Periodic Reporting for period 2 - SUMiDENTITY (Deconstructing the role of SUMO on chromatin in cell identity and tissue repair)
Période du rapport: 2021-07-01 au 2022-12-31
How the complex mammalian body plan develops from a single fertilized egg remains a central question in biology. While much is known about the functions of many individual genes regulating this process, the ways in which these genes are organized into complex functional networks and how, in turn, these are regulated, still remain enigmatic. Many such gene regulatory mechanisms, often called "epigenetic", involve modifications of proteins (or DNA) by small chemical groups or even entire proteins.
One such modification that attaches members of a family of small proteins collectively called SUMOs (for "Small Ubiquitin-like MOdifiers"; SUMOylation) to other proteins has received much recent attention because of its association with many almost all fundamental cellular processes. Since its discovery some twenty years ago, our lab has contributed significantly to the elucidation of SUMO's functions. We have notably shown that SUMO proteins target chromatin, the cell's functionally packaged genetic material. More recently, our work and others' has shown that SUMOylation represents an epigenetic barrier to cell fate change. Using a variety of models, we demonstrated that inhibiting SUMOylation in each case significantly increased the frequency of otherwise rare cellular differentiation or reprogramming events, thus demonstrating that SUMOylation plays an essential role in the maintenance of proper cell identity.
The aim of the SUMiDENTITY project is to elucidate the ways by which SUMO modification ensures the maintenance of cell identity. We will focus particularly on mechanisms operating at the chromatin level, given that chromatin-associated proteins represent the largest fraction of the repertoire of SUMOylated cellular proteins (the "SUMOylome"). For this, we seek to identify the SUMO targets at genes and genomic loci critical for cell identity and cell fate transitions. Reciprocally, we will determine the changes of the SUMO chromatin landscape (i.e. the presence or absence of SUMO-modified proteins at different genes or regions of the genome) and of the SUMOylome that are associated with particular cell states or cell fate transitions. This work principally involves murine embryonic stem cells (mESCs) that offer the advantage of easy manipulation to achieve different cell states and/or developmental trajectories under laboratory (in vitro) conditions. To apply some of the principles from the above, we will also investigate the impact of SUMOylation (or its perturbation) on tissue regeneration. For this, we will focus on the muscle lineage which represents an amenable model system for studying cellular transitions from muscle stem cells ("satellite cells") to regenerated mature muscle fibers upon acute or chronic myopathic injury.
Taken together, the project seeks to decipher the mechanistic underpinnings of SUMOylation, an important regulatory pathway we hypothesize indeed to have evolved to assure the robustness of complex -- at times, fragile and sensitive -- biological systems. We anticipate our findings to have important implications for understanding basic mechanisms of gene regulation and normal development and how these may be corrupted in disease.
One such modification that attaches members of a family of small proteins collectively called SUMOs (for "Small Ubiquitin-like MOdifiers"; SUMOylation) to other proteins has received much recent attention because of its association with many almost all fundamental cellular processes. Since its discovery some twenty years ago, our lab has contributed significantly to the elucidation of SUMO's functions. We have notably shown that SUMO proteins target chromatin, the cell's functionally packaged genetic material. More recently, our work and others' has shown that SUMOylation represents an epigenetic barrier to cell fate change. Using a variety of models, we demonstrated that inhibiting SUMOylation in each case significantly increased the frequency of otherwise rare cellular differentiation or reprogramming events, thus demonstrating that SUMOylation plays an essential role in the maintenance of proper cell identity.
The aim of the SUMiDENTITY project is to elucidate the ways by which SUMO modification ensures the maintenance of cell identity. We will focus particularly on mechanisms operating at the chromatin level, given that chromatin-associated proteins represent the largest fraction of the repertoire of SUMOylated cellular proteins (the "SUMOylome"). For this, we seek to identify the SUMO targets at genes and genomic loci critical for cell identity and cell fate transitions. Reciprocally, we will determine the changes of the SUMO chromatin landscape (i.e. the presence or absence of SUMO-modified proteins at different genes or regions of the genome) and of the SUMOylome that are associated with particular cell states or cell fate transitions. This work principally involves murine embryonic stem cells (mESCs) that offer the advantage of easy manipulation to achieve different cell states and/or developmental trajectories under laboratory (in vitro) conditions. To apply some of the principles from the above, we will also investigate the impact of SUMOylation (or its perturbation) on tissue regeneration. For this, we will focus on the muscle lineage which represents an amenable model system for studying cellular transitions from muscle stem cells ("satellite cells") to regenerated mature muscle fibers upon acute or chronic myopathic injury.
Taken together, the project seeks to decipher the mechanistic underpinnings of SUMOylation, an important regulatory pathway we hypothesize indeed to have evolved to assure the robustness of complex -- at times, fragile and sensitive -- biological systems. We anticipate our findings to have important implications for understanding basic mechanisms of gene regulation and normal development and how these may be corrupted in disease.
