Cell Biology of Genome Maintenance

Cell Biology of Genome Maintenance

Maintaining the integrity of genetic information is essential for cell survival. Mechanisms that counteract DNA damage are important to help maintain cellular homeostasis by suppressing mutagenic events and genome rearrangements that may lead to disease, particularly cancer. In response to DNA damage complex signaling networks mediate cell cycle arrest and repair. Collectively, these pathways are referred to as the DNA damage response (DDR) and they consist of hierarchically organised signaling cascades.

The focus of our lab is to uncover the fundamental principles of DDR regulation in response to DNA double-strand breaks (DSBs), and identify how illegitimate repair contributes to the formation of tumorigenic chromosome translocations. Our studies use an interdisciplinary approach, combining single-cell measurements, advanced microscopy techniques, genomic methods and genetics to dissect the cellular mechanisms that preserve genome integrity. Understanding these aspects of genome maintenance is a vital step towards elucidating the fundamental mechanisms of cancer etiology. 

DSB-repair in the context of chromatin and genome architecture

Tremendous progress has been made in the last decade in identifying the molecular players and pathways that mediate the response to DSBs. These pathways involve the targeting of histone- and chromatin-associated proteins by a plethora of modifications, which trigger nucleosome repositioning and changes in higher-order structure of chromatin. Together, these changes promote the accumulation of repair proteins in large segments of chromatin surrounding DNA lesions, fine-tune the DDR and dictate which repair pathway will be used.

One of the aims of our lab is to understand how these processes are regulated in different cell cycle phases within distinct chromatin domains, and how this regulation influences the DSB-repair outcome. Our studies use state-of-the-art high-throughput microscopy and advanced image analysis and mining methodologies, in combination with novel and established genomic tools. 

Identifying mechanisms of translocation biogenesis

Chromosome translocations are formed by the illegitimate joining of DSBs to produce aberrant fusions with oncogenic potential. While translocations have been extensively characterised and their relevance in cancer is well established, how they form in the context of the intact cell nucleus is remarkably poorly understood.

Figure 1. Capturing and quantifying rare chromosome translocations. An experimental system to integrate key mechanistic steps in the formation of translocations (A; Roukos et al, 2013; Roukos et al, 2014). Engineered ISceI restriction sites adjacent to LacO and TetO array sequences, are integrated on distinct chromosomes, and can be visualised through the LacO/TetO operator/repressor system (red and green spots, respectively). Expression of the endonuclease ISceI induces double-strand breaks (DSBs), which can be tracked in space and in time by live-cell imaging. This established system is used to analyze the DSB-motion properties by mean squared displacement (MSD) analysis, to measure the rate of synapsed double-strand breaks by high-throughput (HT) microscopy and to quantify the frequency of the formed translocations by PCR. Alternatively, high-throughput timelapse microscopy is used to capture the rare joining events that can be seen where the two chromosome ends (red and green spots) become persistently superimposed (yellow spots), as identified by automated image analysis (B, C) (Roukos et al., 2013).

We have recently developed a unique cell-based system that allows us to follow the fate of individual DSBs as they form translocations in living cells (Figure 1) (Roukos et al, 2013). This system can be used as a tool to identify factors that alter translocation frequency and to interrogate key mechanistic steps of translocation formation (Figure 1). Our lab is interested in using this approach to shed light on the molecular and cellular mechanisms that contribute to translocation biogenesis. In addition, our ongoing efforts are focused on modelling recurrent, cancer-initiating chromosome translocations and elucidating the mechanisms underlying their formation.

We are always looking for highly motivated researchers who enjoy working in a multi-disciplinary and collaborative environment to join our group.