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Impact of flanking chromosomal sequences on localization and silencing by the human non-coding RNA XIST.

Kelsey AD, Yang C, Leung D, Minks J, Dixon-McDougall T, Baldry SE, Bogutz AB, Lefebvre L, Brown CJ - Genome Biol. (2015)

Bottom Line: Silencing of flanking reporter genes occurs at all sites, but the spread of silencing to flanking endogenous human genes is variable in extent of silencing as well as extent of spread, with silencing able to skip regions.The non-coding RNA XIST functions as a cis-acting silencer when expressed from nine different locations throughout the genome.A hierarchy among the features of heterochromatin reveals the importance of interaction with the local chromatin neighborhood for optimal spread of silencing, as well as the independent yet cooperative nature of the establishment of heterochromatin by the non-coding XIST RNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada. adkelsey1@gmail.com.

ABSTRACT

Background: X-chromosome inactivation is a striking example of epigenetic silencing in which expression of the long non-coding RNA XIST initiates the heterochromatinization and silencing of one of the pair of X chromosomes in mammalian females. To understand how the RNA can establish silencing across millions of basepairs of DNA we have modelled the process by inducing expression of XIST from nine different locations in human HT1080 cells.

Results: Localization of XIST, depletion of Cot-1 RNA, perinuclear localization, and ubiquitination of H2A occurs at all sites examined, while recruitment of H3K9me3 was not observed. Recruitment of the heterochromatic features SMCHD1, macroH2A, H3K27me3, and H4K20me1 occurs independently of each other in an integration site-dependent manner. Silencing of flanking reporter genes occurs at all sites, but the spread of silencing to flanking endogenous human genes is variable in extent of silencing as well as extent of spread, with silencing able to skip regions. The spread of H3K27me3 and loss of H3K27ac correlates with the pre-existing levels of the modifications, and overall the extent of silencing correlates with the ability to recruit additional heterochromatic features.

Conclusions: The non-coding RNA XIST functions as a cis-acting silencer when expressed from nine different locations throughout the genome. A hierarchy among the features of heterochromatin reveals the importance of interaction with the local chromatin neighborhood for optimal spread of silencing, as well as the independent yet cooperative nature of the establishment of heterochromatin by the non-coding XIST RNA.

No MeSH data available.


Silencing of flanking reporter genes upon XIST expression from various integration sites. a Relative level of Hyg expression after 5 days of XIST expression induced by DOX treatment compared with no DOX levels, measured by q-RT-PCR, for each of nine different integration sites as listed. Error bars show the standard deviation of biological triplicates. A one-way ANOVA with Tukey’s Multiple Comparison Test gives the following differences: 1p and 7q**, 3q and 7q**, 7p and 7q*, 7q and 8p**, 7q and 15q***, 7q and Xq** (*P ≤0.05; **P ≤0.01; ***P ≤0.001). b Map of transgene containing an inducible construct of the 5’A repeats of XIST as well as a DsRed-Express2 reporter gene expressed from a constitutive Pgk1 promoter. c Silencing of DsRed-Express2 relative to no DOX cells, measured by flow cytometry, after 5 or 12 days of DOX induction of 5’A region of XIST (from construct shown in part b) that had been integrated into six different integration sites as listed. The error bars represent ±1 s.d. of the silencing levels of individual single-cell clones (N = 8–11). d DsRed-Express2 expression, measured by flow cytometry, following induction (7 days DOX) and subsequent repression (5 days no DOX) of repeat A of XIST at two integration sites (1p and 3q)
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Fig2: Silencing of flanking reporter genes upon XIST expression from various integration sites. a Relative level of Hyg expression after 5 days of XIST expression induced by DOX treatment compared with no DOX levels, measured by q-RT-PCR, for each of nine different integration sites as listed. Error bars show the standard deviation of biological triplicates. A one-way ANOVA with Tukey’s Multiple Comparison Test gives the following differences: 1p and 7q**, 3q and 7q**, 7p and 7q*, 7q and 8p**, 7q and 15q***, 7q and Xq** (*P ≤0.05; **P ≤0.01; ***P ≤0.001). b Map of transgene containing an inducible construct of the 5’A repeats of XIST as well as a DsRed-Express2 reporter gene expressed from a constitutive Pgk1 promoter. c Silencing of DsRed-Express2 relative to no DOX cells, measured by flow cytometry, after 5 or 12 days of DOX induction of 5’A region of XIST (from construct shown in part b) that had been integrated into six different integration sites as listed. The error bars represent ±1 s.d. of the silencing levels of individual single-cell clones (N = 8–11). d DsRed-Express2 expression, measured by flow cytometry, following induction (7 days DOX) and subsequent repression (5 days no DOX) of repeat A of XIST at two integration sites (1p and 3q)

