Periodic Reporting for period 4 - GENOMIS (Illuminating GENome Organization through integrated MIcroscopy and Sequencing)
Periodo di rendicontazione: 2022-07-01 al 2023-12-31
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.
• 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.
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.