Megadomains and superloops form dynamically but are dispensable for X-chromosome inactivation and gene escape

The mammalian inactive X-chromosome (Xi) is structurally distinct from all other chromosomes and serves as a model for how the 3D genome is organized. The Xi shows weakened topologically associated domains and is instead organized into megadomains and superloops directed by the noncoding loci, Dxz4 and Firre. Their functional significance is presently unclear, though one study suggests that they permit Xi genes to escape silencing. Here, we find that megadomains do not precede Xist expression or Xi gene silencing. Deleting Dxz4 disrupts the sharp megadomain border, whereas deleting Firre weakens intra-megadomain interactions. However, deleting Dxz4 and/or Firre has no impact on Xi silencing and gene escape. Nor does it affect Xi nuclear localization, stability, or H3K27 methylation. Additionally, ectopic integration of Dxz4 and Xist is not sufficient to form megadomains on autosomes. We conclude that Dxz4 and megadomains are dispensable for Xi silencing and escape from X-inactivation.


INTRODUCTION
A longstanding principle in gene regulation invokes significance of higher order chromosome structures and specificity of 3D interactions between distant genetic elements. Advances in genomics have provided new opportunities to probe chromosome architecture and resulted in discovery of three types of long-range intrachromosomal interactions. First, "topologically associating domains" (TADs) define continuous regions with extensive cis-contacts 1 . TADs are usually observed at length scales from 10 4 -10 6 bp 2 , depend on cohesins [3][4][5] ,and are generally flanked by convergent CTCF sites at TAD borders 1,[6][7][8] . TADs are visible as squares along the diagonal of Hi-C contact heat maps. Second, "loops" define enhanced contacts between pairs of distant loci that interact via CTCF. Loops can exist within or between TADs, and are visible as strong "dots" within a TAD square or between separate TADs in Hi-C contact maps 1 .
Third, "compartments" transcend TADs and loops and exist as orthogonal structures formed by interactions between chromatin of similar epigenetic states. A-compartments harbor discontinuous chromosomal regions enriched for active genes, whereas Bcompartments harbor discontiguous regions enriched for repressed genes 1,3,4,[9][10][11] . A/B compartments are visualized in Hi-C correlation maps by alternating "plaid" patterns of strong and weak interactions. Rapid depletion of CTCF 10 or cohesin 3 leads to genomewide loss of TADs and loops, more pronounced A/B compartments (in the case of cohesin depletion) and only modestly affects transcription in the short term [3][4][5]12 . Thus, while loops are thought to be important for long-range gene regulation (such as enhancer-promoter interactions), the functional organization into TADs and compartments is presently less well understood.
Recent conformational studies of the inactive X (Xi) has provided new insight into 3D chromosomal structure-function relationships 13,14 . X-chromosome inactivation (XCI) occurs in female cells as part of a dosage compensation mechanism that equalizes dosage of X-linked genes between males and females [15][16][17] . Chromosome conformation capture studies have demonstrated that, whereas the active X (Xa) resembles autosomes in having defined TADs, loops, and compartments, the Xi adopts a distinct structure seen on no other mammalian chromosome 1,[18][19][20][21][22] . ChIP-seq studies have shown that binding of architectural proteins including CTCF 20,23 and cohesins 20 are relatively depleted on the Xi, providing a mechanistic explanation for the attenuation of TADs. The Xi also lacks the characteristic separation between transcriptionally active A compartments and silent B compartments. Instead, during XCI, the Xi is partitioned into transitional Xist-rich S1 and Xist-poor S2 compartments, which are later merged into a single compartment by the non-canonical SMC protein, SMCHD1 24 . The merging of S1/S2 structure has physiological consequence, as ablating SMCHD1 precludes this fusion and leads to failure of silencing of >40% of genes on the Xi 24 . Thus, on the Xi, compartmentalization appears to play an important role in gene silencing.
On the other hand, the significance of domains on the Xi is under debate. Studies have shown that the Xi folds into two "megadomains" separated by a non-coding locus bearing tandem repeats known as "Dxz4" [20][21][22]25 . In humans, DXZ4 is heterochromatinized and methylated on the Xa but is euchromatic and unmethylated on the Xi, where it binds CTCF 26,27 . Murine Dxz4 is not well-conserved at the sequence level, but the syntenic region harbors a unique tandem repeat harboring strong CTCF binding sites 28 . In both mouse and human, Dxz4/DXZ4 resides at the strong border between the two megadomains of the Xi and binds CTCF and cohesin in an allelespecific manner (Fig. S1). Deleting the Dxz4/DXZ4 region in both species results in loss of megadomains and increased frequency of interaction across the border 22,25,29 . Despite clear disruption of the Xi super-structure, there is presently no agreement regarding functional consequences. One group reported loss of ability of "escapees" to avoid silencing on the Xi 22 . Changes in repressive chromatin marks and accessibility have also been reported in the mouse 22,29 . Still others reported minimal effects, or even opposite effects, such as a partial loss of Xi heterochromatin in human cells 25 . Thus, there exists major disagreement as to whether the Dxz4 region and megadomains enable or oppose silencing.
Additionally, the Xi is characterized by a network of extremely long-range loops termed "superloops" 1 and the importance of these structures is also unknown.
Superlooping occurs between Xist, DXZ4, and another tandem repeat element on the Xi called FIRRE 30,31 , another CTCF-bound noncoding locus (Fig. S1) 1,25,27 . Far longer than almost all other contacts in mammalian genomes, the loops between Dxz4 and Firre extrude up to 25 Mb of DNA, a scale typically seen only in perturbed states, such as between super-enhancers of cohesin-depleted cells 3 . One study suggests that Firre RNA may direct Xist to the perinucleolar space and influence H3K27me3 deposition on the X 32 . However, despite the fact that the Firre locus falls at the border between two TADs and contains many CTCF binding sites, a recent study found that Firre is neither necessary nor sufficient to form borders between TADs, though it is required for superlooping with Dxz4 33 . Here we combine genetic, epigenomic, and cell biological methods and study the impact of large-scale 3D structures on Xi biology.

