Analysis of the molecular function of ATM, ATR and FANCD2 during meiosis in Arabidopsis thaliana

In der Modellpflanze Arabidopsis thaliana hat sich die Erforschung von DNA-Reparatur und Rekombination bewährt, da genetische Veränderungen in diesen biochemischen Prozessen in der Pflanze selten zu Apoptose oder Blockierung des Zellzyklus führen. Zusätzlich werden die meisten humanen DNA-Reparaturgene auch im Arabidopsis-Genom kodiert, darunter auch solche, die z.B. in der Hefe fehlen. Eines dieser Gene ist AtFANCD2, das homologe Arabidopsis-Gen des humanen FANCD2 (Fanconi-Anämie D2). Fanconi-Anämie ist eine genetische Krankheit, die durch Chromosomen-Instabilität und körperliche Fehlbildungen gekennzeichnet wird. Als Antwort auf DNA-Schäden wird das Protein FANCD2 ubiquitinyliert und von den Checkpointkinasen ATM (Ataxia telangiectasia-mutiert) und ATR (ATM und Rad3-bezogen) phosphoryliert. Es konnte bereits gezeigt werden, dass sich das Protein zusammen mit anderen DNA-Reparaturproteinen in Foci im Zellkern befindet, allerdings ist die konkrete Aufgabe von FANCD2 in diesen Foci noch rätselhaft. Mutationen in den Arabidopsis-Genen AtFANCD2, AtATM oder AtATR beeinträchtigen die normale Entwicklung der Pflanzen nicht. Die Fertilität in mutierten Atatm-Pflanzen ist jedoch im Vergleich zu Wildtyp-Pflanzen auf ein Fünftel reduziert. Dieser Fertilitätsdefekt verschlimmert sich in Atatm Atfancd2 Doppelmutanten, und Atatm Atatr Doppelmutanten sind völlig steril. Interessanterweise wurde eine Beteiligung von FANCD2 in meiotischen Prozessen bisher noch nicht beobachtet. Im Einklang mit diesen Ergebnissen zeigte die zytologische Analyse von Pollenmutterzellen keine Veränderungen im Fortschreiten der Meiose bei Atfancd2 und Atatr Mutanten. Atatm und Atatm Atfancd2 Doppelmutanten erscheinen ebenfalls Wildtyp-ähnlich bis zum Pachytän, mit normaler Synapsis (ZYP1-Polymerisation) und Rekombination (RAD51-Foci-Bildung). In der weiteren meiotischen Entwicklung wird jedoch Chromosomen-Fragmentation sichtbar, wiederum verstärkt in der Atatm Atfancd2 Doppelmutante im Vergleich zu Atatm. Die homologen Chromosomen von Atatm Atatr Pollenmutterzellen paaren sich hingegen nicht, obwohl die Zahl der RAD51-Foci nicht reduziert scheint. Massive DNA-Fragmentierung in späteren meiotischen Stadien bedingt die Infertilität dieser Pflanzen. Zwei mögliche Ursachen für die beobachtete Chromosomen-Fragmentation scheinen wahrscheinlich, einerseits unreparierte DNA-Schäden aus premeiotischen Zellstadien, zweitens fehlerhafte Reparatur von Doppelstrangbrüchen (DSBs) der Meiose-spezifischen Nuklease SPO11. Das Einkreuzen einer Atspo11-2 Mutation in Pflanzen des Genotyps Atatm, Atatm Atfancd2 und Atatm Atatr unterdrückt diese Fragmentierung; folglich ist mangelhafte Reparatur von meiotischen DSBs der Grund für die Fertilitätsdefekte. Erstaunlicherweise konnten zahlreiche RAD51-Foci in Atatm Atatr Atspo11-2 Trippelmutanten detektiert werden, was möglicherweise auf premeiotische DNA-Schäden in diesem Genotyp hinweist. Anscheinend werden diese Brüche verlässlich in der Meiose repariert (wahrscheinlich über das Schwesterchromatid), wohingegen die Reparatur AtSPO11-induzierter Brüche von AtATM und AtATR abhängt. Das Einkreuzen einer Atrad51 Mutation in diesen genetischen Kontext soll ermöglichen, die Rolle von RAD51 in der Reparatur von SPO11-unabhängigen meiotischen DSBs zu beleuchten.Arabidopsis has proven to be a powerful organism to study DNA repair and recombination since most genes related to these processes are not essential (e.g. do not lead to apoptosis and cell cycle arrest). Furthermore, most of the DNA repair proteins present in humans are found in the genome of Arabidopsis, including those that are not found in yeast. One of these genes is AtFANCD2, the Arabidopsis homologue of human FANCD2 (Fanconi anemia D2). Fanconi anemia is a genetic disease characterized by chromosome instability and severe pathological conditions. Upon DNA damage the FANCD2 protein is known to be mono-ubiquitinated and phosphorylated by checkpoint kinases ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3 