It is well established that chromosome instability is an underlying cause of several age-related disorders, including cancer and neurodegenerative diseases. Changes in chromosome number and structure can be triggered not only by DNA damaging agents, but also by intrinsically unstable regions of the human genome itself – which we term the ‘enemies within’. The overarching goal of the Center for Chromosome Stability (CCS) is to functionally annotate these ‘enemies within’, and determine how they drive tissue aging and limit cell immortality.
The CCS currently comprises 6 research groups. The group headed by Andres Lopez-Contreras closed during 2020, but Andres will retain an affiliation with the CCS and ICMM. A new research group headed by Tom Miller will join the CCS during 2021. The different research groups possess complementary expertise in genetics, biochemistry and molecular/cell biology. The leaders of the CCS’s research groups are listed below, with a short description of research interest and a link to the home page of the group.
"Mechanism of chromosomal fragile site instability"
Our research is focused on understanding how chromosomal instability impacts on human disease. We study genes in human cells that, when mutated, lead to defective genome maintenance and high rates of cancer, neurodegeneration or infertility.
In recent years we have focused on common fragile sites as a model for understanding how ‘difficult-to-replicate’ regions of the human genome affect propagation of the genome through successive cell divisions. As part of these studies, we identified ultra-fine anaphase bridges (UFBs) which are DNA thread-like structures connecting the separating sister chromatids in the anaphase of mitosis. These UFBs exist in every human cell division but are normally ‘invisible’ due to their lack of staining with DNA dyes. They are coated with specific proteins, including the SNF2 family protein, PICH and the BLM helicase (which when mutated causes Bloom’s syndrome – a cancer predisposition disorder), that allows detection using specific antibodies.
We combine protein biochemistry with molecular/cell biology and high resolution imaging of human cells. Latterly, we have also been developing tools to exploit new developments in microfluidics and single molecule biophysical techniques to reconstitute in vitro defined steps in DNA metabolism.
Homepage for Hickson Group
(Department of Cellular and Molecular Medicine)
"Chromosome dynamics in the germline"
Chromosomes are rearranged and organized into new sets to create diversity as they are passed from parent to offspring through the germline. The genetic changes can be followed in populations, however this represents only a small proportion of the diversity that is generated in our germline. Men produce 500 billion sperm in their lifetime and women are born with two million eggs. Human reproduction is particularly error-prone. 50% of pre-implantation embryos have defects in their development and 20%-85% of human eggs have extra or missing chromosomes (aneuploidy). Maternal age is the strongest risk factor known for aneuploidy and our interests in reproductive aging also includes understanding the decreased quality as well as the decline in the number of eggs as women age.
Our laboratory investigates the role of the DNA damage response and cell cycle proteins in governing the genetic changes that occur in the germline to generate diversity and maintain genome stability. In particular, we focus on those genes that when defective give to reproductive disease or cancer. We use a powerful combination of model organisms (mouse and yeast) as well as human eggs and embryos to explore this poorly understood area of human biology.
Homepage for Hoffmann Group
(Department of Cellular and Molecular Medicine)
"High throughput screening for modulators of genome stability"
In response to DNA damage, the DNA repair machinery is assembled at the site of damage in a highly choreographed manner depending on the chromosomal context, the type of damage, cell cycle phase and other factors.
We study the spatiotemporal organisation of DNA repair processes in the cell nucleus with special focus on homologous recombination (Symington et al. 2014), which plays a key role in the repair of DNA double-strand breaks, restart of stalled or collapsed replication forks, and telomere length homeostasis in telomerase negative cells (Silva et al. 2016). To identify the fundamental biological mechanisms underlying DNA repair, we combine genome-wide cell biological and genetic analyses in the yeast Saccharomyces cerevisiae with more focused studies in vertebrate cell lines (Gallina et al. 2015). In addition to studying the proteins that catalyse DNA repair, we also aim to identify and understand the post-translational modifications that regulate these processes such as sumoylation, phosphorylation, acetylation and ubiquitylation.