Megadomains appear after Xist expression but not before Xi gene silencing
It is presently unknown how the formation of Xi megadomains relates to the timeline of XCI. To assess whether megadomains precede or follow XCI, we performed allele-specific Hi-C in female mouse embryonic stem cells (ES), which model different steps of XCI when they are induced to differentiate in culture. We examined timepoints day 0 (before XCI), day 3 (early XCI), day 7 (mid-XCI), and day 10 (late-XCI) in the mESC line, Tsix TST  For Hi-C analysis, we sequenced to a depth of 25-50 million reads, as megadomains are large (>70 Mb), prominent structures and can be sensitively detected at a resolution of 2.5 megabases (Mb). Allele-specific Hi-C contact maps and corresponding Pearson correlation heatmaps showed that, as expected, megadomains did not appear on the Xa during any stage of differentiation (Fig. S2a,b). Focusing in on the Xi, we observed that, in pre-XCI cells (day 0) and in cells undergoing XCI (day 3), X mus resembled X cas (the Xa) in also lacking detectable megadomains (Fig. 1a,b). RNA FISH analysis of these timepoints showed that 30-60% of cells showed robust Xist RNA clouds by day 3 (Fig. 1c,d). Allele-specific RNA-seq analysis also showed robust upregulation of Xist starting on day 3 and continuing throughout differentiation (Fig. 1e,   Fig S2c,d). Importantly, Xist was upregulated almost exclusively from X mus as expected, consistent with the Tsix TST allele carried in cis 34 . [Note: The nonrandom pattern at day 3 agrees with Tsix being a primary determinant of allelic choice 38 rather than being a secondary selection mechanism following a stochastic choice process 39,40 . A small fraction of reads coming from X cas is likely to be artifactual, as virtually all the X cas reads felll into one peak near the 5' end of Xist, rather than being distributed across the entire gene body (Fig. 1e). This peak fell within a repetitive region of Xist (repeat A) and contained only one SNP (rs225651233) -a 129 G -> Cast/EiJ T variant falling within a low complexity 24 bp poly-T tract. Thus, the X cas reads are likely to be from an improperly-defined SNP.] Despite highly skewed Xist upregulation, allele-specific RNAseq analysis showed that X-linked gene expression remained relatively unskewed (Fig.   1f), implying that de novo silencing or turnover of preexisting mRNA lagged behind Xist upregulation. A Hi-C "mixing" experiment indicated that our allelic Hi-C assay could detect megadomains when present in 25% of cells (Fig. 1h,i). Thus, the fact that megadomains were not readily visible on day 3 suggests that <25% of cells harbored them. Given that Xist spreading had taken place in 30-60% of cells and little silencing had taken place at this time point, megadomains were unlikely to have preceded Xist spreading and gene silencing.
On the other hand, analysis of day 7 cells revealed Xist expression in >80% of cells (Fig. 1c) and robust Xi silencing (Fig. 1f). It was at this timepoint that strong megadomains were first observed (Fig. 1a,b). Analysis of day 10 cells showed similarly strong Xist expression, Xi silencing, and megadomain formation. To quantify megadomain signals, we computed the Pearson correlation for the Xi contact maps and performed principal component analysis (PCA). On day 7 and day 10, there was a sharp transition in the 1 st principal component score (PC1) at Dxz4, indicating changed interaction patterns on each side of Dxz4 at later timepoints but not in days 0 or 3 ( Fig.   1g), consistent with appearance of megadomains. By contrast, the PC1 score distribution for the Xa was nearly identical for all timepoints without a sharp transition at Dxz4 (Fig.1g). In addition, the sharp transition in the PC1 curve is a valid measure of megadomain strength, as our mixing experiment showed that the slope of the curve at Dxz4 was directly proportional to the fraction of day 10 cells in the mixing experiment ( Fig S2e). The dynamics of megadomain formation were highly reproducible between two biological replicates (Fig. S3a). Taken together, these data suggest that megadomains do not precede XCI and appear either concurrently with or (more likely) only after Xist has spread and silenced the Xi.

Time course of TAD attenuation on the Xi
Recent analyses indicate that TADs are not abolished on the Xi but are instead attenuated 24,29 . Here we investigate the time course of TAD attenuation during XCI. To enrich for interactions and obtain higher resolution allele-specific contact maps, we performed Hi-C 2 , a variation of the Hi-C protocol that focuses analysis on defined regions through hybrid capture using high density probe sets 6 . We investigated ~1.5 Mb regions around (i) Dxz4 to assess the behavior of the strong megadomain border and (ii) the TAD harboring the disease locus and inactivated gene, Mecp2, to examine how topological domains are weakened during XCI.
Interestingly, despite megadomains appearing only late during the XCI time course, the Dxz4 region showed a strong boundary during all time points and on both alleles ( Fig. 2a,b). Therefore, Dxz4 acts as a border irrespective of XCI status and presence/absence of megadomains. At days 7 and 10, the proximal TAD flanking Dxz4 strengthened and expanded on the Xi but not the Xa (Fig. 2a,b), leaving only two "boxes" on either side of Dxz4 by day 10, rather than the patchwork of smaller sub-TADs present on the Xa and Xi at earlier timepoints. This finding indicated that the emergence of a megadomain correlates with increased insulation by Dxz4, indicating that Dxz4 insulates interactions at increasingly larger distances when megadomains form. The temporal dynamics of chromatin conformation surrounding Dxz4 were quite similar in two independent replicates of the differentiation timecourse and Hi-C^2 enrichment (Fig.   S3b).
Within the region containing Mecp2, Hi-C^2 contact maps showed that X mus and X cas behaved similarly on days 0 and 3, in that both were organized into several sub-TADs (Fig. 2c,d). However, once Xist spread over X mus and the Xi formed as a consequence on days 7 and 10, both TAD and sub-TAD organization become obscured compared to the Xa, where these domains stayed similar to earlier timepoints. In contrast to a previous analysis performed in neural progenitor cells (NPCs) 22 , we did not observe the persistence of a small domain around Mecp2 in differentiating female ES cells. The loss of domain organization in the Mecp2 region was observed in two distinct biological replicates (Fig. S3c).
To quantify these changes, we computed insulation scores using standard methods 22,41 (Fig. 2e,f). In brief, insulation scores quantify how strongly a given locus acts as a border 41-43 , and are calculated by running sliding windows across a chromosomal region and measuring the log ratio of reads crossing over a locus to reads neighboring a locus (Fig. S3d). Loci in the interiors of a TAD would be expected to have similar numbers of cross-over interactions and local interactions on each side, leading to insulation scores near zero, whereas loci at borders would register as a local minimum of crossover interactions than local interactions, leading to strong negative insulation scores at domain boundaries. We observed several interesting facets of the insulation score curves on the Xa and Xi at Dxz4 and Mecp2 during the differentiation timecourse.
There was a strong decrease in insulation scores near Dxz4 on both alleles at all timepoints, consistent with Dxz4 acting as a boundary throughout differentiation (Fig.   2e,g). The variance of the insulation scores is a measure of the global strength of insulation, with smaller variance corresponding to weaker insulation 44 . Near Dxz4, the variance was slightly smaller on the Xi than the Xa for all timepoints. There was no statistically significant difference in variance of insulation scores on the Xi on day 0 compared with the variance insulation scores on the Xi for the later timepoints (pairwise F-test p-values >0.05 for all comparisons between day 0 Xi and later Xi timepoints) and this observation held across two biological replicates (Fig. 2g, Fig. S3e). Thus, the Dxz4 region is a strong boundary regardless of XCI status, but that Dxz4 insulates interactions from increasingly larger distances to form megadomains.
The Mecp2 region showed a different pattern of insulation score changes across differentiation. The distribution of insulation scores across the Mecp2 region significantly narrowed on days 7 and 10 on the Xi but not Xa (Fig. 2h). Indeed, across the Mecp2 region, the variance on the day 7 or day 10 Xi was significantly lower than on day 0 (day 7 vs day 0 p-value=0.008443; day 10 vs day 0 p-value=0.001819). There was no significant difference in the variance on the Xi between day 0 and day 3 (p=0.7369). This clear decrease in the variance on the Xi at day 7 and day 10 relative to d0 was observed in two biological replicates (Fig. S3f). These results indicate that TAD and sub-TAD structures of the Mecp2-containing TAD region are reduced in strength in the same timeframe that megadomains are gained.

