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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.


Correlation of genomic neighborhood and XIST-induced silencing on chromosome 8. a ChIP-seq changes observed for H3K27me3 on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. H3K27me3 showed significant (P = 3.3e−243, paired t-test) changes for chromosome 8 with the genome showing no significant change. b ChIP-seq changes observed for H3K27ac on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. The chromosome 8 decrease (P = 2.2e−40) as well as the genome-wide increase (P = 2.0e−205) were both highly significant. c Average H3K27ac and H3K27me3 normalized across genes on 8p. Normalized ChIP-seq level are shown across genes and for the 10 kb upstream and downstream of genes before XIST expression (NoDOX) and after XIST expression (DOX) as well as for input, color-coded as outlined. d Total H3K27me3 (blue line) and H3K27ac (pink line) in No DOX overlaid with change in H3K27me3 (blue), H3K27ac (pink) along chromosome 8 after 5 days of DOX induction of XIST. e Change in total reads for genes with average ≥5 FPKM shown as percent silencing. f Density of nuclear contacts with the 8p integration site (AGPAT5) as identified in IMR90 fibroblast cells by HiC [45] in 1 Mb bins (grey shading). Allelic silencing of genes is shown superimposed as green dots. g Density of LINE (blue line) and ALU (green line) repetitive elements per 1 Mb bin. Shown on the axes are the average genome (purple) and X-chromosomal (orange) densities of the elements. h Gene density in 1 Mb bins along the chromosome
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Fig5: Correlation of genomic neighborhood and XIST-induced silencing on chromosome 8. a ChIP-seq changes observed for H3K27me3 on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. H3K27me3 showed significant (P = 3.3e−243, paired t-test) changes for chromosome 8 with the genome showing no significant change. b ChIP-seq changes observed for H3K27ac on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. The chromosome 8 decrease (P = 2.2e−40) as well as the genome-wide increase (P = 2.0e−205) were both highly significant. c Average H3K27ac and H3K27me3 normalized across genes on 8p. Normalized ChIP-seq level are shown across genes and for the 10 kb upstream and downstream of genes before XIST expression (NoDOX) and after XIST expression (DOX) as well as for input, color-coded as outlined. d Total H3K27me3 (blue line) and H3K27ac (pink line) in No DOX overlaid with change in H3K27me3 (blue), H3K27ac (pink) along chromosome 8 after 5 days of DOX induction of XIST. e Change in total reads for genes with average ≥5 FPKM shown as percent silencing. f Density of nuclear contacts with the 8p integration site (AGPAT5) as identified in IMR90 fibroblast cells by HiC [45] in 1 Mb bins (grey shading). Allelic silencing of genes is shown superimposed as green dots. g Density of LINE (blue line) and ALU (green line) repetitive elements per 1 Mb bin. Shown on the axes are the average genome (purple) and X-chromosomal (orange) densities of the elements. h Gene density in 1 Mb bins along the chromosome

Mentions: ChIP-seq for both the archetypical facultative heterochromatic mark H3K27me3 and the active mark H3K27ac showed significant, but opposing changes on chromosome 8 upon DOX induction of XIST from the 8p integration site, and these changes were even more dramatic on the short arm where XIST is integrated (Fig. 5a, b). Surprisingly, DOX treatment significantly increased H3K27ac across the genome, with most chromosomes showing an increase, suggesting a widespread impact of the antibiotic (Fig. 5b). This unexpected change in chromatin accessibility did not extend to any significant change in H3K27me3 across the genome, and the anticipated enrichments in the imprinted regions of KCNQ1 and IGF2R were observed before and after DOX (Additional file 4). When examined across all genes on chromosome 8p, the loss of acetylation was most notable at the promoter, the site of most pre-existing acetylation, but loss was seen throughout the upstream and gene bodies (Fig. 5c). The genes from 8q showed changes similar to that of other autosomes, with an increase only detected at the promoter (Additional file 5). The gain of H3K27me3 on chromosome 8p was observed across both genic and intergenic regions (Fig. 5c), with no change observed for genes on 8q or autosomes (Additional file 5). In order to correlate the change in H3K27me3 and H3K27ac with the silencing observed, as well as other features of the chromosome, we plotted the initial levels (without DOX) as well as the total change in both marks along the chromosome (Fig. 5d). While the loss of H3K27ac could be anticipated to occur from locations where there was acetylation initially, the recruitment of H3K27me3 upon XIST induction also mirrored the pre-existing levels remarkably well (Spearman r = 0.8671; P <0.0001 for 8p). In Fig. 5e we show the average silencing determined from total reads, while in Fig. 5f we show allelic gene silencing. We assessed allelic changes in H3K27ac as well, and the limited informative genes showed a significant correlation between allelic silencing and loss of acetylation (correlation for 29 genes with data for both, r = 0.5904; P = 0.0007). Mouse Xist localizes to sequences that are in contact with the integration site as determined by chromatin conformation capture [43, 44], and therefore we extracted the Hi-C contacts anchored at the 1 Mb domain containing the 8p integration site from the published Hi-C data [45] (Fig. 5f). There are more contacts, as well as stronger silencing, closer to the integration site, confounding the ability to examine correlations. Interestingly, the Hi-C contacts correlated with the pre-existing (No DOX) H3K27me3 levels along the chromosome 8 short arm (Spearman r = 0.4996; P = 0.0006), although we did not identify an association of allelic silencing with domains designated as closed nor open [46] (Fisher’s exact test). Allelic silencing correlated with both the gain of H3K27me3 (Spearman r = 0.4599; P = 0.0003) and loss of acetylation (Spearman r = −0.4557, P = 0.0004). Given the proposed role for repetitive elements in the XCI process [47–49], and the tendency for mouse Xist to first localize to gene-rich regions during early expression in embryonic stem cells [43, 44] we show the LINE and ALU distribution (Fig. 5f) and the gene density (Fig. 5g) along chromosome 8; however, neither feature showed a significant correlation with silencing, although as would be expected, gene density is a major contributor to total levels of H3K27 acetylation. To further explore the relationship of the ability to modify chromatin and the silencing ability of the different chromosomal integration sites we performed immunofluorescence in conjunction with FISH for XIST.Fig. 5


