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. 2022 May 1;149(9):dev200568.
doi: 10.1242/dev.200568. Epub 2022 May 6.

Inversion of a topological domain leads to restricted changes in its gene expression and affects interdomain communication

Rafael Galupa  1 , Christel Picard  1 , Nicolas Servant  2   3 , Elphège P Nora  1 , Yinxiu Zhan  4   5 , Joke G van Bemmel  1 , Fatima El Marjou  6 , Colin Johanneau  6 , Maud Borensztein  1 , Katia Ancelin  1 , Luca Giorgetti  4 , Edith Heard  1   7
Affiliations

Inversion of a topological domain leads to restricted changes in its gene expression and affects interdomain communication

Rafael Galupa et al. Development. .

Abstract

The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors (e.g. CTCF) underlying topologically associating domains (TADs) leads to modest alterations in gene expression, whereas genomic rearrangements involving TAD boundaries disrupt normal gene expression and can lead to pathological phenotypes. Here, we targeted the TAD neighboring that of the noncoding transcript Xist, which controls X-chromosome inactivation. Inverting 245 kb within the TAD led to expected rearrangement of CTCF-based contacts but revealed heterogeneity in the 'contact' potential of different CTCF sites. Expression of most genes therein remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression of Xist was ectopically upregulated. The same inversion in mouse embryos led to biased Xist expression. Smaller inversions and deletions of CTCF clusters led to similar results: rearrangement of contacts and limited changes in local gene expression, but significant changes in Xist expression in embryos. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighboring TADs, highlighting the existence of mechanisms of inter-TAD communication.

