Skip to content
Events

X-chromosome inactivation: a tribute to Mary Lyon

Conference

Starts:

October
032016

09:00

Ends:

October
042016

17:00

Location

The Royal Society, London, 6-9 Carlton House Terrace, London, SW1Y 5AG

Overview

Scientific discussion meeting organised by Professor Edith Heard FRS and Professor Neil Brockdorff FMedSci

Mary Lyon

In 1961, Mary Lyon proposed that one of the two X chromosomes in female cells, selected at random, is stably inactivated during early embryo development, and that the inactive state, once established, is then propagated through cell division throughout the lifetime of the animal. X-chromosome inactivation has provided a powerful model system for understanding epigenetic regulation of the genome. The mechanisms involved in X inactivation, for example non-coding RNAs, chromatin modifications and DNA methylation are of central importance in the processes of differentiation, development and reprogramming in higher organisms. This meeting brings together experts in this exciting field to exchange on the latest breakthroughs in X-inactivation research and also to reflect on the life and work of Mary Lyon, who passed away in 2014.

A publication of this meeting will be made available in due course in Philosophical Transactions of the Royal Society Biological Sciences.

Enquiries: Contact the events team.

Event organisers

Select an organiser for more information

Schedule of talks

03 October

Session 1 09:00-12:30

X inactivation in development

4 talks Show detail Hide detail

09:00-09:50 Activation of X Inactivation, the Rnf12:Rex1 axis

Dr Joost Gribnau, University Medical Center Rotterdam, Netherlands

Abstract

X chromosome inactivation (XCI) is directed by trans-acting XCI activators and inhibitors regulating the key players of XCI, Xist and Tsix. The X-linked XCI activator Rnf12 is located just 500 kb upstream of Xist. Rnf12 encodes an E3 ubiquitin ligase, which catalyzes dose-dependent breakdown of the pluripotency factor REX1 by targeting REX1 for proteasomal degradation. When present at an effective concentration, REX1 inhibits Xist transcription and stimulates Tsix transcription, thereby blocking initiation of XCI. Breakdown of REX1 is more prominent in differentiating female cells, which still have two active copies of Rnf12, resulting in female specific initiation of XCI. The Rnf12-Rex1 axis provides a strong link between female specific initiation of XCI and loss of pluripotency. In this lecture Gribnau will discuss the role of Rnf12 and Rex1 in vitro and vivo, highlighting studies involving Rnf12:Rex1 compound knockout ES cells and mice.

Show speakers

09:50-10:35 How coupled feedback loops could ensure mono-allelic Xist expression in different species

Dr Edda Schulz, Max Planck Institute for Molecular Genetics, Germany

Abstract

Random X-chromosome inactivation is initiated by the long non-coding RNA Xist, which is up-regulated from one out of two X-chromosomes in each female cell. To understand how the mono-allelic and female-specific expression pattern of Xist is set up, mathematical modeling is combined with quantitative experiments to investigate the structure and function of the underlying regulatory network. Female-specific, mono-allelic up-regulation of Xist can be simulated by a model consisting of a positive feedback loop required for mono-allelic expression and a negative feedback, which is responsible to prevent bi-allelic Xist up-regulation. The bistable positive feedback loop could be mediated by mutual repression of Xist and its antisense transcript Tsix, which involves promoter repression and polymerase collisions and which according to the theoretical analysis could also function, if Tsix was truncated such as in the human XIST/TSIX locus. Moreover, direct experimental evidence shows that indeed a negative feedback is in place that will ensure down-regulation of Xist from one X-chromosome, if biallelic expression is ectopically enforced. This suggests that the same network can achieve stable mono-allelic expression by either directly up-regulating Xist from a single X or by resolving an initial bi-allelic pattern to a mono-allelic state. Which of these routes to mono-allelic expression is used depends on the relative time scales of the negative feedback and Xist up-regulation. A small change of these time scales can shift the system from the strictly mono-allelic regime, as it is observed in mice, to the transiently bi-allelic pattern that has been observed in rabbit embryos.

Show speakers

10:35-11:05 Coffee

11:05-11:50 Early events in human X chromosome reactivation induced by pluripotent reprogramming

Professor Amanda Fisher FMedSci FRS, Imperial College London, UK

Abstract

We have used cell fusion to examine the earliest events in reprogramming-mediated human Xi reactivation induced in female fibroblasts. In heterokaryons formed between mouse ESCs and human fibroblasts we observed a rapid and widespread loss of XIST and H3K27me3 from Barr bodies within human nuclei. 

