Molecular Systems

Lead Research Organisation: MRC London Institute of Medical Sciences

Abstract

The human genome contains many sites that look like they disrupt important genes and be might therefore lead to disease. Yet typically, they do not. One important reason is that, looking at the genome sequence on a computer screen, we can see the entirety of genetic information, whereas inside a living cell, a particular portion of the genome can be packaged away into hard-to-access structures or covered by proteins so that the deleterious information cannot be read by the cell.
Our research aims to understand which parts of the genome are visible to the cell and which are not and how we can use this information to discriminate mutations that only look dangerous from those that are actively harmful to the cell.
In order to see what the cell sees and thereby understand how some harmful-looking bits of DNA fail to exert their disruptive effects, we first look at where and when different proteins bind to DNA in different cell types. We then link this to genetic differences within the population to reconstruct how the presence of these binding events has allowed deleterious signals to survive in some regions of the genome but not others. As we aim to understand general principles of how this masking works, we consider not only humans but also species such as yeast and the bacterium E. coli, where we can test predictions experimentally.

Technical Summary

Our aim is to understand how intermolecular interactions inside the cell bias the incidence of mutations, affect their persistence, and ultimately shape patterns of natural variation within and between species.
Specifically, our research focuses on two main areas:
First, we study how the binding of structural chromatin proteins such as histones affects the evolution of the underlying sequence through biasing mutation and repair dynamics. For example, are some genomic regions particularly prone to mutations by virtue of their chromatin architecture? To address these issues, we combine genome-wide data on genetic variation with protein-DNA interaction data (from MNase/Chip-Seq assays) in an evolutionary framework to characterize how genome architecture has shaped evolutionary processes over different time scales (within and between species). Importantly, by integrating further knowledge of cellular processes, such as when certain repair pathways are active, our research is geared towards providing explicit pointers to the molecular mechanisms that link chromatin organization and mutation. In the longer term, this will help us understand and eventually interfere with specific mutational processes including those operating in cancer genomes. As we aim to understand fundamental principles of chromatin-mediated effects, we analyze data from a variety of organism, including humans, bacteria, and archaea, in a comparative manner.

Second, complementing our studies of the biased origin of genetic novelty, we investigate the biased maintenance of genetic variants. Specifically, we focus on how intermolecular interactions can facilitate the maintenance of ostensibly deleterious states. This includes, for example, the capacity of chaperones to buffer the effect of mutations that destabilize protein structure and of RNA-binding proteins to prevent detrimental use of cryptic processing sites such as cryptic polyadenylation signals. Understanding how interactions can mask otherwise deleterious sequence signals is critical for identifying potential disease variants from a frequently vast pool of candidates because signals that are effectively invisible to the cell (and hence not exposed to selection) can easily be misidentified as likely causal variants underlying an observed phenotypic/disease state. Our approach here mirrors our earlier strategy of combining evolutionary sequence data with interaction data (e.g. RNA-protein interaction data from CLIP-Seq experiments) to understand how binding affects the visibility of sequence signals to the cellular machinery and thus mediates whether deleterious signals can persist over time. Currently, our main focus is on characterizing the mutation buffering effect of DEAD-box helicases, a class of RNA chaperones, in E. coli, through a combination of genetic manipulations (gene deletion and overexpression) and competitive fitness assays – conducted in collaboration with Anita Krisko from the Mediterranean Institute for Life Sciences in Split – RNA structural modelling and CLIP-Seq experiments.
 
