Connecting double strand breaks and synaptonemal complex assembly in C.elegans

Das C.elegans him-19 Gen wurde als ein Faktor identifitziert, der in der frühen meiotischen Prophase-1 benötigt wird. Zytologische Beobachtungen zeigten, dass mutierte him-19 Würmer wahrscheinlich fehlerhaft in der Induktion von DNS Doppelstrang-Brüchen (DSB) und im Aufbau des synaptonemalen Komplexes (gemessen an der Polymerisation von SYP-1) sind, welche teilweise durch künstlich induzierte DSBs via gamma-Betrahlung wieder hergestellt werden können. Im Gegensatz dazu wurde berichtet, dass die Induktion von DSB –die Voraussetzung für Rekombination- und der Aufbau des synaptonemalen Komplexes (SC) zwischen den Chromosomen unabhängig voneinander stattfinden in der Meiose von Wildtyp C.elegans. Dennoch zeigten C.elegans him-19 Mutanten, dass DSB und Aufbau des SC in einem neuen Pfad verbunden werden können. Der genetische Hintergrund von him-19 erlaubt mir die Rolle von Rekombinations-Zwischenprodukten in der Förderung oder Hemmung der Polymerisation von SYP-1 zu untersuchen. C.elegans Doppel-Mutanten, welche in verschiedenen Schritten der DSB-Prozessierung beeinträchtigt und hauptsächlich an homologer Rekombination (mre-11, rad-51, msh-5 and zhp-3) und Chromosomen-Bewegung (sun-1) beteiligt sind, zeigten eingeschränkte SYP-1-Polymerisation in einem Hintergrund mit fehlerhaftem him-19 Gen. Auch wenn künstlich induzierte DSB teilweise die Lokalisation von SYP-1 wieder herstellen, umgehen diese nicht die Notwendigkeit von MRE-11, RAD-51 und MSH-5 um die Polymerisation von SYP-1 in der Abwesenheit von HIM-19 einzuleiten. Interressanterweise polymerisiert SYP-1 durch Betrahlung wesentlich ausgedehnter in him-19 zhp-3 verglichen zu him-19 Mutanten. Folglich deckte meine Arbeit eine neue Aktivität des ZHP-3 Proteins in der Beschränkung der SC-Polymerisation auf. Im Gegensatz zu der verbesserten Polymerisation von SYP-1 in bestrahlten him-19 Mutanten zeigten FISH Analysen, dass die homologe Chromosomen-Paarung durch Bestrahlung nicht erhöht werden konnte , was wiederum auf nicht-homologe Synapse oder SC Polymerisation auf individuellen Chromosomenachsen hinweist.The C.elegans him-19 gene is required in early meiotic prophase 1. Cytological observations showed that him-19 mutant worms are likely deficient in double strand break (DSB) induction and synaptonemal complex (SC) formation (as evidenced by SYP-1 polymerization), which can be partially rescued by artificially induced breaks by γ-irradiation. In contrast, DSB induction –the prerequisite for recombination- and synaptonemal complex assembly between chromosomes were reported to take place independently in wild-type C.elegans meiosis. However, the C.elegans him-19 mutant revealed that DSBs and synaptonemal complex assembly can be connected through a new pathway and the him-19 genetic background allowed me to investigate the role of recombination intermediates promoting or inhibiting SYP-1 polymerization. C.elegans double mutants affected in different steps of DSB-processing, mostly concerning the homologous recombination pathway (mre-11, rad-51, msh-5 and zhp-3) and chromosome movement (sun-1) showed restricted SYP-1 polymerization in the him-19 deficient background. Even though artificially induced DSBs partially restore SYP-1 localization onto chromosomes in him-19, they do not circumvent the need of MRE-11, RAD-51 and MSH-5 for triggering SYP-1 polymerization in the absence of HIM-19. Interestingly, SYP-1 polymerized more extensively in irradiated him-19 zhp-3 when compared to him-19 single mutants. Thus my work uncovered a novel activity of the ZHP-3 protein in restricting SC polymerization. In contrast to enhanced SYP-1 polymerization in irradiated him-19 mutants, homologous pairing of chromosomes was not augmented upon irradiation as assessed by FISH analyses, indicating non-homologous synapsis or SC polymerization onto individual chromosome axes

Leptotene/Zygotene Chromosome Movement Via the SUN/KASH Protein Bridge in Caenorhabditis elegans

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2–dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates

HSF1 mediated stress response of heavy metals.

The heat shock response (HSR) pathway is a highly conserved cellular stress response and mediated by its master regulator HSF1. Activation of the pathway results in the expression of chaperone proteins (heat shock proteins; HSP) to maintain protein homeostasis. One of the genes strongest upregulated upon stress is HSPA1A (HSP72). Heavy metals are highly toxic to living organisms and known as environmental contaminants, due to industrialisation. Furthermore, many of them are well-described inducers of the HSR pathway. Here we compare the effect of different heavy metals, concerning their potential to activate HSF1 with a sensitive artificial heat shock reporter cell line, consisting of heat shock elements (HSE). In general the responses of the artificial promoter to heavy metal stress were in good agreement with those of well-established HSF1 target genes, like HSPA1A. Nevertheless, differences were observable when effects of heat and heavy metal stress were compared. Whereas heat stress preferentially activated the HSE promoter, heavy metals more strongly induced the HSPA1A promoter. We therefore analysed the HSPA1A promoter in more detail, by isolating and mutating the HSEs. The results indicate that the importance of the individual binding sites for HSF1 is determined by their sequence similarity to the consensus sequence and their position relative to the transcription start site, but they were not differentially affected by heat or heavy metal stress. In contrast, we found that other parts of the HSPA1A promoter have different impact on the response under different stress conditions. In this work we provide deeper insights into the regulation of HSP72 expression as a well as a method to quantitatively and sensitively evaluate different stressor on their potential to activate HSF1

Mutations in Caenorhabditis elegans him-19 Show Meiotic Defects That Worsen with Age

Faithful meiotic chromosome segregation requires pairing, synapsis and recombination of homologous chromosomes. In mammals, chromosomal non-disjunction increases with age. A mutation in Caenorhabditis elegans him-19 mimics these age-dependent chromosome segregation defects and might therefore further our understanding of this phenomenon