Thereafter, re-expression of a subset of Xi genes was seen in a proportion of cells (30-50%) and reactivation was confirmed by allele-specific RNA analysis, the detection of SNP-associated RNA sequence reads and conventional RNA FISH. Expression of XACT was only evident much later in a minority of hybrid cells after mitosis. These data suggest that stable Xi reactivation is epigenetically regulated at multiple levels that these events are temporally segregated. Interestingly, reactivation was selective for certain genes on the human X and was not limited to genes residing close to (or far from) the XIST locus itself.

Reactivation remained partial even in cells examined up to six days after fusion. Collectively these findings underscore the differential sensitivity of human X loci to reprogramming-mediated reactivation. The implications of these results, as well as novel approaches for examining X reactivation in vivo, will be discussed.

Show speakers

11:50-12:35 Exploring the evolutionary divergence of mammalian X inactivation and lineage specification by transcriptomic analysis of metatherian embryos

Professor James Turner, Francis Crick Institute, UK

Abstract

X chromosome inactivation (XCI) ensures equalisation of X dosage between female and male mammals. Studies in eutherian embryos and ES cells have revealed that XCI is coupled to lineage specification and pluripotency, and is regulated by a suite of non-coding RNAs that comprise the X-inactivation centre (XIC). The extent to which these principles are conserved in more distant mammals has not yet been investigated. Metatherian mammals, which diverged from eutherians 180 Mya, also engage in XCI and therefore represent a unique model system to examine conservation. However, transcriptomic, RNA FISH and genome editing approaches have not yet been applied to metatherians. Here I will describe new work on: 1) the ontogeny of XCI in metatherian embryos and its relationship to lineage specification and pluripotency, 2) the first molecular characterisation of the metatherian XIC, and 3) progress in the application of genome editing to dissect XCI in these organisms.

Show speakers

Session 2 13:30-17:00

Chromosomal studies

4 talks Show detail Hide detail

Chairs

Professor Hunt Willard, Marine Biological Laboratory, USA

13:30-14:15 The structural and functional dynamics of the inactive X chromosome

Professor Edith Heard FRS, Institut Curie and Collège de France, France

Abstract

X-chromosome inactivation represents a remarkable example of chromosome-wide monoallelic gene expression and epigenetic memory.  One of the questions we are interested in is to understand how 3D nuclear and chromosome structural organization relate to the differential treatment of the two X chromosomes in the same nucleus during imprinted and random XCI. This differential treatment initially concerns the Xist gene within the X-inactivation centre (Xic), and subsequently it concerns maintenance of differential X-linked gene expression chromosome-wide. By investigating the regulatory landscape of the Xic locus using chromosome-conformation capture technologies and super-resolution microscopy, we recently uncovered a new level of chromosome folding into topologically associating chromosome domains (TADs), spanning several hundreds of kilobases. Within the Xic, the partitioning of Xist and its antisense transcription unit Tsix into two separate TADs, that may facilitate their monoallelic regulatiom, as well as enabling precise coordination of their expression dynamics during early differentiation. The group’s recent insights into the regulation of Xist in the context of nuclear dynamics and Xic organization will be presented. At the chromosome-wide level, it has also been shown that the inactive X chromosome undergoes major reorganization into two megadomains partitioned by the DXZ4 macrosatellite region. The inactive X is also globally devoid of TADs, except at some regions that escape XCI. Professor Heard present data where the group has further explored the sequences underlying Xi organisation and how this relates to gene expression and dynamic regulation during early development.

Show speakers

14:15-15:00 Genetic and epigenetic influences on inactive X expression

Professor Laura Carrel, Penn State College of Medicine, USA

Abstract

One interest in the lab is why genes variably escape X inactivation on the human X. To explore genetic influences on variable inactive X expression single-cell derived lines from multiple related individuals were isolated and inactive X expression was examined. Somatic and meiotic heritability of inactive X expression for select genes was assessed. These data are also being used to guide efforts to improve estimates of inactive X expression from mosaic RNAseq samples.

