Projects

Prof. Dr. Markus Löbrich (TU Darmstadt, Biologie)

Background: DNA double-strand breaks (DSBs) are arguably the most important lesions induced by ionizing radiation (IR) since unrepaired or mis-repaired DSBs can lead to chromosomal aberrations and cell death (1,2). The two major pathways to repair IR-induced DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Perhaps surprisingly, NHEJ represents the predominant pathway in the G1 and G2 phases of the mammalian cell cycle, but HR also contributes and repairs a subset of IR-induced DSBs in G2. During S-phase, in contrast, HR is required to repair DSBs arising at stalled or collapsed replication forks, and NHEJ is suppressed to avoid the illegitimate joining of incorrect break ends. Little is known about the repair of IR-induced DSBs during S phase. One possibility is that breaks induced in unreplicated genomic regions are repaired by NHEJ since no sister chromatid is available and that DSBs induced in replicated regions are repaired by HR. This is an important issue since strategies to optimize tumor radiotherapy specifically target proliferating S-phase cells.

Aim of the project: This project aims to assess to relative contribution of NHEJ and HR for the repair of radiation-induced DSBs during S-phase. The studies will elucidate the factors determining DSB repair pathway choice with particular emphasis on different genomic regions and processes occurring at the replication fork.

Working strategy: Ionising radiation-induced foci (IRIF), such as H2AX foci, represent cytogenetic markers of DSBs. After irradiation, γH2AX foci arise in a 1:1 relationship to DSBs and the loss of the foci represents repair of the breaks (3). In contrast to H2AX foci, certain other repair foci such as Rad51 or RPA mark only those DSBs which undergo repair by HR. Thus, the immunofluorescence analysis of various IRIF in combination with specific cell cycle markers allows an assessment of the relative contribution of NHEJ and HR in defined cell cycle phases (4).

We will investigate human and rodent cell lines defective in NHEJ or HR proteins and will utilize RNAi approaches to down-regulate specific repair factors. In order to investigate DSB repair processes in defined genomic regions (5), we will apply high resolution microscopy (including confocal laser scanning microscopy and single molecule technology) in cooperation with the Meckel lab (project 1F). Different genomic regions will be visualized in cooperation with the Cardoso and Rapp labs (projects 1B and C). Region-specific induction of DSBs will be achieved in cooperation with the Jakob lab (project 1E) by utilizing heavy ion micro-irradiation. The experimental data obtained during these studies will provide the basis for the theoretical work in the Hamacher and Drossel labs and are important for a detailed understanding of the experimental studies on cell cycle analysis (Thiel and Deckbar labs) and apoptosis (labs of Schmidt, Layer, Fournier and Dencher).

References:

(1) Löbrich und Jeggo (2007) Nat Rev Cancer; 7:861-9.

(2) Deckbar et al. (2007) J Cell Biol; 176:749-55.

(3) Löbrich et al. (2010) Cell Cycle; 9:662-9.

(4) Beucher et al. (2009) EMBO J; 28:3413-27.

(5) Goodarzi et al. (2008) Mol Cell; 31:167-77.

Prof. Dr. M. Cristina Cardoso (TU Darmstadt, Biology)

Background: Methyl-cytosine binding proteins (MeCPs) play a central role in the mediation of epigenetic effects. They recognize methylated DNA and recruit histone deacetylases (1). These epigenetic modifications generate, by as yet unknown mechanism(s), higher-order chromatin structures that might impact on genome expression and stability. We have previously shown that MeCP2 levels increase during cellular differentiation and cause large scale heterochromatin clustering (2,3). The MeCP family includes in addition to MeCP2, four factors sharing the conserved MBD domain – MBD1,2,3,and 4 (1). The latter has been shown to bind repair factors and to exhibit DNA glycosylase activity targeted towards G:T mismatches in the context of CpG sites. Deficiency of MBD4 in mice increases mutation rate at CpG sites and thus MBD4 is thought to be involved in repairing mismatches arising from deamination of methyl-cytosine.

