15 research outputs found
A microscopic screen for mutations affecting synaptonemal complex formation in Budding yeast
Im meiotischen Zellzyklus folgen einer Runde DNA-Replikation zwei Runden Chromosomensegregation in denen zunĂ€chst in der ersten Zellteilung die zwei Paare homologer Schwesterzentromere von einander segregieren, wĂ€hren die Schwesterzentromere in der zweiten Teilung separiert werden. Als Folge wird der Chromosomensatz halbiert und aus einer diploiden Zelle gehen vier Zellen mit einem nun haploiden Genom hervor, als VorlĂ€ufer der Gameten. Im Laufe der meiotischen Prophase 1 bildet sich eine meiosespezifische, hoch konservierten Struktur aus, der sogenannte Synaptonemale Komplex (SC), welcher die Axen gepaarter homologer Chromosomen verbindet. Der SC hat eine dreigliedrige proteinöse Struktur, bestehend aus zwei seitlichen axialen Elementen, verbunden durch eine zentrale Region, dem Transversalfilament. In den meisten Organismen, einschlieĂlich der Hefe Saccharomyces cerevisiae weisen Mutanten, die an der Bildung eines normalen SC scheitern ebenfalls Defekte in der Bildung von âCrossovernâ zwischen den homologen Chromosomen auf, welche notwendig fĂŒr die korrekte Aufteilung der Homologen wĂ€hrend der ersten meiotischen Teilung sind (Börner et al, 2004). Ăber die molekularen Mechanismen der SC-Bildung besteht bisher nur sehr geringe Kenntnis.
Mit dem Ziel jene Gene, welche fĂŒr die Synapsis der Chromosomen in Saccharomyces cerevisiae erforderlich sind umfassend zu identifizieren, habe ich in einem systematischen Screen genomweit, unter Zuhilfenahme visuell-mikroskopischer Methoden, nach Mutationen gesucht, welche die Chromosomenmorphologie beeintrĂ€chtigen.
Von den 4800 nicht essentiellen Genen, reprĂ€sentiert durch eine Gendeletionsbibliothek, habe ich 3630 in einem ersten Lauf analysiert. Nahezu 14.5% (524) der Gendeletionen beeintrĂ€chtigten Synapsis, oder fĂŒhrten zum Unvermögen das meiotische Programm in Gang zu setzen. Beinahe alle Mutanten von Genen â so denn analysiert â, die bereits zuvor als essentiell fĂŒr meiotische Rekombination und SC-Bildung bekannt waren, konnten erneut identifiziert werden. Von den 524 gefundenen Mutanten mit ausgeprĂ€gtem Phenotyp waren 132 nicht in der Lage das meiotische Programm zu initiieren, 90 konnten keinen SC bilden, 102 waren ausschliesslich zu sehr kurzer Synapsis befĂ€higt, und 200 konnten keinen vollstĂ€ndigen oder ausgedehnten SC bilden. Die identifizierten Gene sind breitgefĂ€chert vielen verschiedenen biologischer Prozessen zugeordnet, wie zum Beispiel der Zellatmung, dem RNA und DNA Metabolismus, dem Membranaufbau, oder der Meiose.
FĂŒr eine genauere Charakterisierung wurden 30 der identifizierten Gene, einer breiten Auswahl unterschiedlicher Klassen, in einem reinen SK1 Stammhintergrund deletiert. Die resultierenden Mutanten wurden charakterisiert anhand ihrer Chromosomensynapsis in einer Zeitserie durch die Meiose, und ausgewĂ€hlte Mutanten wurden zudem einer FACS-Analyse und einer physischen Rekombinationsmessung unterzogen. Nur eine dieser Mutanten (rim4â) ist dabei ĂŒberhaupt nicht in der Lage SCs zu bilden, wohingegen drei (ecm11â, ymr196â, und yor296â) im wesentlichen keine vollstĂ€ndige Synapsis zustande bringen. Alle anderen Mutanten zeigen in SK1 vollstĂ€ndige Synapsis, jedoch entweder in zu wenigen Kernen, oder unter aberrantem Verlauf durch die verschiedenen Stadien der Synapsis. Nur vier Deletionen (irc4â, hal5â, rtc1â, and ynl046â) verhielten sich sehr Ă€hnlich zum Wildtyp, wenn auch nicht völlig identisch.
