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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.


Related in: MedlinePlus

Schematic of features examined at the site of XIST RNA induction. Nine different integration sites of XIST were examined, and these were in both G-light (pale gray) and G-dark genomic locations. Upon DOX induction XIST was expressed (intensity of green oval reflects average amount of XIST expression) and increased perinucleolar localization was observed (blue oval intensity reflects increase, with significant changes encircled in black). H2AK119u1 was enriched at all four integration sites examined. The enrichment of chromatin marks or proteins that were variably recruited (see Table 2) is shown as solid (enrichment >25 %), dotted (enrichment between 10 % and 25 %) or unfilled (enrichment ≤10 %). The integration sites are ordered by ranking of gene silencing observed by pyrosequencing (fill of red rectangle reflecting proportion of silenced genes, see Additional file 3)
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Fig7: Schematic of features examined at the site of XIST RNA induction. Nine different integration sites of XIST were examined, and these were in both G-light (pale gray) and G-dark genomic locations. Upon DOX induction XIST was expressed (intensity of green oval reflects average amount of XIST expression) and increased perinucleolar localization was observed (blue oval intensity reflects increase, with significant changes encircled in black). H2AK119u1 was enriched at all four integration sites examined. The enrichment of chromatin marks or proteins that were variably recruited (see Table 2) is shown as solid (enrichment >25 %), dotted (enrichment between 10 % and 25 %) or unfilled (enrichment ≤10 %). The integration sites are ordered by ranking of gene silencing observed by pyrosequencing (fill of red rectangle reflecting proportion of silenced genes, see Additional file 3)

Mentions: We observe a Cot-1 hole and increased perinucleolar association at all integration sites, consistent with previous reports that XIST/Xist expression from autosomes increases perinucleolar association [10, 21]. In our assessment of multiple XIST integrations, we observed heterogeneity in the extent of perinucleolar association, and the correlation with silencing was limited. The three integration sites for which the increase in perinucleolar association was not significant (15q, 3q, and Xq) were all G-dark integration sites; however, the 7p integration was also in a G-dark band yet demonstrated a significant increase in perinucleolar localization, suggesting an incomplete association between perinucleolar association and G-dark integrations with lower XIST levels (Fig. 7).Fig. 7


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)

Schematic of features examined at the site of XIST RNA induction. Nine different integration sites of XIST were examined, and these were in both G-light (pale gray) and G-dark genomic locations. Upon DOX induction XIST was expressed (intensity of green oval reflects average amount of XIST expression) and increased perinucleolar localization was observed (blue oval intensity reflects increase, with significant changes encircled in black). H2AK119u1 was enriched at all four integration sites examined. The enrichment of chromatin marks or proteins that were variably recruited (see Table 2) is shown as solid (enrichment >25 %), dotted (enrichment between 10 % and 25 %) or unfilled (enrichment ≤10 %). The integration sites are ordered by ranking of gene silencing observed by pyrosequencing (fill of red rectangle reflecting proportion of silenced genes, see Additional file 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4591629&req=5

Fig7: Schematic of features examined at the site of XIST RNA induction. Nine different integration sites of XIST were examined, and these were in both G-light (pale gray) and G-dark genomic locations. Upon DOX induction XIST was expressed (intensity of green oval reflects average amount of XIST expression) and increased perinucleolar localization was observed (blue oval intensity reflects increase, with significant changes encircled in black). H2AK119u1 was enriched at all four integration sites examined. The enrichment of chromatin marks or proteins that were variably recruited (see Table 2) is shown as solid (enrichment >25 %), dotted (enrichment between 10 % and 25 %) or unfilled (enrichment ≤10 %). The integration sites are ordered by ranking of gene silencing observed by pyrosequencing (fill of red rectangle reflecting proportion of silenced genes, see Additional file 3)
Mentions: We observe a Cot-1 hole and increased perinucleolar association at all integration sites, consistent with previous reports that XIST/Xist expression from autosomes increases perinucleolar association [10, 21]. In our assessment of multiple XIST integrations, we observed heterogeneity in the extent of perinucleolar association, and the correlation with silencing was limited. The three integration sites for which the increase in perinucleolar association was not significant (15q, 3q, and Xq) were all G-dark integration sites; however, the 7p integration was also in a G-dark band yet demonstrated a significant increase in perinucleolar localization, suggesting an incomplete association between perinucleolar association and G-dark integrations with lower XIST levels (Fig. 7).Fig. 7

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.


Related in: MedlinePlus