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Effect of heterochromatin (com)position on DNA replication timing

Heinz, Kathrin :
Effect of heterochromatin (com)position on DNA replication timing.
[Online-Edition: https://tuprints.ulb.tu-darmstadt.de/8168]
Technische Universität , Darmstadt
[Ph.D. Thesis], (2018)

Official URL: https://tuprints.ulb.tu-darmstadt.de/8168

Abstract

The accurate replication of the genome prior to each cell division is of upmost importance as mistakes are potentially transferred to subsequent generations but can also lead to genetic mutations, karyotype aberrations or to diseases that may result in cancer or cell death. Therefore, the duplication of the genome is one of the most crucial processes in any living organism and thus essential for development and reproduction.\\ The process of DNA replication is a highly organized process, both spatially and temporally, where specific genomic regions undergo DNA replication at distinct times during S-phase (Synthesis-phase). During development of an organism, DNA replication is a flexible process and several chromatin properties have been proposed as potential regulators thereof. Although a lot is known on the composition of the basic DNA replication machinery, it is mostly unclear, how its activity is regulated.\\ In the context of the present thesis, I made use of different targeting and manipulation approaches to elucidate the impact of potential regulators that control DNA replication dynamics in mammalian cells. To this end, I established and validated a novel targeting strategy to manipulate and reposition subcellular structures and molecules.\\ I first took advantage of constitutive heterochromatin as a prominent and well-characterized structure within the mammalian nucleus and repositioned it to the nuclear lamina to assess the nuclear position of DNA as one potential regulator of DNA replication dynamics. Using time-lapse microscopy, I was able to observe that constitutive heterochromatin, known to replicate during late S-phase, was replicated during mid S-phase when repositioned to the nuclear periphery. For this reason, I proposed that constitutive heterochromatin was activated in \textit{trans} according to the domino model of origin activation by nearby (mid S) firing origins. This data provided a novel approach to reposition large genomic regions and to manipulate nuclear DNA position within the nucleus. Here, I established the nuclear position as a novel regulator of DNA replication timing.\\ Secondly, I made use of female \textit{Microtus cabrerae} cells, which are characterized by their very prominent giant sex chromosomes with large coupled heterochromatic blocks. Again, I made use of a well-defined structure within the nucleus and manipulated the histone acetylation level as another potential regulator of DNA replication timing. First, I manipulated the global acetylation level by HDAC inhibition via drug-treatment and secondly, I specifically targeted a histone acetyltransferase to the inactive and active X chromosome. I elucidated the impact of histone acetylation level on DNA replication dynamics in the vole genome. I was able to show that both manipulation assays, global and site-directed, were sufficient to significantly increase the histone acetylation level, resulting in a prolongation of total S-phase duration and substage duration. The DNA replication onset of facultative heterochromatin was shifted toward early S-phase, as DNA content was further increased in early S-phase in treated samples. I was able to detect a negative effect of histone hyperacetylation on DNA replication fork speed. Here, I assessed the validity and reproducibility of DNA replication regulation by histone acetylation level across species.

Item Type: Ph.D. Thesis
Erschienen: 2018
Creators: Heinz, Kathrin
Title: Effect of heterochromatin (com)position on DNA replication timing
Language: English
Abstract:

The accurate replication of the genome prior to each cell division is of upmost importance as mistakes are potentially transferred to subsequent generations but can also lead to genetic mutations, karyotype aberrations or to diseases that may result in cancer or cell death. Therefore, the duplication of the genome is one of the most crucial processes in any living organism and thus essential for development and reproduction.\\ The process of DNA replication is a highly organized process, both spatially and temporally, where specific genomic regions undergo DNA replication at distinct times during S-phase (Synthesis-phase). During development of an organism, DNA replication is a flexible process and several chromatin properties have been proposed as potential regulators thereof. Although a lot is known on the composition of the basic DNA replication machinery, it is mostly unclear, how its activity is regulated.\\ In the context of the present thesis, I made use of different targeting and manipulation approaches to elucidate the impact of potential regulators that control DNA replication dynamics in mammalian cells. To this end, I established and validated a novel targeting strategy to manipulate and reposition subcellular structures and molecules.\\ I first took advantage of constitutive heterochromatin as a prominent and well-characterized structure within the mammalian nucleus and repositioned it to the nuclear lamina to assess the nuclear position of DNA as one potential regulator of DNA replication dynamics. Using time-lapse microscopy, I was able to observe that constitutive heterochromatin, known to replicate during late S-phase, was replicated during mid S-phase when repositioned to the nuclear periphery. For this reason, I proposed that constitutive heterochromatin was activated in \textit{trans} according to the domino model of origin activation by nearby (mid S) firing origins. This data provided a novel approach to reposition large genomic regions and to manipulate nuclear DNA position within the nucleus. Here, I established the nuclear position as a novel regulator of DNA replication timing.\\ Secondly, I made use of female \textit{Microtus cabrerae} cells, which are characterized by their very prominent giant sex chromosomes with large coupled heterochromatic blocks. Again, I made use of a well-defined structure within the nucleus and manipulated the histone acetylation level as another potential regulator of DNA replication timing. First, I manipulated the global acetylation level by HDAC inhibition via drug-treatment and secondly, I specifically targeted a histone acetyltransferase to the inactive and active X chromosome. I elucidated the impact of histone acetylation level on DNA replication dynamics in the vole genome. I was able to show that both manipulation assays, global and site-directed, were sufficient to significantly increase the histone acetylation level, resulting in a prolongation of total S-phase duration and substage duration. The DNA replication onset of facultative heterochromatin was shifted toward early S-phase, as DNA content was further increased in early S-phase in treated samples. I was able to detect a negative effect of histone hyperacetylation on DNA replication fork speed. Here, I assessed the validity and reproducibility of DNA replication regulation by histone acetylation level across species.

