RNAi screen to identify SUN-1 interaction partners in meiosis of Caenorhabditis elegans

Meiose (vom grich. meionon = vermindern ) bezeichnet eine spezielle Form der Zellteilung, durch die es zur Halbierung des ursprünglichen Chromosomensatzes kommt. Diese Reduktionsteilung dient, sich geschlechtlich vermehrenden Organismen zur Erhaltung der Genomgröße über Generationen und der Durchmischung des Erbgutes. Diese Variation, fungiert als Motor der Evolution, da die Anpassungsfähigkeit an veränderte Habitate und Umweltbedingungen gefördert und die Wahrscheinlichkeit für ein Überleben der gesamt Population stark erhöht wird. Die Meiose lässt sich grob in eine prä-meiotische Duplikation und zwei meiotische Teilungen gliedern, wobei die zweite meiotische Teilung grundsätzlich einer mitotischen Teilung entspricht. Anschließend an die Duplikation des Genoms, tritt die Zelle in die sogenannte Prophase der ersten meiotischen Teilung ein. Die wesentlichen Ziele dieser Phase sind die Paarung der homologen Chromosomen und der Austausch genetischen Materials mittels „crossingover“. Die Paarung von Homologen verlangt die gerichtete Bewegung, Erkennung und Synapse der Chromosomen. Dies wird durch das nukleäre Transmembranprotein SUN-1 gefördert. SUN-1 bildet gemeinsam mit ZYG-12 eine Brücke vom Zytoskelett zu den Chromosomen und übermittelt so die zytosolischen Bewegungen in das innere des Zellkerns. Diese Diplomarbeit war der Suche nach potentiellen Interaktionspartnern von SUN-1 gewidmet. Hierfür wurden 39 Kandidaten, welche zuvor in einem Präzipitat mit SUN-1 gefunden wurden, auf eine mögliche Rolle in der Meiose untersucht. Die Genprodukte wurden mittels RNA Interferenz depletiert und die Folgen dieser Depletierung, auf die Meiose, untersucht. Als Modellorganismus wurde der Fadenwurm Caenorhabditis elegans gewählt, da dieser, neben grundlegenden Vorteilen als Modellorganismus, durch den großen Anteil an meiotischen Kernen und ihre außergewöhnliche Anordnung im adulten Tier, einen optimalen Einblick in den zeitlichen Ablauf der Meiose erlaubt.Meiosis (from the greek meionon = decrease) refers to a specialized form of cell division by which the initial set of chromosomes is halved. This reductive division is used by sexually reproducing organisms, in order to maintain genome size over generations and exchange genetic material. The variation of the genome functions as a motor of evolution. Since the ability to adapt to changing habitats and environmental conditions increases and therefore, survival of the total population is greatly increased. Meiosis can be divided into a pre-meiotic duplication and two meiotic divisions, whereas the second meiotic division resembles a mitotic division. Subsequent to the duplication of the genome, the cell enters prophase of the first meiotic division. The main objectives of this phase are the pairing of homologous chromosomes and the exchange of genetic material by "crossing over". The pairing of homologs requires directed movement, recognition and synapsis of chromosomes. The nuclear trans membrane protein SUN-1 supports this essential movement. SUN- 1, together with ZYG-12, forms a bridge between the cytoskeleton and the chromosomes and therefore transmits cytoplasmic forces into the interior of the nucleus. This thesis was devoted to the search for potential interaction partners of SUN-1. For this purpose 39 candidates that were previously found in a precipitate with SUN-1, were examined for a possible role in meiosis. The gene products were depleted by RNA interference and the consequences of this depletion, examined. We chose Caenorhabditis elegans as a model organism. Because, in addition to its fundamental advantages as a model organism, it allows the observation of meiotic stages due to the temporal and special order of germ cells in the gonad

An auto-regulatory module controls fat metabolism in "Caenorhabditis elegans"

