Our projects address how cell-to-cell chromatin heterogeneity enables pathogens to overcome challenges critical for a persistent infection, i.e. adaptation to a new host environment, evasion of the host immune response and differentiation to transmission-competent states that can enter a new host.
ESR 1: Non-coding transcription in the generation of diverse chromatin structures (Mellor)
Main supervisor
Jane Mellor
Location
University of Oxford, UK
Second supervisor
Robert Schneider (Helmholtz Zentrum)
Description
The main objective of this project is to use time-resolved synchronous yeast cells to examine whether non-coding transcripts alter local chromatin structures and to show how this affects phenotypic variability. ESR1 will assess cell-to-cell variability, exploiting the microfluidics and bioinformatics expertise at Helmholtz Zentrum Munich, as environmental conditions, levels of non-coding transcription, or genetic backgrounds vary. Data will be modelled to quantify the relative effects on chromatin and gene expression.
We will exploit environmental conditions to modulate transcription and higher-order structures in the chromatin to reveal underlying interdependencies. Initially, we will map 3C interactions, genome-wide, and relate these to coding and non-coding transcription in the vicinity in cells growing in different conditions or mutant backgrounds and if feasible, using data from a synchronous culture, acting as a virtual single cell. We expect this will define new loci that we can use to validate experimentally these relationships. We will focus on loci where we have already mapped non-coding and coding transcription-dependent chromatin structures, to show how these influence cell-to-cell variability using microfluidics. We expect the microfluidic data will produce quantitative data on variability, which can be modelled to allow us to predict how changes in transcription will influence higher-order structure and variability and to validate this experimentally. Contingency: Should it not be possible to relate chromatin conformations mapped using 3C directly to transcription and phenotypic variation, we will use 4C/capture C to map interactions hubs linked to the GAL locus and the non-coding transcripts in the vicinity. Planned secondments: Month 14-16 to Helmholtz Zentrum Munich to develop the microfluidic analysis of phenotypic variation and to learn advanced bioinformatics
Key techniques
Bioinformatics analysis of 3C chromatin interactions (microC), gene expression, microfluidics, mathematical modeling, yeast genetics, S. cerevisiae
Prerequisites
Master’s degree or four-year bachelor in biological sciences, physics, bioinformatics or bioengineering with practical experience in bioinformatics and molecular biology. Interest in genetics and bioinformatics.
ESR 2: Transcriptional noise in dynamic systems (Schneider)
Main supervisor
Robert Schneider
Location
Institute of Functional Epigenetics, Helmholtz Zentrum Munich, Germany
Second supervisor
Jane Mellor (University of Oxford)
Description
Transcriptional noise is the main source of the variability (noise) in gene expression occurring between isogenic cells within populations. This transcriptional noise is important for survival of cells in changing environments or drug resistance and pathogenicity.
Our lab has recently developed novel microfluids based approaches that allow (i) following (tracking) single, individual budding yeast cells over multiple generations and gene inductions as well as (ii) reproducible media changes to alter gene expression profiles. We will optimize these microfluidics setups to study gene expression noise in dynamic systems (e.g. using reporters during gene induction or repression) over time and to systematically screen the role of chromatin components in transcriptional noise. These studies will be complemented by computational analyses to model transcriptional noise in dynamic systems and to generate testable predictions for the function of chromatin factors in transcriptional noise.
Key techniques
Cell-tracking, design and manufacturing of custom microfluidics chambers, live-cell imaging, single-molecule FISH analysis, pedigree analysis, image segmentation, image analysis, mathematical modeling, yeast genetics
Prerequisites
Master’s degree or four-year bachelor in biological sciences, physics, bioinformatics or bioengineering with practical experience in bioinformatics and molecular biology. Interest in genetics and bioinformatics.
ESR 3: Variability and noise during the cell cycle (Barkai)
Main supervisor
Naama Barkai
Location
Weizmann Institute of Science, Rehovot, Israel
Second supervisor
Robert Schneider
Description
Transcription noise leading to cell-to-cell variability in gene expression or phenotypic response is commonly attributed to stochastic transcription, but can also result from deterministic processes that are not synchronized between cells in the population. Cell cycle may be particularly important in that regards, as cell growth and DNA replication, which occur at different stages of the cell cycle, change protein concentration, cell volume and the epigenetic landscape, all of which are critical for defining gene expression and cell phenotypic response.
