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Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

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Science  28 Oct 2016:
Vol. 354, Issue 6311, pp. 468-472
DOI: 10.1126/science.aae0047
  • Fig. 1 LBR requires its RS motif to interact with Xist and silence transcription.

    (A) Atrx mRNA levels after Xist induction (+dox) relative to pre-Xist (–dox) levels upon knockdown of various nuclear lamina proteins. WT, scrambled small interfering RNA (siRNA) control; siEMD, Emerin knockdown; siLMNB1, Lamin B1 knockdown; sgLBR, knockdown of LBR by using dCas9-KRAB (materials and methods). (B) Xist enrichment after immunoprecipitation of a 3x-FLAG–tagged full-length LBR (WT), ΔRS-LBR, or ΔTM-LBR (materials and methods). Error bars indicate SEM from three independent IP experiments. (C) Relative Atrx mRNA expression upon knockdown of the endogenous LBR and expression of full length LBR (WT), ΔTM-LBR, or ΔRS-LBR. (D) Relative Atrx mRNA expression in Xist-BoxB cells after knockdown of the endogenous LBR and expression of green fluorescent protein (GFP)–λN (control), LBR-λN, or ΔRS-LBR-λN. NS, not significant. ****P < 0.001 relative to [(A) and (B)] wild-type cells, (C) cells transfected with siRNAs alone [shown in (A)], or (D) cells transfected with GFP-λN by means of an unpaired two-sample t test. Error bars indicate SEM across 50 individual cells.

  • Fig. 2 LBR binds to precise regions of the Xist RNA that are required for silencing.

    (A) Cross-linking immunoprecipitation (CLIP) data plotted across the Xist RNA for LBR, SHARP, and PTBP1 proteins. The values represent fold-enrichment at each position on Xist normalized to a size-matched input RNA control. Input represents the total RNA control for the LBR sample. (Bottom) A schematic of the annotated repeat regions on the Xist RNA (WT) and the locations of the deleted regions in ΔA (nucleotides 1–937) and ΔLBS (nucleotides 898–1682). (B) Xist RNA enrichment level measured with quantitative reverse transcription polymerase chain reaction after immunoprecipitation of endogenous LBR or SHARP in wild-type, ΔA, or ΔLBS cells. Error bars indicate SEM from four independent immunoprecipitation experiments. (C) Relative Atrx mRNA expression in wild-type, ΔA, or ΔLBS-Xist cells. (D) Expression of ΔLBS-Xist with a 3x-BoxB fusion (ΔLBS-BoxB) along with expression of GFP-λN (control), EED-λN, SHARP-λN, or LBR-λN. As an additional control, we expressed LBR fused with the bacteriophage MS2 coat protein (LBR-MCP). Error bars indicate SEM across 50 individual cells. NS, not significant. ***P < 0.005, ****P < 0.001 relative to wild-type cells [(B) and (C)], or cells transfected with GFP-λN (D) by means of an unpaired two-sample t test.

  • Fig. 3 Xist-mediated recruitment of DNA to the nuclear lamina is required for transcriptional silencing.

    (A) Images of Xist (red), Lamin B1 (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) across different conditions. Scale bars, 5 μm. (B) The cumulative frequency distribution of normalized distances between Xist and Lamin B1 across 40 individual cells across different conditions. Dashed lines represents a second independent experiment. (C) Relative Atrx mRNA expression in ΔLBS-BoxB cells along with expression of LBR-MCP (control), LBR-λN, or LaminB1-λN. Error bars indicate SEM across 50 individual cells. NS, not significant. ****P < 0.001 relative to cells transfected with LBR-MCP by means of an unpaired two-sample t test.

  • Fig. 4 Recruitment to the nuclear lamina is required for Xist spreading to active genes.

    (A) Xist RNA localization as measured with RNA antisense purification (RAP)–DNA for wild type (top), ΔLBS-Xist (middle), and the smoothed fold change (bottom) across a region of the X chromosome containing active (red) and inactive (blue) genes. The dashed line indicates average Xist enrichment in wild-type cells. (B) Aggregate Xist enrichment relative to the genomic locations of highly active genes [dark red, reads per kilobase per million (RPKM) > 5], all active genes (red, RPKM > 1), and inactive genes (blue) on the X-chromosome for ΔLBS, SHARP knockdown, and ΔLBS-BoxB + LMNB1-λN cells compared with wild-type cells. Shaded areas represent 95% confidence interval. (C) Images of Xist (red), Gpc4 locus (green), and DAPI (blue) across different cell lines (rows) after Xist induction for 1 or 16 hours. Yellow arrowheads indicate the genomic DNA location of Gpc4. Scale bars, 5 μm. (D) The median distance from Gpc4 locus to the Xist-compartment after Xist induction for 1, 3, 6, and 16 hours. Error bars represent the standard error of the median across 50 individual cells. **P < 0.01 relative to 1-hour induction by means of an unpaired two-sample t test. (E) The median distance from the Mecp2 and Pgk1 locus to the Xist compartment after Xist induction for 16 hours across different conditions. Error bars represent the standard error of the median across 50 individual cells. **P < 0.01, ***P < 0.005 relative to siRNAs targeting SHARP (siSHARP) by means of an unpaired two-sample t test. (F) A model for how Xist-mediated recruitment to the nuclear lamina enables spreading to active genes and transcriptional silencing on the X chromosome.

Supplementary Materials

  • Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

    Chun-Kan Chen, Mario Blanco, Constanza Jackson, Erik Aznauryan, Noah Ollikainen, Christine Surka, Amy Chow, Patrick McDonel, Andrea Cerase, Mitchell Guttman

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S19
    • References
     
    Correction (27 October 2016): The authors made some minor changes since the First Release publication: (i) Added the primer sequences used to confirm the ΔLBS deletion. (ii) Added a note in the methods about the random integration into chromosome 12 of the deletion locus in the ΔLBS-BoxB cells. (iii) Added a sentence listing the samples used for RAP-DNA analysis.
    The original version is accessible here.

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