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Nuclear positioning and pairing of X-chromosome inactivation centers are not primary determinants during initiation of random X-inactivation

Nature Genetics volume 51, pages 285–295 (2019)Cite this article

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Abstract

During X-chromosome inactivation (XCI), one of the two X-inactivation centers (Xics) upregulates the noncoding RNA Xist to initiate chromosomal silencing in cis. How one Xic is chosen to upregulate Xist remains unclear. Models proposed include localization of one Xic at the nuclear envelope or transient homologous Xic pairing followed by asymmetric transcription factor distribution at Xist’s antisense Xite/Tsix locus. Here, we use a TetO/TetR system that can inducibly relocate one or both Xics to the nuclear lamina in differentiating mouse embryonic stem cells. We find that neither nuclear lamina localization nor reduction of Xic homologous pairing influences monoallelic Xist upregulation or choice-making. We also show that transient pairing is associated with biallelic expression, not only at Xist/Tsix but also at other X-linked loci that can escape XCI. Finally, we show that Xic pairing occurs in wavelike patterns, coinciding with genome dynamics and the onset of global regulatory programs during early differentiation.

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Fig. 1: Expression of TetR-EGFP-LaminB1 in PGK12.1 XXTetO/XTetOXTetO cells induces relocalization of the Xic-TetO and gene repression in the relocalized Xic.
Fig. 2: Xist upregulation from the Xic-TetO remains unaffected on relocalization to the nuclear lamina.
Fig. 3: Relocalization and tethering of both Xics in PGK12.1 XTetOXTetO reduces the relative number of Xic trans associations but does not affect initiation of XCI.
Fig. 4: Pairing of the Xic is associated with biallelic Tsix expression or expression of Tsix and Xist.
Fig. 5: Homologous Xic pairing can occur in the absence of biallelic Tsix expression.
Fig. 6: Xist expression is not necessary for homologous Xic pairing.
Fig. 7: Other biallelically active loci on the X chromosome show homologous pairing.

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Data availability

All data generated or analyzed during this study are included in the published article (and its supplementary information). The raw datasets generated during and or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the laboratory of E.H. for helpful discussions and input. This work was supported by a Institut Curie PhD student fellowship and a fellowship from Fondation ARC (Aides Individuelles Jeunes Chercheurs) to T.P. E.H. is supported by an ERC Advanced Investigator award (ERC-2014-AdG no. 671027), Labelisation La Ligue, FRM (grant DEI20151234398), ANR DoseX 2017, Labex DEEP (ANR-11-LBX-0044), part of the IDEX Idex PSL (ANR-10-IDEX-0001–02 PSL) and ABS4NGS (ANR-11-BINF-0001). We thank N. Brockdorff (Oxford University), A. Belmont (University of Illinois) and F. Zhang (Broad Institute) for providing materials.

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  1. Tim Pollex

    Present address: European Molecular Biology Laboratory, Heidelberg, Germany

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  1. Mammalian Developmental Epigenetics Group, Genetics and Developmental Biology Unit, Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, France

    Tim Pollex & Edith Heard

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T.P. and E.H. conceived the project and designed the experiments. T.P. carried out all experiments and analyzed the data. T.P. and E.H. wrote the manuscript.

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Correspondence to Edith Heard.

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Supplementary Figure 1 Gene expression in the Xic in PGK12.1 XXTetO TetR-EGFP and PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs.