In this study, we will focus on the epigenetic landscape, examine changes in the landscape during the cell cycle, and ask how these changes affect cellular responses. For this, we will use the budding yeast (S. cerevisiae) model. First, we will rely on our previous data describing the abundance of different chromatin marks along the cell cycle. Second, we will address histone exchange rate, a critical yet poorly explored epigenetic process modulating. To allow this, we established a new method that overcomes major limitations of existing approaches, allowing us to define changes in exchange rate along the cell cycle. Third, we will define the consequences of epigenetic variability on the cellular stress response. Finally, we will examine how variability is buffered, focusing on the role of transcription-factor duplicates revealed in our recent study.
Key techniques
HTP profiling (ChIP-Seq, RNA-Seq), bioinformatics, mathematical modeling
Prerequisites
Master’s degree or equivalent in natural sciences with practical experience in bioinformatics, molecular biology and modeling. Interest in budding yeast.
ESR 4: Cell-to-cell heterogeneity among parasite tissue populations (Figueiredo)
Main supervisor
Luisa Figueiredo
Location
Instituto de Medicina Molecular, Lisboa, Portugal
Second supervisor
Nicolai Siegel (Ludwig Maximilian University of Munich)
Description
The study of protozoan pathogens has been extensively explored often motivated to find suitable targets for new intervention strategies. Using a mouse model of African trypanosomiasis, we have recently discovered that the adipose tissue (fat) is a major reservoir for the extracellular protozoan Trypanosoma brucei and that, within this environment, parasites become phenotypically different from those in the blood. Our study exposed novel biology of the T. brucei life cycle, yet it remains unknown how parasites adapt to the fat tissue environment. Ongoing in vivo imaging studies revealed that the fat harbors parasite populations with distinct motilities, suggesting cell-to-cell functional differences. We hypothesize that fat harbors a subpopulation of slow-growing (or quiescent) parasites that do not cause significant pathology and that can be awakened at later stages of the infection.
The goal of this project is to identify the transcriptomic differences between individual parasites within tissue and between tissues. For this, we will use a mouse model of infection, tissues will be collected and parasites isolated by FACS or microfluidic platforms. In the second phase, we will test if such phenotypic changes are operated epigenetically. Understanding the phenotypic differences between individual parasites will give an unprecedented view of the degree of adaptation that takes place when parasites infect the mammalian host and may reveal novel therapeutic strategies.
Key techniques
single-cell isolation of parasites, computational analysis of single-cell datasets (scRNA-seq and ATAC-seq), live microscopy imaging, mice infection
Prerequisites
Master’s degree or equivalent in biological sciences or bioengineering with practical experience in bioinformatics, molecular biology and animal experimentation. Interest in infectious diseases.
ESR 5: Control of heterogeneity in pathogen populations (Siegel)
Main supervisor
Nicolai Siegel
Location
Ludwig Maximilian University of Munich, Germany
Second supervisor
Maria Colomé-Tatché (Helmholtz Zentrum)
Description
An important strategy used by many pathogens to evade the host immune response is antigenic variation. Antigenic variation refers to the capacity of an infecting organism to systematically alter the identity of proteins displayed to the host immune system, making it difficult or impossible for the host to eliminate the pathogen.
Trypanosoma brucei, a unicellular parasite responsible for lethal and debilitating diseases in humans and animals, has long served as a valuable model to study antigenic variation. Yet, many of the most basic questions have only recently, through different technological advances, become experimentally tractable. Recent data from our lab demonstrate that changes in local chromatin structure and genome architecture can affect which antigen is expressed and present on the surface of the parasite (Müller, Cosentino et al, 2018, Nature). However, a key question that has remained unanswered is why some cells exchange the expressed antigen and others do not (see also the related project of ESR 11).
The goal of this project will be to combine microfluidics and next generation-based sequencing-approaches to establish pipelines to measure antigen switch frequencies of cells at high accuracy.
