Periodic Reporting for period 5 - DissectPcG (Dissecting the Function of Multiple Polycomb Group Complexes in Establishing Transcriptional Identity)
Berichtszeitraum: 2022-09-01 bis 2023-06-30
The establishment of correct cellular fate is at the basis of normal development of any multicellular organism. Developmental processes continue for the entire life of any organisms to sustain homeostasis and tissue regeneration from environmental insults. The correct control of cellular fate is frequently deregulated in diseases where cancer can be considered one of the best examples. Neoplastic development always involves a loss of cell identity constraints and the acquisition of new features that allows disease progression. This entails the acquisition of both genetic and adaptive (epigenetic) features, which frequently comprise the same mechanisms that control development and regenerative proprieties.
Cell identity is established and preserved by the set-up of specific transcription programs allowing a correct use of the genetic information. Simplifying a complex mechanism, this involves both the activation of the “correct” genes while silencing of the rest of the genome. This can be achieved by coordinating the activity of DNA binding transcription factors together with the modulation of the local structure of chromatin.
In such scenario, Polycomb proteins represents the major repressive system by which genes are maintained transcriptionally silenced. This includes a large family of proteins with distinct biochemical features that execute their activity forming large multiprotein complexes that modify the chromatin environment to maintain repression. Their activity is essential for several developmental processes and different members of this family are frequently targeted by mutations that affect their activity in several human diseases. This includes, with high frequency, human tumors where components of the Polycomb machinery are targeted by gain and loss of function mutations in a tissue dependent manner. Although the Polycomb machinery can be reconciled in two major enzymatic activities, these are characterized by an high biochemical heterogeneity. Several distinct forms of Polycomb complexes exist with distinct biochemical features whose role remains poorly understood.
This project aimed to generate new knowledge related to the specific molecular and biological function exerted by these different activities. This has been achieved by combining the use of mouse embryonic stem cells with the development of genetic mouse models to characterize their functional properties in the specific context of adult tissues homeostasis and stem cell identity.
The project has dissected the specific roles of distinct Polycomb ensembles and their molecular mechanisms. The results of this project led to different publications, demonstrating how different activities exert compensatory but also specific roles in regulating gene expression. This work highlighted context specific regulatory mechanisms. In vivo, this translates in specific contributions to define lineage identities and tissue regeneration.
Overall, this work has generated important knowledge advancements related to how developmental and tissue regenerative processes are regulated by repressive chromatin features. Importantly, since these molecular mechanisms play specific roles also in the context of different human pathologies, the uncovering of these molecular details can provide a better disease understanding and uncover new vulnerabilities with therapeutic value.
Cell identity is established and preserved by the set-up of specific transcription programs allowing a correct use of the genetic information. Simplifying a complex mechanism, this involves both the activation of the “correct” genes while silencing of the rest of the genome. This can be achieved by coordinating the activity of DNA binding transcription factors together with the modulation of the local structure of chromatin.
In such scenario, Polycomb proteins represents the major repressive system by which genes are maintained transcriptionally silenced. This includes a large family of proteins with distinct biochemical features that execute their activity forming large multiprotein complexes that modify the chromatin environment to maintain repression. Their activity is essential for several developmental processes and different members of this family are frequently targeted by mutations that affect their activity in several human diseases. This includes, with high frequency, human tumors where components of the Polycomb machinery are targeted by gain and loss of function mutations in a tissue dependent manner. Although the Polycomb machinery can be reconciled in two major enzymatic activities, these are characterized by an high biochemical heterogeneity. Several distinct forms of Polycomb complexes exist with distinct biochemical features whose role remains poorly understood.
This project aimed to generate new knowledge related to the specific molecular and biological function exerted by these different activities. This has been achieved by combining the use of mouse embryonic stem cells with the development of genetic mouse models to characterize their functional properties in the specific context of adult tissues homeostasis and stem cell identity.
The project has dissected the specific roles of distinct Polycomb ensembles and their molecular mechanisms. The results of this project led to different publications, demonstrating how different activities exert compensatory but also specific roles in regulating gene expression. This work highlighted context specific regulatory mechanisms. In vivo, this translates in specific contributions to define lineage identities and tissue regeneration.
Overall, this work has generated important knowledge advancements related to how developmental and tissue regenerative processes are regulated by repressive chromatin features. Importantly, since these molecular mechanisms play specific roles also in the context of different human pathologies, the uncovering of these molecular details can provide a better disease understanding and uncover new vulnerabilities with therapeutic value.
The project has characterized the role played by distinct Polycomb repressive Complexes in embryonic stem cells and in vivo using the adult intestine as proxy. This allowed to dissect the details of their transcriptional repressive features and translated this knowledge in the regulation of adult tissue homeostasis and regeneration.
