X-chromosome inactivation: a historic topic that's still hot

Since 1961, when Mary Lyon first proposed the hypothesis of random X-chromosome inactivation (XCI), now known as Lyonization in her honour, numerous studies have confirmed her hypothesis. XCI consists of the transcriptional silencing of one X chromosome in XX females to compensate for the excess of X-linked gene products compared with XY males (Fig. 1). For many years, researchers questioned the existence of a locus that controls the XCI and expressed from the inactive X chromosome (Xi). This was confirmed by the discovery of a Xi-specific transcript, the Xist/XIST long non-coding RNA, in mice and humans in 1991. Xist/XIST molecules coat the silenced X chromosome (either the maternal or paternal copy) in cis. Xist plays a crucial role in recruiting transcriptional repressive complexes to initiate gene silencing. Molecular mechanisms governing XCI have been deeply dissected in mice; however, much less is known in humans. Furthermore, this epigenetic process is accompanied by the upregulation of the active X chromosome (Xa), referred to as X-chromosome upregulation (XCU), in both female and male somatic cells to maintain a balanced expression with the active pairs of autosomes (Fig. 1).

To celebrate the 30-year anniversary of the discovery of Xist/XIST, a milestone that revolutionized the field of X-chromosome inactivation and enhanced our understanding of epigenetic regulation by long non-coding RNAs, the EMBO workshop organized a special session. This session featured some of the prominent researchers who played a crucial role in the discovery of Xist/XIST (Fig. 2). Andrea Ballabio (TIGEM, Pozzuoli, Italy) and Carolyn Brown (University of British Columbia, Vancouver, Canada) discovered the human XIST, and Neil Brockdorff (University of Oxford, UK) and Sohaila Rastan (independent advisor, UK) discovered Xist in mice. The session started with a brief introduction by Andrea Ballabio, outlining the process of identifying XIST. Subsequently, a discussion followed in which all four of them shared their personal stories behind this ground-breaking discovery. These stories cannot be found in scientific articles and were previously unknown to most of the audience. It was lots of fun to hear about the journeys they embarked on to map Xist/XIST, and the excitement and challenges they encountered. Emmanuel Cazottes (Université Paris Cité/CNRS, Epigenetics and Cell Fate, Paris, France) and Antonia Hauth (EMBL, Heidelberg, Germany), who are PhD students and were brilliant moderators of the session, asked that this historical and unique conference session be concluded with advice for the next generation of scientists. Neil Brockdorff suggested focusing on the questions and their impacts, while Sohaila Rastan advised against getting lost in the techniques. Carolyn Brown encouraged all of us to keep having fun, and Andrea Ballabio emphasized the importance of not throwing anything away. The special session was recorded and is available to watch on the Node (https://thenode.biologists.com/xist-discovery/discussion/).

The EMBO workshop on XCI delivered an extensive program spanning three and a half days, with an impressive line-up of speakers. The event underscored its commitment to diversity in science by including researchers from various career stages, genders and backgrounds. Each presentation was allocated 15 to 20 min, followed by 5 min of discussion, facilitating the sharing of the latest results, often including unpublished results. There was a wide range of topics covered during the presentations, from molecular players and regulatory mechanisms in XCI, evolutionary perspectives in both model and non-model organisms, theoretical modelling of Xist regulation, and sex-specificities in development and disease.

One of the most compelling aspects of the workshop was the integration of state-of-the-art genomic and transcriptomic technologies, advanced microscopy, and cutting-edge in vitro differentiation models, such as mouse and human blastoids. This workshop also highlighted the clinical relevance of XCI, particularly concerning genes that normally evade XCI (‘the escapees’) and their implication in sex-specific conditions. Multidisciplinary approaches have been instrumental in deciphering the complexities of XCI and the workshop encouraged ideas and exchange of methodologies.

