In female mammals, one of the two X chromosomes is inactivated to compensate for gene dosage between males and females (Lyon, 1961), a process termed X-chromosome inactivation (XCI). In mice, there are two successive waves of XCI: imprinted and random. Imprinted XCI starts early in development by specifically inactivating the paternal X chromosome (Xp) (Kay et al., 1994; Okamoto et al., 2004). While the Xp remains silenced in extra-embryonic lineages (Takagi and Sasaki, 1975), it is reactivated in the embryo during implantation, followed by random XCI (Mak et al., 2004). After this decision has been made, the inactive X (Xi) chromosome is epigenetically maintained throughout cell division as a compact chromatin structure known as the ‘Barr body’ (Barr and Bertram, 1949).

Recent studies using chromosome conformation capture (3C) based methods have identified that the Xi folds into two megadomains and forms a network of long-range interactions termed superloops that are directed by the non-coding loci Firre and Dxz4 (Rao et al., 2014; Horakova et al., 2012; Deng et al., 2015). Deletion of Dxz4 in cell lines leads to the loss of both megadomain and superloop (Giorgetti et al., 2016; Bonora et al., 2018; Froberg et al., 2018; Darrow et al., 2016), while deletion of Firre alone disrupts the superloop but has no impact on megadomain formation (Froberg et al., 2018; Barutcu et al., 2018). Moreover, the Firre locus is transcribed into the long non-coding RNA (lncRNA) Firre that escapes XCI and plays a role in nuclear organization (Hacisuleyman et al., 2014; Andergassen et al., 2017; Berletch et al., 2015; Bergmann et al., 2015; Yang et al., 2015). Recent studies in human and mouse cell line models of random XCI, found that deletion of these elements have minimal impact on X chromosome biology beyond the loss of these structures, though the phenotypic consequences of their deletion throughout mammalian development have not been addressed (Giorgetti et al., 2016; Bonora et al., 2018; Froberg et al., 2018; Darrow et al., 2016; Barutcu et al., 2018).

Here, we answer this question by generating mice lacking Firre and Dxz4 and performing an extensive transcriptomic analysis in embryonic, extraembryonic and adult organs. We determined that these elements are dispensable for mouse development and XCI biology. However, the absence of these loci results in organ-specific expression changes on autosomes, suggesting that these regions are involved in autosomal gene regulation rather than XCI biology.

In order to test the in vivo role of Firre and Dxz4 loci both individually and in combination, we generated three knockout (KO) mouse strains: two carrying a single locus deletion (SKO) of either Firre (Lewandowski et al., 2019) or Dxz4 and one carrying a double locus deletion (DKO) of Firre in conjunction with Dxz4 (Figure 1A, Figure 1—figure supplement 1A, Materials and methods). Notably, we targeted the regions that have been previously reported to disrupt the superloop (Barutcu et al., 2018) and megadomain structures (Bonora et al., 2018). All founder mice were screened by PCR using primers that span the deleted region and identified mutants were confirmed by Sanger sequencing (Figure 1—figure supplement 1B–C, Materials and methods).

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We found that homozygous mice of all three strains are viable and fertile, and by crossing males and females carrying a homozygous double deletion we observed the expected litter sizes and sex ratios (Figure 1B). To test whether the absence of the Firre and Dxz4 loci has an impact on random or imprinted (Xp chromosome inactive) XCI in vivo, we collected embryonic day 12.5 female F1 brains (random/skewed XCI), placentas and visceral yolk sac (imprinted XCI) from reciprocal crosses between our KO strains and Mus musculus castaneus (CAST), followed by RNA sequencing (RNA-seq) and allele-specific analysis using the Allelome.PRO pipeline (Andergassen et al., 2015) (Figure 2A). We collected the same organs from males to test the role of these loci on the X chromosome outside of XCI biology. Unsupervised clustering of the sequenced samples confirmed the identity of the tissues (Figure 2—figure supplement 1). We then investigated the Firre expression level in Firre SKO and DKO female brains, and detected approximately half of the wildtype levels as expected for random XCI (Figure 2B, Figure 2—figure supplement 2). Next, we inspected the Firre expression level in the placenta and visceral yolk sac (extra-embryonic organs that undergo imprinted XCI) to distinguish the phenotypic consequences of deletions if they are on the Xa (maternal inheritance of the deletion) versus the Xi (paternal inheritance of the deletion) chromosome. We did not detect Firre expression if the deletion was inherited maternally, while we detected Firre wildtype expression levels in the paternal deletion, demonstrating that Firre is exclusively expressed from the Xa (Figure 2B, Figure 2—figure supplement 2). Thus, in contrast to previous reports that identified Firre as a gene that escapes XCI in cell lines that model random XCI (Hacisuleyman et al., 2014; Andergassen et al., 2017; Berletch et al., 2015), we found that Firre does not escape imprinted XCI. Remarkably, in the Dxz4 SKO we detected a Firre wildtype pattern, suggesting that disruption of the superloop between the two loci or the absence of megadomains has no impact on Firre gene expression in either random or imprinted X inactivation (Figure 2B). We did not observe changes in Xist expression levels in the absence of these loci (Figure 2C).

