Periodic Reporting for period 4 - CHROMTOPOLOGY (Understanding and manipulating the dynamics of chromosome topologies in transcriptional control)
Okres sprawozdawczy: 2020-12-01 do 2021-12-31
Eukaryotic genomes must be tightly packaged in cell nuclei, yet the DNA must be accessible to myriad factors for efficient gene transcription, replication and repair. Past studies have assessed the nuclear organization of specific genomic loci, using microscopic or molecular biology techniques, particularly the chromosome conformation capture techniques coupled to high-throughput sequencing (Hi-C). Collectively, these studies demonstrated a striking correlation between chromatin topology and underlying gene activity (Fig 1a). Distal regulatory elements such as enhancers come into direct contact with their target genes via chromatin loops. At a higher-order scale, metazoan genomes are organized into distinctly folded modules (topologically associated domains; TADs). Many markers of chromatin activity correlate with TAD organization, and the domains appear to delimit the operational range of the regulatory elements contained within them. However, it is still largely unknown whether chromosome folding is a cause or consequence of underlying gene function, nor how the two are mechanistically coupled. In order to fully understand how programmes of gene expression can be controlled and coordinated in response to developmental signals, the CHROMTOPOLOGY project asked four main questions:
• How do chromatin topologies evolve during transcriptional responses during cell differentiation?
• How do “functional” chromatin topologies behave in single cells in real-time?
• Can chromatin loops be engineered de novo, and what are the consequences for transcriptional control?
• What are the minimal DNA sequences and trans-acting factors required to build and stabilize chromosomal domains, such as TADs?
To address these questions, we combined cutting-edge and novel molecular biology and live microscopy tools to explore chromatin loop and TAD dynamics across mouse thymocyte development and on different perturbations of mouse embryonic stem cells. We found that whereas TADs appear stable during development, some can be directly remodelled by transcription. Conversely, chromatin looping interactions with distal elements is extremely dynamic during development and cell type-specific, but topologies are not necessarily linked with transcriptional control. Looking at chromatin topologies in living cells, we find that the spatial separation between genes and enhancers, while often close, does not necessarily correlate with transcriptional status. Instead, local chromatin dynamics are more closely linked: genes and their regulatory elements have much more constrained mobility than “neutral” sequences, and their dynamic properties are specifically modulated on transcriptional perturbation.
• How do chromatin topologies evolve during transcriptional responses during cell differentiation?
• How do “functional” chromatin topologies behave in single cells in real-time?
• Can chromatin loops be engineered de novo, and what are the consequences for transcriptional control?
• What are the minimal DNA sequences and trans-acting factors required to build and stabilize chromosomal domains, such as TADs?
To address these questions, we combined cutting-edge and novel molecular biology and live microscopy tools to explore chromatin loop and TAD dynamics across mouse thymocyte development and on different perturbations of mouse embryonic stem cells. We found that whereas TADs appear stable during development, some can be directly remodelled by transcription. Conversely, chromatin looping interactions with distal elements is extremely dynamic during development and cell type-specific, but topologies are not necessarily linked with transcriptional control. Looking at chromatin topologies in living cells, we find that the spatial separation between genes and enhancers, while often close, does not necessarily correlate with transcriptional status. Instead, local chromatin dynamics are more closely linked: genes and their regulatory elements have much more constrained mobility than “neutral” sequences, and their dynamic properties are specifically modulated on transcriptional perturbation.
We performed Capture Hi-C experiments on mouse thymocyte populations and found that most TAD structures are robust to developmental change. However, some domains were remodelled and we showed using CRISPR-mediated gene induction a direct causal role of transcription in these remodelling events (Fig 1b; Chahar et al., in prep). We also interrogated the dynamics of chromatin loops with all gene promoters in the same thymocyte populations. In order to call interactions robustly, we developed a pipeline for analysing such Capture Hi-C datasets (Ben Zouari et al., 2019; Genome Biol). We identified thousands of both stable and highly dynamic promoter-centred interactions, many as expected carrying the hallmarks of enhancers, but many more with less obvious functional relevance, even though they are reproducible and highly cell type-specific. We also investigated chromatin topology at earlier thymocyte and haematopoietic stem cell stages, requiring us to modify the technology for use in limiting cell numbers. As proof of principle, we identified novel candidate enhancers specific to haematopoietic stem cells, which we validated with CRISPR deletions (Fig 1c; Karasu, Molitor et al., in prep).
