing at least two replication signals were analyzed for each cell line.</p

Cernunnos/XLF mutation leads to increased common fragile site instability.

<p>A. Examples of metaphases from Cernunnos/XLF mutated cells. A cell treated with 0.2 µM aphidicolin for 24 hours, showing a high number of gaps and constrictions (n = 28). The box in the top left is a magnification of the area marked in the picture. Arrows mark gaps and constrictions (top panel). An untreated cell showing no gaps and constrictions (bottom panel). B. Examples of metaphases from Cernunnos/XLF complemented cells. A cell treated with 0.2 µM aphidicolin for 24 hours, showing an average number of gaps and constrictions (n = 6). Arrows mark gaps and constrictions (top panel). An untreated cell showing no gaps and constrictions (bottom panel). C. Number of gaps and constrictions per metaphase in Cernunnos/XLF mutated and complemented cells with or without treatment with 0.2 µM aphidicolin for 24 hours. The data presented is based on two independent experiments. For each experiment, at least 50 metaphases for each condition were analyzed. D. Frequency of chromosomal gaps and constrictions at common fragile sites (FS) FRA3B and FRA16D following treatment with 0.2 µM aphidicolin for 24 hours in Cernunnos/XLF mutated and complemented cells. Bars represent mean and standard error of two independent experiments. For each experiment, at least 50 hybridizations were analyzed.</p

The Cernunnos/XLF mutation lead to an increased activation of Chk1 in response to replication stress.

<p>A. Cernunnos/XLF mutated and complemented cells were treated with 0.5 µM aphidicolin for 24 hours. Protein extracts were prepared and analyzed by immunoblotting with anti- pChk1(S317) antibodies. Untreated cells were analyzed as control. B. Quantification of pChk1(S317) levels in Cernunnos/XLF mutated and complemented cells following 0.5 µM aphidicolin treatment for 24 hours. pChk1(S317) level was normalized to Tubulin control.</p

Cernunnos/XLF mutated cells exhibit Increased accumulation of cells in G2/M following treatment with low aphidicolin concentrations.

<p>A. Cernunnos/XLF mutated and complemented cells were treated with the indicated aphidicolin concentrations for 24 hours, fixed, stained with propidium iodide and analyzed by FACS for DNA content. B. Quantification of the percentage of cells in G2/M following aphidicolin treatment. Bars represent mean and standard error of three independent experiments. The dashed line represents the approximate percentage of cells in G2/M in untreated cells.</p