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.