This work has uncovered both compensatory and specific functions related to the high biochemical heterogeneity that define Polycomb activities using embryonic stem cells. We showed that the activity of specific complexes is not only linked to gene repression but that is also associated with active transcription. This is achieved by functioning in biochemically distinct modules respect to their central enzymatic activity. Using new genetic models, we also provided biological relevance for these molecular features. Using the adult intestine as model, we demonstrated how enzymatically independent modules are critical for specific cell lineage formation.
This project has also allowed to uncover the central role played by the deposition of specific chromatin post-translational modifications in mediating gene repression. This has placed the ubiquitination of histone H2A (H2Aub1) at the center of the machinery where it serves as a “glue” to maintain Polycomb-dependent repressive domains. The project has further extended the role of H2Aub1 demonstrating how a specific Polycomb activity (namely PR-DUB) is devoted to maintain low the diffusion of this modification across the entire genome. This activity preserves chromatin plasticity and allows to concentrate Polycomb repression specifically at the level of gene promoters to reinforce the repressive system. This activity is highly deregulated in cancer by specific mutations. Our work has also contributed, with structural details, to determine the molecular features for its substrate recognition, also showing how oncogenic mutations specifically affect this molecular properties in cancer patients.
The project has also translated this knowledge in vivo establishing various genetic models targeting adult tissues and defined common and specific functional features related to Polycomb heterogeneity in distinct adult stem cell compartments. This has allowed us to define the critical Polycomb activities required to sustain H2Aub1 deposition, the maintenance of transcriptional silencing and the control of stem cell identity in vivo. This work has also uncovered how distinct Polycomb activities exert specific roles in regulating cell identity and lineage choices in a dynamic in vivo developing system. On one hand, this has provided direct biological relevance for the biochemical properties that we have uncovered with the in vitro stem cell models. On the other, the use of dynamic in vivo systems allowed to uncover distinct dependencies between the mechanisms by which pre-established repressive states are maintained respect to the establishment of new transcriptional identities.
This work has uncovered both compensatory and specific functions related to the high biochemical heterogeneity that define Polycomb activities using embryonic stem cells. We showed that the activity of specific complexes is not only linked to gene repression but that is also associated with active transcription. This is achieved by functioning in biochemically distinct modules respect to their central enzymatic activity. Using new genetic models, we also provided biological relevance for these molecular features. Using the adult intestine as model, we demonstrated how enzymatically independent modules are critical for specific cell lineage formation.
This project has also allowed to uncover the central role played by the deposition of specific chromatin post-translational modifications in mediating gene repression. This has placed the ubiquitination of histone H2A (H2Aub1) at the center of the machinery where it serves as a “glue” to maintain Polycomb-dependent repressive domains. The project has further extended the role of H2Aub1 demonstrating how a specific Polycomb activity (namely PR-DUB) is devoted to maintain low the diffusion of this modification across the entire genome. This activity preserves chromatin plasticity and allows to concentrate Polycomb repression specifically at the level of gene promoters to reinforce the repressive system. This activity is highly deregulated in cancer by specific mutations. Our work has also contributed, with structural details, to determine the molecular features for its substrate recognition, also showing how oncogenic mutations specifically affect this molecular properties in cancer patients.
The project has also translated this knowledge in vivo establishing various genetic models targeting adult tissues and defined common and specific functional features related to Polycomb heterogeneity in distinct adult stem cell compartments. This has allowed us to define the critical Polycomb activities required to sustain H2Aub1 deposition, the maintenance of transcriptional silencing and the control of stem cell identity in vivo. This work has also uncovered how distinct Polycomb activities exert specific roles in regulating cell identity and lineage choices in a dynamic in vivo developing system. On one hand, this has provided direct biological relevance for the biochemical properties that we have uncovered with the in vitro stem cell models. On the other, the use of dynamic in vivo systems allowed to uncover distinct dependencies between the mechanisms by which pre-established repressive states are maintained respect to the establishment of new transcriptional identities.
The project was expected to provide new molecular understanding about how distinct Polycomb machineries work in concert with each other to maintain transcriptionally repressive states. The outcome of this project indeed significantly improved this knowledge providing a detailed molecular characterization of the specific roles exerted by these different activities with their related in vivo biological relevance. Although we still far from a complete understanding, the project has delivered a set of new molecular information dissecting the division in labor of biochemically distinct activities and of specific histone repressive modifications in the regulation of cell identity and in vivo lineage determination. Considering that a vast part of these activities is directly mutated in human diseases including cancer, the understanding of their specific molecular contributions to gene transcription and cell identity could also advance our understanding about the etiology of different diseases as well as to envision new molecular strategies for intervention.