Obesity and obesity-related diseases such as type-2 diabetes or metabolic syndrome are on the rise word-wide (Ng et al. 2014). The negative aspects of obesity on the health of individuals is accompanied by an increasing financial burden for the global economy. Excess adipose tissue is often perceived as an indication of poor dietary choices and a sedentary lifestyle. However, compelling evidence from diverse model organisms and humans suggest that genetic make-up influences most aspects of fat metabolism and therefore the likelihood to develop obesity (Min, Chiu, and Wang 2013; Yazdi, Clee, and Meyre 2015). Hence, careful dissection of the underlying genetic regulations of fat metabolism to identify new putative targets for treatment is essential. We successfully used Caenorhabditis elegans as a model organism to uncover and describe a novel auto-regulatory module, which we found is essential for wild-type levels of body fat. Utilizing cold-sensitivity as readout, we identified a hitherto uncharacterized gene, C30F12.1, which we named rege-1 (REGnasE-1 homolog) after its mammalian homolog the PIN-domain endonuclease ZC3H12A/Regnase-1/MCPIP1. Regnase-1 negatively regulates pro-inflammatory cytokines via internal cleavage of their 3` untranslated region (3`UTR) (Iwasaki et al. 2011; Matsushita et al. 2009). Similarly, C. elegans REGE-1 targets the transcription factor ETS-4 and cleaves its mRNA within the first third of its 3`UTR. Knockout of rege-1 causes a strong loss in overall body fat, developmental delay and a transcriptional upregulation of fat metabolic and innate immunity genes, depending on ETS-4. We also provide evidence that ETS-4 transcriptionally activates (directly or indirectly) rege-1, thereby forming an auto-regulatory feedback loop. REGE-1::GFP is mainly localized in the first four cells of the intestine adjacent to the pharynx and the ETS-4/REGE-1 module is transcriptionally upregulated upon re-feeding following long-term starvation. This, together with the observation that metabolic and immunity genes are induced upon loss of rege-1 suggests that the ETS-4/REGE-1 module might have a role in bacteria clearance, nutrient uptake and/or defense against pathogens

Matefin/SUN-1 Phosphorylation Is Part of a Surveillance Mechanism to Coordinate Chromosome Synapsis and Recombination with Meiotic Progression and Chromosome Movement

<div><p>Faithful chromosome segregation during meiosis I depends on the establishment of a crossover between homologous chromosomes. This requires induction of DNA double-strand breaks (DSBs), alignment of homologs, homolog association by synapsis, and repair of DSBs via homologous recombination. The success of these events requires coordination between chromosomal events and meiotic progression. The conserved SUN/KASH nuclear envelope bridge establishes transient linkages between chromosome ends and cytoskeletal forces during meiosis. In <i>Caenorhabditis elegans</i>, this bridge is essential for bringing homologs together and preventing nonhomologous synapsis. Chromosome movement takes place during synapsis and recombination. Concomitant with the onset of chromosome movement, SUN-1 clusters at chromosome ends associated with the nuclear envelope, and it is phosphorylated in a <i>chk-2-</i> and <i>plk-2</i>-dependent manner. Identification of all SUN-1 phosphomodifications at its nuclear N terminus allowed us to address their role in prophase I. Failures in recombination and synapsis led to persistent phosphorylations, which are required to elicit a delay in progression. Unfinished meiotic tasks elicited sustained recruitment of PLK-2 to chromosome ends in a SUN-1 phosphorylation–dependent manner that is required for continued chromosome movement and characteristic of a zygotene arrest. Furthermore, SUN-1 phosphorylation supported efficient synapsis. We propose that signals emanating from a failure to successfully finish meiotic tasks are integrated at the nuclear periphery to regulate chromosome end–led movement and meiotic progression. The single unsynapsed X chromosome in male meiosis is precluded from inducing a progression delay, and we found it was devoid of a population of phosphorylated SUN-1. This suggests that SUN-1 phosphorylation is critical to delaying meiosis in response to perturbed synapsis. SUN-1 may be an integral part of a checkpoint system to monitor establishment of the obligate crossover, inducible only in leptotene/zygotene. Unrepaired DSBs and unsynapsed chromosomes maintain this checkpoint, but a crossover intermediate is necessary to shut it down.</p> </div

Brood size, hatch rate, and X chromosome nondisjunction of <i>sun-1</i> phosphosite mutants.

*<p><i>p</i><0.001 between wild type and the respective mutant in two-tailed <i>t</i>-test.</p><p>Variations correspond to the standard deviation. Data were assessed over the complete self-fertile period of hermaphrodites at 20°C. <i>n</i>, number of hermaphrodites scored.</p

Length of SUN-1 phosphorylation in wild-type and mutant backgrounds.