To track chromatin topology in real-time in single cells, we labelled site-specific regions in mouse embryonic stem cells, in combination with tags for nascent transcription (Fig 1d). Surprisingly, we found that local chromatin dynamics are not uniform across the chromosome but are specific to genomic context (Oliveira et al., 2021; Nat Comm). We then labelled the promoter and distal enhancer of the pluripotency gene Sox2 in mouse embryonic stem cells to follow their spatial communication relative to transcriptional status. Although the two elements were frequently proximal, their exact distance did not correlate with transcription, and was unaltered on treatment with drugs inhibiting transcription. Instead, the local chromatin diffusion dynamics were more closely linked to transcription. Both promoters and enhancers are significantly more constrained than control sequences, perhaps by their confinement within nuclear foci dedicated to transcription, and this confinement was relaxed on perturbation of transcription. Moreover, diffusion of the Sox2 promoter is faster in experimental conditions permitting gene transcription (Platania et al., in prep).
We dissected the Sox2 enhancer with CRISPR-mediated allele-specific deletions and found a remarkable decoupling between transcription and chromatin topology. Whereas transcription was eliminated by deletion of a few transcription factor binding sites, these deletions had no effect on chromatin architecture. Instead, perturbation of the interaction with the gene, and of maintenance of the TAD border, required extensive deletions, suggesting that chromatin topology maintenance is modular and distributed over numerous genomic sites. As a complementary approach, we also perturbed promoter-enhancer interactions within the same locus by engineering new sequences of choice into an intervening region. By engineering CTCF-mediated chromatin loops, we were able to perturb the endogenous promoter-enhancer interaction, but these had no effect on Sox2 transcription (Fig 1e; Taylor, Sikorska, Shchuka et al., 2021; submitted to Genes Dev).
To track chromatin topology in real-time in single cells, we labelled site-specific regions in mouse embryonic stem cells, in combination with tags for nascent transcription (Fig 1d). Surprisingly, we found that local chromatin dynamics are not uniform across the chromosome but are specific to genomic context (Oliveira et al., 2021; Nat Comm). We then labelled the promoter and distal enhancer of the pluripotency gene Sox2 in mouse embryonic stem cells to follow their spatial communication relative to transcriptional status. Although the two elements were frequently proximal, their exact distance did not correlate with transcription, and was unaltered on treatment with drugs inhibiting transcription. Instead, the local chromatin diffusion dynamics were more closely linked to transcription. Both promoters and enhancers are significantly more constrained than control sequences, perhaps by their confinement within nuclear foci dedicated to transcription, and this confinement was relaxed on perturbation of transcription. Moreover, diffusion of the Sox2 promoter is faster in experimental conditions permitting gene transcription (Platania et al., in prep).
We dissected the Sox2 enhancer with CRISPR-mediated allele-specific deletions and found a remarkable decoupling between transcription and chromatin topology. Whereas transcription was eliminated by deletion of a few transcription factor binding sites, these deletions had no effect on chromatin architecture. Instead, perturbation of the interaction with the gene, and of maintenance of the TAD border, required extensive deletions, suggesting that chromatin topology maintenance is modular and distributed over numerous genomic sites. As a complementary approach, we also perturbed promoter-enhancer interactions within the same locus by engineering new sequences of choice into an intervening region. By engineering CTCF-mediated chromatin loops, we were able to perturb the endogenous promoter-enhancer interaction, but these had no effect on Sox2 transcription (Fig 1e; Taylor, Sikorska, Shchuka et al., 2021; submitted to Genes Dev).
The major discovery of this project was the intimate and hitherto unexplored link between chromatin mobility/dynamics and the potential for transcriptional control, and mechanistic exploration of this phenomenon will be a major research axis for our group in the future. In addition, we have uncovered new intricacies in the still unclear relationship between chromatin topologies and gene expression control. On one scale, they appear rather stable and “hard-wired”, yet certain structures can be directly remodelled by induced transcription (to our knowledge, this is the first formal demonstration of such a phenomenon). But on the other hand, promoter-mediated interactions are extremely cell type-specific, to the extent that they can be used to “predict” cell type, yet have only limited correlations with transcriptomes of these cells. Rather, our in-depth study at the Sox2 locus, which uncovered a large decoupling of transcription control and maintenance of chromatin topology, both features of which are completely independent of CTCF, suggests that one cannot make dogmatic conclusions from genome-wide studies, and context dependence is extremely important. This will hopefully inform future studies within the community, which will be aided by the numerous analytical tools we developed in this project, as well as more sophisticated live imaging experiments on more and more model systems.