The first afternoon of the workshop started with a diverse range of talks, spanning evolutionary perspectives to the dissection of XCI players. Although sex chromosome dosage compensation has been extensively studied in mice, Henrik Kaessmann (Heidelberg University, Germany) highlighted the significance of understanding such mechanisms across a broader spectrum of vertebrates (Murat et al., 2023). He gave an overview of the origins and evolution of sex chromosomes across amniotes, assessing mechanisms of dosage compensation in both placental and marsupial mammals, as well as lizards, and highlighting a previously unreported way of dosage compensation in birds that involves both transcriptional and post-transcriptional mechanisms. James Turner delved into the details of the paternal imprinted XCI in marsupials and its master-regulator, the Rsx lncRNA, presenting the development and first results of genome editing in opossums, a marsupial (Mahadevaiah et al., 2020). Another example of non-mammalian X-dosage compensation was presented by Claudia Keller Valsecchi (Institute of Molecular Biology Mainz, Germany) at the end of the day. She highlighted the remarkable case of the SOA gene, which exhibits sex-specific spliced isoforms that regulate dosage compensation in mosquitoes (Kalita et al., 2023). The translational relevance of such an investigation relates to its impact on ecosystems and its role in combating zoonotic diseases such as malaria.

XCI has evolved in mammals to compensate for imbalance of X-linked gene products. David Page (Massachusetts Institute of Technology, Cambridge, MA, USA) explained the similarities of the active X chromosomes in women and men. He underscored the importance of the inactive X and Y chromosomes in influencing autosomal gene expression, and explored the impact of sex-chromosome aneuploidies in development and disease (San Roman et al., 2023).

This first session also featured more mechanistic investigations into Xist RNA regulation and action. Mitchell Guttman (California Institute of Technology, Pasadena, USA) explored how a limited number of Xist molecules could achieve gene silencing across the entire X chromosome, with SPEN/SHARP protein being crucial to this process (Jachowicz et al., 2022). Furthermore, a precise dosage of Xist is also absolutely required for specific silencing of Xi in female cells. Joost Gribnau provided a comprehensive insight into how regulatory elements and transcription factors are involved in Xist regulation, including RNF12/RLIM, as well as previously unidentified factors (Joost Gribnau's laboratory, unpublished).

Day 2 of the EMBO workshop again featured a diverse array of presentations, as well as the first 2 h poster session, a historical session dedicated to the discovery of Xist/XIST and discussion breakout groups (Box 1; Fig. 3). Christine Disteche (University of Washington, Seattle, USA) opened the day with the 4D structure of the X chromosome, and the relationship between chromosome condensation and changes in gene expression (Bonora et al., 2021). Subsequently, Azusa Inoue (RIKEN, Yokohama, Japan) explained how the study of H3K27me3 marks and PRC2 mutants in the oocyte and early mouse embryo has unveiled the maternal imprint of Xist (Matoba et al., 2022). Notably, maternal polycomb complexes, PRC1 and PRC2, protect the maternal X chromosome during the initiation of imprinted XCI. Amanda Fisher and Dounia Djeghloul (University of Oxford, UK) probed the status of Xa and Xi chromosomes during mitosis, with a particular focus on bookmarking proteins (Djeghloul et al., 2023). Their work underscored the significance of active components to the active X chromosome. Complementary to this topic, Rawin Poonperm (RIKEN BDR, Kobe, Japan) delved into DNA replication timing, exploring differences between Xa and Xi, and along the Xi itself, highlighting the importance of SMCHD1 in maintaining late replication timing (Poonperm et al., 2023). Tatsuya Ohhata (Hamamatsu University School of Medicine, Japan) questioned the decreased homologous recombination in XaXa females, compared with XaY and XaXi cells, investigating the potential X-linked gene factor responsible (Tamura et al., 2021).

