Periodic Reporting for period 4 - DYMOCHRO (Dynamics of modified chromatin domains)
Période du rapport: 2024-01-01 au 2024-12-31
The cells of our body contain nearly the same genetic information but greatly differ in function. Accordingly, each cell interprets its genetic information in a distinct way. This involves chemical modifications of the DNA and the histone proteins, which are in close contact with the DNA, and the spatial organization of the genomic DNA within the cell nucleus. These features, which are often called “epigenetic”, are linked to the cellular gene expression program and the cellular identity. Epigenetic dysregulation is often found in diseased cells, indicating that their faithful regulation is important for our health.
DNA and histone modifications as well as the spatial organization of the genome are on the one hand dynamic, as they can change in response to different signals from outside. On the other hand, they are stable enough so that the cellular identity they encode can be maintained over time. How this balance between plasticity and stability is mechanistically regulated is a key question. As many cellular proteins are involved in the regulation of DNA/histone modifications and spatial genome organization, which have multiple functions and are often redundant, it is difficult to study this system using loss-of-function approaches in cells.
The objective of this project was to reconstitute minimal systems to investigate the formation and maintenance of domains of modified chromatin, both in vitro and in living cells. On the one hand, this entailed the development of bulk and single-molecule microscopy assays to visualize chromatin-associated proteins and histone modifications on chromatinized templates in the test tube to assess their biophysical properties. On the other hand, this comprised the implementation of "synthetic biology" approaches to generate ectopic domains in living cells to study their dynamics.
In this project, we have established the above-mentioned assays and have studied the mechanisms underlying the dynamics of modified chromatin domains. Our results have shed light on the question how phase separation and the action of read-write proteins regulate modified chromatin domains, and how this impacts cellular function. We anticipate that our results will be helpful for upcoming applications, for example in the context of condensate-modifying epigenetic drugs.
DNA and histone modifications as well as the spatial organization of the genome are on the one hand dynamic, as they can change in response to different signals from outside. On the other hand, they are stable enough so that the cellular identity they encode can be maintained over time. How this balance between plasticity and stability is mechanistically regulated is a key question. As many cellular proteins are involved in the regulation of DNA/histone modifications and spatial genome organization, which have multiple functions and are often redundant, it is difficult to study this system using loss-of-function approaches in cells.
The objective of this project was to reconstitute minimal systems to investigate the formation and maintenance of domains of modified chromatin, both in vitro and in living cells. On the one hand, this entailed the development of bulk and single-molecule microscopy assays to visualize chromatin-associated proteins and histone modifications on chromatinized templates in the test tube to assess their biophysical properties. On the other hand, this comprised the implementation of "synthetic biology" approaches to generate ectopic domains in living cells to study their dynamics.
In this project, we have established the above-mentioned assays and have studied the mechanisms underlying the dynamics of modified chromatin domains. Our results have shed light on the question how phase separation and the action of read-write proteins regulate modified chromatin domains, and how this impacts cellular function. We anticipate that our results will be helpful for upcoming applications, for example in the context of condensate-modifying epigenetic drugs.
In this project, we have developed methods to study domains of modified histones in vitro and in living mammalian cells. For the in vitro part, we have established microfluidics assays to visualize DNA-based condensates and probe their stability in shear flow. We have found that the length of DNA molecules is a key determinant of the condensate properties. We have also shown how proteins such as linker histone (H1) and heterochromatin protein 1 (HP1) are able to cluster naked and chromatinized DNA. We found that clustering can but does not have to be driven by liquid-liquid phase separation (or phase separation coupled to percolation) of the respective chromatin proteins. In living cells, we have developed several approaches to modulate phase separation of heterochromatin regions and to decorate them with sythetic epigenetic marks. These experiment have allowed us to assess the link between phase separation, chromatin organization and gene expression. We found that clustering of heterochromatin regions can be enforced by the expression of HP1 variants with elevated phase separation potential, and we found that the gene expression changes induced by such spatial rearrangements are relatively mild. Interestingly, HP1 variants with elevated phase separation potential are found in organisms that have appeared earlier in evolution, e.g. fission yeast and fruit fly, while the phase separation potential of mammalian HP1 is comparably lower. This points to an evolutionary adaptation of HP1 function and heterochromatin organization, with a division of labor between HP1 and other chromatin proteins in mammals.
The results obtained in this project have been disseminated in the form of peer-reviewed publications, poster presentations and oral presentations at several meetings, as well as press releases.
The results obtained in this project have been disseminated in the form of peer-reviewed publications, poster presentations and oral presentations at several meetings, as well as press releases.
We have made considerable progress in improving our understanding of modified chromatin domains, elucidating the roles of phase separation and positive feedback loops in the regulation of their dynamics and stability. We have also established several assays to study such domains, including a microfluidic single-molecule assay, a calibrated half-bleach assay, and synthetic biology approaches in living cells, which we anticipate will be useful for the community. Part of the results obtained in this project are published, while another part is in submission.