Dxz4 is necessary but not sufficient for megadomain formation
There presently exist three deletions containing Dxz4/DXZ4 -two in mouse 22,25,29 , one in human 25 . One of the mouse deletions 22 is a large deletion that contains more than just the noncoding element, Dxz4/DXZ4 (Fig. 3a). We generated a new deletion of Dxz4 and its flanking sequences that left untouched a small cluster of CTCF motifs with very high CTCF coverage and an unusual satellite repeat ( Fig. 3a and Fig. S4a) 28 , both of which were deleted in a previous 200-kb Dxz4 deletion. We generated a smaller 100-kb deletion spanning Dxz4 (Dxz4 ∆100 ) and validated our deletion by Sanger sequencing, by DNA fluorescence in situ hybridization (FISH) with a probe internal to the deleted region, and by genomic DNA sequencing to examine read distributions over the deleted region (Fig. S4, Methods). Importantly, to distinguish Xi from Xa, we performed the deletion analysis in Tsix TST /+ mESCs.
To test the impact of removing Dxz4, we differentiated wild-type and homozygously deleted (Dxz4 ∆/∆ ) cells for 10 days and performed Hi-C. Whereas wildtype cells showed strong megadomains, Dxz4 ∆/∆ cells showed disrupted megadomain structures in Hi-C contact maps (Fig. 3b) and corresponding Pearson correlation maps.
Most prominently, the sharp border around Dxz4 was eliminated, though some intramegadomain interactions remained on either side of the deletion. A disrupted megadomain border was confirmed by loss of the sharp transition in PC1 score around the Dxz4 locus (Fig. 4c). This effect was observed in two biological replicates (Fig. S4d).
These results are in agreement with prior reports 22,25,29 that the 200-300 kb region around Dxz4 is required for megadomain organization. Additionally, our work delineates the required region to a 100-kb domain containing the Dxz4 tandem repeat itself (as opposed to the CTCF motif cluster and the proximal satellite repeats).
Given its necessity for megadomain organization, we asked whether Dxz4 is also sufficient to form a megadomain on an autosome when Xist RNA is expressed in cis. We co-transfected a dox-inducible full-length Xist construct along with a BAC containing Dxz4 into male fibroblasts and used RNA and DNA FISH to identify Xist-inducible clones ( Fig. S5a) where both Xist and Dxz4 had co-inserted (XPDxz4.4; Fig. S5b). To localize the transgene, we adapted the 4C technique 45,46 that is ordinarily used to view 3D interactions from a single locus. We reasoned that, by placing the 4C viewpoint anchor at the transgene backbone, we could map the transgene through the pattern of cisinteractions on the same chromosome (Fig. 3d), as interaction frequencies are typically highest near the viewpoint position. This fact has previously been used to aid genome assembly 47-49 . Indeed, in addition to interaction peaks at Xist and Dxz4 as expected (Fig.   S5c), the only other strong 4C peak in the genome appeared on chr14 near Stc1 (Fig.   3e). This finding contrasts with both a control Xist-only transgene line which showed a peak only on chr10 (Fig. 3f) and the parental rtTA fibroblasts which showed no peaks anywhere in the genome. We confirmed insertion of both Dxz4 and the Xist construct into Stc1 by observing co-localization between Stc1, Xist and Dxz4 DNA FISH probes at one spot in the transgenic cell line (Fig. S5d).
To test whether induction of Xist expression could induce megadomain formation at Dxz4 ectopically, we induced Xist and performed Hi-C to determine whether coinsertion of Xist and Dxz4 induced formation of megadomains on transgenic chr14 in fibroblasts. We induced Xist for 2 days because a previous report suggested that induction of Xist from the male X for 2 days was sufficient to at least initiate megadomain formation 22 . No megadomains formed and the overall chr14 contact maps looked highly similar to the non-transgenic and Xist-only controls (Fig. 3g). We then extended the time frame and induced for 9 days, given that our ES cell time course suggested that several days of Xist upregulation were needed to form megadomains on the Xi. Still, no megadomains formed in these post-XCI cells (Fig. 3g). To assess whether Xist and Dxz4 could do so in cells undergoing de novo XCI, we attempted three times to create the Xist-Dxz4 transgene line in a female ES cell background, but such a line could not be generated, due to potential lethal consequences of the Xist transgene. These results indicate that Xist and Dxz4 together are not sufficient for megadomain formation in a cell line that had already undergone XCI (fibroblasts). We conclude that Dxz4 and Xist expression are necessary but not sufficient for megadomain formation in post-XCI cells.