The second day also witnessed in-depth dissections of the initiation of XCI in mice. Guifeng Wei (University of Oxford, UK) explored the post-transcriptional regulation of Xist RNA, reviewing the impact of m6A modification on Xist stability and Xi silencing rate (Wei et al., 2021). Gregor Neuert (Vanderbilt University, Nashville, TN, USA) ventured into the intricate cis-regulation of Xist and Tsix antisense transcription at the single-cell level, using cutting-edge 3D single-molecule quantitative RNA-FISH (Thiemicke et al., 2019). Gemma Noviello (Max Planck Institute for Molecular Genetics, Berlin, Germany) investigated dose-sensitive X-linked activators of Xist in mouse embryonic stem cells (ESCs), developing an exciting tool for analogue tuning of endogenous gene expression (Noviello et al., 2023). Gemma's talk questioned the role of RNF12 as a dose-dependent Xist activator in favour of a previously uncharacterized factor. Further dissection of the RNF12/REX1 axis was conducted by Ingolf Bach (UMass Chan Medical School, Worcester, MA, USA) during early mouse development, in both imprinted and random XCI (Wang et al., 2016). Edith Heard (EMBL Heidelberg, Germany) provided an extensive exploration of the initiation of random XCI, probing gene silencing, Pol II and Xist live imaging, early changes in histone marks, and the pivotal role of SPEN/SHARP (Collombet et al., 2023). Edith also emphasized the significance of escapees (genes not subject to XCI) and the investigation of their unique regulation.

Finally, the relevance of studying dosage compensation beyond the mammalian kingdom was underscored by two excellent presentations. Natali Papanicolaou (Karolinska Institutet, Stockholm, Sweden) examined Z-chromosome upregulation through extensive allele-specific single-cell transcriptomics, providing the first analysis of transcriptional burst kinetics in birds (Björn Reinius' laboratory, unpublished). Barbara Meyer (University of California at Berkeley, USA) shared her ground-breaking discoveries in Caenorhabditis elegans related to sex determination and the Dosage Compensation Complex (DCC), which reduces X expression in XX worms to balance it with X expression from XO worms. Her most recent insights into dosage compensation through DCC recruitment sites (Rex sites), condensin and 3D conformation were presented (Yang et al., 2023).

Scientific excitement remained palpable as the third day of the meeting started, after an engaging evening of informal gathering. The day's agenda was divided between scientific talks in the morning and a free afternoon to explore Berlin or the scientific campus surrounding the Harnack-haus. The highlight of the day was the traditional boat tour on the Spree river, which has become emblematic of XCI meetings (Fig. 3).

Marnie Blewitt (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) conveyed the importance of studying her neomorphic allele of Smchd1 to uncover its in vivo function. Her work is refining our comprehension of the role of SMCHD1 in long-range chromatin looping and maintenance of gene silencing (Tapia Del Fierro et al., 2023). Jop Kind (Hubrecht Institute, Utrecht, The Netherlands) delved into the intriguing antagonism between the repressive histone mark H3K27me3 and its association with the lamina, using cutting-edge single-cell DamID technologies (Jop Kind's laboratory, unpublished). This phenomenon was highlighted during XCI in early mouse embryos, where large genomic regions of the X chromosome appeared to detach from the lamina upon silencing and invasion of H3K27me3. Edda Schulz elegantly dissected the intricate signals regulating the Xist locus, combining CRISPR screens, mathematical modelling and candidate approaches (Gjaltema et al., 2022).