Key techniques
microfluidics, live-cell imaging, single-cell RNA-seq and single-cell ATAC-seq
Prerequisites
Master’s degree or equivalent in biological sciences, physics or bioengineering with practical experience in molecular biology. Interest in microfluidics, live-cell imaging, machine learning and bioinformatics
ESR 6: Linking single-cell transcriptome data with single-cell ATAC-seq data (Sezerman)
Main supervisor
Ugur Sezerman
Location
Epigenetiks (Epigenetiks Genetik Biyoinformatik Yazılım), Istanbul, Turkey
Second supervisor
Jane Mellor (University of Oxford)
Description
While emerging technologies deliver diverse single-cell datasets, algorithms to reveal functional links between different types of datasets are missing. ESR6 will establish pipelines for the analysis of scRNA-seq and scATAC-seq data and develop algorithms to reveal links between transcriptome and chromatin structure from single-cell data.
In this study, we will establish pipelines for the analysis of single-cell RNA-seq and single-cell ATAC-seq data. We will further develop tools to unveil functional links between single-cell datasets derived from different methods.
Key techniques
computational biology, single-cell data analysis, mathematics, and mathematical modeling
Prerequisites
Master’s degree or equivalent in mathematics, physics, bioinformatics, computer engineering. Interest in biology, good knowledge of statistics and very good programming skills.
ESR 7: Single-cell methods to study 3D genome architecture in hosts and pathogens (Bienko)
Main supervisor
Magda Bienko
Location
Karolinska Institute, Stockholm, Sweden
Second supervisor
Angela Taddei (Institut Curie)
Description
The overarching goal for this ESR is to develop tools that can be used to probe genome architecture at the single-cell level, and study the effect of host-pathogen interplay on the 3D genome. Specifically, the ESR will work on two projects:
Project 1: Developing methods for 3D genome reconstruction in single-cell organisms. The ESR will build on our recently developed GPSeq (Girelli et al, in revision for Nat Biotech) and CUTseq (Zhang et al., Nat Commun 2019) methods, and develop new sequencing-based approaches for measuring chromosome contact frequencies and radial positions genome-wide in single cells. Considerable effort will be put in tailoring these methods for single-cell pathogens, whose genome size is 1–2 orders of magnitude smaller compared to one of mammalian cells. The ESR will also attempt to develop a single-cell assay that can simultaneously probe the 3D genome architecture of intracellular pathogens and their host cells.
Project 2: Developing tools for single-molecule DNA and RNA FISH in single-cell organisms. The ESR will use a bioinformatic pipeline already available in the Bienko lab to design: i) DNA FISH probes densely covering the genome of each pathogen studied in the network (this will allow high-resolution visualization of chromosomes and individual loci in single cells); ii) single-molecule RNA FISH (smFISH) probes for each protein-coding transcript in the pathogens studied by the network (this will allow monitoring the expression of individual genes and relate it to their nuclear position and overall chromosome organization). The ESR will then use our recently established iFISH platform (Gelali et al., Nat Commun 2019) to produce the designed probes and create an open-access repository, which will be made available to all the other ESRs in the network. Besides, the ESR will work in conjunction with other ESRs to optimize experimental FISH conditions for applying these probes to different types of cells and tissues.
Key techniques
single-molecule imaging; fluorescence in situ hybridization; single-cell sequencing methods
Prerequisites
Master’s degree or equivalent in biotechnology/bioengineering/biophysics, with demonstrable expertise in common laboratory practices and basic molecular biology methods (PCR, reverse transcription), and a keen interest in methods development and quantitative biology. Knowledge of R/Python is required. Prior experience with fluorescence microscopy and NGS library preparation is a plus.
ESR 8: Dynamics of genome reactivation in relation to chromatin unfolding upon return to growth after quiescence (Taddei)
Main supervisor
Angela Taddei
Location
Institut Curie, Paris, France
Second supervisor
Magda Bienko (Karolinska Institutet)
Description
Unicellular organisms finely tune their growth and behavior to their environment, adapting to nutritional depletion or stresses by engaging specific metabolic and developmental programs. In particular, upon exhaustion of nutrients, a subpopulation of S. cerevisiae reversibly exits the cell cycle and differentiates into a quiescent state (a phenomenon common among pathogens). The genome of these “Q cells” adopts a very specific conformation associated with a massive decrease in transcriptional activity, two features that contribute to Q cell longevity (Guidi et al., 2015; McKnight et al., 2015). Importantly, both transcriptional activity and genome conformation are rapidly restored when nutrients are provided. Return to growth is a key step for survival in a competitive environment and requires restoring the transcriptional program adapted to the available resources.
