Spatial partitioning of the regulatory landscape of the X-inactivation centre

Abstract

In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment. A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the non-protein-coding Xist transcript. The extent of Xic sequences required for the proper regulation of Xist remains unknown. Here we use chromosome conformation capture carbon-copy (5C) and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region including Xist. We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. The Xist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters of Xist and Tsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle of cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre.

Figure 1. Chromosome partitioning into topologically assocating domains (TADs)

a, Distribution of 5C-Forward and 5C-Reverse HindIII restriction fragments across the 4.5 Mb analysed showing positions of RefSeq Genes and known XCI regulatory loci. b, 5C datasets from XY undifferentiated mESCs (E14), displaying median counts in 30kb windows every 6kb. Chromosomal contacts are organised into discrete genomic blocks (TADs A-F). A region containing segmental duplications excluded from the 5C analysis is masked (white). c, Positions of DNA FISH probes. d, Interphase nuclear distances are smaller for probes in the same 5C domain. e, Structured illumination microscopy reveals that colocalisation of neighbouring sequences is greater when they belong to the same 5C domain. Boxplots display the distribution of Pearson correlation coefficient between red and green channels, with whiskers and boxes encompassing all and 50% of values respectively; central bars denote the median correlation coefficient. Statistical significance was assessed using Wilcoxon’s rank-sum test.

Figure 2. Determinants of topologically associating domains

a, Blocks of contiguous enrichment in H3K27me3 or H3K9me2 align with the position of TADs (Chip-chip from) but TADs are largely unaffected in the absence of H3K9me2 in male G9a^{−/ −} cells or H3K27me3 or in male Eed^{−/ −} cells. b, Deletion of a boundary at Xist/Tsix disrupts folding pattern of the two neighboring TADs.

Figure 3. Dynamics of topologically associating domains during cell differentiation

a. Comparison of 5C data from male mESCs (E14), NPCs (E14) and primary MEFs reveals general conservation of TAD positions during differentiation, but differences in their internal organisation (arrows highlight examples of tissue-specific patterns). b Lamina associated domains (LADs, from ref.) align with TADs. Chromosomal positions of tissue-specific LADs reflect gain of lamina association by TADs, as well as internal reorganisation of lamina associated TADs during differentiation.

Figure 4. Transcriptional co-regulation within topologically associating domains

a, Female mESCs were differentiated towards epiblast stem cell lineage for 84 hours. Transcript levels were measured every 4–6 hours at 17 different time points by mircoarray analysis. b, Pearson’s correlation 15 coefficients over all time points were calculated for (1) gene pairs lying in the same TAD, (2) pairs in different TADs and (3) for pairs in randomly defined domains on the X-chromosome that contain a similar number of genes and are of comparable size. Box plots display the distribution of Pearson’s correlation coefficients, with whiskers and boxes encompassing all and 50% of values respectively, and central bars denoting the median correlation coefficient. * represents significant difference with p<10⁻⁷ using Wilcoxon’s ranksum test. c, Pearson’s correlation coefficients for gene pairs in TADs #D, #E and #F with red denoting positive and blue negative correlation. Boxes indicate the TAD boundaries.

Figure 5. 5C maps reveal new regulatory regions in the Xic

(a) 5C maps at the restriction fragment resolutions (male E14 mESCs) show that Tsix and Xist promoters belong to two distinct neighbouring TADs, respectively spanning >200kb and >550kb (male E14 mESCs). b, Corresponding statistically significant looping events (5C peaks) for restriction fragments within Xite, Tsix promoter or Xist promoter within their respective TAD. The Tg80 YAC transgene lacks genomic elements found to interact physically with Xite/Tsix that are present in Tg53. c, RNA FISH analysis of Tsix Tsix expression is detected in the inner cell masses of heterozygous transgenic male E4.0 embryos by RNA FISH from single-copy paternally inherited Tg53 but not Tg80 transgenes. Transgenic (star) and endogenous Tsix alleles (arrowhead) were discriminated by subsequent DNA FISH as in supplementary Fig. 5. n=20 ICM cells (2 embryos each). d, Linx transcripts (green, wi1–1985N4 probe) are expressed in both E4.0 ICM cells and mESCs, together with Tsix (red, DXPas34 probe), and unspliced transcripts accumulate locally in a characteristic cloud-like shape. e, RNA FISH in differentiating female mESCs revealing synchronous downregulation of Linx and Tsix with concomitant up-regulation of Xist (detected with a strand-specific probe). Barrs are the standard deviation around the mean of three experiments. f, Triple-color RNA FISH allowing simultaneous detection of Linx, Tsix and Xist RNAs. Scoring of Xist-negative alleles demonstrates that prior to Xist up-regulation Tsix expression is more frequent from Linx-expressing alleles than from Linx non-expressing alleles, at all time-points tested. Presented is the mean of three experiments. Statistical differences were assessed using Fisher’s exact test. Cells were differentiated in monolayers by LIF withdrawal.