Inducible XIST-dependent X-chromosome inactivation in human somatic cells is reversible - PubMed (original) (raw)

Inducible XIST-dependent X-chromosome inactivation in human somatic cells is reversible

Jennifer C Chow et al. Proc Natl Acad Sci U S A. 2007.

Abstract

During embryogenesis, the XIST RNA is expressed from and localizes to one X chromosome in females and induces chromosome-wide silencing. Although many changes to inactive X heterochromatin are known, the functional relationships between different modifications are not well understood, and studies of the initiation of X-inactivation have been largely confined to mouse. We now present a model system for human XIST RNA function in which induction of an XIST cDNA in somatic cells results in localized XIST RNA and transcriptional silencing. Chromatin immunoprecipitation and immunohistochemistry shows that this silencing need only be accompanied by a subset of heterochromatic marks and that these can differ between integration sites. Surprisingly, silencing is XIST-dependent, remaining reversible over extended periods. Deletion analysis demonstrates that the first exon of human XIST is sufficient for both transcript localization and the induction of silencing and that, unlike the situation in mice, the conserved repeat region is essential for both functions. In addition to providing mechanistic insights into chromosome regulation and formation of facultative heterochromatin, this work provides a tractable model system for the study of chromosome silencing and suggests key differences from mouse embryonic X-inactivation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Inducible XIST cDNA construct results in localized focus of XIST and silencing. (A) RNA-FISH analysis of the 293 cell line with the XIST transgene integrated into the FRT site. An ectopic focus of XIST expression (arrow) is observed in addition to the large endogenous signals and increases substantially after induction (+DOX, Right). The transgene signal is distinguished by hybridization with the vector. (B) HT1080 cells containing the XIST cDNA transgene after DOX induction for the times listed. (C) Cohybridization to detect XIST and chromosome 3 DNA show colocalization of the RNA with the chromosome from which it is expressed. Panels show the merged signal as well as only the chromosome paint and the XIST RNA signals. (D) Induction of XIST results in a focus of XIST expression that colocalizes to a reduction in Cot1 staining of the cell. Cells shown were induced for 25 days with DOX. (E) Flow cytometry plots of GFP fluorescence after different days of DOX induction. Multiple plots of fluorescence levels (log scale) from FloJo software for various times of XIST induction are shown. The vertical bars indicate that there is signal that has reached the lower baseline. (F) Bar chart of average fluorescence levels at various time points of DOX induction and then after 31 days of DOX induction, subsequent days without DOX (paler green). As in E plots showed a single peak of fluorescence suggesting that level is due to reduction in amount of GFP rather than percent of cells expressing GFP. (G–I) The 293 cells after 15 days of DOX induction hybridized to detect XIST RNA, H3K27m3 (G), H4K20m1 (H) or macroH2A (I) by immunofluorescence (IF); showing colocalization of the RNA and IF signal.

Fig. 2.

Heterochromatic changes associated with XIST expression. (A–F) The amplification of the EGFP promoter in the HT1080 cells was compared by Q-PCR after ChIP relative to the input for the times of DOX induction indicated. The antibodies used for ChIP recognized: histone H4 acetylated at lysines 5, 8, 12, and 16 (A); histone H3 dimethylated at lysine 4 (B); histone H3 trimethylated at lysine 4 (C); histone H4 monomethylated at lysine 20 (D); HP1-γ (E); and histone H3 dimethylated at lysine (F). (G) Schematic showing CpG dinucleotides upstream of the CMV promoter driving EGFP expression. Open circles represent unmethylated Cs detected by bisulphite conversion, and closed circles demarcate methylated Cs resistant to bisulphite conversion. Each line represents an independent clone derived from PCR products of bisulphite modified DNA from uninduced (upper cluster) or 30-day inductions of the HT1080-inducible XIST clone (lower cluster).

Fig. 3.

Deleted XIST constructs show differential silencing and localization. (A) Schematic of the inducible XIST construct shows the regions deleted in each construct. (B) FACS scans of the three XIST deletion constructs induced with DOX for the days listed. (C) Representative images of nuclei after RNA FISH for XIST in the three XIST deletion constructs in HT1080 cells. (D) Quantitative PCR of XIST levels relative to β-actin after actinomycin treatment for the times listed on the x axis. Levels were normalized to paired cultures treated with DMSO as the actinomycin is dissolved in DMSO. No significant difference was observed for the full XIST construct (diamonds) and the 5′ repeat deletion construct (squares). (E) The total level of XIST relative to actin was compared between the full XIST construct (left) and the 5′ repeat deletion (right).

References

1. Heard E. Curr Opin Genet Dev. 2005;15:482–489. - PubMed
1. Migeon BR. Cytogenet Genome Res. 2002;99:8–16. - PubMed
1. Migeon BR. Nat Genet. 2003;33:337. - PubMed
1. Chadwick BP, Willard HF. Sem Cell Dev Biol. 2003;14:359–367. - PubMed
1. Wutz A, Jaenisch R. Mol Cell. 2000;5:695–705. - PubMed

Inducible XIST-dependent X-chromosome inactivation in human somatic cells is reversible - PubMed (original) (raw)