The goal of this project is to study the mechanisms underlying the dynamics of genome reactivation in relation to chromatin unfolding upon return to growth. Another goal is to investigate whether and how cell to cell heterogeneity contributes to the population’s ability to adapt to new conditions. To this end, the ESR will apply super-resolution microscopy (Photoactivated localization microscopy, PALM; Multi-focus microscopy, MFM), chromatin profiling (ChIP-seq), and multiplex RNA-FISH (in collaboration with the Bienko lab).
Key techniques
live-cell imaging, single-molecule (super-resolution) microscopy, RNA-FISH, genetics, and ChIP-Seq (chromatin immunoprecipitation sequencing)
Prerequisites
Master’s degree or equivalent in biological sciences, physics or bioengineering with practical experience in microscopy and/or molecular biology. Interest in chromatin and nuclear organization.
ESR 9: Spatial regulation of subtelomeric chromatin and gene expression in response to environmental stress (Braun)
Main supervisor
Sigurd Braun
Location
Ludwig Maximilian University of Munich, Germany
Second supervisor
Angela Taddei (Institut Curie)
Description
The nuclear envelope is a specific nuclear subcompartment that promotes the establishment of repressed (‘silent’) chromatin, and re-localization of stress-inducible genes often correlates with transcriptional activation. However, we still have little knowledge of what is the trigger and the underlying mechanism that releases stress-regulated genes from the nuclear periphery.
The fungus Schizosaccharomyces pombe (aka fission yeast) is an ideal model system for studying gene repression and nuclear architecture. We recently demonstrated that the inner nuclear membrane protein Lem2 plays a critical role in heterochromatin localization and silencing (Barrales et al., Genes & Dev 2016). Preliminary data indicate that Lem2-dependent silencing is altered upon stress, making this nuclear membrane protein a prime candidate for studying stress-dependent gene regulation.
The goal of this project is to use super-resolution microscopy and genetics combined with a highly sensitive reporter gene approach to study chromatin organization and to identify factors involved in Lem2-dependent stress-regulation at the single-cell level. Since Lem2 is highly conserved among eukaryotes, regulation of chromatin organization by this inner nuclear membrane protein is likely preserved among other species including Trypanosomes and Plasmodium spp.
Key techniques
automated large-scale genetic screens, flow cytometry-based monitoring of gene expression at single-cell resolution, live-cell imaging and super-resolution microscopy
Prerequisites
Master’s degree or equivalent in biological sciences, physics or bioengineering with practical experience in molecular biology and cell biology. Interest in genetics, microscopy and bioinformatics.
ESR 10: Single-cell isolation of yeast and unicellular parasites (Szabó)
Main supervisor
Bálint Szabó
Location
Cellsorter Company of Innovations, Budapest, Hungary
Second supervisor
Luisa Figueiredo (Instituto de Medicina Molecular)
Description
We developed a fully automated micropipette with a precision of less than 1 nanoliter improving the efficiency of imaging-based single-cell isolation to above 90%. This improvement is crucial when sorting rare or precious cells, especially in medical applications. We envision that this new technology will shortly become a standard tool for single-cell manipulations in medical diagnostics, e.g., circulating tumor cell isolation (https://www.singlecellpicker.com/).
The goal of the current project is to establish strategies to analyze the same cell by imaging and single-cell sequencing. Such an analysis will link transcriptome data with phenotypic characteristics. We will develop new methods and protocols to automatically recognize and isolate single yeast or T. brucei cells for subsequent DNA/RNA sequencing.
Key techniques
Robotics, computer vision, and live-cell imaging
Prerequisites
Master’s degree or equivalent in engineering or biological/physical sciences. Practical experience in optical microscopy or robotics or software development. Interest in microscopy, computer vision and engineering. Experience with image segmentation and/or AI is an advantage.