Aim of the project: We want now to test whether MBD4, similarly to MeCP2, has the ability to reorganize heterochromatin and consequently either protects it from damage and/or hinders the access of repair factors to the condensed chromatin. We will test the ability of MBD4 to aggregate and to compact heterochromatin in a dose-dependent mode. In addition, we will test the recruitment and the kinetics of MBD4 at DNA damage sites and compare it to other repair factors. We will further test its dependency from specific repair pathways and specific epigenetic modifications, namely DNA methylation. Finally, we will test the potential dominant negative effect of a naturally occurring frameshift mutation in humans causing a truncated MBD4 with MBD domain but no glycosylase activity for its effect on chromatin aggregation, susceptibility to damage and repair of DNA damage. The planed experiments will combine molecular biology methods, with multidimensional live-cell confocal microscopy and quantitative image analysis.

References:

(1) Brero, A., Leonhardt, H. and Cardoso, M. C. Replication and Translation of Epigenetic Information. (2006). In Current Topics in Microbiology and Immunology, W. Doerfler, P. Böhm, eds., vol.301, p.21-44 , Springer-Verlag, Heidelberg.

(2) Brero, A., Easwaran, H. P., Nowak, D., Grunewald, I., Cremer, T., Leonhardt, H., and Cardoso, M. C. (2005). Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation. J Cell Biol 169: 733-743.

(3) Agarwal, N., Hardt, T., Brero, A., Nowak, D., Rothbauer, U., Becker, A., Leonhardt, H., Cardoso, M. C. (2007). MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation. NAR 35: 5402-5408.

Dr. Alexander Rapp (TU Darmstadt, Biologie)

Background: Both DNA damage induction as well as subsequent repair are dependent on the chromatin organisation. One important aspect is chromatin compaction and an other the presence of bound non-histone chromatin components for the recognition and procession of the DNA lesion. One predominant factor for chromatin packaging are histone modifications, and the question whether these affect DNA damage induction and/or whether these modifications are in turn affected by induced DNA lesions. E.g. a hyperacetylation upon damage induction is expected to loosen the chromatin packaging and thus allowing easier repair. An other aspect so far not well understood is the question how -after DNA repair- the epigenetic marks are restored or if they are not repaired how this affects the overall radiation induced effect on the cell.

In direct interplay with the histone modifications we are also aiming to study DNA methylation as an other level of epigenetic information. Here there exists an established model that neoplastic transformation leads to a specific change in methylation patterns throughout the genome, the question whether this is somehow connected to DNA damage is so far not understood.

Aim of project: This project is aimed to identify effects of different kinds of radiation (ionizing radiation, heavy ions and UV radiation) on histone modification patterns both on a whole genome scale or within defined genomic loci. To achieve this we use a combination of cell biology (live cell imaging together with microirradiation, immuno-fluorescence), biochemistry (protein analysis) and molecular biology (Chromatin immuno precipitation (ChIPqPCR of ChIPseq)). Interaction and collaboration with several other partners within the Graduiertenkolleg are already established and should be used to address these questions. The main partners are AG Löbrich (1A), AG Cardoso (1B), AG Deckbar (1D), AG Jakob 1E and AG Drossel (3A).

References:

(1) Vidanes et al. (2005) Cell; 121:973-6.

(2) Peterson und Lanie (2004) Curr Biol; 13:1029-37.

(3) Downs et al. (2004) Mol Cell; 16:979-90.

(4) Goodarzi et al. (2008) Mol Cell; 31:167-77.

(5)Ehrlich (2002) Oncogene; 21:5400-13.

Dr. Dorothee Deckbar (TU Darmstadt, Biologie)

Background: After the occurrence of DNA double-strand breaks (DSBs), different signaling cascades are activated in order maintain genomic integrity. Cell cycle checkpoints slow down or halt cell cycle progression to provide time for the repair mechanisms to remove the damages. Biochemically, the signaling cascades resulting in the inactivation of the CDK/Cyclin complexes which trigger proliferation are well characterized. In contrast, less is known about the downstream events leading to checkpoint induction and the consequences on the cellular level (1).

