Illuminating GENome Organization through integrated MIcroscopy and Sequencing

Project Information

GENOMIS

Grant agreement ID: 715727

DOI

10.3030/715727

Project closed

EC signature date 7 October 2016

Start date 1 January 2018

End date 31 December 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 808,00

EU contribution

€ 1 499 808,00

Coordinated by

KAROLINSKA INSTITUTET
Sweden

Periodic Reporting for period 4 - GENOMIS (Illuminating GENome Organization through integrated MIcroscopy and Sequencing)

Reporting period: 2022-07-01 to 2023-12-31

Summary of the context and overall objectives of the project

In the cell nucleus, DNA is not randomly placed but is folded into a complex three-dimensional (3D) structure that is believed to play a crucial role in regulating gene expression and, ultimately, cell identity. Indeed, alterations of the physiological 3D genome structure have been linked to a variety of disorders, including neurodevelopmental disorders and cancer. While various types of 3D genome structural modules have been identified and extensively characterized in a variety of organisms and cell types — including chromatin loops, so-called topologically associating domains (TADs) and larger chromatin compartments — how they are spatially arranged and reciprocally positioned within the cell nucleus has remained largely unexplored, setting the stage for the GENOMIS project.
In GENOMIS, we have taken an innovative approach to characterize the higher-order spatial organization of chromatin, by first developing a set of novel experimental and computational methods and then applying them to explore how DNA is radially organized from the nuclear periphery inwards, as well as to visualize the topology of chromosomes in interphase cells.
Our new methodology allowed us to create the first-ever highly-resolved genome-wide map of the radial placement of DNA loci in the nucleus of human cells. We used this map to reveal how various genomic and epigenomic features are distributed along the periphery-center axis of the nucleus.
By developing innovative tools and datasets and making them publically available following the principles of open science, GENOMIS has contributed to advance the rapidly expanding field of 3D genome biology. Importantly, two of the methods developed in this project (iFISH and Deconwolf) bear clear applicability beyond basic research, as they can be translated to applied research settings, including in vitro diagnostics.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

• We developed a conceptually novel genome-wide method named Genomic loci Positioning by Sequencing or GPSeq (https://doi.org/10.1038/s41587-020-0519-y) which allows us to create genome-wide maps of the radial placement of DNA in the cell nucleus. We have now applied GPSeq to create first-ever high-resolution radial maps of DNA in variety of human and mouse cell lines, as well as in unicellular organisms such as S. cerevisiae and P. falciparum.

• Together with GPSeq, we developed an advanced software, named Chromflock (https://doi.org/10.1038/s41587-020-0519-y) for simulating how the 3D genome structure might look like in single cells. Using Chromflock, we were able to study how different chromosomes might be positioned along the nuclear radius in single nuclei.

• In parallel to sequencing methods, in GENOMIS we also developed a variety of DNA/RNA fluorescence in situ hybridization (FISH) methods to visualize DNA loci, chromosomal topologies, or individual transcript molecules, potentially in thousands of single cells. Specifically:

1. We developed iFISH (https://doi.org/10.1038/s41467-019-09616-w) a freely available platform for designing and producing oligonucleotide-based DNA and RNA FISH probes. By applying iFISH to a variety of human and mouse cell lines, we were able to identify, among other features, different shapes and spatial arrangements of chromosomes or specific sub-chromosomal regions, discovering an intriguing pattern of high inter-chromosome admixture in human embryonic stem cells.

2. We developed FRET-FISH (https://doi.org/10.1038/s41467-022-34183-y) an innovative method combining DNA FISH with fluorescence resonance energy transfer (FRET) to probe the compaction of chromatin at defined DNA loci. Using FRET-FISH, we were able to detect, for the first time, inter-allelic differences in chromatin compaction within the same cell and study relationship between DNA compaction and accessibility.

3. Lastly, we developed Deconwolf (https://deconwolf.fht.org/) a freely available, user-friendly, highly computationally efficient software for deconvolution of any type of fluorescence microscopy image. In particular, we demonstrated that Deconwolf can greatly improve the resolution and amount of information that can be extracted from images obtained with high-throughput FISH and spatial omics assays, such as in situ spatial transcriptomics and OligoFISSEQ.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The GPSeq and FRET-FISH methods developed in GENOMIS clearly go beyond the state-of-the-art, as they are the first methods capable of probing, respectively, genome radiality and local chromatin compaction. Importantly, GPSeq is part of a growing repertoire of methods for probing 3D genome organization, which are not derived from Hi-C. The latter has been instrumental in unraveling the fundamental design principles of genome organization across multiple species and cell types. However, neither Hi-C nor the dozens of Hi-C-derived methods that have been developed over the past decade can accurately capture certain important aspects of the higher-order organization of chromatin in the nucleus, such as the intra-nuclear placement of DNA loci. At the same time, it has been demonstrated that the localization of genes inside the nucleus affects their expression levels, indicating a strong link between spatial localization patterns of chromatin and transcription. In this sense, GPSeq fills in the previous knowledge gap by probing for larger-scale structural aspects in comparison to Hi-C and methods alike.

iFISH and Deconwolf build on pre-existing methods, however, they bring unique spins and improvements that make these tools not ‘just the next obvious step’, but rather a significant step forward, especially in terms of applications enabled by them. In particular, we believe that Deconwolf is the first tool that can truly democratize the use of fluorescence image deconvolution in multiple bioimaging applications, particularly in the rapidly growing field of imaging-based spatial omics.

In sum, GENOMIS has been a successful technology-driven project that has spun a set of innovative and impactful methods, which can now be leveraged by the 3D genome biology community and beyond.

Multiple chromosomes visualized by iFISH probes in human embryonic stem cells