related). It was shown to co-localize together with DNA repair proteins and form nuclear foci, but the molecular function of the protein is still unclear. In Arabidopsis, mutations in AtFANCD2, AtATM or AtATR do not lead to obvious growth defects, but mutant Atatm plants show only 20% fertility when compared to wild type. This fertility defect is further exacerbated in Atatm Atfancd2 double mutants and complete sterility is observed in Atatm Atatr double mutants. A role of FANCD2 in meiosis has so far not been recognized. Consistent with these observations, cytological analysis of pollen mother cells (PMCs) shows wild type-like meiotic progression for Atfancd2 and Atatr. Atatm and the Atatm Atfancd2 double mutant appear normal until pachytene, showing regular synapsis (ZYP1-polymerization) and recombination (RAD51-foci formation), but in later stages chromosome fragmentation and anaphase bridges become visible, being more pronounced in the double mutant. In contrast, meiotic chromosomes in Atatm Atatr PMCs do not pair and synapse, but the number of AtRAD51 recombination foci are not reduced. In later meiotic stages of Atatm Atatr, massive DNA fragmentation accounts for the complete sterility of these plants.There are two possible explanations for the observed chromosome fragmentation in Atatm single and double mutants, namely persisting DNA damage from pre-meiotic stages or unrepaired DNA double-strand breaks generated by the meiosis-specific SPO11 nuclease. Introducing an Atspo11-2 mutation into the Atatm, Atatm Atfancd2 and Atatm Atatr plants reveals that DNA fragmentation is suppressed in all three plant lines. Interestingly, the recombinase AtRAD51 appears in numerous foci in an Atatm Atatr Atspo11-2 triple mutant, indicative for DNA lesions from pre-meiotic stages. Obviously, these breaks are reliably repaired during meiosis (most probably from the sister chromatid), whereas the breaks generated by AtSPO11 depend on AtATM and AtATR for repair during meiosis. Introducing an Atrad51 mutation into the afore-mentioned backgrounds will help us understand the requirement for repair of SPO11 independent DNA breaks during meiosis

ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesinSTAG1 from WAPL.

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity

Rapid movement and transcriptional re-localization of human cohesin on DNA

The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion

Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation

Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer, and reduced fertility. So far, 19 genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homolog of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here, we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE81 (AtMUS81), known for its role in the formation of noninterfering COs. AtFANCD2 promotes noninterfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously

Experimental and computational framework for a dynamic protein atlas of human cell division.

Essential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, the timing of interactions and changes in cellular structure are all crucial to ensure the correct assembly, function and regulation of protein complexes(1-4). Imaging of live cells can reveal protein distributions and dynamics but experimental and theoretical challenges have prevented the collection of quantitative data, which are necessary for the formulation of a model of mitosis that comprehensively integrates information and enables the analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries. Here we generate a canonical model of the morphological changes during the mitotic progression of human cells on the basis of four-dimensional image data. We use this model to integrate dynamic three-dimensional concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy(5). The approach taken here to generate a dynamic protein atlas of human cell division is generic; it can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types, and can be conceptually transferred to other cellular functions