X chromosome inactivation initiated by dysfunctional Xist RNA
Professor Takashi Sado, Kindai University, Japan
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.
A cell genetics approach to understanding gene repression by Xist RNA
Professor Anton Wutz, ETH Zurich, Switzerland
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.
Uncover the mechanism of Xist-mediated silencing
Chun-Kan Chen, California Institute of Technology, USA
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.
Polycomb recruitment by Xist RNA
Professor Neil Brockdorff FMedSci, University of Oxford, UK
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.