<p>Dissected gonads were measured from meiotic entry (TZ) to beginning of cellularization. Relative percentage of cell rows with SUN-1 phosphorylation was assessed and normalized to the length of the meiotic gonad from meiotic entry to cellularization. When >50% of nuclei in a cell row were phosphorylated on SUN-1, it was counted as phosphorylated. Variations correspond to the standard deviation. <i>p</i> values indicate comparison of percentage of cell rows with SUN-1 phosphorylation between wild type and the respective mutant in a two-tailed <i>t</i>-test. <i>n</i>, number of hermaphrodites scored; WT, wild type.</p

Effect of <i>sun-1</i> phosphosite mutations on the duration of meiotic stages, DSB turnover, SUN-1 aggregates, chromosome movement, and synapsis.

<p>(A) TZ nuclei of <i>sun-1(wt); sun-1(ok1282</i>) (top), and <i>sun-1(allA); sun-1(ok1282)</i> (bottom) hermaphrodite gonads stained with DAPI (left; blue in merged picture) and anti-GFP (middle; green in merged picture). Scale bars, 10 µm. (B) First frame of <i>in vivo</i> time-lapse GFP-recorded TZ nuclei of <i>sun-1(wt); sun-1(ok1282)</i> (top, left) and <i>sun-1(allA); sun-1(ok1282)</i> (bottom, left) hermaphrodite gonads. Displacement tracks of SUN-1 aggregates represent 2D plotted chromosome end movements over 3 min (right). Average speed of SUN-1 aggregates in TZ (<i>n</i> = 9 nuclei, followed over 3 mins). Scale bar, 2 µm. (C) Schematic of a wild-type hermaphrodite gonad with nuclei in the corresponding zones, as used in the quantifications in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003335#pgen-1003335-g003" target="_blank">Figure 3D</a>: chromatin (blue) and SUN-1 morphology (green). Gonad is subdivided into ten zones, as used for the RAD-51 foci quantification in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003335#pgen-1003335-g003" target="_blank">Figure 3E</a>. Meiotic entry and beginning of cellularization are indicated. (D) Representation of relative time of residency in different meiotic stages, as assessed by presence of SUN-1 aggregates in different mutant backgrounds, normalized to the length of the gonad (from meiotic entry [0%] to start of meiocyte cellularization at diplotene [100%]). Nuclei were sorted into three categories: “TZ” (dark green, more than one SUN-1 aggregate), “early pachytene” (light green, one SUN-1 aggregate), and “late pachytene” (red, no SUN-1 aggregate). Categories were assigned once ≥50% of nuclei in a cell row fulfilled one of these criteria. At least eight gonads were counted per genotype. (E) Average numbers of RAD-51 foci per nucleus in the hermaphrodite gonad of different <i>sun-1</i> phosphosite mutants. Gonads were divided into ten zones, as schematically indicated in (B). Three gonads per genotype were counted. (F) Nuclei from early and late pachytene zones of <i>sun-1(wt); sun-1(ok1282)</i> (top) and <i>sun-1(allA); sun-1(ok1282)</i> (bottom) hermaphrodite gonads stained with anti-HTP-3 (left, green in merged) and anti-SPY-1 (middle, red in merged). Scale bars, 10 µm.</p

SUN-1 S12 phosphorylation in male meiosis.