This meeting showcased a growing interest in escapee genes, which evade XCI in females or are reactivated from the Xi in specific contexts. This phenomenon potentially explains gene dosage imbalances in females compared with males and sheds light on sex specificities observed in development and disease. Xinxian Deng (University of Washington, Seattle, USA) explored the persistence of escape from the Xi with regard to the 3D conformation of the genome, with the Xi-specific environment restraining the expression of specific escapees (Fang et al., 2023 preprint). Carolyn Brown concentrated on defining the requisite elements for escape in humans, using human transgenes integrated into mouse X chromosomes beside a gene subject to silencing (Peeters et al., 2023). Antonio Adamo (King Abdullah University of Science and Technology, Thuwal, Saudi Arabia) investigated the impact of X and Y aneuploidies in human brain organoids (Antonio Adamo's laboratory, unpublished). This impressive work led to the hypothesis that aberrant expression of escapees might be responsible for impaired neuronal development. Advancements in XCI regulation went a step further towards translation applications with Jeanne Lawrence's (University of Massachusetts Chan Medical School, Worcester, MA, USA) work, wherein an XIST mini-gene was used to silence an extra copy of chromosome 21 in Down syndrome cells (Czermiński and Lawrence, 2020).

Advancing our comprehension of XCI regulation in humans, Kathrin Plath (University of California, Los Angeles, USA) explored gene dosage regulation by dampening X-linked gene expression in naïve human pluripotent stem cells and highlighted the pivotal role of XIST in this process (Sahakyan et al., 2017). A surprising function of XIST on trans-regulating autosomal genes was discussed, which may be related to sex-specificities. Claire Rougeulle also delved into the biology of the human XCI and X-dampening. Her elegant dissection of the roles of XACT and XIST in human ESCs and early embryos unveiled different contributions of XIST to X-chromosome dosage compensation, depending on the cellular context (Vallot et al., 2017). Charbel Alfeghaly (Université Paris Cité/CNRS, Epigenetics and Cell Fate, Paris, France) further dissected the regulation of human X chromosome dosage compensation in naïve human ESCs, with an engineered XIST knockout, assessing its impact on gene dosage, distribution of histone marks and SPEN/SHARP function (Alfeghaly et al., 2023 preprint).

Finally, the day witnessed exciting progress in in vitro culture of human blastoids, facilitating the visualization and study of early XCI in human development. Ana Elisa Ribeiro Orsi (University of Sao Paulo, Brazil) presented impressive human blastoids that follow the progressive display of XCI in both embryonic-like and extra-embryonic-like lineages (Ana Elisa Ribeiro Orsi, Carlos A. Pinzon-Arteaga, Lygia V. Pereira and Jun Wu, unpublished). Vincent Pasque (University of Leuven, Belgium) enthusiastically shared his integrative work on both mouse and human models, exploring the X-chromosome dosage compensation. From the investigation of XCU via large genomic deletions to the development of novel human trophoblast, extra-embryonic mesoderm and human blastoid models, the team advanced our understanding of X-dosage in naïve cells and early human development (Pham et al., 2022).

As the EMBO workshop was coming to an end, it was evident that X-chromosome inactivation continued to fuel enthusiastic discussions and raised intriguing questions. One notable aspect of this XCI meeting was the emphasis on topics related to sex-specific susceptibility to disease.

Montserrat Anguera (University of Pennsylvania, Philadelphia, USA) delved into the unique dynamics of XCI and XIST in the naïve and activated lymphocyte B cells (Sierra et al., 2022 preprint). Howard Chang (Stanford University, CA, USA) highlighted the significance of the Xist ribonucleoprotein (RNP) complex, primarily found in females for XCI, in driving sex-biased autoimmunity, which could exacerbate autoimmune diseases (Yu et al., 2021). Irene Cantone (University of Naples Federico II, Italy) provided valuable insights into the use of single cell genomics and high content image analysis to explore the impact of X chromosome escape and/or reactivation in the sex bias of human autoimmune diseases, such as multiple sclerosis, with potential clinical applications (Irene Cantone's laboratory, unpublished). James Cleland (German Cancer Research Center and EMBL Heidelberg, Germany) also highlighted the use of these four core genotype mice in untangling the contribution of XX content and hormonal influences in driving sex-based differences in the liver, using comprehensive single-cell transcriptomics (Duncan Odom's and Edith Heard's laboratories, unpublished).