Dxz4, Firre, and Xi-specific superloops
In addition to serving as the border between the megadomains, Dxz4 has been shown to form extremely long (>10 Mb) looping interactions with other loci on the human Xi 1,25,27 . To further dissect the role of Dxz4 in establishing the large-scale structure of the Xi, we performed 4C using a viewpoint within the core of the Dxz4 tandem repeats in post-XCI fibroblasts to identify interacting loci that may be important for helping to establish the unique structure of the mouse Xi. Dxz4 generally interacted with the chromosome telomeric to Dxz4 and formed few long-range interactions towards the centromeric side of the chromosome (Fig. 4a, Fig. S6a). However, Dxz4 interacted strongly with another non-coding tandem repeat, Firre (Fig. 4a). The two loci formed an extremely strong loop despite the fact that Firre is 25 Mb centromeric to Dxz4. The strength of their interaction was equivalent to that of two loci separated by < 200 kb (data not shown). To verify the Dxz4:Firre interaction, we performed a reciprocal 4C using a viewpoint within the core of the Firre tandem repeats and confirmed a strong reciprocal interaction (Fig. 4a). On the Xa, Firre also formed a broad domain of interactions with nearly all sequences within several Mb of itself, as reported for the Firre RNA contact map previously 30 . In contrast to this prior study, however, we did not observe any evident interchromosomal contacts from either Xa or Xi allele in fibroblasts.
Our allele-specific analysis revealed that the Dxz4:Firre interaction is primarily detected on the Xi (mus) allele. Indeed, when we repeated this reciprocal 4C experiment in another hybrid fibroblast line that chose to inactive X cas , the Dxz4:Firre interaction was detected on X cas , rather than X mus . For these experiments, we used both a unique 4C anchor in the 3' flanking region of Firre that provides allelic information, as well as an allele-agnostic anchor in the core of Firre repeat. Because Dxz4 and Firre are both highly repetitive, we also examined multiply-aligning reads. The Firre-Dxz4 interaction was only observed on the Xi regardless of whether the Xi was the mus or cas chromosome ( Fig. 4b-d). Thus, Firre and Dxz4 formed an Xi-specific superloop conserved between mouse and primate 1,25 .
Other superloops have been identified on the human Xi using high-resolution Hi-C. FIRRE, DXZ4, XIST, ICCE and X75 are all repetitive loci that bind CTCF on the Xi in human cells, and all form long-range interactions with each other 1,25 . We examined whether these superloops also occur in mouse cells. Indeed, in addition to Firre, we observed elevated interaction frequencies between Dxz4 and a region spanning Xist to Ftx (Fig. S6a,b) and a region syntenic with human X75 (Fig. S6a,c). However, the Xist-Dxz4 and x75-Dxz4 contacts were less prominent than the Firre-Dxz4 contact.

Firre is predominantly expressed from the Xa
Previous reports have suggested that Firre escapes from X-inactivation 30,32,50 , and that Firre RNA is necessary for Xist localization and deposition of H3K27me3 on the Xi 32 . By allele-specific RNA-seq, we observed Firre reads from both Xa and Xi during differentiation, and expression appeared to be predominantly though not exclusively exonic Fig. 5a,b). Because Firre is highly repetitive, SNP calls may be not be fully reliable. To confirm allele-specific expression, we used genetic means to examine expression from the Xa and Xi. With allele-specific guide RNAs, we generated an Xi-specific Firre deletion in Tsix TST /+ ("Firre Xi∆/+ clone D1"), an Xa-specific Firre deletion in Tsix TST /+ ("Firre Xa∆/+ clone H6"), and a homozygous deletion ("Firre ∆/∆ ") in female ES cells ( Fig. S7). We then measured Firre expression on day 10 of differentiation using 4 published sets of primers 30,32 and one new intronic primer set and deduced the expressed allele(s) by examining differences in expression pattern between the reciprocal heterozygous clones. First, by quantitative RT-PCR of wild-type female ES cells, we inferred that Firre expression was expressed at <10% of Xist RNA overall. The variability between amplicons suggested that there could be multiple isoforms of Firre  superloop may work together with Dxz4 to strengthen the megadomain structure.

Megadomains and superloops can be uncoupled from XCI and escape
Recent studies have diverged on the effect of deleting the Dxz4 region on XCI, as one study suggested a loss of escape from XCI for many escapees in mouse NPCs 22 and another suggested a partial loss of H3K27me3 on the human fibroblast Xi 25 . A prior report also suggested that knockdown of Firre RNA disrupts localization of the Xi to the nucleolus and maintenance of H3K27me3 on the Xi 32 . Here we assessed the effect of deleting Dxz4, Firre, or both on various aspects of XCI. First, we examined effects on the Xist RNA cloud that normally forms over the Xi, but observed no obvious changes in Xist cloud morphology or number of cells exhibiting Xist upregulation on day 10 of differentiation in Dxz4 ∆/∆ , Firre Xi∆/+ , Firre Xa∆/+ , Dxz4 ∆/∆ :Firre Xi∆/+ or Firre ∆/∆ (Fig. 7a,b, Fig.   S9a,b) versus wildtype female cells. There was also no effect on the characteristic localization of Xist RNA/Xi to the perinucleolar region 52 (Fig.7a,b, Fig. S9a,b). We also observed no difference in the enrichment of the H3K27me3 repressive mark on the Xi after 7 days of differentiation in any of the Dxz4 or Firre deletions (Fig. 7a,c) or after 10 days in the Dxz4 and Xi-specific Firre deletions (Fig. S9a,c). This suggests that neither Firre nor Dxz4 is required for Xist to be expressed, localized, and deposit H3K27me3 on the Xi. To test whether there is a partial loss of H3K27me3 across a macroscopic region of the Xi, as observed in a human DXZ4 deletion 25 , we produced metaphase spreads in WT and Dxz4 ∆/∆ cells and performed H3K27ac and H3K27me3 immunofluoresence to visualize the Xi. The Xi stood out as the chromosome with almost no H3K27ac signal and very strong H3K27me3 signal 53 (Fig. S9d). However, we observed no obvious difference between the H3K27me3 banding pattern on the WT or Dxz4 ∆/∆ Xi in metaphase spreads, suggesting no loss of H3K27me3 across a large region of the mouse Xi following Dxz4 deletion (Fig. S9e).
We next used ATAC-seq 54 to assay chromatin accessibility on the Xi. In wild-type cells, ATAC signal was indeed heavily skewed towards the Xa, with >85% of all peaks on the X binding specifically to the Xa (Fig. 7d). There were ~20 biallelic sites (e.g., promoters of escapee genes) and only one Xi-specific peak (at Firre) (Fig. 7e). If Dxz4 impairs X-chromosome accessibility as previously proposed for escapee genes 22 , we would expect nearby biallelic peaks near in wild-type to become Xa-specific in the deletion. On the other hand, if Dxz4 or Firre were required to inhibit chromatin accessibility, we would expect many ATAC-peaks to appear on the Xi near genes subject to XCI. Significantly, the overall ATAC-seq patterns were highly similar between wild-type and all mutant genotypes -Dxz4 ∆/∆ , Firre Xi∆/+ , and Dxz4 ∆/∆ :Firre Xi∆/+ (Fig. 8a).
We did not observe any "restored" sites on the mutant Xi, when plotting mutant Xi read counts vs. wild-type Xa read counts for peaks that reached at least one half of the wildtype Xa read count ( Fig. 8b-d, Fig. S10a). We also compared the Xi read counts for biallelic peaks and observed no changes in Dxz4 ∆/∆ cells (Fig. 8e), Firre Xi∆/+ cells, and Dxz4 ∆/∆ : Firre Xi∆/+ cells (Fig. 8f, Fig. S10b). Thus, we found no decrease in chromatin accessibility at escapee genes, in contrast to a previous study 22 . Altogether, our results demonstrate that deletion of either Dxz4, Firre, or both tandem repeats has no impact on chromatin accessibility on the Xi, at either inactive genes or escapees.
Finally, we asked whether deleting both Dxz4 and Firre disrupts the pattern of gene silencing or escape on the Xi. We performed allele-specific RNA-seq in wild-type Tsix TST /+ and Dxz4 ∆/∆ :Firre Xi∆/+ and looked for changes on either a genome-wide or Xiscale in the mutant relative to wildtype. Surprisingly, no significant differences were detected on a global or Xi-wide scale ( Fig. 8g-i), in contrast to previous deletions of Dxz4 22 . Cumulative frequency plots showed balanced X-to-autosomal gene dosages when comparing mutant to wildtype cells (Fig. 8g). The overall number of genes escaping XCI was similar in wild-type and Dxz4 ∆/∆ :Firre Xi∆/+ , with about half of the escapees being shared between them Fig. 8h). Examination of allelic contributions to overall X-chromosomal expression showed a predominance of Xa expression in both wild-type and Dxz4 ∆/∆ :Firre Xi∆/+ , with the pattern being highly similar in two biological replicates ( Fig. 8i; p > 0.3 for all pairwise comparisons). We conclude that neither Firre nor Dxz4 significantly perturbs Xi silencing and escape from silencing. Thus, the unique superloops of the Xi can be uncoupled from XCI biology.