Mentions: We previously reported silencing of a flanking EGFP reporter gene at the 3q integration site [20, 40]. At the other FRT sites we did not co-integrate a reporter construct; however, the integration of XIST into the FRT site results in expression of an upstream Hygromycin (Hyg) gene. Robust silencing of Hyg was observed at all integrations after 5 days of XIST expression (Fig. 2a), suggesting that XIST is able to silence a virally-derived promoter (SV40), consistent with our previous demonstration that an EGFP reporter driven by the CMV promoter could be silenced [20]. The 7q integration site showed significantly less silencing than the 1p, 3q, 7p, 8p, 15q, and Xq integration sites (P ≤0.01). As only the repeat A region of XIST is required for silencing of the flanking reporter genes [40], we generated a construct containing the XIST repeat A and a DsRed reporter driven by the mouse Pgk1 gene promoter, which is normally X-linked and subject to XCI (Fig. 2b). We integrated this construct into six of the integration sites and again observed consistent silencing of greater than 90 % (Fig. 2c), suggesting both viral and mouse-derived promoters could be silenced by XIST in the HT1080 cells. Consistent with previous results with the EGFP reporter gene at 3q, silencing of the dsRed reporter was reversible when induction of XIST expression was stopped by removal of DOX (Fig. 2d).Fig. 2


Impact of flanking chromosomal sequences on localization and silencing by the human non-coding RNA XIST.

Kelsey AD, Yang C, Leung D, Minks J, Dixon-McDougall T, Baldry SE, Bogutz AB, Lefebvre L, Brown CJ - Genome Biol. (2015)

Silencing of flanking reporter genes upon XIST expression from various integration sites. a Relative level of Hyg expression after 5 days of XIST expression induced by DOX treatment compared with no DOX levels, measured by q-RT-PCR, for each of nine different integration sites as listed. Error bars show the standard deviation of biological triplicates. A one-way ANOVA with Tukey’s Multiple Comparison Test gives the following differences: 1p and 7q**, 3q and 7q**, 7p and 7q*, 7q and 8p**, 7q and 15q***, 7q and Xq** (*P ≤0.05; **P ≤0.01; ***P ≤0.001). b Map of transgene containing an inducible construct of the 5’A repeats of XIST as well as a DsRed-Express2 reporter gene expressed from a constitutive Pgk1 promoter. c Silencing of DsRed-Express2 relative to no DOX cells, measured by flow cytometry, after 5 or 12 days of DOX induction of 5’A region of XIST (from construct shown in part b) that had been integrated into six different integration sites as listed. The error bars represent ±1 s.d. of the silencing levels of individual single-cell clones (N = 8–11). d DsRed-Express2 expression, measured by flow cytometry, following induction (7 days DOX) and subsequent repression (5 days no DOX) of repeat A of XIST at two integration sites (1p and 3q)
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Related In: Results  -  Collection

