Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CHROMTOPOLOGY (Understanding and manipulating the dynamics of chromosome topologies in transcriptional control)

Teaser

Eukaryotic genomes must be tightly folded and packaged to be contained within cell nuclei, yet the DNA must be accessible to myriad factors for efficient gene transcription, replication and repair. Over the past decades, many studies have assessed the spatial proximity and...

Summary

Eukaryotic genomes must be tightly folded and packaged to be contained within cell nuclei, yet the DNA must be accessible to myriad factors for efficient gene transcription, replication and repair. Over the past decades, many studies have assessed the spatial proximity and nuclear organization of specific genomic loci, using microscopic techniques, such as fluorescent in situ hybridization, 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. 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), whereby genomic interactions are strong within a domain, but are sharply depleted on crossing the boundary between two domains. Many markers of chromatin activity correlate very well 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 asks 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 combine 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. Collectively, CHROMTOPOLOGY will provide groundbreaking mechanistic insights into how chromatin structures are made, the dynamics of such structures, and to what extent they are causal in gene expression control.

Work performed

We have performed high-resolution Capture Hi-C experiments on key mouse thymocyte populations and have uncovered an extensive network of both developmentally stable and dynamic chromatin loops with gene promoters.
Conversely, using a different Capture Hi-C design, we also found that most TAD structures are robust to large transcriptional changes associated with development. However, some TAD borders and sub-domains were remodelled, and we were able to demonstrate the causal role of transcriptional induction for some of these events.
We have developed ChiCMaxima, a simple yet robust pipeline for analysing and visualising Capture Hi-C datasets, which performs favourably to the few existing methods for this novel data type (https://www.biorxiv.org/content/early/2018/10/16/445023; in review). A similar browser for visualisation of similar chromatin interaction datasets (4C) has also been developed.
We have optimised conditions for labelling two site-specific genomic regions of choice in mouse embryonic stem cells, and for tracking the nuclear localisation of these regions in real-time light microscopy (the ANCHOR technology, pioneered by a collaborator). Conditions for a third label have also been optimised, and we are currently perfecting simultaneous triple-labelling conditions.
More than ten different knock-in lines have been constructed from this labelling system to study chromatin interaction dynamics in real-time, and are starting to bear fruitful insights. Preliminary results include direct visualisation of enhancer-promoter contacts and determining that the chromatin motion of TAD borders is not inherently different from regions elsewhere within a TAD.
A functional screen to rapidly identify functional TAD borders in mouse embryonic stem cells is in development, and many technical hurdles to this challenging task have been overcome.

Final results

With further analysis and perturbation studies leading from the Capture Hi-C results, we have the potential to better understand what determines when and how a regulatory element contacts its target gene promoter, and the mechanistic consequences for transcriptional firing of this interaction. Already, it is clear from our results that classical enhancers are only a small subset of the reproducible promoter interactions tested, providing a rich source from which novel or underappreciated classes of genetic regulatory elements may be uncovered.

Ongoing technical improvements and analytical method development give great potential for the ANCHOR system as a tool for the scientific community to study any pairwise or three-way chromatin interactions in real time and in single cells. Within the scope of the CHROMTOPOLOGY project, we anticipate a deeper understanding of various fundamental and outstanding questions, not limited to:
• How frequently and for how long a gene needs to engage its distal regulatory elements in order for control to be inferred.
• The true behaviour of spatial chromatin domains, such as TADs, in single cells, including their persistence throughout the cell cycle, and whether they genuinely exist in the majority of the cells of a population.
• Insight into how chromatin and interaction dynamics are affected by transcriptional changes and developmental cues.
Budding projects within CHROMTOPOLOGY, whose details cannot be divulged for confidentiality reasons, have the potential to answer more fundamental questions about chromosome architecture and its potential for gene expression control:
• How are TADs formed, maintained and broken down? Can TADs be engineered de novo for perfect “insulation” of inserted transgenes?
• What are the mechanistic links between an enhancer-promoter interaction and instructive transcriptional firing?
• Can transcriptional control be switched by ectopic, engineered re-wiring of chromatin interactions?

Website & more info

More info: http://www.igbmc.fr/research/department/2/team/118/.