Show speakers

15:00-15:30 Tea

15:30-16:15 Structural aspects of the inactive X chromosome

Professor Christine Disteche, University of Washington, USA

Abstract

Mary Lyon based her X inactivation hypothesis in part on the observation of a condensed heterochromatic body - the Barr body - in female cells. She also suggested that the inactive X may occupy a preferred site in the nucleus. To compare the 3D structure of the inactive and active X chromosomes within the nucleus we have mapped allele-specific chromatin contacts in female cells and tissue using in situ Hi-C assays in mouse F1 hybrid systems (brain and Patski cells). We have found that the inactive X is uniquely composed of two condensed superdomains of frequent long-range intrachromosomal contacts separated by a hinge region, a configuration conserved in human. Fewer specific short-range intrachromosomal contacts, resulting in fewer topological associated domains, are found on the inactive X compared to the active X. Genes that escape X inactivation are preferentially located at the periphery of the inactive X. The hinge region contains the conserved long noncoding RNA Dxz4 locus that specifically binds CTCF on the inactive X. Hi-C analyses of allele-specific CRISPR/cas9 deletions and inversions within the hinge region show that deletion of Dxz4 alone is sufficient to disrupt the bipartite structure. New contacts between superdomains and loss of contacts within superdomains are apparent in the deleted cells, as well as minor disruptions in X inactivation and escape as measured by allele-specific gene expression studies. In addition to CTCF the hinge region also binds nucleophosmin and appears to represent a nucleolus-associated domain. We have found that the non-coding RNA Firre locus may help the perinucleolar location of the inactive X and the maintenance of repressive chromatin marks.

Show speakers

16:15-17:00 Analysis of X-inactivation in homozygous Xist mutant embryonic stem cells

Professor Barbara Panning, UCSF School of Medicine, USA

Abstract

The silencing of the inactive X chromosome (Xi) can be divided into (at a minimum) two stages, establishment and maintenance. Establishment is the transition from an active to silent state and maintenance is the stable propagation of the silent state. These two stages may have differing requirements. For example, during imprinted X-inactivation Xist is not necessary for establishment of silencing, but is essential for robust maintenance.  While forced Xist expression is sufficient for chromosome-wide silencing, it is not known whether Xist is necessary for establishment of silencing during random X-inactivation. To examine the role of Xist in random X-inactivation, we created mouse female Xisthomozygous mutant induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). We are currently characterizing markers of X-inactivation during directed differentiation into epiblast-like cells.

Show speakers

04 October

Session 3 09:00-12:30

Non-coding RNAs in X inactivation

4 talks Show detail Hide detail

Chairs

Professor Carolyn Brown, University of British Columbia, Canada

09:00-09:45 RNAs in nuclear chromosome architecture and regulation: more the rule than the expection

Professor Jeanne Lawrence, Massachusetts Medical School, USA

Abstract

XIST RNA established the precedent for a “chromosomal RNA”, which spreads and stably associates with the nuclear chromosome to induce X-chromosome inactivation.  Although XIST evolved to control a singular chromosome, we find XIST has a remarkably comprehensive ability to inactivate an autosome (Chr21), indicating a mechanism for RNA regulation available genome-wide.  There has been growing interest in the possibility that many large non-coding RNAs function in chromatin, yet for very few specific examples has this been established.   Recent findings from our lab indicate that a diversity of highly repeat-rich RNAs collectively is very abundant across euchromatin.  These repeat-rich RNAs tightly localize in cis across the nuclear chromosome territory, and the RNA territory persists long after transcriptional arrest (by multiple methods). Understanding how RNAs are localized and maintained on the nuclear chromosome territory can illuminate the “black box” of higher-order chromosome architecture.  Our analysis of protein factors, particularly SAF-A, thought to control XIST RNA’s chromosomal association indicates that current concepts of how RNA is tethered to chromatin require major revision.  These and other findings will be presented in the context of a model whereby RNA is not an occasional modifier of chromatin state, but an integral component of nuclear chromosome structure, with different classes of RNA impacting the condensation state of distinct regions.  Sequencing and cytological data points to the prevalence of repetitive sequence RNA, which may have unusual properties to contribute to chromosome structure and function.

Show speakers

09:45-10:30 Exploring the contribution of long non-coding RNAs to X-chromosome inactivation diversity in mammals

Professor Claire Rougeulle, University of Paris, France

Abstract

Sex-chromosome dosage compensation is essential in most metazoan yet the developmental timing and the underlying strategies are remarkably variable, even amongst placental mammals. X chromosome inactivation (XCI), the mammalian dosage compensation system, is triggered by the accumulation of the long non-coding RNA (lncRNA) XIST on the chromosome, which is responsible for the silencing and heterochromatinization of the inactive X. In the mouse, the up-regulation of Xist on one of the X-chromosomes systematically triggers the XCI process. Strikingly, in human pre-implantation embryos, XIST accumulates on every X chromosomes in both males and females without inducing XCI. This unusual configuration suggests the existence of human-specific mechanisms preventing XIST-mediated silencing.

