Periodic Reporting for period 2 - XCYCLE (Toward a comprehensive analysis of the dynamics and mechanisms of X chromosome inactivation in Humans)
Berichtszeitraum: 2023-06-01 bis 2024-11-30
The X chromosome is subjected to a formidable degree of concerted gene regulation, with appropriate dosage of X-linked expression in female mammals being ensured by the stable yet reversible inactivation of one of the two copies. The process of X chromosome inactivation is critical for female development and linked to sex-specific traits and susceptibilities to various diseases. Well characterized in rodents, there is remarkably little knowledge of how X chromosome inactivation is articulated and controlled in humans, despite its major implication for human health.
Here we proposed to model in vitro the whole X-inactivation cycle in humans to gain novel and high-resolution insights into the key steps of the process and to identify critical actors and mechanisms. We proposed to exploit human embryonic stem cells and recent culture methods to mimic early developmental stages as well as more advanced organogenesis. This allows us to investigate the four phases of X-inactivation: (i) the steps that precede X chromosome inactivation (ii) its initiation, (iii) its maintenance in complex tissues and (iv) its reversion in the germline. We are implementing a combination of exploratory and mechanistic approaches integrating single cell analyses, CRISPR/Cas9 genome editing and genetic screens to build a comprehensive spatiotemporal map of X-chromosome activity across the human life cycle, to screen for regulators of each phase and to assess the importance of X-inactivation and appropriate X-linked gene dosage to tissue development and homeostasis. Using an innovative set of approaches, we hope to challenge the general principles of X-inactivation that were formerly defined in model organisms and to unravel human-specific features of the process that could serve as new research paradigms in developmental epigenetics. The molecular and cellular understanding of human X-inactivation that this project should provide is a fundamental pre-requisite to further understand the principles of human development and sex-specific diseases.
Here we proposed to model in vitro the whole X-inactivation cycle in humans to gain novel and high-resolution insights into the key steps of the process and to identify critical actors and mechanisms. We proposed to exploit human embryonic stem cells and recent culture methods to mimic early developmental stages as well as more advanced organogenesis. This allows us to investigate the four phases of X-inactivation: (i) the steps that precede X chromosome inactivation (ii) its initiation, (iii) its maintenance in complex tissues and (iv) its reversion in the germline. We are implementing a combination of exploratory and mechanistic approaches integrating single cell analyses, CRISPR/Cas9 genome editing and genetic screens to build a comprehensive spatiotemporal map of X-chromosome activity across the human life cycle, to screen for regulators of each phase and to assess the importance of X-inactivation and appropriate X-linked gene dosage to tissue development and homeostasis. Using an innovative set of approaches, we hope to challenge the general principles of X-inactivation that were formerly defined in model organisms and to unravel human-specific features of the process that could serve as new research paradigms in developmental epigenetics. The molecular and cellular understanding of human X-inactivation that this project should provide is a fundamental pre-requisite to further understand the principles of human development and sex-specific diseases.
During the period covered by the report, we have first investigated the status of the X chromosome in the developmental stage that precedes the establishment of X chromosome inactivation (corresponding to preimplantation embryos). We knew that X chromosomes at this stage are mainly active, yet are covered by the XIST long noncoding RNA, which is known to trigger X chromosome inactivation (at least this was shown in the mouse). Such uncoupling between XIST accumulation and the actual silencing of the chromosome was a puzzling observation that we aimed to understand molecularly.
For this we have worked with human embryonic stem cells that mimic, in vitro, this developmental period and have combined molecular investigation at the chromosome level to functional approaches. We have shown that the X chromosomes, which are expressed in these cells, display chromatin features that are typical of inactive chromosomes. We demonstrated that these features are put in place by the XIST long noncoding RNA, which, in addition, slightly attenuates the expression levels of most, but not all X-linked genes. This attenuation also involves SPEN, a protein known to interact with XIST and to mediate X chromosome inactivation. In summary, our work reveals that during female human development, X chromosome activity can be modulated to different extents (partial attenuation of expression of full silencing) depending on the context, but through the same machinery. This work, which has been published, raises questions at to the functional significance of X chromosome attenuation, as well as to the switch in the way X chromosome activity is regulated (attenuation versus inactivation).
We have also studied the initiation of X chromosome inactivation during developmental progression, using a model of extraembryonic differentiation. This part of the work, which is not yet published, highlighted the importance of X chromosome dosage in the development of extraembryonic annexes, with potential consequences for pregnancy success.
We will be comparing our results on the dynamics and importance of X chromosome inactivation in the differentiation of extraembryonic lineages to that of embryonic tissues, using model systems that we are currently implementing. We have also set up systems that allow us to tackle X-inactivation maintenance in complex tissues and its reactivation in the germline.
For this we have worked with human embryonic stem cells that mimic, in vitro, this developmental period and have combined molecular investigation at the chromosome level to functional approaches. We have shown that the X chromosomes, which are expressed in these cells, display chromatin features that are typical of inactive chromosomes. We demonstrated that these features are put in place by the XIST long noncoding RNA, which, in addition, slightly attenuates the expression levels of most, but not all X-linked genes. This attenuation also involves SPEN, a protein known to interact with XIST and to mediate X chromosome inactivation. In summary, our work reveals that during female human development, X chromosome activity can be modulated to different extents (partial attenuation of expression of full silencing) depending on the context, but through the same machinery. This work, which has been published, raises questions at to the functional significance of X chromosome attenuation, as well as to the switch in the way X chromosome activity is regulated (attenuation versus inactivation).
We have also studied the initiation of X chromosome inactivation during developmental progression, using a model of extraembryonic differentiation. This part of the work, which is not yet published, highlighted the importance of X chromosome dosage in the development of extraembryonic annexes, with potential consequences for pregnancy success.
We will be comparing our results on the dynamics and importance of X chromosome inactivation in the differentiation of extraembryonic lineages to that of embryonic tissues, using model systems that we are currently implementing. We have also set up systems that allow us to tackle X-inactivation maintenance in complex tissues and its reactivation in the germline.
3. Progress beyond the state of the art and expected results until the end of the project
We have provided a new understanding of how X chromosome activity is controlled in early human development and of the extent to which it differs from what was learnt using mice as a model. We also reported the first evidence of the importance of X-dosage for proper human development, which may have major impact for pregnancy success.
Further results will be obtained until the end of the project:
1/ on the mechanisms and importance of X-inactivation for the differentiation of embryonic tissues
2/ on the way X-inactivation is reversed in the germline, and whether this is relevant for the formation of gametes
3/ on X-inactivation maintenance in tissues and how this might impact cellular homeostasis and contribute to sex dimorphisms
Resolving the enigmatic mechanisms behind X-chromosome inactivation/reactivation in humans are expected to have broad implications in medicine and for research on developmental processes.
We have provided a new understanding of how X chromosome activity is controlled in early human development and of the extent to which it differs from what was learnt using mice as a model. We also reported the first evidence of the importance of X-dosage for proper human development, which may have major impact for pregnancy success.
Further results will be obtained until the end of the project:
1/ on the mechanisms and importance of X-inactivation for the differentiation of embryonic tissues
2/ on the way X-inactivation is reversed in the germline, and whether this is relevant for the formation of gametes
3/ on X-inactivation maintenance in tissues and how this might impact cellular homeostasis and contribute to sex dimorphisms
Resolving the enigmatic mechanisms behind X-chromosome inactivation/reactivation in humans are expected to have broad implications in medicine and for research on developmental processes.