DISCUSSION
An outstanding question in genome and nuclear organization is how higher-order chromatin structure regulates gene expression. Here, using the Xi as a model, we have tested the relevance of two higher order structures -superloops and megadomainsfor the biology of XCI. While indeed Dxz4 is required to form megadomains and Firre is required for superloops and for full strength of megadomains, we unexpectedly observed that abolishing these structures had no impact whatsoever when assaying a range of XCI phenotypes, including (i) chromosome-wide silencing as determined by RNA-seq, (ii) ability of escapees to avoid inactivation, (iii) subnuclear localization of the Xi, (iv) enrichment of H3K27me3 mark along the Xi, and (v) general chromatin accessibility as measured by ATAC-seq. By analyzing the time course of megadomain and superloop formation, we determined that these structures do not precede Xist spreading or XCI.
Instead, they either occur concurrently with Xist spreading and XCI, or they may be a consequence thereof. We also observe that Dxz4 and Firre may work together to strengthen megadomains through superloop formation: Deleting Firre weakens intramegadomain interactions without affecting the strong Dxz4 border, deleting Dxz4 abolishes the sharp megadomain border, and deleting both loci has a more severe effect on overall megadomain organization than deleting either singly. The significance of attenuated megadomains is unclear, however, given that there was no perturbation to XCI when either Dxz4 or Firre or both were deleted. Taken together, our data argue that the superstructures are not necessary for XCI biology, at least in the ex vivo cellular context.
Our findings therefore beg a number of interesting questions. First, what is the purpose of Dxz4, Firre, megadomains, and superloops, and why are they conserved across 80 million years of mammalian radiation? While our observation that Dxz4 is required for megadomains on the Xi is in agreement with three other studies 22,25,29 , our results are at odds with the previous proposal that Dxz4 enables genes to escape XCI 22 .
Our study also finds no loss of accessibility on ~35 escapee genes when Dxz4 is deleted on the Xi. The different conclusions may result from either use of different cell types (mESC versus NPCs) or clonal variation. Notably, the previous study observed the effects only in one of four NPC clones and the clone showed an unusually high number of escapees -~100 escapees, a number that is 2-4 times greater than reported by any other study 50,55,56 . If this NPC clone were an oddity, comparing its expression state to a Whatever function Dxz4 might serve, our study indicates that it is necessary but not sufficient for megadomain formation in post-XCI cells, even when Xist is present ectopically together with Dxz4. Because we could not derive the transgenic line in a female ES cell background, we do not formally know whether Dxz4 and Xist together might be sufficient during de novo XCI in an ectopic context. However, given that Dxz4 has no impact on XCI and escape in any measurable way, the sufficiency during the XCI establishment phase seems moot.
Firre was also of interest. What is its function and why do superloops form on the Xi via this CTCF-enriched repeat? Although we failed to find an XCI-related function for Firre after ablating it on the Xi, we found that Firre, Dxz4, Xist, and X75 form a conserved network of superloops on the mammalian Xi, in agreement with a previous study 25  Could megadomains and superloops be default consequences of Xist-mediated attenuation of TADs and compartments? It is important to note that our study follows XCI only in the ex vivo cellular context. It is possible that Dxz4, Firre, megadomains, and superloops play an important role in long-term maintenance of the Xi and that this role would only be revealed by following mice over their lifespan. It is also possible that these Xi megadomains and superloops are incidental organizational structures with no primary impact on gene regulation. While these structures do not disrupt XCI, other macrostructures do. In particular, the recently identified S1/S2 compartments that are revealed by loss of SMCHD1 function play an essential role during de novo Xi silencing 24 . Why the Xi would be folded in these ways with or without function is unclear.
For Dxz4 and Firre superloops, a role in a non-XCI pathway -critical in a wholeorganism context and not measurable by our present assays -must also be entertained. Irrespective of function, the megadomains and superloops represent the largest architectural structures identified by Hi-C to date in mammals, and both are clearly unique to the Xi. Their evolutionary conservation across 80 million years suggests that the superstructures likely persist for reasons that will only become clear with further study.                                             OptiMEM was added to 0.5 uL PLUS reagent. The OptiMEM+PLUS mix was added to a mixture of 250 ng of each gRNA, then the OptiMEM+LTX mix was added and incubated at room temperature for 5 minutes to generate the transfection mixture. Meanwhile, 2x10 5 mESCs were harvested by trypsinization and brought to a volume of 900 uL ES+LIF media. Once the transfection mixture was ready, the mESCs were layered dropwise on top of it and allowed to incubate for 20 minutes at room temperature.
Following incubation, the entire transfection mixture was added to one well of a 12-well dish containing feeders and 1 mL ES+LIF media. The transfected cells were allowed to grow for 16-48 hours.
To screen for Cas9-transfected cells, the transfected cells were harvested with trypsin, washed 2X in PBS and resuspended in 300 uL FACS media (1X Leibowitz's+5% FBS) and passed through a cell strainer. The GFP-positive cells were isolated by FACS selection and plated on 10 cm feeder plates (~2000-10,000 GFP+ cells/plate). The FACS-sorted GFP positive cells were allowed to grow into large colonies, typically after about 6-8 days of growth. 192 colonies were manually picked and transferred to 96 well plates covered in feeders. Once the 96 well plates were nearly confluent, they were passaged onto 3 new gelatinized plates (no feeders). Freezing media (MEF media+ final concentration 10% DMSO) was added to two plates and they were left at -80°C for storage. The third plate was grown until most wells were fully confluent.
We used a PCR screen to identify Dxz4 or Firre deletion clones. Genomic DNA was prepared from the colonies by incubating them overnight in Laird buffer+proteinase K (50 uL buffer per well) at 55°C. The genomic DNA was transferred to a new 96 well plate and diluted it 1:10 in H2O, then heated at 95°C for 10 minutes to denature it and inactivate the proteinase K. Next, PCR reactions using primers flanking Dxz4 or Firre were prepared in 96 well plates using 20 uL PCR mix+2 uL denature genomic DNA. 40 cycles of amplication were used, and the PCR reactions were run on 2% agarose gels and visualized by ethidium bromide staining. Deletion clones were identified by PCR reactions that produced a band at the expected size. Deletion clones were thawed onto 12-well plates with feeders, and deletions were verified by Sanger sequencing the PCR product, performing DNA FISH using a fosmid probe entirely within the deleted region, and examining reads over the deleted regions from our genomics experiments.
To generate Xa-specific and homozygous Firre deletions, we employed a restriction assay to determine whether clones carried a deletion on the Xa or Xi (or both).
We took advantage of a cas-(Xa-) specific polymorphism that creates a new TaqI restriction site within the Firre deletion PCR product to screen clones for deletions on particular alleles. We performed PCR amplification as before, but then added 30 uL of 1X Cutsmart buffer+10U TaqI (NEB) to each PCR reaction, then incubated the reactions at 65°C for 45 minutes before running the reactions on a 2% agarose gel.

