Periodic Reporting for period 4 - EpiScope (Epigenomics and chromosome architecture one cell at a time)
Reporting period: 2022-03-01 to 2023-08-31
In Eukaryotes, cellular identity and tissue-specific functions are linked to the epigenetic landscape and the multi-scale architecture of the genome. The packing of DNA into nucleosomes at the ~100 bp scale and the organization of whole chromosomes into functional territories within the nucleus are well documented. At an intermediate scale, chromosomes are organized in megabase to sub-megabase structures called Topologically Associating Domains (TADs). Critically, *TADs are highly correlated to patterns of epigenetic marks determining the transcriptional state of the genes they encompass.* Until now, the lack of efficient technologies to map chromosome architecture and epigenetic marks at the single-cell level have limited our understanding of the molecular actors and mechanisms implicated in the establishment and maintenance of the multi-scale architecture of chromosomes and epigenetic states, and the interplay between this architecture and other nuclear functions such as transcription.
Epigenomic modifications are at the source of many health related disorders. For instance, the epigenome of cancer cells carries a fingerprint of the cell type that originated the cancer. Thus, understanding the epigenomic organisation of single cells will be key in determining the underlying disease mechanisms and designing targeted diagnosis and treatments. The characterisation of the epigenomic organisation at the single-cell level will be key in the future to understand cell differentiation, the mechanisms linking higher-order chromosome structure to transcription, as well as the onset and progression of disease. Finally, the results and technologies developed in EpiScope will impact personalised medicine by improving the speed of diagnosis methods and the testing of treatments.
The overall aim of EpiScope is to unveil the functional, multi-scale, 3D architecture of chromatin at the single-cell level while preserving cellular context. Our findings should lead to a better understanding of the spatiotemporal mechanisms involved in transcriptional regulation and shed light into the interplay between key nuclear functions, such as transcription or chromatin remodeling, and the multi-scale, 3D folding of the chromosome.
Epigenomic modifications are at the source of many health related disorders. For instance, the epigenome of cancer cells carries a fingerprint of the cell type that originated the cancer. Thus, understanding the epigenomic organisation of single cells will be key in determining the underlying disease mechanisms and designing targeted diagnosis and treatments. The characterisation of the epigenomic organisation at the single-cell level will be key in the future to understand cell differentiation, the mechanisms linking higher-order chromosome structure to transcription, as well as the onset and progression of disease. Finally, the results and technologies developed in EpiScope will impact personalised medicine by improving the speed of diagnosis methods and the testing of treatments.
The overall aim of EpiScope is to unveil the functional, multi-scale, 3D architecture of chromatin at the single-cell level while preserving cellular context. Our findings should lead to a better understanding of the spatiotemporal mechanisms involved in transcriptional regulation and shed light into the interplay between key nuclear functions, such as transcription or chromatin remodeling, and the multi-scale, 3D folding of the chromosome.
We used super-resolution microscopy to visualize the 3D folding of TADs in single cells. We used stochastic optical reconstruction microscopy to observe the folding of repressed TADs in Drosophila cells and could demonstrate that they form discrete nano-compartments interspersed by less condensed active regions (Szabo, et al, Science Adv, 2018; Cattoni, et al, Nat Comm, 2017).
We developed a new family of technologies, termed Hi-M, based on the sequential imaging of tens-to-hundreds of different DNA species (Cardozo, et al, Mol Cell, 2019; Cardozo et al, Nat Protocols, 2020). We developed several software packages to control the robotized HiM microscope and to perform automated analysis (Barho, Open Research Europe 2022; Rombouts, Open Research Europe 2023; Rombouts, Nat. Comm 2023; https://github.com/marcnol/pyHiM).
Next, we used HiM also to investigate chromatin organization at the TAD level at the early stages of embryonic development (Espinola, Nat Genetics, 2021; Goetz, Nat Comm, 2022), the roles of insulators in this process (Messina, Nat. Comm, 2023), and the long range organization of Pc domains (Gurgo, BioRxiv, 2022). Finally, we also applied HiM to investigate how E-P interactions and PRE loops are linked to the regulation of transcription and chromosome structure in this model system using in part our HiM technology (Denaud, et al, 2022).
We developed a new family of technologies, termed Hi-M, based on the sequential imaging of tens-to-hundreds of different DNA species (Cardozo, et al, Mol Cell, 2019; Cardozo et al, Nat Protocols, 2020). We developed several software packages to control the robotized HiM microscope and to perform automated analysis (Barho, Open Research Europe 2022; Rombouts, Open Research Europe 2023; Rombouts, Nat. Comm 2023; https://github.com/marcnol/pyHiM).
Next, we used HiM also to investigate chromatin organization at the TAD level at the early stages of embryonic development (Espinola, Nat Genetics, 2021; Goetz, Nat Comm, 2022), the roles of insulators in this process (Messina, Nat. Comm, 2023), and the long range organization of Pc domains (Gurgo, BioRxiv, 2022). Finally, we also applied HiM to investigate how E-P interactions and PRE loops are linked to the regulation of transcription and chromosome structure in this model system using in part our HiM technology (Denaud, et al, 2022).