We recently demonstrated that the G2/M checkpoint is initiated very rapidly but is terminated before DSB repair is completed resulting in cells entering mitosis with unrepaired DSBs (2,3). As the coordinated interplay between the different cell cycle checkpoints is an important requirement for the maintenance of the genomic integrity, our current work focuses on the regulation of the G1/S checkpoint. Here, we demonstrated that the induction of DSBs by ionizing radiation (IR) in normal human slows down S phase entry but cells are still able to enter S phase for several hours (4). These cells progress through S phase with high numbers of unrepaired DSBs and exhibit chromosomal aberrations in the subsequent G2 phase and in mitosis compromising genomic stability. After its full induction 4-6 h after IR, the G1/S checkpoint is very sensitive and only releases single cells into S phase with unrepaired DSBs. To elucidate the consequences of the limitations of the G1/S checkpoint with regard to the development of cancerous growth it is important to obtain a better understanding for its regulation with respect to the DNA damage level.

Aim of the project: This project aims to elucidate the mechanisms underlying the limitations of the G1/S checkpoint with special emphasis on the interplay of DSB repair mechanisms and checkpoint activation.

Work plan: Within this project, the differences between the early insensitive response and the late sensitive response will be investigated. It could be demonstrated that both processes are ATM-dependent and that the early response additionally depends on the checkpoint kinase Chk2. Now, a more detailed characterization of the components involved in the regulation of the G1/S checkpoint as well as a comparison to the insensitive intra-S checkpoint is required.

First results of our group indicate that UV irradiation harbors the potential to directly abolish S phase entry after irradiation. UV does not induce DSBs but mainly photoproducts. This raises the question in which way the checkpoint-inducing signaling cascades differ between IR and UV. Because contradictory reports can be found in the literature regarding the potential of photoproducts to activate checkpoint-inducing signaling cascades (5), we now aim to shed light on this issue and to uncover potential differences between IR and UV. Furthermore, the consequences of the different regulatory mechanisms will be measured and compared on the cellular level.

The applied methods reach from biochemical and flow-cytometrical approaches to immuno-fluorescence microscopy and life cell imaging. This project is methodologically closely related to the projects 1A Löbrich, 1B Cardoso and 1C Rapp and there will be a close interrelationship with the groups Thiel and Schmidt. Furthermore, the expected results from this project will provide important information for the clinically oriented projects.

References:

(1) Löbrich and Jeggo (2007) Nat Rev Cancer; 7:861-9.

(2) Deckbar et al. (2007) J Cell Biol; 176:749-55.

(3) Krempler et al. (2007) Cell Cycle; 6:1682-6.

(4) Deckbar et al. (2010) Cancer Res; 70:4412-21.

(5) Callegari and Kelly (2007) Cell Cycle; 6:660-6.

Dr. Burkhard Jakob (GSI, Biophysik)

Background: Radiation induced chromosomal translocations generated by misrejoining of DNA double strand breaks (DSBs) may lead to failure in protein expression or regulation. The involvement of genetic regions coding for oncogenes in radiation induced translocations has the potential of cancer development. Beside radiation quality, the mobility of broken chromatin ends represents a crucial factor regarding the connection of incorrect DNA sequences. A local accumulation of DNA DSB ends as it might occur after high LET particle irradiation in connection with an increased chromatin mobility is expected to lead to an increased risk of chromosomal translocations.

First studies in our group indicate a non altered dynamic behaviour of damaged chromatin after high local DNA damage induced by particle irradiation (1). However, in the literature, examples of increased mobility of chromatin are reported under conditions of reduced or altered expression level of repair related proteins (2). In addition, the mobility of DSBs is of special interest for heterochromatic DSBs, as their processing was recently shown to take place at the boundary of Eu- and Heterochromatin, implying a small scale motion from the site of their origin (3; own unpublished results). Heterochromatin plays a prominent role in the maintenance of genomic integrity. Thus, a deeper understanding of the dynamics and spatiotemporal organisation of the repair of DNA-damage should shed light on the initial events potentially leading to the development of cancer.

The aim of the project: The impact of early acting repair factors like Parp1, 53BP1 and Ku80 on the mobility of DNA DSBs should be determined after sparsely and densely ionising irradiation. In case of a significant change in DSB mobility, the impact on the repair of heterochromatic DSBs will be examined Furthermore, the recruitment and binding behaviour of NBS1 (early DNA repair protein mutated in the disease Nijmegen Breakage Syndrome) will be addressed in the light of the afore mentioned factors. A long term goal aims at the connection between changes in the chromatin mobility and the formation of chromosomal translocations.