ESR 11: Visualization the nuclear organization during a switch in antigen expression (Siegel)
Main supervisor
Nicolai Siegel
Location
Ludwig Maximilian University of Munich, Germany
Second supervisor
Magda Bienko (Karolinska Institutet)
Description
An important strategy used by many pathogens to evade the host immune response is antigenic variation. Antigenic variation refers to the capacity of an infecting organism to systematically alter the identity of proteins displayed to the host immune system, making it difficult or impossible for the host to eliminate the pathogen.
Trypanosoma brucei, a unicellular parasite responsible for lethal and debilitating diseases in humans and animals, has long served as a valuable model to study antigenic variation. Yet, many of the most basic questions have only recently, through different technological advances, become experimentally tractable.
Genome-wide Hi-C analyses have begun to uncover the 3D organization of chromosomes at high resolution and have highlighted the critical role of DNA architecture in the regulation of gene expression and recombination. Recent Hi-C and single-cell RNA-seq data from our lab (Müller, Cosentino et al, 2018, Nature) indicate that the deletion of certain histone variants can lead to changes in the genome architecture and a switch in antigen expression. This finding led us to hypothesize that spatial proximity between different antigen-coding genes is critical for a successful switching event.
The goal of this project is to test this hypothesis by visualization of the genome architecture during an antigen switch. To isolate cells that are in the process of changing their surface coat, we will employ intelligent image-activated cell sorting followed by FISH or Cas9-based DNA imaging approaches.
Key techniques
intelligent image-activated cell sorting, super-resolution FISH, live-cell imaging of genomic loci using CRISPR-Cas9-based approaches
Prerequisites
Master’s degree or equivalent in biological sciences, molecular biology, biomedicine or biochemistry with practical experience in molecular biology and fluorescence microscopy. Interest in developing FISH and CRISPR-Cas9-based imaging approaches.
ESR 12: Computational and mathematical modeling of cell-to-cell chromatin heterogeneity (Colomé-Tatché)
Main supervisor
Maria Colomé-Tatché
Location
Helmholtz Zentrum Munich, Institute of Computational Biology, Germany
Second supervisor
Nicolai Siegel (Ludwig Maximilian University of Munich)
Description
To study and understand how pathogens use variation in chromatin structure and gene expression to achieve successful infection, many members of this consortium will measure single-cell open chromatin as well as single-cell transcriptomics data. The goal of this project is to develop computational methods for the analysis of that single-cell open chromatin data, and its integration with single-cell transcriptomics data, in different species of pathogens. These include a feature selection method to identify the most relevant multi-omics features that drive heterogeneity, as well as downstream analysis methods such as lineage tracing, clustering, pseudotemporal ordering and differential analysis.
Moreover, in this project, we will develop mathematical models that will provide an understanding of the role of cell-to-cell heterogeneity in response to stress, such as nutrient depletion or host immune response upon infection. The model parameters will be informed by the levels of cell-to-cell heterogeneity measured by other consortium members, as well as the observed epimutation rates (antigenic switching for example).
Key techniques
computational biology, single-cell data analysis, mathematics, and mathematical modeling
Prerequisites
Master’s degree or equivalent in mathematics, physics, bioinformatics, computer engineering. Interest in biology, good knowledge of statistics and very good programming skills.
ESR 13:Dynamics of heterochromatin spreading at subtelomeric chromatin (Braun)
Main supervisor
Sigurd Braun
Location
Ludwig Maximilian University of Munich, Germany
Second supervisor
Bassem Al-Sady (University of California San Francisco, UCSF)
Description
Active transcriptionally euchromatin and repressed (‘silent’) heterochromatin are separated by boundary elements, often encoded by specific DNA elements. However, subtelomeric heterochromatin in Schizosaccharomyces pombe (aka fission yeast) lack these typical DNA elements, resulting in high variability in the extent of heterochromatin expansion (‘negotiable boundaries’). This situation is reminiscent of the variable boundary regulation in the malaria parasite Plasmodium falciparum.
The Al-Sady lab recently developed a single-cell heterochromatin spreading sensor (HSS). The goal of this project is to introduce this system into S. pombe subtelomeres for determining the spatial and temporal dynamics of subtelomeric heterochromatin by flow-cytometry-based sorting and single-cell tracking. Potential regulators (trans-factors) that control these behaviors will be isolated by genetic high-throughput screens using genome-wide mutant libraries. These studies will be complemented by computational analysis to identify common genomic features enriched at identified negotiable boundaries.