Preparation of high molecular weight DNA
Briefly, 500 mL cultures of E. coli containing either Xist+P or RP23-161K4 were grown and spun at 4000 rpm for 15 minutes. Alkaline lysis was performed by re-supsending in 20 mL Buffer 1, aliquoting the cell suspension into two Oak Ridge polypropylene centrifuge tubes, adding 10 mL of Buffer 2 to each tube and inverting 20 times to mix, then adding 12 mL buffer 3 and inverting 20 times and incubating on ice for 5-10 minutes. Protein and genomic contaminants were removed by centrifugation at 10,000 rpm in a JA-20 rotor at 4°C. DNA was precipitated by adding 35 mL isopropanol to 15 mL centrifuged lysate in a 50 mL Falcon tube, incubating 20 minutes at room temperature, then spinning at 3500 rcf for 20 minutes at 4°C. Pellets were resuspended in 500 uL TE+1%SDS and 15 uL 20 mg/mL Proteinase K was added and the DNA mixture was incubated at 55°C for 1.5 hours to remove protein contaminants. The DNA was phenol:choloroform extracted by adding phenol:cholorform:isoamyl alchohol and shaking by hand for 20 seconds, then the DNA was precipitated with 40 uL 3M NaOAc and 1 mL isopropanol per 500 uL DNA mixture for 10 minutes at -20°C. At this time, the precipitated DNA formed a stringy white mass, the excess liquid was removed from this mass and 1 mL 70% ethanol was added to the DNA. The DNA was centrifuged for 5 minutes at 16300 g, the supernatant removed and 1 mL 70% ethanol was added to the pellet and the pellet was spun again for 5 minutes at 16300 g. Supernatant was removed and excess ethanol was allowed to evaporate for 5 minutes, then the DNA pellet was resuspended in 200 uL 10 mM Tris by gentle pipetting with a cut tip.

Generation of a Xist+Dxz4 transgene
To generate an autosomal Xist+Dxz4 transgene, we co-transfected a doxycyclineinducible Xist construct and a BAC containing mouse Dxz4 into male fibroblasts containing rtTA 2 . First, we prepared DNA from our "Xist+P" construct and the Dxz4containing BAC RP23-161K4 using a custom high-molecular weight purification protocol. Electroporated cells were plated onto 3 10 cm dishes in MEF media made with tet-free FBS and grown for one day. The Xist+P construct contains a hygromycin selectable marker, and to select for Xist transgenes, starting one day after transfection, we added 200 ug/mL hygromycin to the media and changed the media every day for 10 days.
Once colonies were grown, we manually picked them and transferred them to 96-well plate. We only obtained about 10 hygromycin resistant colonies. Once confluent, we split colonies onto 3 wells of a 24-well plate. One well was kept for maintenance, the other two were used for screening.
To screen for transgenic lines with both inducible Xist and Dxz4 inserted at the same ectopic site, we used the following strategy. We induced each clone with 1000 ug/mL doxycycline overnight and performed Xist RNA FISH to test if Xist could be induced. We also performed Xist RNA FISH in the same clones without dox induction to ensure Xist expression is insducible. We kept clones that could induce robust Xist RNA FISH clouds. We then used DNA FISH to check whether the Xist+P construct inserted at the same site as the Dxz4-containing BAC. We simultaneously performed DNA FISH using an Xist probe, a probe within the Xist construct backbone, and a fosmid probe against Dxz4. We obtained one clone where all 3 probes co-localize at one spot, indicating co-insertion of Xist and Dxz4 into an autosome. We then used 4C to localize the candidate insertion site into Stc1 on chr14. We then performed DNA FISH using a fosmid probe overlapping Stc1 combined with a Dxz4 fosmid and a probe overlapping the backbone of the Xist transgenic construct to confirm co-localization of Xist, Dxz4 and Stc1 at one spot.

