Periodic Reporting for period 2 - CellSex (The importance of cellular sex in physiology and the underlying mechanisms)
Okres sprawozdawczy: 2021-11-01 do 2023-04-30
The difference between males and females constitutes the largest phenotypic dimorphism in any given species. In humans, this variation accounts for differences seen in the risk, incidence, and response to treatment for a plethora of diseases. While reproductive organ-derived sex hormones play key roles in sculpting and maintaining such sex differences, our recent work highlighted the importance of cell-intrinsic mechanisms involving the sex chromosomes. In fact, we demonstrated that the sex of intestinal stem cells plays a key role in the adult gut, both for the organ size and for the sex-specific pre-disposition to tumours. While these findings establish the proof-of-principle of the influence of sex chromosomes in adult cells, essential gaps remain to be filled. Indeed, the full range of phenotypic consequences of the presence of sex chromosomes in somatic cells, the genes, the mechanisms involved, and their sites of action remain entirely elusive. Our research aims to understand how cellular sex impacts physiology across the body using Drosophila as an in vivo model. This question has been poorly investigated in part due to the practical difficulties of studying sex chromosome effects. Fruit flies offer the remarkable possibility to generate mosaic animals in which sex chromosomes can be genetically manipulated in defined organs.
We are combining classical fly genetics, novel genetic methods, and cutting-edge genomic techniques to 1. characterise new pathways driving sex differences, 2. achieve, for the first time, conditional and organ-specific Y chromosome deletion in vivo, and 3. use this new method to study the role of the Y chromosome in sex gap in longevity.
Results from our research should have a major impact on our understanding of the importance of cellular sex in physiology and disease.
We are combining classical fly genetics, novel genetic methods, and cutting-edge genomic techniques to 1. characterise new pathways driving sex differences, 2. achieve, for the first time, conditional and organ-specific Y chromosome deletion in vivo, and 3. use this new method to study the role of the Y chromosome in sex gap in longevity.
Results from our research should have a major impact on our understanding of the importance of cellular sex in physiology and disease.
Our research program aims to understand how cellular sex impacts physiology across the body using Drosophila as an in vivo model.
Aim 1.1: Discover cell-type specific importance of chromosomal sex and the underlying mechanisms.
People involved: Chloe Herault (Ph.D. 2020-2024), Renald Delanoue (Inserm permanent researcher, CR1), Marion Rameau (University permanent technician), and Charlene Clot (Ph.D. 2020-2024).
While there is mounting evidence of the importance of cellular sex, critical gaps remain to be filled about: 1) the spatial and temporal influence of cellular sex outside the sex organs, 2) the underlying molecular mechanisms, 3) and the whole repertoire of physiological significance. Our experiments have already established a definitive organism-wide map of tissues and organs where the presence of two X chromosomes converts into active production of the fly female-specific sex determinant, TransformerF (TraF) (i.e. potential to sexually differentiate using unknown sex pathways). We have also comprehensibly identified the cellular contexts where TraF expression matters functionally for sex gaps in body size, weight, and predisposition to gut tumours. We now will focus on these genetically-defined TraF-positive cell subpopulations, to characterize the molecular mechanisms driving sex differences. Using cutting-edge genomic/RNA techniques, we plan to isolate sex-specific RNA transcripts under the direct control of the female-specific TraF splicing factor. We will then analyse the contribution of sex-specific splicing isoforms to our sex phenotypes. This workflow should identify novel sex pathways, driving sexual dimorphisms in body size, weight, and predisposition to tumours.
Chloe Herault and Charlene Clot presented these works at 4 international conferences.
Aim 1.2: Metabolic control of germline stem cell differentiation
People involved: Charlotte Francois (Post-doc, 2020-2023), and Thomas Phil (technician, 2020-2023).
We recently described (Hudry et al., 2019 Cell) a new way in which sex differences in physiology arise, involving bidirectional communication between the testis and the intestine; the testis “talks” (through a cytokine and not the classical steroid hormones) to a particular portion of the intestine to change its carbohydrate handling. In response to the testis signal, this intestinal portion secretes citrate. These findings prompted us to investigate the molecular mechanisms that connect cellular metabolism and cell fate decisions, as cells differentiate. Using fly spermatogenesis, we investigated how metabolic signals contribute to stem cell differentiation and germline homeostasis. We discovered that external citrate supply fuels Acetyl-CoA production and is essential for the commitment to the final stage of germline differentiation. In contrast to known metabolic control of gene regulation, high Acetyl-CoA level promotes NatB-dependent N-terminal protein acetylation. Genetic and biochemistry experiments establish that the critical role of N-terminal acetylation is shielding proteins from proteasomal degradation by the specific ubiquitin ligase, Ubr1. This work uncovers that a protein post-translational modification couples the dynamics of stem cell differentiation to the metabolic state, revealing that N-terminal protein acetylation is physiologically essential for animal germline homeostasis. We plan to explore if the instructive role of N-terminal acetylation in controlling protein stability is significant in more biological contexts. We have also performed a genetic screen and identified multiple uncharacterised metabolic genes essential for male germline stem cell differentiation. We plan to study how those genes control cell differentiation.