XACT is a recently identified X-linked lncRNA that coats active X chromosomes in human pluripotent stem cells. XACT is not or weakly conserved across mammals and is absent from the mouse, suggesting that it could fulfill primate-specific function. The Rougeulle lab is now addressing the potential role of XACT during the establishment of the XCI process. Single-cell RNA-sequencing and imaging revealed co-activation and accumulation of XACT and XIST on active X chromosomes in early human pre-implantation embryos. In these contexts, the XIST RNA adopts an unusual, highly-dispersed organization, which may underlie its inability to trigger X chromosome inactivation. Functional studies further demonstrated that XACT influences XIST accumulation in cis. These findings suggest an antagonist action of XIST and XACT in controlling X chromosome activity specifically in human, which highlights the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms.

Show speakers

10:30-11:00 Coffee

11:00-11:45 Genome regulation by long noncoding RNAs

Professor Howard Chang, Stanford University School of Medicine, USA

Abstract

The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation. This talk will discuss genome-scale strategies to discover and characterize lncRNAs, including RNA chemical modifications and RNA structures.  An emerging theme from multiple model systems is that LncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators, and target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, long noncoding RNAs can function as modular scaffolds to specify higher order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs in human diseases.

Show speakers

11:45-12:30 Dissecting the molecular functions of Xist RNA

Professor Kathrin Plath, University of California, USA

Abstract

I will address the state of the X chromosome in human embryonic stem cells (ESCs). It is well established that conventionally-cultured female human ESCs can carry active X-chromosomes (Xa) or an XIST-RNA-coated inactive X-chromosome (XiXIST+). Additionally, many ESC lines are known to have abnormal X-chromosome-inactivation (XCI) states where the Xi no longer expresses XIST RNA and has transcriptionally active regions (eroded Xi=Xe). The fate of each XCI state upon differentiation has remained unclear because individual lines often contain a mixture of XCI-states. We established homogeneous XiXa, XeXa, and XaXa ESC lines and found that they were unable to initiate XIST-expression and X-chromosome-wide silencing upon differentiation such that the ESC XCI state is maintained in differentiated cells. Thus, conventionally-cultured ESCs are not suitable to study the initiation of human XCI. Notably, conventional human ESCs are in a primed pluripotent state. To enable studies of initiation of XCI and overcome the epigenetic instability of the X of primed ESCs, we got interested in naive ESCs. Previously, it had been established that naïve pluripotent cells of the female human blastocyst have two active X-chromosomes that express the XIST RNA but whether this state can be captured in ESCs has remained unclear. We found that naïve ESCs can be established that resemble the unique X-chromosome state of the pre-implantation blastocyst, and thereby identified a cell culture system for studying X-chromosome dosage compensation mechanisms in early human development. Additionally, we demonstrate that this naïve state resets epigenetic abnormalities of the Xi of primed ESCs.

Show speakers

Session 4 13:30-17:00

Xist mediated chromosome silencing

4 talks Show detail Hide detail

Chairs

Dr Philip Avner, EMBL Monterotondo, Italy

13:30-14:15 X chromosome inactivation initiated by dysfunctional Xist RNA

Professor Takashi Sado, Kindai University, Japan

Abstract

Xist RNA plays a pivotal role in silencing and heterochromatinization of the inactive X chromosomes in female mammals. It has been shown that the A-repeat, one of the conserved repeats present in Xist RNA among many mammalian species, is essential for the silencing function of the RNA in differentiating ES cells. The group had previously attempted to explore the role of the A-repeat in vivo by targeted deletion of the corresponding genomic sequence in the mouse. This unexpectedly abolished transcriptional upregulation of the mutated Xist allele, precluding the further analysis for the behavior of the Xist RNA lacking the A-repeat in vivo. Here, a new Xist allele is introduced lacking the A-repeat under the control of a constitutively active promoter in the mouse. When this allele was paternally transmitted, the mutated Xist RNA was successfully expressed and coated the paternal X chromosome, inducing apparent heterochromatinization albeit defective in silencing in the embryo. A detail analysis of transcriptional state of the mutated X is currently underway. Sado will discuss the behavior of the mutated Xist RNA lacking the A-repeat in vivo and its effects on the X chromosome silencing.