Place of Publication: Darmstadt
Divisions: 10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
Date Deposited: 18 Nov 2018 20:55
Official URL: https://tuprints.ulb.tu-darmstadt.de/8168
URN: urn:nbn:de:tuda-tuprints-81688
Referees: Cardoso, Prof. Dr. M. Cristina and Laube, Prof. Dr. Bodo
Refereed / Verteidigung / mdl. Prüfung: 26 October 2018
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Alternative Abstract:
Alternative abstract Language
Die akkurate und fehlerfreie Duplikation des Genoms vor jeder Zellteilung ist von gro\ss{}er Bedeutung, da potenzielle Fehler an Nachfolgegenerationen weitergegeben werden können. Potenzielle Fehler können zu genetischen Mutationen, karyotypischen Aberrationen und Krankheiten wie Krebs oder zum Zelltod führen. Die Replikation des Genoms ist folglich einer der wichtigsten Prozesse in jedem lebenden Organismus und essentiell für dessen Entwicklung und Reproduktion. Der Prozess der DNA Replikation ist räumlich und zeitlich hoch organisiert, da spezifische genomische Regionen in festen Zeitfenstern während der S-Phase (Synthese-Phase) dupliziert werden. Während der Entwicklung ist die Duplikation des Genoms ein flexibler Prozess und verschiedenste epigenetische Eigenschaften des Chromatins wurden als potenzielle Regulatoren postuliert. Obwohl die grundlegende Maschinerie der DNA Replikation bekannt ist, ist die eigentliche Regulation ihrer Aktivität weitestgehend unbekannt.\\ Im Rahmen dieser Dissertation habe ich mich mit verschiedenen Targeting- und Manipulationsmethoden befasst, um gezielt verschiedene potenzielle Regulatoren der DNA Replikation in Säugerzellen zu manipulieren, um deren Effekt und Einfluss zu bewerten. Hierzu habe ich eine neue Targetingstrategie etabliert sowie validiert, um subzelluläre Strukturen und Moleküle zu manipulieren und zu repositionieren.\\ Somit habe ich mir zunächst die prominente und gut charakterisierte Struktur des konstitutiven Heterochromatins in Mauszellen zu Nutze gemacht und dieses an die nukleare Lamina transferiert. Diese Repositionierung vom Inneren des Zellkerns an die Peripherie wurde genutzt, um den Einfluss der Position von DNA auf ihren DNA Replikationszeitpunkt zu evaluieren. Mittels Lebendzellmikroskopie und Zeitrafferaufnahmen war ich in der Lage zu beobachten, dass repositioniertes konstitutives Heterochromatin, welches eigentlich erst in der späten S-Phase repliziert wird, durch die Repositionierung bereits in der mittleren S-Phase repliziert wird. Daher nahm ich an, dass die Replikation des transferierten konstitutiven Heterochromatins in \textit{trans} aktiviert wurde, gemä\ss{} dem sogenannten Domino Model. Dieses Model postuliert die Aktivierung von feuernden Replikationsursprüngen durch naheliegende feuernde Startpunkte der mittleren S-Phase. Meine Ergebnisse indizieren nicht nur eine Aktivierung in \textit{cis} entlang des Chromosoms, sondern auch in \textit{trans} über verschiedene Chromosomen hinweg. Diese Daten validieren die Wirksamkeit der Methode, um die Position von DNA gezielt zu manipulieren und demonstrieren zudem den Einfluss der Lokalisation von DNA auf ihren Zeitpunkt der DNA Replikation.\\ Außerdem machte ich mir weibliche \textit{Microtus cabrerae} Zellen zu Nutze, welche ebenfalls eine prominente Struktur aufweisen und zwar die der gigantischen Sexchromosomen, die gro\ss{}e heterochromatische Blöcke haben. Ich manipulierte das Histonacetlyierungslevel als potenziellen Regulator der Organisation der DNA Replikation. Ich erhöhte global mittels HDAC Inhibitor und durch gezieltes Targeting einer Histonacetyltransferase an die Sexchromosomen das Histonacetylierungslevel in dieser Spezies. Ich war in der Lage zu demonstrieren, dass beide Methoden zu einer Hyperacetylierung führen und die Dauer der gesamten S-Phase sowie der einzelnen Subphasen verlängern. Dies ging einher mit einer Erhöhung der genomischen DNA bereits in der frühen S-Phase in behandelten Zellen. Dies zeigte eine verfrühte DNA Replikation des fakultativen Heterochromatins, das eigentlich erst in der mittleren S-Phase repliziert. Die globale Hyperacetylierung hatte au\ss{}erdem einen negativen Effekt auf die Geschwindigkeit der Replikationsgabel. Mit dieser Studie demonstriere ich den spezies-übergreifenden Einfluss der Histonacetylierung als Regulator der DNA-Replikation.German
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