This project will lead to one publication in a high-impact journal (currently under redaction). Charlotte Francois presented this work at 2 international conferences.
Aim 2: Create conditional and cell-type specific Y chromosome elimination/truncations to uncover how the Y chromosome controls the sex gap in longevity.
People involved: Renald Delanoue (Inserm permanent researcher, CR1), and Charlene Clot (Ph.D. 2020-2024).
While sex chromosomes carry sex-determining genes, they also often differ from autosomes in size and composition, consisting mainly of silenced heterochromatic repetitive DNAs. Even though Y chromosomes show structural heteromorphism, the functional significance of such differences remains elusive. Correlative studies suggest that the amount of Y chromosome heterochromatin might be responsible for several male-specific traits, including sex-specific differences in longevity observed across a wide spectrum of species, including humans. However, experimental models to test this hypothesis have been lacking. We are using the Drosophila Y chromosome to investigate the relevance of sex chromosome heterochromatin in somatic organs in vivo. Using CRISPR-Cas9, we have already generated a library of Y chromosomes with variable levels of heterochromatin. We have shown that these different Y chromosomes can disrupt gene silencing in trans, on other chromosomes, by sequestering core components of the heterochromatin machinery. This effect is proportional to the level of Y heterochromatin. We now plan to leverage these unique genetic tools to investigate if the ability of the Y chromosome to affect genome-wide heterochromatin is used to generate physiological sex differences, including sexual dimorphism in longevity.
This project will lead to one publication currently under revision in a high-impact journal (Nature Ecology and Evolution). Renald Delanoue presented this work at one international conference.
Aim 1.1: Discover cell-type specific importance of chromosomal sex and the underlying mechanisms.
People involved: Chloe Herault (Ph.D. 2020-2024), Renald Delanoue (Inserm permanent researcher, CR1), Marion Rameau (University permanent technician), and Charlene Clot (Ph.D. 2020-2024).
While there is mounting evidence of the importance of cellular sex, critical gaps remain to be filled about: 1) the spatial and temporal influence of cellular sex outside the sex organs, 2) the underlying molecular mechanisms, 3) and the whole repertoire of physiological significance. Our experiments have already established a definitive organism-wide map of tissues and organs where the presence of two X chromosomes converts into active production of the fly female-specific sex determinant, TransformerF (TraF) (i.e. potential to sexually differentiate using unknown sex pathways). We have also comprehensibly identified the cellular contexts where TraF expression matters functionally for sex gaps in body size, weight, and predisposition to gut tumours. We now will focus on these genetically-defined TraF-positive cell subpopulations, to characterize the molecular mechanisms driving sex differences. Using cutting-edge genomic/RNA techniques, we plan to isolate sex-specific RNA transcripts under the direct control of the female-specific TraF splicing factor. We will then analyse the contribution of sex-specific splicing isoforms to our sex phenotypes. This workflow should identify novel sex pathways, driving sexual dimorphisms in body size, weight, and predisposition to tumours.
Chloe Herault and Charlene Clot presented these works at 4 international conferences.
Aim 1.2: Metabolic control of germline stem cell differentiation
People involved: Charlotte Francois (Post-doc, 2020-2023), and Thomas Phil (technician, 2020-2023).
We recently described (Hudry et al., 2019 Cell) a new way in which sex differences in physiology arise, involving bidirectional communication between the testis and the intestine; the testis “talks” (through a cytokine and not the classical steroid hormones) to a particular portion of the intestine to change its carbohydrate handling. In response to the testis signal, this intestinal portion secretes citrate. These findings prompted us to investigate the molecular mechanisms that connect cellular metabolism and cell fate decisions, as cells differentiate. Using fly spermatogenesis, we investigated how metabolic signals contribute to stem cell differentiation and germline homeostasis. We discovered that external citrate supply fuels Acetyl-CoA production and is essential for the commitment to the final stage of germline differentiation. In contrast to known metabolic control of gene regulation, high Acetyl-CoA level promotes NatB-dependent N-terminal protein acetylation. Genetic and biochemistry experiments establish that the critical role of N-terminal acetylation is shielding proteins from proteasomal degradation by the specific ubiquitin ligase, Ubr1. This work uncovers that a protein post-translational modification couples the dynamics of stem cell differentiation to the metabolic state, revealing that N-terminal protein acetylation is physiologically essential for animal germline homeostasis. We plan to explore if the instructive role of N-terminal acetylation in controlling protein stability is significant in more biological contexts. We have also performed a genetic screen and identified multiple uncharacterised metabolic genes essential for male germline stem cell differentiation. We plan to study how those genes control cell differentiation.