Work plan: The effect of early repair factors (involved in holding together the DNA ends) on the mobility of radiation-damaged chromatin will be determined by modulating these proteins using siRNA in mammalian cells. Alternatively, established knockout cell lines or chemical inhibition (e.g. 3_AB or PJ-34 or new inhibitors developed in project 2E Schmidt) will be applied. Cells will be irradiated with charged particles or x-rays,to evaluate the influence of lesion density, . The recruitment of the repair protein NBS1 can be determined in real time at the beamline microscope (4). In addition confocal live cell microscopy will add to the analysis of the mobility of radiation induced foci. Special attention will be turned to differences in the dynamic behaviour of eu- and heterochromatin using murine cell lines utilizing the special nuclear morphology of these cells (chromocenter). The obtained recruitment data will be directly implemented in the modelling of the repair factors in the project 3A (Drossel).

In addition, this project is linked via the exchange of knowledge to other members of the GRK, especially to the ones dealing with high resolution fluorescence microscopy or quantitative image analysis (1A Löbrich, 1B Cardoso, 1D Deckbar, 1F Meckel). The methods of this project comprise both molecular biology but also radiation biophysics and modern microscopic techniques. Physicists with interest in biology are highly welcome to apply.

References:

(1) Jakob et al. (2009) Proc Natl Acad Sci USA; 106:3172-7.

(2) Dimitrova et al. (2008) Nature; 456:524-8.

(3) Goodarzi et al. (2008) Mol Cell; 31:167-77.

(4) Jakob et al. (2005) Radiat Res; 163:681-90.

Dr. Tobias Meckel (TU Darmstadt, Biologie)

Background: The attachment of cells to the extracellular matrix (ECM) is a highly regulated process, which is carried out by integrin receptors. It plays an essential role at cellular processes like polarization, mobility, viability, proliferation, and differentiation. Hence, the ECM is also regarded a critical element in carcinogenesis and metastasis (1). In presence of an ECM cells have an elevated capacity to repair radiation-dependent damage and to continue cell division. Here, the beta1-Integrin mediated signal cascade is known to be a key component (2). Consequently, the impact of radiation on cellular processes needs to be investigated in consideration of the ECM.

The attachment of integrins and their spatial distribution in the plasma membrane of cells is directed by the micro architecture of the ECM: Chemical composition, the spatial distribution of its components, as well as the physical properties of the ECM (e.g. density and elasticity) plays an important role (3). Cells, which make direct use of the spatial distribution of their integrin receptors to orient themselves, are able to recognize differences in ligand distributions as small as 1nm (4).

Both X-ray and alpha-radiation can cause changes to the ECM. Collagen cross-links increase, elastin-chains break, and hyaluronic acid depolymerizes (5). Hence, radiation affects the micro architecture of the ECM and is therefore likely to change the distribution of integrin receptors, causing further cellular reactions.

Aim of the project: This project aims to investigate the impact of radiation in relation to the ECM. How radiation-dependent changes of the ECM cause changes in the spatial distribution, mobility, and activity of beta1-integrin will show, whether cells are able to recognize extracellular radiation-dependent damage.

Work plan: A549 cells (human lung carcinoma) will be cultured on selected ECM components (i.e. collagen, fibronectin, laminin) in 2D, as well as in ECM-polymers in 3D (in collaboration with project 2B, Dr. Fournier). Beta1-integrins will be fused to photoactivatable fluorescent proteins, to localize their positions and follow their mobility with very high spatial (<30 nm) and temporal (<5 ms) precision using single molecule microscopy. The ECM-reproductions will then be exposed to radiation of different quality and intensity, both in presence and absence of A549 cells. Thereby, effects on cells can be separated from effects on the matrix.

In the course of the project, the existing single molecule setup will be extended to locate and track single molecules in 3D. In addition, a simple fluorescent microscope will be constructed, which allows for the observation of cells during X-ray radiation (in collaboration with project 2A, Prof. Thiel).

References:

(1) Barcellos-Hoff et al. (2005) Nat Rev Cancer; 5:867-75.

(2) Nam et al. (2009) Int J Radiat Biol; 85:923-8.

(3) Cavalcanti-Adam et al. (2007) Biophys J; 92:2964-74.

(4) Arnold et al. (2008) Nano Lett; 8:2063-9.

(5) Mohamed et al. (2007) Nuclear Instruments and Methods in Physics Res Section A; 580:566 – 569.