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Since Firre is only expressed from the Xa in the placenta and visceral yolk sac (imprinted XCI), we can use these tissues to disentangle the functional roles of Firre RNA from megadomain and superloop structures that only exist on the Xi. We hypothesized that: (1) females and males carrying a maternal Firre single or Firre-Dxz4 double deletion (deletion on Xa) lack the Firre lncRNA, (2) females carrying a paternal Firre deletion (deletion on Xi) lack the superloop and (3) females carrying a paternal Dxz4 single or Firre-Dxz4 double deletion (deletion on Xi) lack both the superloop and megadomains (Figure 3A left panel). To identify dysregulated X-linked genes for each possible combination of the deletion, we performed differential expression analysis and found that the only dysregulated gene on the X chromosome was the lncRNA Firre (FDR ≤ 0.1 and |log2FC| ≥ 1), suggesting that the mega-structures and the Firre lncRNA have no impact on imprinted XCI (Figure 3A right panel, Supplementary file 1 sheet A-B). Notably, by using the same criteria we detected only a few autosomal genes dysregulated in the DKO that were not changed in the SKO, suggesting that the mega-structures and the lncRNA Firre have no impact on autosomal gene regulation in the placenta (Supplementary file 1 sheet A-B). DNA methylation levels on CpG islands were also not affected in the absence of the mega-structures or the lncRNA Firre, suggesting that the epigenetic processes involved in establishing the inactive X proceed normally without either (Figure 3—figure supplement 1, Supplementary file 1 sheet C).

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Next, we performed an allele-specific expression analysis to test for deviations of the expected maternal ratios or a gain of gene escape in the presence of the deletions. We found that the median allelic ratio of all the X-linked genes was unchanged regardless if the deletions were on Xa or Xi (Figure 3B). Moreover, deletion of these loci on the Xa or Xi did not result in increased escape in the placenta or visceral yolk sac (Figure 3C, Supplementary file 1 sheet D-E). Of note, allele-specific analysis revealed strain-specific escaping with a greater number of gene escape from CAST Xi compared to Bl6 Xi (Figure 3C, Figure 3—figure supplement 2A, Supplementary file 1 sheet D-E). Random XCI in the brain also did not appear to be affected, since we detect the expected XCI skewing ratios in the presence of the deletions, a well-documented effect in female cells from crosses between Bl6 and CAST that results in the predominant inactivation of the Bl6 X chromosome (Calaway et al., 2013) (Figure 3—figure supplement 2B left panel). However, our DKO animals show significant skewing of the Xist allelic ratios (t-test, FDR-adjusted p-value=0.0108) (Figure 3—figure supplement 2B right panel), suggesting that the Bl6 chromosome is further biased toward silencing in mice lacking both Firre and Dxz4. In contrast, for autosomal genes we detect the expected biallelic ratios across all samples (Figure 3—figure supplement 2C).

To address whether the absence of both the mega-structures and Firre RNA has an impact on gene expression in an organ-specific manner, we generated a transcriptomic bodymap from adult females carrying a homozygous double Firre-Dxz4 deletion (Figure 4A, Figure 4—figure supplement 1A–B). We then performed differential expression analysis of spleen, brain, kidney, heart, lung and liver and found that most of the dysregulated genes show organ-specific expression changes, primarily on autosomes (autosomes: 98.15% n = 372 chrX: 1.85% n = 7), with only a few overlapping genes across organs (Figure 4B, Supplementary file 1 sheet F). The highest number of differentially expressed genes was detected in the spleen (n = 239, compared to the rest of the organs that on average had 30 dysregulated genes).

