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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.


Immunofluorescence shows features enriched at site of XIST RNA. The XIST RNA is identified by RNA FISH (green, except for H2AK119u1 (uH2A) photos, shown in greyscale) after 5 days DOX induction for the Xq integration clone. Co-IF with the antibodies listed was performed (shown in grayscale), with the merged image in color. Bars to the right show the proportion of XIST-positive HT1080 cells (integration into Xq) displaying enrichment of the indicated chromatin modification/protein
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Fig6: Immunofluorescence shows features enriched at site of XIST RNA. The XIST RNA is identified by RNA FISH (green, except for H2AK119u1 (uH2A) photos, shown in greyscale) after 5 days DOX induction for the Xq integration clone. Co-IF with the antibodies listed was performed (shown in grayscale), with the merged image in color. Bars to the right show the proportion of XIST-positive HT1080 cells (integration into Xq) displaying enrichment of the indicated chromatin modification/protein

Mentions: In addition to H3K27me3, the Xi is known to be enriched for additional histone marks and proteins associated with repressed chromatin; however, the hierarchy of recruitment of these heterochromatic features by XIST has not been clarified. Therefore, we examined the co-localization of these features with XIST expressed from various integration sites (Table 2). The extent of co-localization can vary with the cell cycle, and therefore we categorize the co-localization as positive (consistently observed in greater than 25 % of XIST-positive cells), negative (consistently observed in less than 10 % of cells) or +/− (in between 10 % and 25 % co-localization, or variable between replicates). No recruitment of H3K9me3 was observed at the four sites examined, while H2AK119u1 was observed at all four examined integration sites. The integration onto Xq showed recruitment of all marks (with the exception of H3K9me3) and is shown in Fig. 6. Recruitment was still less than was seen for a female cell line – in IMR90 we routinely detect over 80 % co-localization. H3K27me3 was not recruited to the 1p or 3q integration sites, while SMCHD1 was not enriched at 1p, 3q, and also 4q integration site clones. The integration clone at 3q did however recruit macroH2A, which was not seen to be enriched at the 1p or 7q integration clones. Overall the integration at 1p showed the least enrichment of marks with only variable recruitment of H4K20me1 in addition to H2AK119u1. To further examine dependence on integration site, independent clones integrated into 7q, Xq, and 8p were compared by IF-FISH for H3K27me3 and/or H4K20me1. Of the six side-by-side comparisons of independent clones at the same integration site, enrichment of marks were very comparable showing an average of 6 % difference in enrichment and all falling within the same category (except for 8p with H4K20me1 which spanned the +/− and +).Table 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)

Immunofluorescence shows features enriched at site of XIST RNA. The XIST RNA is identified by RNA FISH (green, except for H2AK119u1 (uH2A) photos, shown in greyscale) after 5 days DOX induction for the Xq integration clone. Co-IF with the antibodies listed was performed (shown in grayscale), with the merged image in color. Bars to the right show the proportion of XIST-positive HT1080 cells (integration into Xq) displaying enrichment of the indicated chromatin modification/protein
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591629&req=5

Fig6: Immunofluorescence shows features enriched at site of XIST RNA. The XIST RNA is identified by RNA FISH (green, except for H2AK119u1 (uH2A) photos, shown in greyscale) after 5 days DOX induction for the Xq integration clone. Co-IF with the antibodies listed was performed (shown in grayscale), with the merged image in color. Bars to the right show the proportion of XIST-positive HT1080 cells (integration into Xq) displaying enrichment of the indicated chromatin modification/protein
Mentions: In addition to H3K27me3, the Xi is known to be enriched for additional histone marks and proteins associated with repressed chromatin; however, the hierarchy of recruitment of these heterochromatic features by XIST has not been clarified. Therefore, we examined the co-localization of these features with XIST expressed from various integration sites (Table 2). The extent of co-localization can vary with the cell cycle, and therefore we categorize the co-localization as positive (consistently observed in greater than 25 % of XIST-positive cells), negative (consistently observed in less than 10 % of cells) or +/− (in between 10 % and 25 % co-localization, or variable between replicates). No recruitment of H3K9me3 was observed at the four sites examined, while H2AK119u1 was observed at all four examined integration sites. The integration onto Xq showed recruitment of all marks (with the exception of H3K9me3) and is shown in Fig. 6. Recruitment was still less than was seen for a female cell line – in IMR90 we routinely detect over 80 % co-localization. H3K27me3 was not recruited to the 1p or 3q integration sites, while SMCHD1 was not enriched at 1p, 3q, and also 4q integration site clones. The integration clone at 3q did however recruit macroH2A, which was not seen to be enriched at the 1p or 7q integration clones. Overall the integration at 1p showed the least enrichment of marks with only variable recruitment of H4K20me1 in addition to H2AK119u1. To further examine dependence on integration site, independent clones integrated into 7q, Xq, and 8p were compared by IF-FISH for H3K27me3 and/or H4K20me1. Of the six side-by-side comparisons of independent clones at the same integration site, enrichment of marks were very comparable showing an average of 6 % difference in enrichment and all falling within the same category (except for 8p with H4K20me1 which spanned the +/− and +).Table 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.