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«Titel der Diplomarbeit Cell cycle and DNA damage dependent control of transcription of the DNA repair gene AtCOM1 Verfasser Stefan Bailey ...»

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DIPLOMARBEIT

Titel der Diplomarbeit

Cell cycle and DNA damage dependent control of transcription

of the DNA repair gene AtCOM1

Verfasser

Stefan Bailey

angestrebter akademischer Grad

Magister der Naturwissenschaften (Mag. rer. nat.)

Wien, 2012

Studienkennzahl lt. Studienblatt: A 490

Studienrichtung lt. Studienblatt: Diplomstudium Molekulare Biologie

Betreuerin / Betreuer: Dr. Peter Schlögelhofer

Table of contents 1

Abstract

6 2 Zusammenfassung 7 3 Introduction 9

3.1 The Cell Cycle 10

3.2 Endoreduplication 12

3.3 Promoters and Transcriptional Regulation 13

3.4 E2F transcription factors 15

3.5 DNA damage signalling 20

3.6 Somatic DNA double strand break repair 26

3.7 Meiosis 31

3.8 The DNA repair protein COM1/SAE2/CtIP 36

3.9 Analysis of the AtCOM1/GR1 promoter 38

3.10 Objectives 42 4 Results 44

4.1 The E2F binding site is necessary for AtCOM1 44 promoter activity in intact plants

4.2 The AtCOM1 promoter is active in dividing cells and 51 this basal promoter activity is independent of ATM

4.3 AtCOM1 expression in first true leaves 54

4.4 Reduction of RBR mRNA level increases AtCOM1 56 expression

4.5 E2Fa is enriched at the E2F binding site of the 58 AtCOM1 promoter

4.6 AtCOM1 expression in transgenic E2F lines 61 5 Discussion 64

5.1 AtCOM1 is expressed in dividing cells 64

5.2 AtCOM1 is regulated by E2F transcription factors 65

5.3 A model for the regulation of AtCOM1 expression 69

5.4 Experimental outlook 72 Further ChIP experiments 72 Further qPCR experiments 72 Immunostaining of root tips 73 6 Materials and methods 73

–  –  –

1 Abstract The maintenance of genomic integrity is of crucial importance for all living organisms.

The genome is however constantly threatened by a multitude of different DNA lesions, that can be caused by internal stresses, or exogenous genotoxic agents.

Therefore, the ability to sense and repair DNA damage in a timely manner is essential. A large variety of proteins are involved in this DNA damage response.

COM1, which is highly conserved from yeast (COM1/SAE2) to humans (CtIP), is one of these proteins. The plant homologue AtCOM1 is essential for homologous recombination (HR) and meiotic progression. It is expressed at basal levels in mitotic cells and at strongly enhanced levels in cells undergoing the endocycle. Its transcription is strongly enhanced in response to ionizing radiation depending on the checkpoint kinase ATM (Ataxia telangiectasia mutated). It is essential for HR and acts in cooperation with the MRN/X complex (Mre11, Rad50, Nbs1/Xrs1), in response to DNA double strand breaks (DSBs). Atcom1 mutants are sterile due to defects in meiotic double strand break (DSB) processing and subsequent DNA repair and are sensitive to the interstrand crosslinking agent Mitomycin C.

This study presents data about the cell cycle and tissue specific, regulatory dynamics of AtCOM1 expression. Evidence is provided, that an E2F transcription factor (TF) binding site in the promoter is essential for the transcription of AtCOM1 and that, in the absence of genotoxic stress, the gene is expressed in mitotic cells, independent of ATM at a basal level. Ionizing radiation (IR) leads to a strongly enhanced expression of AtCOM1, that depends on ATM. The AtCOM1 promoter activity is however restricted to the meristems. At least one of the six plant E2Fs, E2Fa is shown to be enriched at the E2F transcription factor binding site of the AtCOM1 promoter. Alterations of the expression levels of different E2F TFs lead to altered expression levels of AtCOM1.

The results presented in this thesis lead to the hypothesis, that the transcription of the DNA repair gene AtCOM1 depends on E2F and ATM dependent cell cycle control. AtCOM1 promoter activity seems to be restricted to the meristems and is strongly upregulated in response to IR. This upregulation of AtCOM1 is dependent of ATM. These results suggest, that IR-induced DSBs lead to an ATM dependent switch from proliferation to endocycle in meristematic cells. In endocycling cells E2Fa activity is strongly enhanced, leading to the upregulation of AtCOM1.