Description Imperial College Junior Research Fellowship
Amount £150,000 (GBP)
Organisation Imperial College London 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description Integrative Experimental and Computational Biology Studentship
Amount £69,125 (GBP)
Organisation Imperial College London 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 03/2018
 
Description Marie Sklodowska-Curie Individual Fellowship
Amount € 183,454 (EUR)
Funding ID MuRChap - DLV-747199 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 09/2017 
End 08/2019
 
Description Comparative models for sickle cell disease 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Collection of deer blood/tissue samples. Sequencing, assembly, and evolutionary analysis of beta-globin orthologs to understand the genetic basis and evolution of sickling phenotype. Manuscript preparation.
Collaborator Contribution MRC HGU - computational modeling of protein structures and the impact of individual mutations on sickling propensity. Imperial - tissue samples.
Impact Esin et al (2018) The genetic basis and evolution of red blood cell sickling in deer. Nature Ecology and Evolution 2:367
Start Year 2016
 
Description Comparative models for sickle cell disease 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom 
Sector Public 
PI Contribution Collection of deer blood/tissue samples. Sequencing, assembly, and evolutionary analysis of beta-globin orthologs to understand the genetic basis and evolution of sickling phenotype. Manuscript preparation.
Collaborator Contribution MRC HGU - computational modeling of protein structures and the impact of individual mutations on sickling propensity. Imperial - tissue samples.
Impact Esin et al (2018) The genetic basis and evolution of red blood cell sickling in deer. Nature Ecology and Evolution 2:367
Start Year 2016
 
Description Mutation buffering in RNA 
Organisation Imperial College London
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Re-sequencing of E. coli strains and bioinformatic analysis of mutations. RNA-sequencing/proteomics of mutant S. cerevisiae strains and analysis of differential gene expression. Conceptualisation and planning of experiments. Manuscript writing
Collaborator Contribution Conducted various experiments to characterize the phenotypic impact of mutations found in two mutator strains, including fitness assays. Phenotyping of mutant S. cerevisiae strains. Construction of S. cerevisiae mutants.
Impact Rudan et al (2015) RNA chaperones buffer deleterious mutations in E. coli. eLIFE 4:e04745 Rudan et al (2018) Normal mitochondrial function in Saccharomyces cerevisiae has become dependent on inefficient splicing. eLIFE:e35330
Start Year 2013
 
Description Mutation buffering in RNA 
Organisation Mediterranean Institute for Life Sciences (MedILS)
Country Croatia 
Sector Charity/Non Profit 
PI Contribution Re-sequencing of E. coli strains and bioinformatic analysis of mutations. RNA-sequencing/proteomics of mutant S. cerevisiae strains and analysis of differential gene expression. Conceptualisation and planning of experiments. Manuscript writing
Collaborator Contribution Conducted various experiments to characterize the phenotypic impact of mutations found in two mutator strains, including fitness assays. Phenotyping of mutant S. cerevisiae strains. Construction of S. cerevisiae mutants.
Impact Rudan et al (2015) RNA chaperones buffer deleterious mutations in E. coli. eLIFE 4:e04745 Rudan et al (2018) Normal mitochondrial function in Saccharomyces cerevisiae has become dependent on inefficient splicing. eLIFE:e35330
Start Year 2013
 
Description Structural bioinformatic tools for predicting epistasis 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom 
Sector Public 
PI Contribution This project, led by UKRI Innovation Fellow Jacob Swadling, is looking to establish whether intramolecular epistasis (in the context of protein structures) can be adequately predicted using state-of-the-art molecular dynamics simulations and, if not, whether we can develop these tools further to allow adequate predictions to be made. We are then looking to model the structural effects of human genetic variation on a population-wide scale, including hypothetical situations where multiple existing variants are combined in the same molecule. We provide expertise in human variant data and epistasis.
Collaborator Contribution Our collaborator, Joseph Marsh, provides expertise in computational structural biology.
Impact None at present
Start Year 2018
 
Description Imperial CREST 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 4 A-level students developed their own research project - on methylation and epigenetic inheritance in bacteria - that is now being implemented by them in my lab (during supervised fortnightly visits).
Year(s) Of Engagement Activity 2017,2018
 
Description Skype a Scientist 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact I participated in "Skype a Scientist", a US-/Canada-focused outreach event in which schools are connected, on a subject-specific basis, to researchers who go on to Skype into the classroom to discuss their research, career, and scientific subjects of interests to the pupils. I participated in 3 Skype calls this year.
Year(s) Of Engagement Activity 2018
URL https://www.skypeascientist.com/