As highlighted in this Meeting Review, there is a growing focus on the X-linked genes that escape silencing, both in normal development and in disease. Furthermore, there are specific contexts in which the entire Xi is reactivated, such as in primordial germ cells, which are the precursors of future gametes. Bernhard Payer (Centre of Genomic Regulation, Barcelona, Spain) compiled evidence of X-chromosome reactivation, using an in vitro culture system, for female germline competency (Severino et al., 2022). We also presented our recent findings on the dynamics of X-linked gene reactivation and chromatin changes during in vivo primordial germ cell development (Roidor et al., 2023 preprint). Additionally, Sven Falk (Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany) presented intriguing work on human neuronal organoids, suggesting that Xi reactivation is an integral part of human female brain development and protects females from neurodevelopmental diseases (Käseberg et al., 2023 preprint).

Beyond Xist, several interacting factors play crucial roles in XCI. Teresa Robert-Finestra (Erasmus MC Rotterdam, The Netherlands) shared her latest research in the search for the SPEN/SHARP interactome and recently identified proteins associated with XCI (Robert-Finestra et al., 2021). Tim Hasenbein (Technische Universität München, Germany) conducted a multi-omic study to characterize mouse models lacking X-linked loci with prominent sex-specific epigenetic signatures (Tim Hasenbein, Sarah Hoezl and Daniel Andergassen, unpublished). Interestingly, the study revealed an interplay between the Crossfirre and Firre loci that regulate autosomal genes instead of XCI biology. Interestingly, these deletions did not impair mouse female development or fertility. Elsie Jacobson (University of California, Los Angeles, USA) used a mouse Xist mutant with defective X-inactivation to investigate the impact of increased X-linked gene dosage on placental development, with implications for mammalian evolution and human disease (Kathrin Plath's laboratory, unpublished). The meeting closed with three renowned scientists in the field of XCI. Takashi Sado (Kindai University, Osaka, Japan) discussed SCHMD1 function in XCI maintenance (Ichihara et al., 2022). Jeannie T. Lee (Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA) delved into the world of G-quadruplexes (G4) and their connection to Xist silencing (Jeannie T. Lee's laboratory, unpublished). Finally, Neil Brockdorff delivered a captivating finale in music! He featured the latest development in fast live-cell imaging using super-resolution microscopy to study Xist association with chromatin and newly synthesized Xist molecules (Rodermund et al., 2021). The incorporation of music enhanced the microscopy movies, creating a memorable conclusion to the EMBO Workshop.

Finally, we would like to mention the EMBO policy lecture titled ‘Chromosome, sex and gender: it's complicated’, which took place on the fourth day, after the second and final poster session. Sabine Oertelt-Prigione (Radboudumc, Nijmegen, The Netherlands and University of Bielefeld, Germany) delivered an incredible talk addressing the importance of considering sex and gender in medicine and science. Her presentation served as a poignant reminder of the significance of our topics and the necessity to continually raise awareness within both the general population and the scientific community, emphasizing the importance of examining both aspects: sex and gender.

The organizers of the workshop deserve immense credit for orchestrating a remarkable meeting that spanned the historical origin of XCI discovery to its most recent advances. They supported inclusivity and fostered meaningful discussions, while providing valuable support to attendees with limited access, including travel grants, registration fee waivers, childcare grants and online access to talks. We thank the organizing team, their support team, EMBO and all the sponsors. A special round of applause is reserved for the participants of the poster sessions, whose presentations were enthusiastic and engaging, and for the poster prize winners (Fig. 3).

As the EMBO meeting ended, it was clear that XCI research is poised for an exciting future. Attendees were already eagerly anticipating the next XCI meeting, scheduled in 2026, possibly in Japan for the first time.

We thank Simão Teixeira da Rocha for proofreading the manuscript and for important input. We also thank the organizers and all the participants for creating together an exciting meeting, and we apologize to our colleagues for not being able to cite all relevant publications owing to space constraints.