Our discovery that the SUMO chromatin landscape differs greatly when comparing differentiated cells (mouse embryo fibroblasts, MEFs) with stem cells (mESCs), led us to hypothesize similarly important differences in the composition of the SUMOylome between these cell types. Using a sensitive mass-spectroscopy approach, we identified the SUMOylated proteins in MEFs and found these to be largely comprised of proteins functioning in a variety of "normal" cellular processes that regulate MEF-specific gene expression. By contrast, mESCs displayed a highly interconnected network of SUMO targets involved in maintaining densely-compacted chromatin ("heterochromatin") usually associated with inactive (repressed) genes. These results indicate that SUMOylation contributes to the maintenance of the fragile stem cell state by globally repressing the activity of genes associated with cell differentiation, or even de-differentiation (reversion) to an earlier ("totipotent") cell state and further imply a massive rewiring of the cellular SUMOylome that accompanies changes in cell fate.
A most unexpected discovery was made when we treated mESCs at two intervals with a pharmacological SUMOylation inhibitor. Here, we found these cells to aggregate into spheroidal structures that self-organized into gastrula-like, patterned assemblages which, in turn, developed into "embryo-like structures" (ELSs) when cultured in a specially-designed droplet microfluidic device (see Figure). These structures possess a marked anterior-posterior axis and to contain properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures, thus recapitulating salient features of 8 day-old natural embryos. While these ELSs remain far from perfect in lacking, for example, proper dorso-ventral patterning and primordial germ cell clusters, these results indicate that pulsed SUMOylation inhibition, i.e. by only targeting an epigenetic regulator, can drive a single cell type, ESCs, into multiple, "self-organized" developmental trajectories that go well beyond the simple transitions of one cell type into another seen previously.
To extend the above principles to a tissue context -- muscle differentiation and regeneration after injury, our in vitro (ex vivo) and in vivo experiment have yielded preliminary results indicating that SUMOylation-compromised conditions yield higher numbers of satellite cells and that in a chronic injury model, mice from a SUMOylation-compromised genetic background display fewer muscular lesions and larger muscle fibers than control animals. While these results need to be confirmed, they tend to indicate that lowering SUMOylation levels may increase muscle regenerative capacity.
A most unexpected discovery was made when we treated mESCs at two intervals with a pharmacological SUMOylation inhibitor. Here, we found these cells to aggregate into spheroidal structures that self-organized into gastrula-like, patterned assemblages which, in turn, developed into "embryo-like structures" (ELSs) when cultured in a specially-designed droplet microfluidic device (see Figure). These structures possess a marked anterior-posterior axis and to contain properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures, thus recapitulating salient features of 8 day-old natural embryos. While these ELSs remain far from perfect in lacking, for example, proper dorso-ventral patterning and primordial germ cell clusters, these results indicate that pulsed SUMOylation inhibition, i.e. by only targeting an epigenetic regulator, can drive a single cell type, ESCs, into multiple, "self-organized" developmental trajectories that go well beyond the simple transitions of one cell type into another seen previously.
To extend the above principles to a tissue context -- muscle differentiation and regeneration after injury, our in vitro (ex vivo) and in vivo experiment have yielded preliminary results indicating that SUMOylation-compromised conditions yield higher numbers of satellite cells and that in a chronic injury model, mice from a SUMOylation-compromised genetic background display fewer muscular lesions and larger muscle fibers than control animals. While these results need to be confirmed, they tend to indicate that lowering SUMOylation levels may increase muscle regenerative capacity.
Previous models for the elaboration of "synthetic embryos" relied either on mixing different ES cell types or on the use of added developmental regulators. our finding, here, that targeting an essentially epigenetic mechanism, SUMOylation, achieves similar outcomes, represents an unexpected breakthrough. We anticipate that further work aimed at improving the culture conditions will likely enhance the fidelity of the obtained embryo-like structures. Such embryoid systems, much like existing organoid systems, could then be employed to examine the behaviour of exogenously added cells to better understand the interactions of cells with their cellular microenvironment, with, for example, important implications for studying how cancer cells interact with their niche. Additionally, this model could be used for rapid (animal-free) in vitro testing of pharmacological molecules for teratogenicity and embryonic lethality.
At the molecular level, we expect the identification of SUMO substrates and their sites of action (the affected genes and genomic loci) critical for regulated cell fate change to uncover novel fundamental principles of gene regulation, thus paving the way towards the identification of novel signals regulating the function of such regulatory complexes.
Finally, the work on muscle regeneration after injury will affirm or refute the hypothesis that SUMO-inhibited conditions may increase the regenerative capacity of satellite cells and their progeny by enhancing their phenotypic plasticity.
At the molecular level, we expect the identification of SUMO substrates and their sites of action (the affected genes and genomic loci) critical for regulated cell fate change to uncover novel fundamental principles of gene regulation, thus paving the way towards the identification of novel signals regulating the function of such regulatory complexes.
Finally, the work on muscle regeneration after injury will affirm or refute the hypothesis that SUMO-inhibited conditions may increase the regenerative capacity of satellite cells and their progeny by enhancing their phenotypic plasticity.