DNA is organized into TADs and TADs into chromosomes, but how this process occurs at the single-cell level was unknown until recently. One main reason was the lack of appropriate tools. For example, conventional microscopies lacked the resolution or specificity to visualize TAD structures in single cells. To solve these limitations, we developed two different methodologies. In the first, we adapted two-color stochastic optical reconstruction microscopy (STORM) to visualize active and repressed TADs in single cells, and used 3D-structured illumination microscopy (3D-SIM) to measure for the first time absolute interaction frequencies within a single TAD. In the second, we developed a unique combination of experimental and modeling to show that repressed TADs form a succession of discrete nanocompartments and are highly structurally heterogeneous.
Super-resolution-based methods allowed us to visualize the structures of TADs in single cells, however these technologies allow only for the visualization of a limited number of targets (1-2). Thus, while we could super-resolve the volume occupied by a TAD in a single cell, we could not restore the connectivity between genomic elements critical to understanding function (e.g. enhancers, promoters). Sequencing-based methods, in contrast, can access multiple interactions at once, but are difficult to perform in single cells and lack spatial resolution. To uplift these limitations of conventional microscopies and next generation sequencing-based techniques, we developed Hi-M. Hi-M provides the ability to simultaneously monitor transcription and chromosome organization in single cells while retaining spatial information within the cell and the organism. Critically, Hi-M can be performed in whole-mount embryos and in tissue sections. For these reasons, Hi-M has a tremendous potential to make transformative changes to fundamental and applied biomedical research, for example by providing new ways of understanding disease progression at the single-cell level, and by deriving new diagnostic tools. In the near future, we will use Hi-M to elucidate mechanisms of transcriptional activation, and repression, and characterize the intrinsic heterogeneities of DNA organization in single cells.
First, we leveraged the unique advantages of HiM to investigate how chromosomes folded within single cells during early Drosophila embryogenesis. We were particularly interested in testing if preferential contacts between enhancers and promoters within a model TAD interacted together and whether these interactions were conductive of activation. Surprisingly, we found that regulatory interactions are present in both transcriptionally active and inactive cells, supporting a model in which occupation of transcription factors is the main regulator of enhancer activity. We also applied Hi-M to shed light into the role of chromatin insulators in the folding of Drosophila TADs.
Next, we studied the functional importance of cis regulatory elements (PREs, enhancers) for the structure and function of Polycomb TADs. We showed that PcG dependent silencing and chromatin marks can be established in the absence of PRE looping, but 3D contacts between PREs might stabilize PcG-mediated gene repression during development. Also, we showed that PREs interact both in tissues where expression is active or repressed, and that insulators produce small changes to this organization, but have important transcriptional consequences. Finally, we investigated long-range Pc domain organization using HiM, and found that Pc domains colocalize in 3D sporadically and mostly in pairs. This data is in contrast to current models for human cells suggesting Pc domains form phase separated compartments.
Super-resolution-based methods allowed us to visualize the structures of TADs in single cells, however these technologies allow only for the visualization of a limited number of targets (1-2). Thus, while we could super-resolve the volume occupied by a TAD in a single cell, we could not restore the connectivity between genomic elements critical to understanding function (e.g. enhancers, promoters). Sequencing-based methods, in contrast, can access multiple interactions at once, but are difficult to perform in single cells and lack spatial resolution. To uplift these limitations of conventional microscopies and next generation sequencing-based techniques, we developed Hi-M. Hi-M provides the ability to simultaneously monitor transcription and chromosome organization in single cells while retaining spatial information within the cell and the organism. Critically, Hi-M can be performed in whole-mount embryos and in tissue sections. For these reasons, Hi-M has a tremendous potential to make transformative changes to fundamental and applied biomedical research, for example by providing new ways of understanding disease progression at the single-cell level, and by deriving new diagnostic tools. In the near future, we will use Hi-M to elucidate mechanisms of transcriptional activation, and repression, and characterize the intrinsic heterogeneities of DNA organization in single cells.
First, we leveraged the unique advantages of HiM to investigate how chromosomes folded within single cells during early Drosophila embryogenesis. We were particularly interested in testing if preferential contacts between enhancers and promoters within a model TAD interacted together and whether these interactions were conductive of activation. Surprisingly, we found that regulatory interactions are present in both transcriptionally active and inactive cells, supporting a model in which occupation of transcription factors is the main regulator of enhancer activity. We also applied Hi-M to shed light into the role of chromatin insulators in the folding of Drosophila TADs.
Next, we studied the functional importance of cis regulatory elements (PREs, enhancers) for the structure and function of Polycomb TADs. We showed that PcG dependent silencing and chromatin marks can be established in the absence of PRE looping, but 3D contacts between PREs might stabilize PcG-mediated gene repression during development. Also, we showed that PREs interact both in tissues where expression is active or repressed, and that insulators produce small changes to this organization, but have important transcriptional consequences. Finally, we investigated long-range Pc domain organization using HiM, and found that Pc domains colocalize in 3D sporadically and mostly in pairs. This data is in contrast to current models for human cells suggesting Pc domains form phase separated compartments.