In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.

SUMMARYRecQ helicases are a conserved group of proteins with a role in the maintenance of genome integrity. In Saccharomyces cerevisiae (budding yeast), meiotic recombination is increased in the absence of the RecQ helicase Sgs1. Here we investigated the potential meiotic role of the Sgs1 homologue AtRECQ4A and the closely related AtRECQ4B. Both proteins have been shown to function during recombination in somatic cells, but so far their meiotic role has not been investigated. Both AtRECQ4A and AtRECQ4B were expressed in reproductive tissues. Although immunolocalization studies showed that AtRECQ4A associates with recombination intermediates, we found no evidence that its loss or that of AtRECQ4B had a significant effect on meiotic cross-overs, suggesting functional redundancy with other RECQ family members. Nevertheless, pollen viability decreased in Atrecq4A, resulting in a reduction in fertility, although this was not the case in Atrecq4B. Cytological analysis revealed chromatin bridges between the telomeres of non-homologous chromosomes inAtrecq4A at metaphase I, in some instances accompanied by chromosome fragmentation at anaphase I. The bridges required telomeric repeats and were dependent on meiotic recombination. Immunolocalization confirmed the association of AtRECQ4A with the telomeres during prophase I, which we propose enables dissolution of recombination-dependent telomeric associations. Thus, this study has identified a hitherto unknown role for a member of the RECQ helicase family during meiosis that contributes to the maintenance of chromosome integrity. As telomere structure is generally conserved, it seems likely that these associations may arise during meiosis in other species, where they must also be removed.

Hematopoietic stem cell transplantation (HSCT) is often exploited to treat hematologic disease. Donor HSCs must survive, proliferate and differentiate in the damaged environment of the reconstituting niche. Illuminating molecular mechanisms regulating the activity of transplanted HSCs will inform efforts to improve HSCT. Here, we report that GPRASP proteins function as negative regulators of HSCT. Silencing of Gprasp1 or Gprasp2 increased the survival, quiescence, migration, niche retention and hematopoietic repopulating activity of hematopoietic stem and progenitor cells (HSPCs) post-transplant. We further show that GPRASP1 and GPRASP2 promote the degradation of CXCR4, a master regulator of HSC function during transplantation. CXCR4 accumulates in Gprasp-deficient HSPCs, boosting their function post-transplant. Thus, GPRASPs negatively regulate CXCR4 stability in HSCs. Our work reveals GPRASP proteins as negative regulators of HSCT and CXCR4 activity. Disruption of GPRASP/CXCR4 interactions could be exploited in the future to enhance the efficiency of HSCT.

Although hematopoietic stem cell transplantation (HSCT) is used routinely to cure hematologic disease, the efficacy of transplantation is limited by the paucity of HSC. One way to overcome this is to increase the efficiency of HSC engraftment. Thus, we executed a functional screen for novel regulators of HSCT. Murine HSC were lentivirally transduced with shRNAs targeting prioritized gene candidates prior to transplantation into cohorts of lethally irradiated recipient mice. In total, around 1300 mice were transplanted to assess a putative role for about 50 genes in HSCT. We thereby identified Gprasp2 and Armcx1 as putative negative regulators of HSCT. When transplanted at a 1:4 disadvantage relative to control, recipients of either Gprasp2 or Armcx1 shRNA-treated CD45.2 Lineage- Sca-1+ c-Kit+ (LSK) cells displayed 3.12 (p=0.024) and 2.8 (p=0.04) fold enhanced CD45.2 chimerism in peripheral blood (PB) at 16 weeks post-transplant, respectively, relative to mice transplanted with CD45.2 LSK cells treated with control shRNAs. Although loss of each gene did not favor a particular PB lineage, CD45.2 chimerism was enhanced in all bone marrow (BM) HSC and progenitor (HSPC) compartments in these recipients, correlating with their enhanced PB chimerism. qRT-PCR reveals that both murine Armcx1 and Gprasp2 are highly enriched for expression in LSK CD150+CD48- cells relative to all downstream hematopoietic progeny. Further, HemaExplorer, a bioinformatics database of human hematopoietic gene expression, suggests that GPRASP2 and ARMCX1 are also highly expressed in human HSC. This prediction is currently being validated by qRT-PCR. Interestingly, Gprasp2 and Armcx1 both belong to the G protein-coupled receptor associated sorting protein (GASP) gene family, which has never before been implicated in HSC function. The closely related GASP family member, Gprasp1, sorts G protein-coupled receptors (GPCR) to lysosomes for degradation. As Gprasp1 and Gprasp2 both contain GPCR-binding domains and ~70% amino acid sequence conservation in their C-termini, Gprasp2 may also regulate GPCR trafficking and degradation in HSC. Although Gprasp1 was not tested in our screen, qRT-PCR analysis reveals that it is also highly expressed by murine HSC relative to downstream progeny, suggesting that it too may play a role in HSC function. We are currently assessing this using Gprasp1-shRNAs and competitive transplantation. In contrast, Armcx1 lacks the GPCR binding domain and contains both a nuclear and mitochondrial localization signal and has been shown to localize to mitochondrial networks when expressed in HEK-293 cells, suggesting a role in mitochondrial/nuclear communication. To determine how loss of Gprasp2 and Armcx1 promotes HSC engraftment, we are currently employing transplantation and ex vivo culture assays to analyze the effect of their loss on cell cycle, apoptosis, migration, and adhesion of HSPC post-transplant. Our work may help elucidate the mechanisms underlying efficient engraftment, adhesion, and retention of HSPC in the BM niche, which in turn may shed light on novel pathways that could be targeted to promote the efficiency of HSCT in the clinic. Disclosures No relevant conflicts of interest to declare.

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