<p>(A) Representative TZ nuclei of a <i>sun-1(wt)</i> hermaphrodite gonad (upper panel) and a <i>sun-1(wt)</i> male gonad (lower panel) to highlight SUN-1 (anti-GFP, green), anti-Him-8 (red), and DAPI (blue). (B) Representative nuclei from wild-type hermaphrodite TZ (upper panel) and wild-type male TZ (lower panel) stained with anti-SUN-1 S12Pi (green), anti-HIM-8 (red), and DAPI (blue). (B′) Representative nuclei from <i>him-3 (gk149)</i> hermaphrodite TZ (upper panel) and <i>him-3 (gk149)</i> male TZ (lower panel) stained with anti-SUN-1 S12 Pi (green), anti-HIM-8 (red), and DAPI (blue). (B″) Representative nuclei from <i>sun-1(jf18)</i> hermaphrodite TZ (upper panel) and <i>sun-1(jf18)</i> male TZ (lower panel) stained with anti-SUN-1 S12 Pi (green), anti-HIM-8 (red), and DAPI (blue). (C) Representative wild-type TZ nuclei of a hermaphrodite gonad (upper panel) and a male gonad (lower panel) costained with anti-PLK-2 (green), anti-Him-8 (red), and DAPI (blue). (D) Relative time of residency in different meiotic stages in different <i>sun-1</i> phosphorylation mutant backgrounds in males, assessed by presence of SUN-1 aggregates; zones were normalized to the individual length of the scored gonad (from meiotic entry to diplotene). Nuclei were sorted into two categories: “TZ” (dark green, more than one SUN-1 aggregate) and “pachytene” (red, no SUN-1 aggregates). (E) Pachytene zone nuclei of <i>sun-1(wt)</i> and <i>sun-1(6E)</i> stained with anti-HIM-8 (blue), anti-SYP-1 (yellow), and DAPI (red). Note that the single male X chromosome was unsynapsed in both WT and <i>6xE</i> substitution lines.</p

Prolonged SUN-1 phosphorylation correlates with meiotic failure.

<p>(A) Wild-type (WT) hermaphrodite gonad stained with DAPI (top and blue in merge), anti-SUN-1 S43Pi (middle and green in merge), and anti-SUN-1 S12Pi (bottom and red in merge). Arrow highlights a nucleus in mid/late pachytene zone with clustered chromatin and phosphorylated SUN-1. Magnifications at the bottom of representative TZ or early pachytene nuclei to highlight differences in SUN-1 phosphorylation patterns. Schematics on top (in A, C, D, and E) delineate quantifications of nuclei with and without phosphorylation of SUN-1 (S8, S12, S24, and S43) in the meiotic part of the gonad (quantified from meiotic prophase entry to beginning of cellularization/diplotene in WT, marked with dotted lines in DAPI channels). Orange represents cell rows with ≥50% of nuclei with SUN-1 phosphorylation. <i>n</i>, number of gonads scored for each genotype. (B) Wild-type hermaphrodite gonad stained with DAPI (left and blue in merge), anti-SUN-1 S43Pi (middle and green in merge), and anti-RAD-51 (red in merge). Box, upper right: magnification of representative nuclei in late pachytene with clustered chromatin, phosphorylated SUN-1, and numerous RAD-51signals. Red channel boosted in merge picture to better visualize RAD-51 foci in all nuclei. (C and D) <i>syp-2(ok307)</i> (C) and <i>rad-51(ok2218)</i> (D) mutant hermaphrodite gonad stained with DAPI (top and blue in merge), anti-SUN-1 S43Pi (middle and green in merge), and anti-SUN-1 S12Pi (bottom and red in merge). (E) Wild-type hermaphrodite gonad dissected 90 min after 90 Gy gamma irradiation stained with DAPI (top and blue in merge), anti-RAD-51 (middle and green in merge), and anti-SUN-1 S12Pi (bottom and red in merge). (F) Wild-type hermaphrodite gonad dissected 27 h after 70 Gy gamma irradiation; anti-SUN-1 S12Pi (red), anti-SUN-1 S43Pi (green), and DAPI (blue). Scale bars, 10 µm.</p

Effect of nonphosphorylatable SUN-1 on aggregate persistence, PLK-2 localization, and chromosome mobility beyond TZ.

<p>Hermaphrodite gonad of <i>sun-1(wt); sun-1(ok1282), syp-2(ok307)</i> (top) and <i>sun-1(allA); sun-1(ok1282); syp-2(ok307)</i> (bottom) stained with DAPI (blue), anti-GFP to highlight SUN-1 (green), and anti-PLK-2 (red). Dark green and light green frames highlight zones with distinct chromosome movement patterns: magnifications of nuclei in the corresponding zones below, showing displacement tracks of 2D plotted chromosome end movements over 3 min (left) and PLK-2 (red) and DAPI (blue). Bottom: average number of aggregates, aggregate velocity (in nm/sec), and fusion/split events (per nucleus/min) for nuclei from corresponding zones. Scale bar, 10 µm.</p