2 Zusammenfassung Die Aufrechterhaltung der genomischen Unversehrtheit ist von entscheidender Bedeutung für alle lebenden Organismen. Das Genom wird jedoch permanent von einer Vielzahl verschiedener DNA Schäden bedroht, die interne Ursachen haben, oder von exogenen, genotoxischen Stoffen verursacht werden können. Daher ist die Fähigkeit DNA Schäden schnellstmöglich zu erkennen und zu beseitigen entscheidend. Eine Vielzahl an Proteinen sind an dieser Antwort auf DNA Schädigungen beteiligt. COM1, das von der Hefe (COM1/SAE2) bis zum Menschen konserviert ist, ist eines dieser Proteine. Das homologe Pflanzenprotein AtCOM1 ist essentiell für homologe Rekombination und den Ablauf der Meiose. Eine basale Expression von AtCOM1 findet in mitotischen Zellen statt und eine stark erhöhte AtCOM1 Expression findet in Zellen statt, die eine besondere Form des Zellzyklus durchlaufen, das Endocycle. AtCOM1 wird, als Reaktion auf DNSDoppelstrangbrüche, abhängig von der Checkpoint Kinase ATM (Ataxia telangiectasia mutated) aktiviert und treibt im Zusammenspiel mit dem MRN/X Komplex (Mre11, Rad50, Nbs1/Xrs1) die homologe Rekombination voran. Atcom1 Mutanten sind steril, aufgrund von Störungen bei der Prozessierung von DNSDoppelstrangbrüchen und der darauffolgenden DNS Reparatur, außerdem reagieren sie sensitiv auf die Substanz Mitomycin C, die einander gegenüberliegende DNA Stränge quervernetzt.





Diese Arbeit präsentiert Daten über die Zellzyklus – und gewebsspezifische, regulatorische Dynamik der AtCOM1 Expression. Es werden Hinweise dafür gegeben, daß eine E2F Transkriptionsfaktorbindestelle im AtCOM1 Promotor entscheidend für die Transkription des Gens ist. Außerdem wird gezeigt, daß AtCOM1, in Abwesenheit von genotoxischem Stress, in mitotischen Zellen, unabhängig von ATM auf einem basalen Niveau exprimiert wird. Ionisierende Strahlung führt zu einer stark erhöhten, ATM abhängigen Expression von AtCOM1.

Die Aktivität des AtCOM1 Promotors ist jedoch auf die Meristeme beschränkt. Es wird nachgewiesen, daß zumindest einer, der sechs E2F Faktoren, nämlich E2Fa an der E2F Bindestelle des AtCOM1 Promotor angereichert ist. Durch die Veränderung der Expressionsniveaus, unterschiedlicher E2F Faktoren, wird die Expression von AtCOM1 differentiell modifiziert.

Die Ergebnisse, die in dieser Arbeit präsentiert werden, führen zu der Hypothese, daß die Transkription des DNS-Reparatur-Gens AtCOM1 durch die E2F- und ATMabhängige Kontrolle des Zellzyklus reguliert wird. Die Aktivität des AtCOM1 Promotor scheint auf die Meristeme beschränkt zu sein und wird als Antwort auf ionisierende Strahlung, in Abhängigkeit von ATM stark hochreguliert. Diese Ergebnisse führen zu der Annahme, daß durch ionisierende Strahlung verursachte DNSDoppelstrangbrüche zu einem ATM-abhängigen Einsetzen des Endocycle in Zellen der Meristeme führen. In diesen Zellen ist die E2Fa Aktivität stark erhöht, was der Grund für die vermehrte AtCOM1 Expression sein könnte.

3 Introduction In contrast to animals, plant development is largely post-embryonic, as plants have to be able to flexibly adapt to environmental conditions, given their sessile lifestyle.

New organs, such as roots, stems, leaves, and flowers, originate from life-long iterative cell divisions followed by cell growth and differentiation (Inze and De Veylder 2006). These cell divisions take place at specialized zones, called meristems.

Flowers and leaves are produced at the floral and shoot meristems, respectively.

Root meristems continuously add new cells to the growing root. The cells at the meristems are pluripotent, so their progeny can become committed to a variety of developmental fates (Inze and De Veylder 2006). Many differentiated plant cells have the ability to de-differentiate, thereby requiring pluripotentiality (Steward 1970; Grafi and Avivi 2004). Quiescent root pericycle cells, for example, can be stimulated to undergo cell divisions and to form lateral roots de novo (Casimiro, Beeckman et al.

2003; Inze and De Veylder 2006).

Another aspect, making plant development unique, is the fact, that plant cells are surrounded by rigid cell walls, preventing any cell migration. The number of cells produced at the meristems and the cell division plane is thus important for determining the organization of plant tissues (Di Laurenzio, Wysocka-Diller et al.

1996; Inze and De Veylder 2006). Another interesting aspect of plants, is that they do not develop tumors, except as specific responses to certain pathogens (Doonan and Hunt 1996).