DNA FISH
BAC or fosmid DNA was prepared using the high molecular weight DNA preparation procedure. Probes were labeled using the Roche Nick Translation kit. 75,000-150,000 cells were cytospun onto slides for 5 minutes at 1000 rpm. Cells were pre-extracted and fixed by passing the slides through CSK-T for 3 minutes at 4°C, CSK for 3 minutes at 4°C, 1X PBS+4% formaldehyde for 10 minutes at room temperature. RNA was removed by digestion with 0.1 mg/mL RnaseA in 1X PBS for 1 hr at 37 degrees. Slides were dehydrated by passage through 70%, 90%, 100% ethanol for two minutes at each concentration, then allowed to dry. Probe was added to hybridization mix (50% formamide, 2X SSC, 10% dextran sulfate, 0.1 mg/mL mouse Cot-1 DNA) and added directly to the slides. Slides were denatured at 92°C for 10 minutes on a PCR block, then incubated in a humid chamber at 37°C overnight. Slides were washed once in 2X SSC, once in 2X SSC+Hoechst 33342 and once in 2X SSC. Mounting media was added and the slides were imaged.

RNA FISH
Slides were prepared for RNA FISH using the same protocol as for DNA FISH but with the RnaseA treatment omitted. Xist RNA FISH was performed using a mixture of Cy3labeled DNA oligos covering Repeats A, B and C within Xist. The RNA FISH protocol was the same as the DNA FISH protocol, except that the denaturing step was omitted and the hybridization buffer+probe mixture was heated at 92°C for 5 minutes then 37°C for 5 minutes and then added directly to the slides. Slides were incubated at 42°C for 4-8 hours and then were washed once in 2X SSC, once in 2X SSC+Hoechst 33342 and once in 2X SSC. Mounting media was added and the slides were imaged.
Immunofluoresence 75,000-150,000 cells were cytospun onto slides for 5 minutes at 1000 rpm. Slides were washed once with 1X PBS, then 1X PBS+4% formaldehyde was added for 10 minutes at room temperature, then 1X PBS+0.5% Triton-X 100 for 10 minutes at room temperature to remove un-crosslinked proteins. Slides were washed once in 1X PBS, excess buffer was removed from cell spots and 1% BSA in 1X PBS was added for 45 minutes. Block solution was removed and a 1:200 dilution of H3K27me3 antibody (Active Motif 39155) in 1X PBS+1% BSA was added for 1 hour. Slides were washed 3X in 1X PBS+0.02% Tween-20. Excess liquid was removed and a 1:2000 dilution of goat-Anti-Rabbit Alexa 555 conjugated antibody (ThermoFisher) was added for 1 hour in the dark. Slides were washed once in 1X PBS+0.02% Tween-20, then twice in 1X PBS and then imaged.

ImmunoFISH
Slides were prepared the same way as for immunofluorescence; with 0.5 U/uL Protector RNase Inhibitor (Sigma) added to the blocking buffer. To visualize the nucleolus, we used a 1:200 dilution of Nucleophosmin antibody (abcam 10530) in blocking solution as the primary antibody. After immunofluorescence, we post-fixed the slides for 10 minutes in 4% formaldehyde+PBS, and then Xist RNA FISH was performed starting at the dehydration step.

Metaphase immunofluorescence
We added 50 ng/mL Karyomax to the media of day 10 differentiating embryoid bodies for 4 hours to arrest cells in metaphase. We harvested the cells via trypsinization, and trypsin was quenched by addition of media. We spun the cells at 1000 rpm for 5 minutes, aspirated the media, then washed twice in 1X PBS. Cells were then resuspended to a concentration of 5x10 5 cell/mL in 75 mM KCl, and placed at 37°C for 10 minutes for swelling. 1x10 5 cells were then cytospun onto a microscope slide at 1000 rpm for 5 minutes. The cells were fixed in PFA and immunofluorescence was performed as described for interphase cells. We stained H3K27me3 with a 1:200 dilution of Active Motif 39535 and H3K27ac with a 1:200 dilution of Cell Signaling D5E4.

Hi-C library preparation
We used the in situ Hi-C method of Rao et al. 3 to prepare all libraries, using 5-10 million cells. Importantly, we sequenced 20-40 million reads per library. This is a lower sequencing depth than many published Hi-Cs, however since the megadomains are large and prominent feature of the organization of the Xi, this depth is appropriate for detecting the megadomains efficiently and economically. We performed a timecourse of

Hi-C analysis
Hi-C alignment to mm9 was performed according to the method of Minajigi & Froberg et al. 4 The allele-specific Hi-C reads were filtered for quality and uniqueness with HOMER.
Custom scripts were used to convert HOMER tag directories into the format accecpted by Juicebox; contact maps were generated using the Juicer tools 'pre' command. All Hi-C contact maps visualized in this study are KR-normalized contact maps generated by Juicebox.
The first principal component of the Hi-C correlation matrix has been used as a quantitative measure of the presence or absence of megadomains 5 . We used R to generate the Pearson correlation of 1Mb KR-normalized allele-specific chrX Hi-C matrices, and we plot the first principal component as a function of position along the Xchromosome. Hi-C matrices with a megadomain exhibit a sharp transition in the first principal component score at the bin containing Dxz4.

Hi-C mixing experiment
We mixed together aligned reads from the day 0 and day 10 Hi-C libraries such that 0%, 10%, 25%, 50%, 75% and 100% of reads were from the day 10 Hi-C. We the generated HOMER tag directories and normalized contact maps in Juicebox as described for the Hi-C experiments. We plotted PC1 scores across the Xi at 1Mb resolution, and defined the PC1 slope at Dxz4 as the PC1 score @ bin 74 -PC1 score @ bin 72.

HYbrid Capture Hi-C (Hi-C 2 )
HYbrid Capture Hi-C (Hi-C 2 ) probes were designed and hybridization to in-situ Hi-C libraries carried out as described previously 6  Hi-C 2 libraries were sequenced to a depth of 8-15 million 50 bp paired-end reads. Reads were trimmed using cutadapt with the options --adapter=GATCGATC (MboI ligation junction) and --minimum-length=20. Reads of each pair were individually mapped to the mus and cas reference genomes using novoalign and merged into Hi-C summary files and filtered using HOMER as previously described 4 . For the chrX:70,370,161-71,832,975 captures, 3-4% of mapped and paired reads fell within the target region (0.05% expected based on size of capture region versus genome) and for the chrX:71,832,976-73,511,687 captures, 1-2% of mapped and paired reads fell within the target region (0.06% expected based on size of capture region versus genome). To avoid computational complexities arising from normalization of sparse, non-enriched regions in the Hi-C contact map, only Hi-C interactions falling within the capture region were analyzed further. For each capture, a custom script was used to pull out the filtered Hi-C interactions falling within the target region from the HOMER tag directories. Hi-C contact maps of the capture regions were then generated from these HOMER tags using the 'pre' command of Juicer tools 7 . The resulting Hi-C contact maps in .hic format were visualized and normalized with the 'Coverage (Sqrt)' option in Juicebox 8 .