a, Depicted are colonies of PGK12.1 XXTetO or XTetOXTetO cell lines stably expressing TetR-EGFP fusion proteins. The imaged cells were fixed and the EGFP fluorescence signal was acquired. b, DNA FISH for the TetO array locus (green) and the Xist/Tsix region in the Xic (red). Displayed is a cell in which the TetO array locus localized to pericentric heterochromatin (DAPI-dense region, chromocenter) as depicted in the magnified panels next to the projection displaying the entire nucleus. c, Quantification of pericentric heterochromatin association in undifferentiated bound and control PGK12.1 XXTetO TetR-EGFP-LaminB1 cells. Scored was the overlap of the TetO array DNA FISH signal with DAPI-dense regions. d, Mean relative gene expression level (normalized to Arp0 expression levels) assessed by qRT–PCR in control (empty bars) and bound (empty bars, lighter shade) PGK12.1 XXTetO TetR-EGFP cells (*P < 0.05, **P < 0.01, t-test (unpaired, two-tailed); error bars indicate s.d.; n = 3; individual data points are depicted next to the respective bar as filled and empty circles). e, Mean relative gene expression level (normalized to Arp0 expression levels) assessed by qRT–PCR in control (empty bars) and bound (empty bars, lighter shade) PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 cells (*P < 0.05, **P < 0.01, t-test (unpaired, two-tailed); error bars indicate s.d.; n = 3; individual data points are depicted next to the respective bar as filled and empty circles). f, RNA FISH was performed in control and bound PGK12.1 XXTetO TetR-EGFP-LaminB1 ESCs for nascent Tsix transcripts. Depicted is a single cell with biallelic Tsix expression. g, Quantification of the presence of focal RNA FISH signals indicating the presence of nascent Tsix transcripts. h, Sequential RNA and DNA FISH for Tsix (red) and Linx (white) transcripts (RNA FISH) as well as for the TetO array locus (green) and the Xist/Tsix region in the Xic (red) (DNA FISH). The second focal signal/pinpoint in the green channel is due to cross-hybridization of the TetO plasmid probe (backbone) with the stably inserted pBROAD3-TetR-EGFP-LaminB transgene. i, Logarithmic ratio of the integrated intensity of the RNA FISH signal of Tsix (left) and Linx (right) transcripts on Xic-TetO to the wildtype Xic in control and bound PGK12.1 XXTetO TetR-EGFP-LaminB1 cells based on sequential RNA–DNA FISH as depicted in h (*P < 0.05, KS test). (n is the number of cells; scale bar, 2 µm; representative cells for the respective condition are depicted in a, b, f and h.)

Supplementary Figure 2 Gene expression in bound and control PGK12.1 XXTetO TetR-EGFP-LaminB1cells during differentiation.

Mean relative gene expression assessed by qRT–PCR (normalized to Arp0 expression levels) in control (filled circles) and bound (empty circles) PGK12.1 XXTetO TetR-EGFP-LaminB1 ESCs and their differentiating counterparts (n = 6; error bars indicate s.d.; individual data points are depicted as smaller filled and empty circles in a lighter color shade).

Supplementary Figure 3 Xic–Xic distance distribution in PGK12.1 XXTetO and PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs and their differentiating counterparts.

a, DNA FISH for different regions of the Xic (green, BAC 7; red, BAC 8; white, BAC 5) in bound PGK12.1 XXTetO TetR-EGFP-LaminB1 ESCs. (Scale bar, 2 µm; one representative cell is depicted.) b, Cumulative frequency plot of Xic–Xic distances (Xist/Tsix region, BAC 8) in control (dotted line, n = 834) and bound (continuous line, n = 799) PGK12.1 XXTetO TetR-EGFP-LaminB1 ESCs assessed by 3D distance measurement after DNA FISH as depicted in a (P = 0.007, Kolmogorov–Smirnov test; pooled results of three replicates). c, Distribution of Xic–Xic distances (Xist/Tsix region, BAC 8) in control (dotted line) and bound (continuous line) PGK12.1 XXTetO TetR-EGFP-LaminB1 ESCs assessed by 3D distance measurement after DNA FISH as depicted in a (500 nm binning). d, DNA FISH for various parts of the Xic (green, BAC 7; red, BAC 8; white, BAC 5) in bound PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs. (Scale bar, 2 µm; one representative cell is depicted.) e, Cumulative frequency plot of Xic–Xic distances (Xist/Tsix region, BAC 8) in control (dotted line, n = 611) and bound (continuous line, n = 733) PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs assessed by 3D distance measurement after DNA FISH as depicted in d (P = 1.57 × 10–4, Kolmogorov–Smirnov test; pooled results of three replicates). f, Distribution of Xic–Xic distances (Xist/Tsix region, BAC 8) in control (dotted line) and bound (continuous line) PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs assessed by 3D distance measurement after DNA FISH as depicted in d (500 nm binning). g, Table depicting pairing frequencies observed in previous studies. h,i, Xic–Xic distance distribution in control (h) and bound (i) PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs and their differentiating counterparts depicted as a box plot (the box shows the 0.25 quartile to the 0.75 quartile; median Xic–Xic distances are connected by a red line). j, Median Xic–Xic distance distribution in control PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs and their differentiating counterparts from a single biological replicate. k, Relative gene expression assessed by qRT–PCR (normalized to Arp0 gene expression) in control (filled circles) and bound (empty circles) PGK12.1 XTetOXTetO TetR-EGFP-LaminB1 ESCs and their differentiating counterparts (n = 3; error bars indicate s.d.; individual data points are depicted as smaller filled and empty circles in a lighter color shade).