Ebenso habe ich zu der Charakterisierung 22 weiterer Mutanten beigetragen, welche zuvor in einem Pilot-Screen identifiziert wurden, durchgefĂŒhrt in unserem Labor durch A. Woglar (Diplomarbeit, 2008). Rad6 und Bre1 werden fĂŒr die Monoubiquitinierung von Histon H2B an Lysin 123 (K123) benötigt (Robzyk et al., 2000). Ich konnte zeigen, dass der Synapsisdefekt einer htb1-K123R htb2-K123R Doppelmutante, bre1â und rad6â, oder lge1â Ă€hnlich, jedoch nicht so ausgeprĂ€gt ist. Dies weist auf eine wesentliche Rolle von Histon H2B-K123 Ubiquitinierung fĂŒr Synapsis hin, deutet aber auch zusĂ€tzliche Funktionen von Rad6/Bre1 an. Monoubiquitinierung von H2B-K123 ist wiederum notwendig fĂŒr Trimenthylierung von Histon H3K4 durch COMPASS (Set1), beziehungsweise H3K79 durch Dot1 (Nakanishi et al., 2009). Interessanterweise fĂŒhrt die gemeinsame Eliminierung von SET1 und DOT1, welche auch H3K4 und H3K79 Trimethylierung eliminieren sollte, nicht zum Verlust von ausgedehnter Synapsis, obgleich es zu einer Verzögerung von ungefĂ€hr zwei Stunden kommt. Diese Beobachtungen legen nahe, dass H2B-K123 Monoubiquitinierung ĂŒber einen anderen Weg als H3K4/K79 Trimethylierung an Synapsis beteiligt ist.
Der PP4 Phosphatasekomplex (Pph3, Psy2 und Psy4) wirkt der Phosphorylierung zahlreicher Substrate der Checkpointkinasen ATM/ATR (Tel1/Mec1) entgegen, inklusive Histon H2A Serin 129 (S129), und erlaubt somit den Austritt aus dem Checkpointarrest nach erfolgter Reparatur von DNA-SchĂ€den (Keogh et al, 2006). Wir haben fĂŒr pph3â Defekte in der pre-meiotischen DNA Replikation, Synapsis, der Bildung von Crossovern, und den meiotischen Kernteilungen gefunden (Woglar A., Diplomarbeit; diese Arbeit), welche auch durch andere nachgewiesen wurden (Falk et al., 2010). Interessanterweise beobachteten wir, dass pch2â bezĂŒglich Synapsis epistatisch ĂŒber pph3â ist, und in pph3â Synapsis unter Ăberkompensierung wieder herstellt. Auch die Deletion von SWC2 erlaubt wieder ausgedehnte Synapsis in pph3â Mutanten. Dagegen bewirkt eine Deletion von MEK1 oder FPR3 die Suppression des Kernteilungsdefektes der pph3â Mutanten. Bemerkenswerterweise liessen sich in der Tripelmutante pph3â pch2â fpr3â die meiotischen Defekte der pph3_ Mutante praktisch vollstĂ€ndig aufheben. pph3â pch2â fpr3â durchlĂ€uft nahezu normal das meiotische Programm, hat Synapsis, und produziert lebensfĂ€hige Sporen. Wir schliessen daraus, dass Pch2 Chromosomensynapsis als Antwort auf ATM/ATR CheckpointkinasenaktivitĂ€t verhindert, und dass PP4 die Funktion einnimmt dieses Checkpointsignal nach erfolgter DNA-Reparatur zu eliminieren um Pch2 zu inaktivieren und Synapsis zu erlauben. Wir legen damit nahe, dass es sich hierbei um ein funktionelles Modul handelt, welches Synapsis mit der VervollstĂ€ndigung der DNA-Reparatur koordiniert. Fpr3 hingegen wirkt als Inhibitor auf einen anderen Antagonisten von ATM/ATR, PP1 (Glc7-Phosphatase), welche normalerweise erst kurz vor der Kernteilung aktiviert wird.During the meiotic cell cycle, one round of DNA replication is followed by two rounds of chromosome segregation in which the two pairs of homologous sister centromeres segregate from each other during the first division while sister centromeres are partitioned in the second division. Hereby, the chromosome set of a diploid cell is reduced to half and four haploid cells are generated as precursors of gametes. A highly conserved meiosis-specific chromosomal structure called synaptonemal complex (SC) is formed during meiotic prophase I, connecting the axes of paired homologous chromosomes. The SC has a tripartite proteinaceous structure consisting of two axial elements connected by a central region called transverse filament. In most organisms including the budding yeast Saccharomyces cerevisiae, mutants that fail to form mature SC are also defective in crossover formation between the homologous chromosomes, which is required for accurate segregation of the homologs during the first meiotic division (Börner et al, 2004). The molecular mechanism of SC formation is not understood very well.
