In budding yeast, inactivation of telomerase and ensuing telomere erosion cause relocalization of telomeres to nuclear pore complexes (NPCs). However, neither the mechanism of such relocalization nor its significance are understood. We report that proteins bound to eroded telomeres are recognized by the SUMO (small ubiquitin-like modifier)-targeted ubiquitin ligase (STUbL) Slx5-Slx8 and become increasingly SUMOylated. Recruitment of Slx5-Slx8 to eroded telomeres facilitates telomere relocalization to NPCs and type II telomere recombination, a counterpart of mammalian alternative lengthening of telomeres (ALT). Moreover, artificial tethering of a telomere to a NPC promotes type II telomere recombination but cannot bypass the lack of Slx5-Slx8 in this process. Together, our results indicate that SUMOylation positively contributes to telomere relocalization to the NPC, where poly-SUMOylated proteins that accumulated over time have to be removed. We propose that STUbL-dependent relocalization of telomeres to NPCs constitutes a pathway in which excessively SUMOylated proteins are removed from "congested" intermediates to ensure unconventional recombination.

DAP12 is an ITAM-containing adaptor molecule conveying activating properties to surface receptors on many cell types. We show here that DAP12 paradoxically down-modulates plasmacytoid dendritic cell (pDC) cytokine production in vivo during murine CMV (MCMV) infection. Higher levels of IFN-αβ and IL-12 were detected upon MCMV infection or CpG treatment in DAP12-deficient (DAP12°) mice as compared with wild-type (WT) mice. This resulted from altered homeostasis and enhanced responsiveness of pDCs in DAP12° animals. Increased numbers of pDCs were observed in the periphery of both naive and MCMV-infected DAP12° mice. A higher proportion of pDCs was activated in infected DAP12° mice, as demonstrated by intracellular staining using an optimized protocol for simultaneous detection of IFN-α and IFN-β. The homeostasis of WT and DAP12° pDCs did not differ in mixed bone marrow chimeric mice. In addition, a similar efficiency of pDC differentiation was observed in vitro in Fms-like tyrosine kinase receptor 3 ligand cultures of WT and DAP12° bone marrow cells. This suggests that DAP12 signaling effects on pDC homeostasis are indirect. In contrast, in response to CpG, DAP12-mediated effects on both IL-12 and IFN-αβ production were intrinsic to the pDCs. However, in response to MCMV, only IL-12 but not IFN-αβ production was affected by pDC-intrinsic DAP12 signaling. Thus, DAP12 signaling in pDCs can mediate different regulatory effects on their functions, depending on the mechanisms of pDC activation. The potential implications of the regulation of pDC functions by DAP12 for promoting health over disease are discussed.

Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1–3-like tracts present between subtelomeric X and Y′ elements, which is followed by BIR-mediated non-reciprocal translocation of Y′ element and terminal TG1–3 repeats from the donor end onto the shortened telomere. We found that choice of the Y′ donor was not random, since both engineered telomere VII-L and native VI-R acquired Y′ elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y′ translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1–3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y′ translocation events taking place during presenescence are genetically separable from Rad51-dependent Y′ amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y′ translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.

Summary Functional telomeres in yeast lacking telomerase can be restored by rare Rad51- or Rad59-dependent recombination events that lead to type I and type II survivors, respectively. We previously proposed that polySUMOylation of proteins and the SUMO-targeted ubiquitin ligase Slx5-Slx8 are key factors in type II recombination. Here, we show that SUMOylation of Rad52 favors the formation of type I survivors. Conversely, preventing Rad52 SUMOylation partially bypasses the requirement of Slx5-Slx8 for type II recombination. We further report that SUMO-dependent proteasomal degradation favors type II recombination. Finally, inactivation of Rad59, but not Rad51, impairs the relocation of eroded telomeres to the Nuclear Pore complexes (NPCs). We propose that Rad59 cooperates with non-SUMOylated Rad52 to promote type II recombination at NPCs, resulting in the emergence of more robust survivors akin to ALT cancer cells. Finally, neither Rad59 nor Rad51 is required by itself for the survival of established type II survivors.

Introduction Somatic mutations acquired in key signalling pathway, transcription factor, spliceosome, epigenetic and tumor suppressor genes are of central importance in the development and progression of myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To date, in order to evaluate all relevant genetic alterations, multiple tests are needed, requiring large amounts of DNA. As tests are typically performed sequentially this unnecessarily extends the time between sample acquisition and mutation detection. The QIAact Myeloid DNA UMI Panel in combination with the QIAGEN GeneReader NGS System provides a single solution to simultaneously test for actionable mutations, whilst also saving sample material (only 40ng DNA input required per sample), shortening test time and enabling simplification of lab operations. The QIAact Myeloid DNA UMI Panel is a multi-gene targeted sequencing panel designed to detect complex mutations throughout the most informative genes linked to myeloid disease. This allows reliable and sensitive detection of single nucleotide variants (SNV) and large Insertion/Deletion (InDel) mutations. Methods The QIAact Myeloid DNA UMI Panel targets 25 genes known to be important in myeloid leukemia. A key feature of the panel is the addition of a unique molecular index (UMI) to tag each individual original DNA molecule prior to target enrichment by PCR. UMIs enable sequencing and PCR bias corrections, allowing sensitive detection of mutations. To assess the assay performance, reference standards and blood and bone marrow samples were used. Following target enrichment, libraries were sequenced on the GeneReader NGS System and mutations analyzed using the QIAGEN Clinical Insight (QCI) Analyze software suite. Results To confirm DNA mutation detection, Horizon Discovery and SeraCare Reference Standards containing variants typical of myeloid malignancies were used. The anticipated DNA mutations were consistently identified both within and between runs. The samples used, including blood and bone marrow samples, demonstrated the ability of the assay to detect important large indels (52 bp deletion CALR type 1 variant) and key SNVs down to a minor allele fraction (MAF) of 1% for JAK2 (e.g. exon 12, 13, 14 & 15) and KIT (exon 8, 9, 10, 11 & 17). A sensitive variant detection of allele frequency <0.5% for the KIT D816V was also achieved. For the other genes covered by the panel, including ASXL1, RUNX1, NPM1, DNMT3A, IDH1/2, results show sufficiently uniform amplification and sequencing coverage to support mutation detection with a MAF of 5%. Conclusion The QIAact Myeloid DNA UMI Panel in combination with the QIAGEN GeneReader NGS System offer a fully integrated DNA to variant detection and interpretation solution. The optimized chemistry allows superior analytical sensitivity resulting in accurate and efficient mutation detection of highly relevant genetic alterations for myeloid malignancy research. Disclosures Laloux: QIAGEN France S.A.S: Employment. Biglia:HalioDx: Employment. Bona:HalioDx: Employment. Lafi:HalioDx: Employment. Charifi:HalioDx: Employment. Larsen:QIAGEN Aarhus: Employment. Lueerssen:QIAGEN Manchester Ltd: Employment. Gupta:QIAGEN Aarhus: Employment. Lauber:QIAGEN GmbH: Employment. Hughes:QIAGEN Manchester Ltd: Employment.