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Unravelling the functional role of 3D chromosome folding in nuclear defense systems

English title Unravelling the functional role of 3D chromosome folding in nuclear defense systems
Applicant Grob Stefan
Number 200704
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pflanzen- und Mikrobiologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Molecular Biology
Start/End 01.08.2021 - 31.07.2025
Approved amount 766'606.00
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All Disciplines (4)

Discipline
Molecular Biology
Biochemistry
Genetics
Embryology, Developmental Biology

Keywords (5)

genetically modified organisms; gene silencing; transgene; epigenetics; chromatin architecture

Lay Summary (German)

Lead
Chromosomen sind lange Moleküle, die dicht gepackt, ähnlich einem Knäuel, im Zellkern untergebracht sind. Die 3D Struktur, die sie dabei einnehmen, ist jedoch keineswegs zufällig. Wir untersuchen die 3D Struktur der Chromosomen und wie sie molekulare Prozesse im Zellkern beeinflusst.
Lay summary
Inhalt und Ziele des Forschungsprojekts:

Der Chromosomensatz eines Zellkerns kann mehr als einen Meter lang werden. Da Chromosomen jedoch in einem Zellkern von nur 10 Mikrometer Durchmesser Platz finden müssen, werden sie nach einem komplexen System gefaltet. Im Zellkern der Ackerschmalwand berühren sich aufgrund dieser Faltung spezifische chromosomale Regionen und bilden so eine knotenartige Struktur. Diese spielt eine wichtige Rolle in der Verteidigung des Zellkerns gegen invasive DNA-Elemente (z.B. Transgene oder springende Gene/Transposons). Wenn ein solches Element diese 3D Struktur berührt, wird es als fremd erkannt und dessen Transkription gehemmt. Wir untersuchen die molekularen Grundlagen dieser Verteidigungsstrategie und wollen herausfinden, wie der Zellkern zwischen eigener und fremder DNA unterscheiden kann.

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts:

Wir betreiben Grundlagenforschung, die mehrere Gebiete miteinschliesst: Phytopathologie, (Epi-) Genetik und 3D Chromosomenfaltung. Da die Integration von DNA-Elementen ins Genom zu weitreichenden Mutationen führen kann, wird dieser Prozess von der Natur stark reguliert. Solche Mutationen sind nicht nur in der Entstehung von Tumoren involviert, sondern können auch nützlich sein, etwa um die genetische Vielfalt zu vergrössern, die eine fundamentale Voraussetzung für die Pflanzenzucht ist. Das Verständnis, wie der Zellkern invasive DNA-Elemente reguliert, ist daher von breitem Interesse.

Direct link to Lay Summary Last update: 07.07.2021

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Abstract

Invasive DNA elements, such as viruses and transposable elements, are major contributors to evolution and their mobilization can trigger large chromosomal rearrangements that can severely affect the host’s fitness. To safeguard the genome from this thread, a multitude of mechanisms have evolved to prevent the mobility of invasive elements. This defense comprises two main steps: 1) the identification of foreign DNA and 2) the transcriptional or post-transcriptional silencing of the invasive element. Whereas step 2 has been studied extensively and many silencing mechanisms are known, step 1, the initial targeting of the invasive element, is much less understood, but addresses the fundamental question: what is self and what is foreign?I have discovered a novel genome defense system, termed KNOT-linked silencing (KLS). In KLS, both invasive element detection and silencing are accompanied by specific and discernable signatures concerning alterations of the three-dimensional chromosomal (3D) architecture of the invasive element’s insertion site. This enables investigating the element’s uncovering as well as its silencing. Diagnostic for KLS are ectopic 3D chromosomal contacts between the transgene insertion site and a 3D nuclear structure termed the KNOT. The contact strength between the transgene insertion site and the KNOT likely determines, whether the invasive element is subject to step 1, identification, to step 2, silencing, or in case no contacts are formed, whether it remains hidden from the defence machinery. The KNOT is formed by tight contacts among at least ten KNOT Engaged Elements (KEEs), genomic elements found distributed across all five Arabidopsis thaliana chromosomes. KEEs share sequence homology among each other due to the presence of specific transposon fragments and show an enrichment in associated small RNAs. In three separate aims, we will unravel the mechanism of KLS, employing genetic, biochemical, and cytological tools to reveal players involved in KLS and its dynamics across generations in order to elucidate how invasive elements are detected and silenced. As invasive element model, I will study transgenes in Arabidopsis that originally stem from naturally occurring tumor-inducing Agrobacterium plasmids.Aim A focuses on the identification of factors genetically interacting with KLS. We will conduct a forward-genetic screen for loss-of-KLS to unambiguously isolate genes involved KLS initiation and maintenance. Additionally, we will perform reverse genetic experiments and test candidate genes for their involvement in KLS. Thus, Aim A is designed to reveal the genetic basis of KLS.Aim B is dedicated to the isolation of proteins physically associated with KLS-involved DNA elements, using a cutting-edge reverse chromatin immuno precipitation method and we will characterize proteins physically bound to small RNAs associated with KEEs using a previously established RNA-protein pull down technique. In Aim B, we aim to decipher the molecular partners directly associated with KLS.Aim C seeks to elucidate the establishment and maintenance of KLS, using a combination of genetic, transcriptomic, and cytological methods. We will create an innovative KNOT-reporter system based on ANCHOR technology, which allows differential labelling of single genetic loci and their cytological study using microscopy. We will additionally use cytological and genetic means to study KLS establishment and perform transcriptome profiling aiming at identifying factors involved in KLS initiation. The study of invasive element defense and the involvement of 3D chromosomal architecture is original and timely. It allows us to make a substantial contribution to understanding on genome defense systems in general and to the role of 3D chromosome architecture in gene regulation. The novelty of the findings gives us the decisive intellectual and technological advantage to study KLS and allows our team to become a significant contributor to the field.
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