In order to identify a comprehensive set of genes required for chromosome synapsis, I have performed a systematic, genome-wide visual screen for mutations affecting chromosome morphology. Of the 4800 non-essential ORFs represented in the deletion library, I have analyzed 3630 in a primary round. Nearly 14.5% (524) of the deletions affected synapsis or were unable to induce the meiotic program. Nearly all mutants previously known to be essential for meiotic recombination and SC formation were re-identified, if analyzed. Of the 524 identified mutants with strong phenotypes, 132 were unable to initiate the meiotic program, 90 failed to undergo synapsis, 102 only underwent exclusively very short synapsis and 200 failed to form complete or nearly complete SC. The identified ORFs had been annotated with a wide variety of biological processes, such as respiration, RNA and DNA metabolism, membrane biology or meiosis.
For closer characterization, a set of 30 identified ORFs from various classes were deleted in the SK1 strain background. The respective mutants were characterized for chromosome synapsis during a meiotic time course and selected mutants were subjected to FACS analysis and to physical recombination assays. Only one of these (rim4â) is unable to synapse at all, whereas 3 (ecm11â, ymr196â, and yor296â) show nearly no complete synapsis. All other ORFs, show complete synapsis in SK1, but either in too few nuclei, or they show an aberrant progression through the different stages of synapsis. Only 4 deletions behaved very similar to wild type (irc4â, hal5â, rtc1â, ynl046â), although not perfectly identical.
I also contributed to the characterization of 22 mutants, which were previously identified in a pilot screen carried out in our lab by A. Woglar (master thesis 2008). Rad6 and Bre1 are required for the monoubiquitination of histone H2B at lysine 123 .(Robzyk et al., 2000). I showed that the synapsis defect in htb1-K123R, htb2-K123R double mutants is similar to, but not as rigorous as, in bre1â and rad6â, or lge1 mutants. This indicates a major role of histone H2B-K123 ubiquitination in synapsis, but also hints at additional roles of Rad6/Bre1. H2B-K123 monoubiquitination is required for histone H3K4 and H3K79 trimethylation by COMPASS (Set1) and Dot1 (Nakanishi et al., 2009) respectively. Interestingly, elimination of both, SET1 and DOT1, which should eliminate H3K4 and H3K79 trimethylation, does not eliminate extensive synapsis, although it causes ca. a 2 hours delay. These observations suggest that H2B-K123 monoubiquitination may promote synapsis through an additional pathway, other than H3K4,K79 trimethylation.
The PP4 phosphatase complex anatagonizes the phosphorylation of various targets by he checkpoint kinases ATM/ATR (Tel1/Mec1) including histone H2A-S129, enabling checkpoint recovery after DNA damage (Keogh et al, 2006). We found pph3â to be defective in pre-meiotic DNA replication, chromosome synapsis, crossover formation and nuclear division (Woglar A, master thesis; this work), observations also made by others (Falk et al., 2010). Interestingly, pch2â is epistatic to pph3â concerning synapsis, overcompensating the synapsis defect. Also deletion of SWC2 can restore extensive synapsis in pph3â mutants. In contrast deletion of MEK1 or of FPR3 suppressed the nuclear division defects of pph3â mutants. Interestingly, the triple mutant pph3â, pch2â, fpr3â progressed through meiosis almost normally, underwent synapsis and produced viable spores. We conclude, that Pch2 prevents synapsis in response to the ATM, ATR mediated checkpoint response, and that the role of PP4 is to downregulate that signal upon repair, to inactivate Pch2 and to allow synapsis. We propose that this constitutes a module to coordinate synapsis with completion of DSB repair. Fpr3 rather is an inhibitor of an alternative way to antagonize ATM/ATR, that is PP1 (Glc7-phosphatase), normally only activated shortly before the division
Proof of Concept of Culturomics Use of Time of Care
