Periodic Reporting for period 5 - ChromADICT (Chromatin Adaptations through Interactions of Chaperones in Time)
Periodo di rendicontazione: 2022-07-01 al 2023-06-30
The primary role of chromatin is to compact the genome within the cell nucleus, but it also provides a large repertoire of information in addition to that encoded genetically. Chromatin organization contributes to genome functions such as gene expression and stable transmission through cell divisions. Additionally Chromatin structure and organization changes over time and in different tissues and adapts to physiological conditions. These dynamics are critical for development and for the maintenance/plasticity of cell fate over the course of life.
Understanding how to build this organization to ensure the establishment, maintenance, and propagation of a cellular identity in a given cell lineage has been a challenge for the field. The major protein bricks in this organization are the histones. In metazoans, they exist as histone variants. These variants, along with post-translational modifications provide a marking for distinct chromosomal landmarks important for both genome stability and expression. This is exemplified by the presence of the centromeric H3 variant (CenH3) at the centromere, a key chromosomal region enabling proper chromosome segregation during cell division.
Mechanistic aspects of histone deposition have been documented over the past years. However, understanding at a molecular level how histone variant choice and targeting to particular loci is controlled remains a major challenge. As the architects and bricklayers of the genome, histone chaperones escort histones throughout their cellular life and they represent key candidates to link the choice of variants to particular chromatin assembly pathways, and possibly their targeting to particular loci.
Our objective is to understand how the dosage of histones and their chaperones can affect cell fate. Using advanced genomics and imaging technologies in model systems, our aim is to characterize the dynamics of molecular complexes in which they are engaged and the changes that occur at places they mark in the genome. By extending the approach to distinct developmental transitions we wish to apply our findings to whole organisms. Ultimately we hope to establish a system to intervene and control these events in real time in developing organism.
Understanding how to build this organization to ensure the establishment, maintenance, and propagation of a cellular identity in a given cell lineage has been a challenge for the field. The major protein bricks in this organization are the histones. In metazoans, they exist as histone variants. These variants, along with post-translational modifications provide a marking for distinct chromosomal landmarks important for both genome stability and expression. This is exemplified by the presence of the centromeric H3 variant (CenH3) at the centromere, a key chromosomal region enabling proper chromosome segregation during cell division.
Mechanistic aspects of histone deposition have been documented over the past years. However, understanding at a molecular level how histone variant choice and targeting to particular loci is controlled remains a major challenge. As the architects and bricklayers of the genome, histone chaperones escort histones throughout their cellular life and they represent key candidates to link the choice of variants to particular chromatin assembly pathways, and possibly their targeting to particular loci.
Our objective is to understand how the dosage of histones and their chaperones can affect cell fate. Using advanced genomics and imaging technologies in model systems, our aim is to characterize the dynamics of molecular complexes in which they are engaged and the changes that occur at places they mark in the genome. By extending the approach to distinct developmental transitions we wish to apply our findings to whole organisms. Ultimately we hope to establish a system to intervene and control these events in real time in developing organism.
A central question in chromatin biology is how to both structurally organize the genome and mark specific regions with a choice of histone variants. Understanding how to establish and maintain, but also how to change chromatin states is a fundamental challenge. Although histone modifications have been studied intensively, less is known about mechanisms concerning histone variant placement. Histone chaperones are escort factors that regulate the supply, loading, eviction, and degradation of histone variants. They are key in the placement of histone variants at specific chromatin landmarks and bridge organization scales from the nucleosome to higher order structures. Indeed, selective chaperone-variant partnerships have been documented in multicellular organisms, yet recently, dosage imbalances occurring in natural and pathological contexts underline plasticity in these interactions. Considering the known changes in histone dosage during development, we aimed to re-evaluate chaperone function not as individual fixed modules, but rather as a dynamic circuitry that adapts to cellular needs during the cell cycle, DNA replication and repair, differentiation, development, and pathology.Our project CHROMatin ADaptation through Interactions with Chaperones in Time (ChromAdICT) has built on our proposed concept by which a partial interchangeability in chaperones and histone variants provides unique possibilities to respond to cellular demands during the cell cycle and during development. A control of these dynamics at distinct chromatin regions could thus contribute to not only establish and maintain functional domains, but also to a plasticity enabling to adapt properties locally. We thus explored how this balance in chromatin stability and plasticity could be tuned in distinct cell types depending on context. We explored the underlying mechanisms enabling adaptability to naturally as well as experimentally induced chaperone and variant dosage changes over time. Our dissection of how chaperone-histone complexes act as a coordinated team of chromatin architects enabled four complementary and interconnected achievements.
1.We engineered a toolbox for probing the chaperone-variant partnership nature and plasticity.
2. We assessed the dynamics of histone variants promoted by chaperones during DNA replication and damage assessing the fate of new and old histones.