This project will lead to one publication in a high-impact journal (currently under redaction). Charlotte Francois presented this work at 2 international conferences.
Aim 2: Create conditional and cell-type specific Y chromosome elimination/truncations to uncover how the Y chromosome controls the sex gap in longevity.
People involved: Renald Delanoue (Inserm permanent researcher, CR1), and Charlene Clot (Ph.D. 2020-2024).
While sex chromosomes carry sex-determining genes, they also often differ from autosomes in size and composition, consisting mainly of silenced heterochromatic repetitive DNAs. Even though Y chromosomes show structural heteromorphism, the functional significance of such differences remains elusive. Correlative studies suggest that the amount of Y chromosome heterochromatin might be responsible for several male-specific traits, including sex-specific differences in longevity observed across a wide spectrum of species, including humans. However, experimental models to test this hypothesis have been lacking. We are using the Drosophila Y chromosome to investigate the relevance of sex chromosome heterochromatin in somatic organs in vivo. Using CRISPR-Cas9, we have already generated a library of Y chromosomes with variable levels of heterochromatin. We have shown that these different Y chromosomes can disrupt gene silencing in trans, on other chromosomes, by sequestering core components of the heterochromatin machinery. This effect is proportional to the level of Y heterochromatin. We now plan to leverage these unique genetic tools to investigate if the ability of the Y chromosome to affect genome-wide heterochromatin is used to generate physiological sex differences, including sexual dimorphism in longevity.
This project will lead to one publication currently under revision in a high-impact journal (Nature Ecology and Evolution). Renald Delanoue presented this work at one international conference.
We generated Drosophila tissues and lines in which we directly manipulated the size and the number of Y chromosomes using a novel method. Using this genetic approach, we discovered that the presence of a Y chromosome does not result in reduced longevity in males. These findings refute the widespread and popular hypothesis that the Y chromosome has a toxic effect and shortens the male lifespan. We used the same genetic manipulations to show that the Y chromosome is dispensable for other key physiological sex differences. We investigated for example the impact of the Y chromosome on sex differences in adult somatic stem cell behaviours and sexually dimorphic intestinal cancers. The implications of our findings are far-reaching because sex differences in longevity are observed in most animals, including humans, and plants.
The “toxic Y” hypothesis, which our data argue against, is one of the major ideas used to explain the sex gap in lifespan. Instead, we use the expression of a single master-switch gene to show that these differences in longevity are determined by phenotypic sex. Genetic manipulations of this factor allowed bidirectional transformation in longevity in both sexes independently of the chromosomal sex. This discovery raises the possibility that other sex determinants play equivalent roles in other/most species.
To make these observations, we developed a new method using CRISPR-Cas9 that will be of interest to the broader research community, and that can be applied in any species with heteromorphic sex chromosomes. The X and Y chromosomes represent the biggest genetic difference in any given species but their potential effects are still routinely ignored. For the first time, we offer a method to explore sex-biased phenotypes from a mechanistic perspective by directly engineering the size and the number of Y chromosomes in contrast to classical approaches manipulating single Y-linked genes.
The “toxic Y” hypothesis, which our data argue against, is one of the major ideas used to explain the sex gap in lifespan. Instead, we use the expression of a single master-switch gene to show that these differences in longevity are determined by phenotypic sex. Genetic manipulations of this factor allowed bidirectional transformation in longevity in both sexes independently of the chromosomal sex. This discovery raises the possibility that other sex determinants play equivalent roles in other/most species.
To make these observations, we developed a new method using CRISPR-Cas9 that will be of interest to the broader research community, and that can be applied in any species with heteromorphic sex chromosomes. The X and Y chromosomes represent the biggest genetic difference in any given species but their potential effects are still routinely ignored. For the first time, we offer a method to explore sex-biased phenotypes from a mechanistic perspective by directly engineering the size and the number of Y chromosomes in contrast to classical approaches manipulating single Y-linked genes.