Claire Rougeulle

X-chromosome inactivation (XCI) is essential for extraembryonic tissue growth and embryo development in mice. However, species-specific differences in its timing and regulation have left its role in human development unclear. Here, we show that the in vitro differentiation of naive human pluripotent stem cells to trophoblast stem cells and extraembryonic mesoderm cells is accompanied by XCI. The inactive X chromosome displays an atypical chromatin state, lacking DNA methylation and canonical heterochromatin marks. We demonstrate that extraembryonic differentiation and XCI are temporally linked. Using loss of function approaches, we found that XIST is required for XCI establishment in humans. Furthermore, we show that XCI is needed for the survival of human female extraembryonic cells. Together, these findings link XCI to the formation of human extraembryonic lineages, with important implications for human reproductive biology.

Unraveling how gene regulations are remodeled during evolution is central to understanding how biological processes evolve. We explored this question in the frame of X-chromosome inactivation, a process under strong selective constraint, governed by the XIST lncRNA and its cis-regulators. Using functional approaches on closely related primate species, we show that XIST regulation has uniquely diverged over a short evolutionary timescale. In human and marmoset embryonic stem cells (ESCs), the JPX lncRNA gene is a major regulator of XIST expression. In contrast, JPX has a minor effect on XIST in macaque ESCs, where it acts together with a macaque-specific enhancer. This occurs within a reshuffled 3D organization of the XIST neighborhood triggered by the insertion of a HERVK transposon in the macaque lineage. Retrospective sequence comparisons revealed that many XIST regulators are not evolutionarily constrained, supporting the hypothesis that neutrally evolving noncoding elements harbor adaptive potential. These results illuminate how evolutionary recent elements are integrated into preexisting regulatory landscapes.

X chromosome inactivation (XCI) is a mammalian dosage compensation mechanism that ensures balanced expression of X-linked genes between males and females. Research using rodent models has led to major discoveries regarding XCI mechanisms and dynamics, in addition to the molecular actors involved in this process, including the long noncoding RNA Xist and its protein partners. However, several features of XCI vary significantly across mammalian species, not only between marsupials and placental mammals, but also within the latter. This review discusses the fundamental aspects of XCI from an evolutionary perspective, highlighting both conserved features and species-specific variations across mammalian species.

X-chromosome inactivation (XCI) is initiated during early mammalian embryogenesis by a long non-coding RNA (lncRNA) XIST , which coats one of the two X-chromosomes and facilitates epigenetic transcriptional silencing. A second, evolutionarily recent primate-specific lncRNA XACT was proposed to antagonize XIST ’s ability to induce XCI. XACT expression is restricted to pluripotent states and early embryonic stages and coats the active X-chromosome in both females and males. Here, we report a novel XACT transcript expressed in normal and cancerous somatic cells from both the inactive (Xi) and active (Xa) X chromosomes. It coexists with XIST on the Xi without affecting XIST expression or (re)activating X-linked genes inactivated by XCI. During hematopoietic stem cell (HSC) differentiation, XACT is primarily expressed in myeloid progenitors in both sexes. XACT expression is activated in HSCs and peaks in megakaryocyte-erythrocyte progenitor cells (MEPs) before rapidly declining as the MEPs differentiate into megakaryocytes or erythrocytes. By combining CRISPR-based XACT perturbation with epigenomic and transcriptional studies, we revealed the key role of XACT in the self-renewal and differentiation of erythroid progenitors into erythrocytes, by recruiting cis-regulatory proteins and regulating transcription through ETS and AP-1 transcription factors. Furthermore, XACT is expressed in a subset of acute myeloid leukemia (AML) patients, with high levels found in erythroid-megakaryocytic blast cells; suggesting XACT ’s potential as a marker for AML subtypes. Thus, we identified a novel XACT transcript with a unique expression profile and important roles in normal and cancer cells, revealing XACT lncRNA functions beyond its previously proposed role in XCI during embryogenesis.

XIST (X-inactive specific transcript) long noncoding RNA (lncRNA) is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female preimplantation embryos without triggering X chromosome silencing. The XACT (X-active coating transcript) lncRNA coaccumulates with XIST on active X chromosomes and may antagonize XIST function. Here, we used human embryonic stem cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during preimplantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and dampens the transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional before XCI, confirms the existence of a transient process of X chromosome dosage compensation and reveals that XCI and dampening rely on the same set of factors.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist , in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti–nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

Unravelling how gene regulatory networks are remodelled during evolution is crucial to understand how species adapt to environmental changes. We addressed this question for X-chromosome inactivation, a process essential to female development that is governed, in eutherians, by the XIST lncRNA and its cis -regulators. To reach high resolution, we studied closely related primate species, spanning 55 million years of evolution. We show that the XIST regulatory circuitry has diversified extensively over such evolutionary timeframe. The insertion of a HERVK transposon has reshuffled XIST 3D interaction network in macaque embryonic stem cells (ESC) and XIST expression is maintained by the additive effects of the JPX lncRNA gene and a macaque specific enhancer. In contrast, JPX is the main contributor to XIST expression in human ESCs but is not significantly involved in XIST regulation in marmoset ESCs. None of these entities are however under purifying selection, which suggests that neutrally evolving non-coding elements harbour high adaptive potentials.

XIST long non-coding RNA is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female pre-implantation embryos without triggering X chromosome silencing. The long non-coding RNA XACT co-accumulates with XIST on active Xs and may antagonize XIST function. Here we used human ES cells in a naïve state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during pre-implantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and attenuates transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional prior to XCI, confirms the existence of a transient process of X chromosome dosage compensation, and reveals that X chromosome inactivation and dampening rely on the same set of factors.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism that ensures the silencing of one X in females, may participate in these sex-biases. Here, we perturbed the expression of the trigger of XCI, the non-coding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signalling pathway, in monocyte/macrophages, dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal centre B cells and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signalling is dysregulated in macrophages, which leads to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity. Teaser: The reason why autoimmunity mostly affects women is unclear. Here, we show that aberrant expression of genes on the X induces signs of lupus in female mice.