Keywords: Gene expression; Genomic engineering; TADs; X-inactivation; Xist.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Strategy for inverting almost the entire Tsix-TAD. (A) Topological organization of the Xic (top) and chromatin ChIP-seq profiles (bottom; see Materials and Methods for sources); the Xist/Tsix locus lies at the boundary between two TADs. The red and blue arrowheads indicate the orientation of the CTCF motif (orientated left or right, respectively). (B) Targeting strategy for inverting the ∼245 kb region comprising most of the Tsix-TAD, except Tsix and its known regulator Xite, and leaving the boundaries intact. (C) PCR strategy (bottom) and gel results (top) for detecting the inversion events. E14 is the wild-type (WT) parental cell line. Cl.1 and Cl.2 are the two clones that were generated and analyzed throughout the study.
Fig. 2.
Fig. 2.
Rearrangement of contacts within the TAD and increased insulation with neighboring TAD following 245 kb intra-TAD inversion. (A) 5C profiles of wild-type (WT; two replicates pooled) and 245 kb-INV mutant (two clones pooled) mESCs. The mutant map is corrected for inversion and gray pixels represent filtered contacts (see Materials and Methods). (B) Detailed view of the Tsix-TAD. The small red and blue arrowheads indicate the orientation of the CTCF motif (orientated left and right, respectively). The large black and large blue arrowheads indicate specific contacts that are gained or lost in the mutant, respectively (more details in the text). Gray pixels represent filtered contacts. (C) View of the Tsix- and Xist-TADs, and differential map representing the subtraction of z-scores calculated for WT and 245kb INV mutant maps separately. Gray pixels represent filtered contacts. (D) Insulation scores across the Xic TADs and downstream TADs based on 5C profiles for WT and 245 kb-INV mutant mESCs. The ‘troughs’ represent TAD boundaries.
Fig. 3.
Fig. 3.
Inversion leads to transcriptional changes of specific genes within the TAD and of Xist across the TAD boundary. (A) Schematic of the mESC-to-epiblast-like stem cell (EpiLSC) differentiation and time points analyzed by Nanostring nCounter (see Materials and Methods). (B) Gene expression analysis during differentiation (d0-d2.5). Data are normalized to wt-d0 for each gene, and represent the mean±s.d. of two biological replicates (wild type) or of two independent clones (mutant). Data were analyzed with a two-tailed paired Student's t-test (*P<0.05; **P<0.01; ***P<0.001). (C) RNA FISH for Huwe1 (X-linked gene outside of the Xic) and Xist (exonic probe) on mESCs differentiated to d1.5. The percentage of cells with Xist RNA accumulation is indicated and represents the mean from two independent experiments, where N indicates the number of experiments and n indicates the number of nuclei counted. Scale bars: 2 µm.
Fig. 4.
Fig. 4.
Female embryos with the 245 kb-INV allele show a bias in Xist expression. (A,D) Crosses used for the analysis of RNA allelic ratios in female hybrid embryos inheriting the Mus musculus domesticus allele either paternally (A; blue) or maternally (D; red). Tables summarize the number of embryos collected. (B,C,E,F) RNA allelic ratios for the X-linked gene Atp7a (B,E) and Xist (C,F). Each black dot corresponds to a single female embryo. Box-and-whisker plots indicate median, interquartile range and min/max values, respectively, with blue and red plots indicating paternally or maternally inherited alleles, respectively. Data were analyzed using the Mann–Whitney U test (*P<0.05).
Fig. 5.
Fig. 5.
Inversion of Linx cluster of CTCF sites leads to Xist upregulation in cis. (A) The Linx locus, CTCF binding, and orientation of CTCF motifs associated with CTCF ChIP-seq peaks. The red and blue arrowheads indicate the orientation of the CTCF motif (orientated left or right, respectively). The targeted inversions Linx-25 kb-INV and Linx-51 kb-INV are indicated. (B) 5C profiles (Tsix-TAD zoom-in) of wild-type (WT; two replicates pooled) and Linx-51 kb-INV (two clones pooled) mESCs, and 5C differential map, representing the subtraction of z-scores calculated for WT and Linx-51 kb-INV maps. The large black and large blue arrowheads indicate specific contacts that are gained or lost in the mutant, respectively (more details in the text). (C) (Left) 5C profile of Linx-51 kb-INV mESCs (two clones pooled); the map is corrected for inversion and gray pixels represent filtered contacts (see Materials and Methods); (Right) 5C differential map, representing the subtraction of z-scores calculated for WT and Linx-51 kb-INV maps separately. (D) Insulation scores across the Xic TADs and downstream TADs based on 5C profiles for WT and Linx-51 kb-INV mESCs. The ‘troughs’ represent TAD boundaries. (E) Gene expression analysis during differentiation (d0-d2.5). Data are normalized to wt-d0 for each gene, and represent the mean±s.d. of two biological replicates (WT) or of two independent clones (mutant). Data were analyzed with a two-tailed paired Student's t-test. (F,G) (Left) Crosses used for analysis of RNA allelic ratios in female hybrid embryos inheriting the Mus musculus domesticus allele either paternally (F; blue) or maternally (G; red). Tables summarize the number of embryos collected. (Right) RNA allelic ratios for Xist and the X-linked gene Atp7a. Each black dot corresponds to a single female embryo. Box-and-whisker plots indicate median, interquartile range and min/max values, with blue and red plots indicating paternally or maternally inherited alleles, respectively. Data were analyzed using the Mann–Whitney U test (*P<0.05; **P<0.01).
Fig. 6.
Fig. 6.
Deletion of Chic1 cluster of CTCF sites leads to Xist downregulation in cis. (A) The Chic1 locus, CTCF binding and orientation of CTCF motifs associated with CTCF ChIP-seq peaks. The targeted deletions Chic1-4 kbΔ and Chic1-14 kbΔ are indicated. The red and blue arrowheads indicate the orientation of the CTCF motif (orientated left or right, respectively). (B,C) (Top) 5C profiles of Chic1-4 kbΔ (B, two clones pooled) and Chic1-14 kbΔ (C, one clone, two replicates pooled). (Middle) 5C differential maps, representing the subtraction of z-scores calculated for wild-type (WT) and deletion maps. Gray pixels represent filtered contacts (see Materials and Methods). (Bottom) Differential maps for the Tsix-TAD. (D,E) Gene expression analysis during differentiation (d0-d2.5). Data are normalized to wt-d0 for each gene, and represent the mean±s.d. of two biological replicates (WT and Chic1-14 kbΔ) or of two independent clones (Chic1-4 kbΔ). Data were analyzed with a two-tailed paired Student's t-test (*P<0.05; **P<0.01; ****P<0.0001). (F) (Top) RNA allelic ratios for Xist and the X-linked gene Atp7a. Each black dot corresponds to a single female embryo. Box-and-whisker plots indicate median, interquartile range and min/max values. Data were analyzed with a Mann–Whitney U test (**P<0.01; ***P<0.001; ****P<0.0001). (Bottom) Crosses used for the analysis of RNA allelic ratios in female hybrid embryos inheriting the Mus musculus domesticus allele paternally. Tables summarize the number of embryos collected.
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