To understand the role of cell division in plant development and growth, it is required to understand the cell cycle and the basic machinery controlling it. Given its importance for growth and development, the cell cycle is one of the most comprehensively studied biological processes (Inze and De Veylder 2006).

In response to DNA damage the cell cycle is stopped, giving a complex DNA repair machinery the time to repair the DNA damage, before replication and cell division continue. This study is about the cell cycle dependent regulation of the expression of a specific DNA repair factor.

3.1 The Cell Cycle The eukaryotic cell cycle is divided into four distinct phases. In the G1 (gap1) phase cells increase in size and prepare for DNA replication, which occurs during S (synthesis) phase. After DNA replication, the cell enters G2 (gap2) phase, in which cell growth continues and preparations for cell division are made. Cell division is taking place during M (mitosis) phase. (Figure 1).

Figure 1 – Schematic overview of the cell cycle and its four distinct phases (Furler 2012) The cell cycle is tightly regulated, as alterations of the cell cycle machinery can cause severe developmental defects via unrecognized replication errors, or uncontrolled cell division, for instance. In all eukaryotes, cyclins and cyclin dependant kinases (CDKs) are two major classes of regulatory molecules, that determine a cell´s progression through the cell cycle. In plants there are two different classes of CDKs: CDKAs play a pivotal role in both the G1/S and G2/M transitions, whereas CDKBs accumulate at the G2- and M-phase and are essential for regulating the G2/M transition (Porceddu, Stals et al. 2001). These CDKs require binding of cyclins for their activity. Plants contain many more of these regulatory proteins, than previously described in other organisms - Arabidopsis thaliana contains at least 32 cyclins with a putative role in cell cycle progression(Inze and De Veylder 2006). Dtype cyclins (CYCD) are conserved between plants and animals and are responsible for triggering the G1/S transition through their association with CDKAs (Dewitte, Scofield et al. 2007). One of the targets of CDK-CYCD complexes is the retinoblastoma related protein (RBR). Upon phosphorylation through CDKA, RBR dissociates from E2F transcription factors, which, in their RBR-free form, activate genes required for S-phase entry. B-type cyclins on the other hand play an important role in the G2/M transition and intra M-phase control, while A-type cyclins are reported to regulate the S-to-M phase control.

The activity of CDK/cyclin complexes is regulated by phosphorylation and dephosphorylation, interaction with regulatory proteins and protein degradation (Pines 1995; Zhao, Harashima et al. 2012). Yeast CDK/cyclin complexes are inhibited by phosphorylation of an N-terminal Tyr residue in the CDK partner. In vertebrates this CDK phosphorylation occurs on an N- terminal Tyr and a Thr residue. Tyr phosphorylation is catalyzed by the WEE1 kinase and is counteracted by the phosphatase CDC25 (Inze and De Veylder 2006). Plants also possess a WEE1 kinase, that is putatively involved in the inhibitory phosphorylation of CDKs (Sorrell, Marchbank et al. 2002; Vandepoele, Raes et al. 2002). However in Arabidopsis and other plants, whose entire genome sequences are available, no CDC25 gene, encoding a CDK-phosphatase could be identified (Vandepoele, Raes et al. 2002;

Bisova, Krylov et al. 2005). Cell cycle control is a very complex network of many proteins working in concert, the exact molecular events controlling the cell cycle still need to be subject of further studies.

The cell cycle is an ordered and unidirectional process, that cannot be reversed. At certain positions, checkpoints prevent the cycle from further progression, to ensure that all requirements for the next phase are met. Several checkpoints were designed to make sure, that damaged, or incomplete DNA is not passed on to daughter cells.

The two main checkpoints are the G1/S checkpoint and the G2/M checkpoint, while the former is the rate-limiting step of the cell cycle, also known as restriction point.

Cyclin-dependant kinase inhibitors (CKIs) can inhibit progression of the cell cycle, by binding and inhibiting CDKs. Plant CKIs all share a C-terminally located 31-aminoacid domain, involved in the binding of CDKs and cyclins, that is essential for the inhibitory activity of the proteins (Wang, Fowke et al. 1997; De Veylder, Beeckman et al. 2001; Jasinski, Perennes et al. 2002; Coelho, Dante et al. 2005).

Cell cycle regulation is of pivotal importance for plant growth and development.

Plants contain more genes encoding core cell cycle regulators than other organisms (Dewitte, Scofield et al. 2007). The large number of regulatory genes might reflect the high developmental plasticity of sessile plants to respond to both intrinsic developmental signals and extrinsic environmental cues. The different cyclins might posses a wide range of expression patterns and confer different substrate specificities (Inze and De Veylder 2006).



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