3. We explored the Impact of the chaperone-histone partnership on functional chromosome landmarks – with a particular focus on centromeres, telomeres, heterochromatin, and DNA regulatory elements.
4. We evaluated the plasticity and adaptability of histone dynamics during differentiation and T cell commitment in mice and during development in Xenopus.
Taken together these results obtained within the project illustrate the importance of characterizing the relationships between histones and their dedicated chaperones, including their dosage and dynamics, to understand how these can impact on cell fate, survival and maintenance of chromatin marks supported by histone variants. Now these findings prove their importance for medical applications and have led to innovation strategies.
1.We engineered a toolbox for probing the chaperone-variant partnership nature and plasticity.
2. We assessed the dynamics of histone variants promoted by chaperones during DNA replication and damage assessing the fate of new and old histones.
3. We explored the Impact of the chaperone-histone partnership on functional chromosome landmarks – with a particular focus on centromeres, telomeres, heterochromatin, and DNA regulatory elements.
4. We evaluated the plasticity and adaptability of histone dynamics during differentiation and T cell commitment in mice and during development in Xenopus.
Taken together these results obtained within the project illustrate the importance of characterizing the relationships between histones and their dedicated chaperones, including their dosage and dynamics, to understand how these can impact on cell fate, survival and maintenance of chromatin marks supported by histone variants. Now these findings prove their importance for medical applications and have led to innovation strategies.
We developed models to control histone variants and dosage of chaperones to study cell fate and plasticity using imaging and genome wide methods.
During T cell differentiation in mice, we identified a key role for the histone H3 lysine-9 methyl transferase, Suv39h1, in the control of stemness in CD8+ T cell with immunologists (Pace et al., 2018) and explored how Suv39h1 modulates CD8+T cell responsiveness to anti- PD-1 in mouse models of melanoma (Niborski et al., 2022; two patents and Mnemo therapeutics company by our collaborators).
Unexpectedly, we revealed a critical role in H3.3 with a single amino acid (serine 31) important during gastrulation in the amphibian, Xenopus (Sitbon et al., 2020). While the replicative and non-replicative histone variants were considered as exchangeable, differing only by their deposition mode, this finding highlighted the unique importance of an amino-acid only present in the H3.3 variant. In the context of oncohistone mutations.
We revealed unique H3 variants dynamics in chromatin during transcription and replication: (i) during transcription, an important recycling of old histones relies on the histone chaperones HIRA and ASF1. The HIRA chaperone ensures a coordinated incorporation of new H3.3 histones and their recycling during transcription (Ray-Gallet et al., 2018; Torné et al., 2020); (ii) during replication, using super-resolution imaging, we showed the spatial distribution of H3 variants and their recycling during replication (Clément et al., 2018). (iii) Using genome-wide mapping we revealed how new histone deposition shape replication in mammals involving the chaperone HIRA (Gatto et al., 2022).
We showed how the overexpression of CENP-A in cancer impacts tumor progression and the response to treatments (Filipescu et al., 2017; Jeffery et al., 2021);(iii) We identified a distinct subnuclear CENP-A pattern predicting curability by chemoradiation therapy in a patient cohort of locally advanced head and neck cancer patients (Verrelle et al., 2021, patent).
During T cell differentiation in mice, we identified a key role for the histone H3 lysine-9 methyl transferase, Suv39h1, in the control of stemness in CD8+ T cell with immunologists (Pace et al., 2018) and explored how Suv39h1 modulates CD8+T cell responsiveness to anti- PD-1 in mouse models of melanoma (Niborski et al., 2022; two patents and Mnemo therapeutics company by our collaborators).
Unexpectedly, we revealed a critical role in H3.3 with a single amino acid (serine 31) important during gastrulation in the amphibian, Xenopus (Sitbon et al., 2020). While the replicative and non-replicative histone variants were considered as exchangeable, differing only by their deposition mode, this finding highlighted the unique importance of an amino-acid only present in the H3.3 variant. In the context of oncohistone mutations.
We revealed unique H3 variants dynamics in chromatin during transcription and replication: (i) during transcription, an important recycling of old histones relies on the histone chaperones HIRA and ASF1. The HIRA chaperone ensures a coordinated incorporation of new H3.3 histones and their recycling during transcription (Ray-Gallet et al., 2018; Torné et al., 2020); (ii) during replication, using super-resolution imaging, we showed the spatial distribution of H3 variants and their recycling during replication (Clément et al., 2018). (iii) Using genome-wide mapping we revealed how new histone deposition shape replication in mammals involving the chaperone HIRA (Gatto et al., 2022).
We showed how the overexpression of CENP-A in cancer impacts tumor progression and the response to treatments (Filipescu et al., 2017; Jeffery et al., 2021);(iii) We identified a distinct subnuclear CENP-A pattern predicting curability by chemoradiation therapy in a patient cohort of locally advanced head and neck cancer patients (Verrelle et al., 2021, patent).