Supplementary Figure 4 Expression of Xist and Tsix in induced and uninduced TX1072 ESCs and their differentiating counterparts.

a, Expression of nascent Xist transcripts assessed by the presence of focal RNA FISH signals in control and differentiating TX1072 cells with either uninduced or induced Xist expression by addition of doxycycline to the culture medium 1 d prior to initiation of differentiation or simultaneous to the initiation of differentiation. b, Expression of nascent Tsix transcripts assessed by the presence of focal RNA FISH signals in control and differentiating TX1072 cells with either uninduced or induced Xist expression by addition of doxycycline to the culture medium 1 d prior to initiation of differentiation or simultaneous to the initiation of differentiation.

Supplementary Figure 5 Gene expression during differentiation of XXTetO ΔXist DKO ESCs.

a, Schematic representation of a wildtype Xist/Tsix locus and the introduced deletion. qPCR amplicons for Tsix 5ʹ, Tsix 3ʹ and Xist are indicated in the scheme. b, Depicted is the mean relative level of gene expression of selected pluripotency genes as well as Xist and Tsix assessed by qRT–PCR (normalized for Arp0 gene expression) during differentiation by LIF withdrawal in PGK12.1 XXTetO wildtype cells (filled circles) and PGK12.1 XXTetO ΔXist DKO cells (empty circles). (n = 3; error bars represent s.d.; individual data points are depicted as smaller filled and empty circles in a lighter color shade.)

Supplementary Figure 6 Gene expression during differentiation of PGK12.1 XXTetO cells.

Depicted is the mean relative level of gene expression of selected genes assessed by qRT–PCR (normalized for Arp0, Rrm2 and Actb gene expression) during differentiation by LIF withdrawal in PGK12.1 XXTetO cells. Blue, pluripotency factor genes; green, lamin genes; red, clock genes; orange, Glut8. Note that Lmna (laminA/C) is not highly expressed in ESCs and during early differentiation. Lmnb expression increases and decreases in a wave-like pattern during differentiation. Expression of Glut8 and the clock gene Per2 appears to initiate cycling after day 2 of differentiation. (n = 3; error bars represent s.d.; individual data points are depicted as smaller filled circles in a lighter color shade.)

Supplementary Figure 7 Vector maps of the expression plasmids used for the generation of stably expressing cell lines.

a,b, Vector maps of pBROAD3-TetR-EGFP (a) and pBROAD3-TetR-EGFP-LaminB1 (b) were generated using Geneious (Biomatters Limited) and SnapGene Viewer (GSL Biotech).

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Pollex, T., Heard, E. Nuclear positioning and pairing of X-chromosome inactivation centers are not primary determinants during initiation of random X-inactivation. Nat Genet 51, 285–295 (2019). https://doi.org/10.1038/s41588-018-0305-7

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