Insulation score analysis with Hi-C 2 data
We computed insulation score across the Mecp2 and Dxz4 regions to quantitatively measure changes in domain organization during the timecourse of X-inactivation. To do this, we output the 'Coverage (Sqrt)' normalized Hi-C contact maps at 25 kb resolution across either the Mecp2 or Dxz4 regions using Juicer tools 'dump' command. We used custom shell and R scripts to convert the densematrix format output from Juicer into the full matrix format accepted by the cworld suite of Hi-C tools (https://github.com/dekkerlab/cworld-dekker). We computed insulation scores across the captured regions using the cworld perl script 'matrix2insulation.pl' using the parameters '-v --is 125000 --ids 75000 -im sum'. This set of options uses a smaller number of bins to calculate insulation scores, which we found to be optimal for analyzing insulation over small regions with just a few dozen bins. We plotted the distribution of insulation scores across each region and each timepoint. We evaluated changes in insulation across regions by testing whether there was a difference in the variance of insulation scores between timepoints or between the Xa and the Xi using the F-test. This is appropriate as a loss of insulation by definition is a decrease in the variance of insulation across a region 9 , which can be visualized as a "flatter" insulation score curve. To generate violin plots and calculate F-test p-values, we excluded the 6 bins on the left and right edges of each Hi-C 2 region because the windows used to calculate insulation score at these loci fall partially outside the region covered by Hi-C 2 probes and have far less read coverage than the regions covered by the probes.

4C library preparation and analysis
We previously developed a modified 4C protocol 10 to examine chromatin conformation from repetitive viewpoints. Our protocol has several advantages over existing 4C profiles: 1.) It sequences the genomic region amplified by the 4C primers, ensuring that on-target priming events can be identified and filtered from numerous off-target priming events 2.) Sequencing the viewpoint allows every read to be assigned to a particular allele if the viewpoint is near a variant, 3.) We use a random barcode to identify PCR duplicates, which previously has not been possible in 4C experiments. We performed our modified 4C using the protocol and analysis pipeline previously described for viewpoints within PAR-TERRA repeats 10 . We used it for several viewpoints within Firre and Dxz4. Some viewpoints were in the core tandem repeats. For these viewpoints, we use the read outside the viewpoint for allelic determination. Others were in unique regions near the tandem repeats; for these we could use known variants to assign every read to the Xa or the Xi. We performed our analysis in two fibroblast lines, one where the mus X is inactive (mus Xi cas Xa), the other where the cas X is inactive (mus Xa cas Xi).

ATAC-seq analysis
Attack seq alignment to mm9 was performed exactly as ChIP-seq alignment was performed in Minajigi & Froberg et al. 4 . Peaks were called using macs2 with default parameters. Biallelic peaks were identified as peaks with at least 10 alleleic reads in a sample and an Xi:Xa ratio greater than 1/3. Xi-specific peaks were defined as peaks with at least 10 allelic reads and a Xi:Xa ratio less than 1/3. To test whether Xi-specific peaks in wild-type are "restored" (that is: acquire appreciable accessibility on the Xi) in either the Dxz4 or Firre deletion, we plot the wild-type Xa reads on the x-axis and the deletion Xi reads on the y-axis and identify peaks where the deletion Xi/wild-type Xa ratio is greater than ½ (these are peaks where the deletion accessibility level reaches at least half the wild-type accessibility ratio). We also examine the biallelic peaks and plot the wild-type Xi reads on the x-axis and the deletion Xi reads on the y-axis to determine whether the accessibility on the Xi changes for the peaks that are bi-allelic in wild-type.

RNA-seq library preparation
Total RNA was isolated from 2-5 million trypsinized cells using trizol extraction. polyA+ mRNA was isolated using the NEBNext NEBNext® Poly(A) mRNA Magnetic Isolation Module using 5 ug of total RNA as input. Isolated mRNA was reverse-transcribed using Superscript III and actinomycin D to inhibit template switching. Second-strand synthesis was performed using the NEBNext Ultra Directional RNA Second Strand Synthesis Module. Library preparation and NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina. A USER enzyme treatment was performed following adaptor ligation to specifically degrade the second strand and allow a stranded analysis. Libraries were amplified for 10-15 cycles of PCR using Q5 polymerase and NEBNext multiplex oligos.

RNA-seq analysis
RNA-seq reads were aligned to the cas (Xa) and mus (Xi) genomes allele-specifically using a previously published pipeline 4,11-13 . Following alignment, gene expression levels for each gene were defined using HOMER. Differential expression and fold changes between conditions were calculated using DESeq2. We plotted the cumulative distributions of fold changes for autosomal and X-linked genes and evaluated the significance of any differences between the distributions of the fold changes using the Kolmogorov-Smirnov (KS) test. To examine allele-specific expression from the Xa and the Xi, we summed together allelic reads across both biological replicates and filtered for genes with at least 12 allelic reads in both wild-type and Dxz4 ∆/∆ :Firre Xi∆/+ and fpm > 0 in all replicates. We also used RNA-seq done in pure hybrid mus or cas fibroblasts to identify and eliminate genes that have incorrect SNP information. We defined escapee genes in a particular condition as genes where at least 10% of allelic reads came from the Xi in either replicate of that condition. We plotted the distribution of expression levels from the Xi (Xi/(Xi+Xa) read counts) for all genes passing our filtered for each replicate.
We evaluated the significance in differences of the mean expression level from the Xi using the Wilcoxon Signed Rank Test with Bonferroni correction for multiple hypothesis testing.

qRT-PCR
Total RNA was isolated from cells using trizol extraction. 500 ng RNA was heated at 70 degrees C for 10 minutes then cooled to 4 degrees in the presence of 50 ng random primers in 5 uL total volume. The RNA was reverse-transcribed in a 10 uL reaction containing 1X First Strand buffer, 10 mM DTT, 500 uM dNTPs, 6U Protector RNase inhibitor and 100U Superscript III. The reaction was incubated for 5 minutes at 25 degrees, then 1 hr at 50 degrees and 15 minutes at 85 degrees. Reverse transcription reactions were diluted to 100 uL with water before qPCR. 500 ng RNA was added to 100 uL water as a -RT control. 1 uL template was used per 15 uL qPCR reaction prepared with 1X Taq UniverSYBR Green (BioRad) master mix and 200 nM primers, and reactions were performed in triplicate. All qPCR primers were run using an annealing temperature of 55 degrees.