If chromosome aberrations are not repaired in a timely manner prior to cell division, they may lead to DNA anaphase bridges in mitosis (see illustration below), chromosome breakage, and ultimately missegregation of genetic material (Germann et al. 2014; Pedersen et al. 2015). DNA anaphase bridges have been linked to chromosomal fragile sites (see the Hickson Lab website), which are chromosome regions prone to exhibit gaps and breaks on metaphase chromosomes, especially when cells are challenged by DNA replication stress. Notably, more than 50% of recurrent cancer mutations have been linked to fragile sites. Our aim is to identify the proteins and mechanisms responsible for sensing and resolving DNA anaphase bridges in a manner that preserves chromosome integrity and to understand the coordination of these processes with mitosis.
Homepage for Michael Lisby
Our group aims to understand the processes cells use to counteract replication stress caused by either internal factors (i.e. oncogene activation), or external factors (i.e. folate deficiency), and why some regions in the genome are particularly susceptible to those factors (e.g. common or rare fragile sites). Specifically, we focus on the following two areas to achieve our goals. We employ techniques in the fields of cellular and molecular biology, cytogenetics, biochemistry, advanced imaging, whole genome sequencing, and mass spectrometry-based proteomics.
1) The characterization of helicases that play a role in resolving DNA or RNA associated secondary structures
It is well established that incomplete replication can cause a delay in chromatin condensation that leads to the ‘expression’ of common fragile sites (CFSs) [1, 2]. CFSs are hot spots for deletions and chromosome rearrangements in cancer . We previously discovered that a process called mitotic DNA synthesis (MiDAS) that operates in mitosis is a strategy used by human cells to rescue the incomplete replication at those loci, particularly in cancers cells [4-7]. Our current findings demonstrate that RTEL1, a DNA helicase, can prevent the accumulation of G-quadruplex-associated R-loops at difficult-to-replicate loci including CFSs in the human genome in S phase, and can facilitate MiDAS in M phase . We are now investigating how RTEL1 accomplish these important roles using in vitro or in vivo assays. (Figure 1)
2) The analysis of folate deficiency induced genome instability
Folate deficiency is known to be associated with a diverse range of human disorders including fetal neural tube defects, age-associated dementia, infertility, and some type of cancers. Intriguingly, folate deficiency is known can cause the expression of group of rare fragile sites, all of which contain long stretch of CGG simple repeats. The most well studied locus of this kind is called FRAXA that is associated with Fragile X syndrome (FXS). Using FXS cells as a model, we have demonstrated that (i) folate deprivation triggers the extensive missegregation and aneuploidy of chromosome X , and (ii) MiDAS at the FRAXA locus occurs via break-induced DNA replication (BIR) and this process requires the SLX1/SLX4 endonuclease complex, the RAD51 recombinase and POLD3 . We are currently investigating other regions that are vulnerable to folate deficiency in the human genome, and the strategies cells employ to maintain the stability of those regions. (Figure 2)
Homepage for Liu Group
(Department of Cellular and Molecular Medicine)
"DNA damage and replication stress signaling"
A key focus of the Mailand lab (The Novo Nordisk Foundation Center for Protein Research) is to understand how regulatory signaling processes promote cellular responses to DNA damage and replication stress to protect chromosome stability in mammalian cells. While much is known about the basic machinery underlying DNA repair pathways, the regulatory framework that controls and coordinates these processes is less well understood. To remedy this knowledge gap, we are combining innovative systems-wide screening approaches with focused cell- and biochemistry-based studies in order to identify and functionally characterize new factors and signaling processes that protect the integrity of the genome following genotoxic insults.
Within the Center for Chromosome Stability (CCS), our major focus is to characterize the signaling processes that enable cells to overcome replication stress, a major driver of genome instability. To understand the molecular mechanisms employed by cells to mitigate the threat posed by replication stress, we are employing cutting-edge strategies to enrich and map the factors that are recruited to the replication machinery when it encounters DNA lesions or other impediments to its continued progression. In one approach, we are using the BioID method for biotinylation and isolation of factors acting directly in the context of replication forks under various conditions. In another approach, we combine Xenopus egg extracts, a powerful cell-free system for biochemical studies of DNA transactions, with mass spectrometry to identify the spectrum of cellular proteins undergoing enrichment at the replication machinery upon DNA damage or replication stress. Using such strategies we are now able to obtain detailed, systematic insights into the signaling processes that operate directly in the context of stalled replication forks to counteract the deleterious consequences of replication stress. In collaboration with other groups within CCS, we are currently studying the functions and physiological importance of several newly identified components of replication stress-responsive pathways in protecting chromosome stability.
