Structure–dynamics–function relationships in the eukaryotic nucleus

The mammalian genome is highly organized in both space and sequence. Preferred positions in the nucleus are observed from whole chromosomes to individual loci and appear closely related to many DNA-based biological processes (transcription, replication, repair …etc). This multi-level 3D arrangement of chromosomes is dynamic, cell-type dependent, altered in response to external signals, and perturbed in many diseases.

Unraveling the physical causes and biological consequences of such dynamic organization is key for understanding the genome. Our team studies both aspects:

• Physical principles governing the organization and dynamics of chromosome
  Mechanisms have been identified to explain how the exceedingly long molecules that are chromosomes can fold into different structures at various spatial and genomic scales (territories, compartments, TADs, loops). In this context, the physical nature of chromosomes plays a central role: their polymeric nature imposes topological constraints and complex dynamic properties, their material state (liquid, gel-like, ...etc) governs how they self-organize and deform, and the action of many energy-dependent molecular motors drives and maintains conformations away from equilibrium. We aim to understand how these physical aspects of chromosomes contribute to their folding and dynamics in the mammalian nucleus.
   
• Functional implications of genome conformation for gene regulation
  Absolute and relative positions of genes in 3D space and along the 1D genome are closely linked to their regulation. Indeed, genes tend to show physical proximity even if distant in sequence when co-regulated by the same TFs. At a smaller scale in cis, TADs are thought to expose genes to a common regulatory landscape of enhancers and are known to often harbor functionally-related genes. We aim to understand how local domain-wide regulatory mechanisms taking place in nuclear microenvironments and local conformational structures can functionally co-regulate the expression of multiple genes.

An interdisciplinary and quantitative approach

Our interdisciplinary team, at the interface between physics and biology, includes members from various backgrounds and disciplines (molecular and cellular biology, microscopy, biochemistry, bioengineering, theoretical physics and computer science). We rely on in-house technique development and state-of-the-art approaches in:

• Advanced microscopy – including single-molecule imaging techniques, in both fixed and live cells,
• Mechanical micro-manipulation – in particular, a new tool we developped for manipulating chromosomes in living cells,
• Physical modeling – polymer modeling and non-equilibrium statistical physics.

Projects

We highlight below two examples of ongoing projects in the team.

Probing chromosome mechanics inside living cells by magnetic micro-manipulation

Over the past decades, our understanding of the physical principles that organize genomes in 4D has improved tremendously. However, fundamental questions about the physical state of chromosomes remains open, largely due to the inability to make direct mechanical perturbations and measurements on interphase chromosomes in live cells.

To address this gap, together with collaborators, we developed a new tool to actively manipulate a genomic locus in the nucleus of living cells [Keizer et al. (2022) Science, 377:6605]. It consists in targeting iron-containing nanoparticles to a genomic locus of interest and applying a controlled magnetic field with a microfabricated magnet.

With this new technology, we were able to perform the first-ever measurements of how a genomic locus, in its native nuclear context, responds to a point force. By observing the displacement and recoil of the locus, we measure signature of chromosome rheology and access key physical parameters, giving unprecedented insight into the physical nature of interphase chromatin and chromosomes. Our measurements provide a basis for developing new physical models of chromosomes. More information here.

Our new technology offers an unprecedented way of perturbing biological genomic processes, with potential applications in all fields of genome biology – including transcription, replication, DNA repair, and chromosome segregation. In addition, it also opens unique avenues in mechanobiology, in particular for understanding how gene expression may be affected by the mechanical signals transmitted from the cytoskeleton to the nucleus.

Local gene co-regulation within the 3D genome

At the sub-megabase scale, chromosome conformation capture techniques have revealed that the genome is partitioned into topologically associating domains (TADs), i.e. self-interacting regions of the genome. TADs are thought to functionally influence gene regulation by exerting spatial constraints on enhancer-gene communication. In line with this view, genes from the same TAD tend to show correlated transcription across cells and tissues, and during development. These results depict TADs as ‘regulatory units’ of the genome where enhancers would co-regulate multiple genes domain-wide. Interestingly, correlated expression is often seen between functionally related genes, e.g. coding for proteins of a complex. This effect is stronger for nearby genes along the genome, raising the possibility that local 3D genome organization could play a role in co-regulating functionally related genes. However, this ‘TAD-wise’ logic in gene co-regulation remains poorly understood.

To study the role of local genome folding in gene co-regulation, we have optimized and combined on the same sample single-molecule RNA FISH and oligopaint/iFISH-based DNA FISH. With this 6-color RNA/DNA FISH approach, we measure the coordinated activity of single genes at individual loci and infer, based on perturbations of the local architecture of the locus, the principles the govern local and domain-wide gene regulation. Our goals are to understand how a set of enhancers can locally co-regulate multiple genes in parallel at a single endogenous locus and how genome organization into TADs influences this co-regulation.