Structural organization of the inactive X chromosome in the mouse
Authors: Job Dekker
Howard Y Chang
Chong Jian Chen
Mikael Attia
Edith Heard
Ye Zhan
Noam Kaplan
Jin Xu
Bryan R Lajoie
Luca Giorgetti
Ava C Carter
Journal: Nature (2016)
Discipline: Multidisciplinary
Keywords: X-chromosome inactivation; Xist RNA; chromosome conformation capture; DXZ4 macrosatellite; chromatin accessibility

Abstract:  X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions,remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes indifferent neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors. 

Retracted: No

PubPeer comments:
 dXZ4 is a tandem repeat that is getting a lot of attention recently. Seems that everybody agrees that it causes a bipartite X chromosome structure (2014 Rao). In the Giorgetti paper (2016 Nature), the novel claim is that dXZ4 is required for ability of escapee genes to dodge X inactivation. However, there are now several papers, listed below, that refute this major claim. Is there now a consensus that dXZ4 does not affect X expression? Can someone comment - what in the analysis of Giorgetti that misled them, which Darrow, Froberg, and Bonora were not able to replicate? Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture. Darrow EM, Huntley MH, Dudchenko O, Stamenova EK, Durand NC, Sun Z, Huang SC, Sanborn AL, Machol I, Shamim M, Seberg AP, Lander ES, Chadwick BP, Aiden EL. Proc Natl Acad Sci U S A. 2016 Aug 2;113(31):E4504-12. doi: 10.1073 pnas.1609643113. Megadomains and superloops form dynamically but are dispensable for X-chromosome inactivation and gene escape. Froberg JE, Pinter SF, Kriz AJ, Ju00e9gu T, Lee JT. Nat Commun. 2018 Nov 27;9(1):5004. doi: 10.1038 s41467-018-07446-w. Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome. Bonora G, Deng X, Fang H, Ramani V, Qiu R, Berletch JB, Filippova GN, Duan Z, Shendure J, Noble WS, Disteche CM. Nat Commun. 2018 Apr 13;9(1):1445. doi: 10.1038 s41467-018-03694-y. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Rao SS, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden EL. Cell. 2014 Dec 18;159(7):1665-80. doi: 10.1016 j.cell.2014.11.021. , 
 The consensus is that DXZ4 does not play a major role in X inactivation. Giorgetti s conclusion is based on only one cell line, the other cell lines showed negative results. Nature s reviewers evidently overlooked this not so minor detail. , 
 More evidence against DXZ4 from Rinn lab deletions of DXZ4 in mice. DXZ4 is not required for X chromosome inactivation or escaping genes, mice are normal, but there seeems to be some autosomal disregulation. Andergassen D, Smith ZD, Lewandowski JP, Gerhardinger C, Meissner A, Rinn JL. In vivo Firre and Dxz4 deletion elucidates roles for autosomal gene regulation. Elife. 2019 Nov 18;8:e47214. doi: 10.7554 eLife.47214. PMID: 31738164; PMCID: PMC6860989. Abstract. Recent evidence has determined that the conserved X chromosome mega-structures controlled by the Firre and Dxz4 loci are not required for X chromosome inactivation (XCI) in cell lines. Here, we examined the in vivo contribution of these loci by generating mice carrying a single or double deletion of Firre and Dxz4. We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus-dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology. ,