Homepage for Mailand Group
(Novo Nordisk Foundation Center for Protein Research)
"Regulatory fundaments of DNA replication"
In our recently established lab we are interested in the molecular mechanisms that allow mammalian cells to fully and faithfully replicate their DNA content. In every division cells across species face a plethora of challenges in order to successfully duplicate their genome. Aberrant structures or modifications on the DNA molecule can act as roadblocks that stall and even dismantle replication forks. A lot of focus has been set in the past years on understanding how, in response to these obstacles, cells elicit the so called replication stress responses. Various molecular pathways allow cells not only to restore the damage but to temporarily adapt the replication machinery so that DNA synthesis can be resumed.
In our lab we focus on understanding how cells have solved a more fundamental level of challenges that are inherent to the architecture of the DNA replication machinery. Specially for higher eukaryotes, DNA replication requires a great level of regulation both locally (at the level of the replisome) and globally (throughout the nucleus) that we still poorly understand. At the replisome multiple proteins act together and cooperate to carry out different functions necessary for DNA synthesis in perfect synchrony. Among these, we want to understand how DNA unwinding is coupled with DNA synthesis, which implies a very different task at the leading and at the lagging strand. Nucleus wide, DNA replication has to be tightly regulated to proceed at multiple sites simultaneously with perfect temporal and spatial coordination. How this is achieved in a three dimensional space so that replication proceeds orderly following the so called replication program is still an outstanding question in the field. When any of these multiple levels of regulation is disrupted, cells can suffer endogenous replication stress, which can increase the rate of genomic aberrations favouring malignant transformation but also lead to irrecoverable DNA damage and cell death. Thus understanding these processes and how cells deal with the different sources of exogenous replication stress is highly relevant for human disease. Importantly, the fundamental challenges that cells encounter during DNA replication can be exploited to develop new strategies to treat diseases like cancer, which is the ultimate goal of our lab. For that, we also carry out small molecule screenings to boost the discovery of novel therapeutic targets that could be used in the treatment of proliferative disorders.
We are a cell biology lab with a taste for modern technologies. We use state of the art methodologies such as high content microscopy to analyse cell biology with quantitative detail in single cell resolution. Our research is performed in transformed and primary human cell lines, where we apply classical and modern genetic manipulation approaches (siRNA and CRISPR), intervention with small molecules, and quantitative mass spectrometry to dissect novel regulatory mechanisms and the key players involved.
Homepage for Toledo Group
(Department of Cellular and Molecular Medicine)
Affiliated Research Group
"Mouse models of DNA replication stress"
Chromosome instability leads to genetic alterations that can cause cancer and other diseases. To limit these alterations, cells have evolved complex mechanisms that coordinate cellular responses altogether known as the DNA Damage Response (DDR). However, in many cases the DDR is not sufficient to protect our genomes from either exogenous or endogenous insults such as replication-borne DNA damage. The overall goal of our laboratory is to increase our knowledge of the mechanisms that regulate the DDR and the consequences of their deregulation. This will help us to dissect and understand the underlying causes of a number of diseases and to identify novel therapeutic strategies.
With this purpose, we are utilizing a wide range of approaches to investigate chromosomal instability from the underlying molecular mechanisms to its ultimate consequences on health. We are using cellular-based systems to perform screens, gain mechanistic insight and analyse cellular phenotypes in different genetic contexts. In addition, we will generate transgenic mouse models to address the physiological impact of specific genetic alterations and, in particular, their influence on cancer and aging. For both, cellular and mouse models-based studies, we are using the novel CRISPR/Cas9 technology to efficiently manipulate the genome. We also employ high content microscopy (HCM) as a routine approach to quantify alterations in the DDR in our different models. In addition, HCM will allow us to set-up different genetic and drug screenings to identify novel therapeutic targets or compounds with clinical interest. Finally, in collaboration with groups at the Center for Protein Research (CPR), we will perform a number of proteomic studies to investigate novel regulatory networks and the interplay between different proteins involved in the DDR. The most relevant findings will be validated and further characterized using mouse models.