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Fig2: Silencing of flanking reporter genes upon XIST expression from various integration sites. a Relative level of Hyg expression after 5 days of XIST expression induced by DOX treatment compared with no DOX levels, measured by q-RT-PCR, for each of nine different integration sites as listed. Error bars show the standard deviation of biological triplicates. A one-way ANOVA with Tukey’s Multiple Comparison Test gives the following differences: 1p and 7q**, 3q and 7q**, 7p and 7q*, 7q and 8p**, 7q and 15q***, 7q and Xq** (*P ≤0.05; **P ≤0.01; ***P ≤0.001). b Map of transgene containing an inducible construct of the 5’A repeats of XIST as well as a DsRed-Express2 reporter gene expressed from a constitutive Pgk1 promoter. c Silencing of DsRed-Express2 relative to no DOX cells, measured by flow cytometry, after 5 or 12 days of DOX induction of 5’A region of XIST (from construct shown in part b) that had been integrated into six different integration sites as listed. The error bars represent ±1 s.d. of the silencing levels of individual single-cell clones (N = 8–11). d DsRed-Express2 expression, measured by flow cytometry, following induction (7 days DOX) and subsequent repression (5 days no DOX) of repeat A of XIST at two integration sites (1p and 3q)
Mentions: We previously reported silencing of a flanking EGFP reporter gene at the 3q integration site [20, 40]. At the other FRT sites we did not co-integrate a reporter construct; however, the integration of XIST into the FRT site results in expression of an upstream Hygromycin (Hyg) gene. Robust silencing of Hyg was observed at all integrations after 5 days of XIST expression (Fig. 2a), suggesting that XIST is able to silence a virally-derived promoter (SV40), consistent with our previous demonstration that an EGFP reporter driven by the CMV promoter could be silenced [20]. The 7q integration site showed significantly less silencing than the 1p, 3q, 7p, 8p, 15q, and Xq integration sites (P ≤0.01). As only the repeat A region of XIST is required for silencing of the flanking reporter genes [40], we generated a construct containing the XIST repeat A and a DsRed reporter driven by the mouse Pgk1 gene promoter, which is normally X-linked and subject to XCI (Fig. 2b). We integrated this construct into six of the integration sites and again observed consistent silencing of greater than 90 % (Fig. 2c), suggesting both viral and mouse-derived promoters could be silenced by XIST in the HT1080 cells. Consistent with previous results with the EGFP reporter gene at 3q, silencing of the dsRed reporter was reversible when induction of XIST expression was stopped by removal of DOX (Fig. 2d).Fig. 2

Bottom Line: Silencing of flanking reporter genes occurs at all sites, but the spread of silencing to flanking endogenous human genes is variable in extent of silencing as well as extent of spread, with silencing able to skip regions.The non-coding RNA XIST functions as a cis-acting silencer when expressed from nine different locations throughout the genome.A hierarchy among the features of heterochromatin reveals the importance of interaction with the local chromatin neighborhood for optimal spread of silencing, as well as the independent yet cooperative nature of the establishment of heterochromatin by the non-coding XIST RNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada. adkelsey1@gmail.com.

ABSTRACT

Background: X-chromosome inactivation is a striking example of epigenetic silencing in which expression of the long non-coding RNA XIST initiates the heterochromatinization and silencing of one of the pair of X chromosomes in mammalian females. To understand how the RNA can establish silencing across millions of basepairs of DNA we have modelled the process by inducing expression of XIST from nine different locations in human HT1080 cells.

Results: Localization of XIST, depletion of Cot-1 RNA, perinuclear localization, and ubiquitination of H2A occurs at all sites examined, while recruitment of H3K9me3 was not observed. Recruitment of the heterochromatic features SMCHD1, macroH2A, H3K27me3, and H4K20me1 occurs independently of each other in an integration site-dependent manner. Silencing of flanking reporter genes occurs at all sites, but the spread of silencing to flanking endogenous human genes is variable in extent of silencing as well as extent of spread, with silencing able to skip regions. The spread of H3K27me3 and loss of H3K27ac correlates with the pre-existing levels of the modifications, and overall the extent of silencing correlates with the ability to recruit additional heterochromatic features.

Conclusions: The non-coding RNA XIST functions as a cis-acting silencer when expressed from nine different locations throughout the genome. A hierarchy among the features of heterochromatin reveals the importance of interaction with the local chromatin neighborhood for optimal spread of silencing, as well as the independent yet cooperative nature of the establishment of heterochromatin by the non-coding XIST RNA.

No MeSH data available.