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To validate and categorize the dysregulated genes identified in the DKO into superloop, megadomain, and Firre locus-dependent gene sets, we sequenced the spleen and the liver from independently generated Firre and Dxz4 SKO animals (Figure 4C). We hypothesized that dysregulated gene sets that are: (1) shared across the three strains are superloop specific (2) shared between Dxz4 SKO and DKO are megadomain-dependent and (3) shared between Firre SKO and the DKO are Firre locus specific. To identify gene sets for these categories, we selected the genes dysregulated in at least one of the three KO strains where the direction of the expression change in either one of the single KO agrees with that of the DKO (|log2FC| ≥ 1). We first used this approach for the liver, an organ with a low number of differentially expressed genes in the DKO, and identified 15 megadomain, four superloop, and 4 Firre locus dependent genes (Figure 4—figure supplement 2A, Supplementary file 1 sheet G). Notably, without applying the fold change cutoff, we observe megadomain dependence of Xist, which is significantly upregulated in the DKO brain and kidney, as well as in the Dxz4 SKO spleen (mean upregulation 30%, Figure 4—figure supplement 2B).

We next applied the same approach to the spleen, the organ with the highest number of differentially expressed genes in the DKO, and identified seven megadomain (4%), 26 superloop (14.8%) and 142 Firre locus (81.1%) dependent genes (Figure 4D, Supplementary file 1 sheet H). The Firre locus dependent gene set—the largest group—contains only downregulated genes, including the gene Ypel4 that was previously described to form interchromosomal interactions with the Firre locus (Hacisuleyman et al., 2014; Maass et al., 2018).

By gene set enrichment analysis (GSEA), we find that the top Firre SKO and DKO enriched gene sets are almost identical, sharing downregulated gene sets involved in chromosome structure and segregation (Figure 4—figure supplement 2C, Supplementary file 1 sheet I). Genes extracted from the enriched gene sets identified in the Firre SKO and DKO share a similar pattern of downregulation, which was not observed in the Dxz4 SKO (Figure 4E). In addition, fold changes in Firre SKO and DKO are strongly correlated (spearman rho = 0.87, p-value<2.2*10⁻¹⁶), indicating that the Firre locus is the main driver for downregulation of these gene sets (Figure 4E). To test whether this molecular phenotype can be uncoupled form X inactivation, we performed the same analysis in DKO male spleens. We found a similar pattern of downregulation as observed in females (Figure 4E). Across all strains carrying the Firre locus deletion, the enriched gene sets share the majority of genes with the greatest degree of dysregulation (Supplementary file 1 sheet I). Taken together these findings point to the Firre locus, independently of X inactivation, as the main driver of these autosomal expression signatures.

The Firre and Dxz4 loci provide the platform for the formation of the X chromosome mega-structures and have been extensively studied in cell lines modeling random XCI (Rao et al., 2014; Horakova et al., 2012; Deng et al., 2015; Giorgetti et al., 2016; Bonora et al., 2018; Froberg et al., 2018; Darrow et al., 2016; Barutcu et al., 2018). Here we addressed the in vivo role of these elements by generating mice carrying a single or double deletion of these loci. In agreement with previous in vitro studies, we find that the loss of these loci in vivo does not affect random XCI. The lack of dysregulated X-linked genes in adult organs suggests that these mega-structures may also not be important for long-term maintenance of the Xi chromosome. Moreover, by studying the placenta and the visceral yolk sac we were able to show for the first time that loss of these loci also does not affect imprinted XCI.

Remarkably, deletion of these loci results in reproducible organ-specific expression changes on autosomes suggesting that structural changes of the Barr body may lead to autosomal gene dysregulation. Indeed, crosstalk between autosomes and the X chromosome has been proposed as a mechanism for X inactivation counting (Rastan, 1983). Whether these changes are directly regulated by the Barr body structure remains to be investigated. The largest transcriptional effect on autosomes is Firre locus-dependent and X inactivation-independent, suggesting a role for the Firre RNA or DNA locus in autosomal gene regulation. The major function of these dysregulated genes appears to be in ontologies associated with chromosome structure and segregation, which is in line with the known role for Firre lncRNA in nuclear organization (Hacisuleyman et al., 2014). This relationship points to an RNA dependent role, which may be either direct or indirect, on autosomal gene regulation. Collectively, our results indicate that the X-linked loci Firre and Dxz4, are involved in autosomal gene regulation rather than XCI biology in vivo.

Sequence data and alignments have been submitted to the Gene Expression Omnibus (GEO) database under accession code GSE127554.

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Author details

1. Daniel Andergassen

   Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1196-4289

2. Zachary D Smith

   Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States

   Contribution

   Conceptualization, Resources, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Jordan P Lewandowski

   Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States

   Contribution

   Resources, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Chiara Gerhardinger

   Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States

   Contribution

   Conceptualization, Supervision, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Alexander Meissner

   1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States
   2. Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—review and editing

   For correspondence

   meissner@molgen.mpg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8646-7469

6. John L Rinn

   Department of Biochemistry, University of Colorado Boulder, Boulder, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review and editing

   For correspondence

   john.rinn@colorado.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7231-7539

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