Key techniques
computational analysis of flow cytometry datasets and single-cell imaging traces, bioinformatics analysis of genomic features, chromatin immunoprecipitation (ChIP), automated large-scale genetic screens
Prerequisites
Master’s degree or equivalent in biological sciences, physics or bioengineering with practical experience in bioinformatics and molecular biology. Interest in genetics and bioinformatics.
ESR 14: “Breaking” heterochromatic boundaries of the malaria parasite (Bártfai)
Main supervisor
Richárd Bártfai
Location
Radboud University, Nijmegen, The Netherlands
Second supervisor
Till Voss (Swiss Tropical and Public Health Institute)
Description
Despite decades of elimination efforts malaria parasites still kill about half a million people every year. Phenotypic variability between parasite strains and individual parasites majorly contributes to the success of this pathogen and hinders elimination efforts. Due to our work and result from other groups, we know that difference in chromatin organization exists between parasite strains (Fraschka et al., Cell Host & Microbe, 2018) and results in differential expression of genes involved in antigenic variation or transmission of the parasites between the human host and the mosquito vector (Filarsky et al., Science, 2018). Interestingly, this variation is a feature of genes that are located close to the boundaries between densely packed heterochromatin and more open, transcriptionally active chromatin regions. However, how the location these boundaries are defined is not understood.
The goal of this project is to understand the mechanisms that maintain and alter the location of heterochromatic boundaries. We will use single-cell transcriptomic and develop single-cell epigenomic approaches to measure variability in chromatin organization and gene expression. Furthermore, we will identify proteins that are involved in maintaining these boundaries using both candidate approaches and unbiased genetic/chemical screens. Collectively, this project will further our understanding of heterochromatic boundaries in a deadly human pathogen and start dissecting the mechanisms that lead to their maintenance and heterogeneity. It also has the potential to identify drug candidates influencing heterochromatin boundaries of the parasite.
Key techniques
parasite culturing, single-cell analysis of transcriptome (RNA-sequencing) and the chromatin structure (Chromatin Immunoprecipitation sequencing), CRISPR-Cas9-mediated genome editing, genetic and chemical screening
Prerequisites
Master’s degree or equivalent in biology, molecular life sciences or bioengineering with practical experience in molecular or systems biology. Interest in molecular parasitology, gene regulation and BIG data analysis.
ESR 15: Sex-specific biology of malaria parasites (Voss)
Main supervisor
Till Voss
Location
Swiss Tropical and Public Health Institute/University of Basel, Switzerland
Second supervisor
Magda Bienko (Karolinska Institute)
Description
Malaria is caused by unicellular parasites of the genus Plasmodium that multiply in the human bloodstream through repeated cycles of red blood cell invasion and intracellular replication. During each round of replication, a small proportion of parasites exit the cell cycle and differentiate into either male or female gametocytes. Gametocytes are strictly required to undergo the obligate sexual reproduction step in the mosquito vector and are therefore essential for malaria transmission. The switch from asexual proliferation to sexual differentiation is regulated epigenetically and informed by environmental cues. The factors and molecular processes underlying sex determination and gender-specific biology of male and female gametocytes, however, remain almost entirely unknown.
The goal of this project is to use a chemical screening approach combined with high content microscopy and flow cytometry analyses of genetically engineered fluorescent reporter lines to identify environmental triggers and molecular pathways of sex determination in P. falciparum. In addition, we aim to use bulk and single-cell RNA-seq approaches to study the sex-specific transcriptional landscapes in differentiating male and female gametocytes. We anticipate the results of this project will deliver unprecedented insight into the sex-specific biology of malaria parasites.
Key techniques
molecular and cell biology, CRISPR-Cas9, Plasmodium falciparum cell culture, bulk and single-cell RNA-seq, fluorescence microscopy, high content imaging, and flow cytometry
Prerequisites
Master’s degree or equivalent in molecular life sciences with practical experience in molecular and cell biology. Interest in molecular parasitology, fluorescence microscopy, next-generation sequencing and bioinformatics.
This project has received funding from the European Union’s Horizon 2020 Research and Innovation programme under the Marie Skłodowska-Curie grant number 860675.