Culturomics, a high throughput culture method with rapid identification of the colonies by Matrix Assisted Laser Desorption Ionization/Time Of Flight Mass Spectrometry (MALDI-TOF MS), has demonstrated its contribution to the exploration of the gut microbiota over the past 10 years. However, the cost, work time and workload, considerably limit its use on a large scale or emergency context. Here, by testing two different stool samples, including a stool sample from a patient requiring rapid immunotherapy treatment, we tested a new fast culturomic protocol using two pre-incubation media, blood culture bottle and YCFA modified medium. Both media were supplemented with 2 ml of rumen fluid filtered at 0.2 mu m and 2 ml of defibrinated and sterile sheep blood. Unlike the standard culturomics, subculturing of blood culture bottle were performed at reduced incubation time (3 h, 6 h, 9 h, 24 h) and at a longer incubation time (3 days, 7 days, and 10 days) at 37 degrees C. By testing 5,200 colonies per MALDI-TOF MS and obtaining a comparable number of cultured bacterial species (131 to 143) in a stool sample, this new protocol reduced the number of colonies tested by 57%, working time by 78.6% and cost by 72.2%. In addition, we highlighted that the proportion of strict anaerobic species has increased by 24%, known to be the preferential targets for biotherapy, including Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Phascolarctobacterium faecium. Finally, this work showed that some bacterial species grew earlier but disappeared with prolonged incubation times
Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification
Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD 'code' for co-transcriptional histone modifications
Molecular drivers of emerging multidrug resistance in Proteus mirabilis clinical isolates from Algeria
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The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast
<div><p>Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast <i>Schizosaccharomyces pombe</i> that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.</p></div
Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification
Prf1 and PAF pathways have opposing biological effects.
<p>(<b>A</b>) Quantification of the abnormal septation patterns in the indicated strains. Strains carrying the <i>cdk9<sup>as</sup></i> allele were cultured in DMSO (â) or 2 ”M 3-MB-PP1 (+) for 15 hours prior to fixation for microscopy. Error bars denote standard deviations from 3 independent experiments. (<b>B</b>) ChIP of RNAPII was carried out in the indicated strains and quantified by qPCR using primers specific to the <i>nup189</i><sup>+</sup> gene. Values were normalized to that for primer pair 1. Error bars denote standard deviations from three independent experiments. Significant differences from wild-type values (unpaired t-test) are indicated.</p
Prf1 does not stably associate with the PAF complex.
<p>(<b>A</b>) TAP purification was carried out using whole cell extracts from the indicated strains and purified material was analyzed by SDS-PAGE and silver staining. The proteins contained in the labeled bands were identified by mass spectrometry (as indicated on the right). âCBPâ denotes the residual fusion to the calmodulin binding peptide resulting from the TAP procedure. Leo1 was detected in two bands that also contained either Paf1-CBP (in the <i>paf1-TAP</i> lane) or Paf1 (in the <i>tpr1-TAP</i> lane). Molecular weight standards (in kD) are denoted on the left. (<b>B</b>) Single-step TAP purifications were performed using extracts from the indicated strains. 5% of input fractions (âinputâ) and 50% of bead-bound fractions (âbeadsâ) were analyzed by SDS-PAGE and western blotting.</p
Model depicting the roles of the Prf1/Rtf1 and PAF pathways in RNAPII elongation.
<p>The Prf1 pathway, involving direct association of Prf1 with phosphorylated Spt5 CTD, is labeled â1.â The PAF pathway, involving multiple Cdk9 targets, is labeled â2.â Potential crosstalk between the two pathways is indicated by the broken double arrow. See text for details.</p
Prf1 and PAF are recruited to chromatin via alternate mechanisms.
<p>(<b>A</b>) Strains used for ChIP with IgG resin (recognizing the TAP tag) are indicated at the bottom. All strains also harbored the <i>cdk9<sup>as</sup></i> allele and were treated with either DMSO (â) or 20 ”M 3-MB-PP1 (+) for two hours before crosslinking for ChIP (also indicated by â3MBâ at the far right). Assays were quantified by qPCR using primers specific for the <i>act1</i><sup>+</sup> (left) or <i>adh1</i><sup>+</sup> (right) genes. Lengths of the gene coding regions (in base pairs) and positions of PCR amplicons are indicated at the top. Numbering of the datasets for the corresponding PCR amplicons shown for <i>act1</i><sup>+</sup> (left) is used throughout the figure. Error bars denote standard deviations from 2â3 independent experiments. (<b>B and C</b>) Strains of genotypes indicated at the bottom were used for ChIP of either Prf1-TAP (left) or Tpr1-TAP (right). Cultures were treated with DMSO or 3-MB-PP1 as in (A).</p