Show speakers

14:15-15:00 A cell genetics approach to understanding gene repression by Xist RNA

Professor Anton Wutz, ETH Zurich, Switzerland

Abstract

X chromosome inactivation requires the Xist gene, which is exclusively expressed in female cells and encodes a long RNA that associates with the inactive X chromosome (Xi). Xist accumulation  leads to changes in chromatin composition and causes repression of a majority of genes on the Xi. A long standing question relates to the molecular mechanism of the gene repression pathway of Xist. Recently, factors could be identified by different research groups using genetic and biochemical strategies. In order to identify genetically required factors for Xist mediated gene repression screening in haploid mouse embryonic stem cells carrying an engineered Xist expression system was performed. Half a dozen high confidence candidate factors could be identified including genes that have been implicated in promoter regulation, histone modification and chromatin assembly. Ongoing efforts focus on validating these candidates and characterizing their function with the view of defining the underlying pathways in chromatin regulation and transcriptional repression. Although it appears likely that our screen has not reached saturation in a technical meaning, it remains unclear if additional components can be identified through further genetics efforts such as CRISPR nuclease based approaches. Independently, other laboratories have discovered additional and non-overlapping factors using biochemical strategies. It is anticipated that the combined data will contribute to advance our understanding of the molecular mechanisms underlying chromosome wide silencing. The results of our genetic screen and the conclusions that can be drawn at the time will be discussed.

Show speakers

15:00-15:30 Tea

15:30-16:15 Uncover the mechanism of Xist-mediated silencing

Chun-Kan Chen, California Institute of Technology, USA

Abstract

Xist initiates XCI by spreading across the future inactive X-chromosome, excluding RNA Polymerase II (PolII), recruiting the polycomb repressive complex and its associated repressive chromatin modifications, and repositioning active genes into a transcriptionally silenced nuclear compartment. While much is known about the events that occur during XCI, the mechanism by which Xist carries out these various roles remains unclear. We used RAP-MS to identify proteins that directly associate with Xist, and we further show that 3 of these proteins are required for Xist-mediated transcriptional silencing. One of these proteins, SHARP, which is known to interact with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA Polymerase II (PolII) from the inactive X. We also show that both SMRT and HDAC3 are required for Xist-mediated silencing, PolII exclusion, and PRC2 recruitment. Another of these proteins, LBR, is required for Xist-mediated silencing but not for PolII exclusion or PRC2 recruitment. We further demonstrate that Xist, through its direct interaction with LBR, recruits the inactive X chromosome to the nuclear lamina and through this leads to changes in the 3-dimensional structure of DNA in the nucleus. This process enables Xist to spread across the entire X chromosome to achieve its essential role in embryonic development. Specifically, by reorganizing nuclear structure, Xist changes the accessibility of DNA and thereby enables the Xist RNA to spread across the entire X chromosome and achieve chromosome-wide silencing. Together, these results present an integrative picture of how Xist can scaffold multiple proteins to orchestrate the complex functions required for the establishment of the inactive X-chromosome.

Show speakers

16:15-17:00 Polycomb recruitment by Xist RNA

Professor Neil Brockdorff FMedSci, University of Oxford, UK

Abstract

The Polycomb repressive complexes PRC1 and PRC2 play a key role in developmental gene regulation, functioning primarily by catalysing the histone modifications, H2AK119ub1 and H3K27me3 respectively.  Canonical PRC1 complexes bind to PRC2 mediated H3K27me3, and this interaction has been suggested to account for PRC1 localisation to target loci. In mammals Polycomb target loci include the promoter regions of a large number of developmental regulator loci and also, in female cells, the inactive X chromosome. In the latter model it has been proposed that Xist RNA directly recruits PRC2, with the resultant deposition of H3K27me3 accounting for subsequent recruitment of PRC1.   However, in a previous study we found that variant PRC1 complexes (varPRC1), localise to targets independently of H3K27me3, including on the inactive X chromosome, suggesting Polycomb recruitment mechanisms are more complex than had been thought.  Accordingly, we and others recently demonstrated that PRC1 mediated H2AK119ub1 can recruit PRC2 to target loci, the reverse of the classical recruitment model.   Building on these findings we now show that a varPRC1 complex, PCGF3/5-PRC1, initiates both PRC1 and PRC2 recruitment by Xist RNA.  Specifically, PCGF3/5-PRC1 mediated H2AK119ub1 acts as a signal to recruit other varPRC1 complexes via interaction of the Ranbp2 zinc finger in the core subunit Rybp/YAF2, thus amplifying H2AK119ub1 deposition over the inactive X.  H2AK119ub1 additionally recruits PRC2, in large part via direct interaction with a ubiquitin binding domain that we have identified in the PRC2 cofactor Jarid2.    Our findings thus overturn the prevailing model for Polycomb recruitment by Xist RNA and provide a novel paradigm for chromatin modification by Xist RNA.

Show speakers
X-chromosome inactivation: a tribute to Mary Lyon The Royal Society, London 6-9 Carlton House Terrace London SW1Y 5AG UK