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)

Correlation of genomic neighborhood and XIST-induced silencing on chromosome 8. a ChIP-seq changes observed for H3K27me3 on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. H3K27me3 showed significant (P = 3.3e−243, paired t-test) changes for chromosome 8 with the genome showing no significant change. b ChIP-seq changes observed for H3K27ac on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. The chromosome 8 decrease (P = 2.2e−40) as well as the genome-wide increase (P = 2.0e−205) were both highly significant. c Average H3K27ac and H3K27me3 normalized across genes on 8p. Normalized ChIP-seq level are shown across genes and for the 10 kb upstream and downstream of genes before XIST expression (NoDOX) and after XIST expression (DOX) as well as for input, color-coded as outlined. d Total H3K27me3 (blue line) and H3K27ac (pink line) in No DOX overlaid with change in H3K27me3 (blue), H3K27ac (pink) along chromosome 8 after 5 days of DOX induction of XIST. e Change in total reads for genes with average ≥5 FPKM shown as percent silencing. f Density of nuclear contacts with the 8p integration site (AGPAT5) as identified in IMR90 fibroblast cells by HiC [45] in 1 Mb bins (grey shading). Allelic silencing of genes is shown superimposed as green dots. g Density of LINE (blue line) and ALU (green line) repetitive elements per 1 Mb bin. Shown on the axes are the average genome (purple) and X-chromosomal (orange) densities of the elements. h Gene density in 1 Mb bins along the chromosome
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Fig5: Correlation of genomic neighborhood and XIST-induced silencing on chromosome 8. a ChIP-seq changes observed for H3K27me3 on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. H3K27me3 showed significant (P = 3.3e−243, paired t-test) changes for chromosome 8 with the genome showing no significant change. b ChIP-seq changes observed for H3K27ac on each chromosome, with chromosome 8 also subdivided into 8p (arm with integration) and 8q. The chromosome 8 decrease (P = 2.2e−40) as well as the genome-wide increase (P = 2.0e−205) were both highly significant. c Average H3K27ac and H3K27me3 normalized across genes on 8p. Normalized ChIP-seq level are shown across genes and for the 10 kb upstream and downstream of genes before XIST expression (NoDOX) and after XIST expression (DOX) as well as for input, color-coded as outlined. d Total H3K27me3 (blue line) and H3K27ac (pink line) in No DOX overlaid with change in H3K27me3 (blue), H3K27ac (pink) along chromosome 8 after 5 days of DOX induction of XIST. e Change in total reads for genes with average ≥5 FPKM shown as percent silencing. f Density of nuclear contacts with the 8p integration site (AGPAT5) as identified in IMR90 fibroblast cells by HiC [45] in 1 Mb bins (grey shading). Allelic silencing of genes is shown superimposed as green dots. g Density of LINE (blue line) and ALU (green line) repetitive elements per 1 Mb bin. Shown on the axes are the average genome (purple) and X-chromosomal (orange) densities of the elements. h Gene density in 1 Mb bins along the chromosome
Mentions: ChIP-seq for both the archetypical facultative heterochromatic mark H3K27me3 and the active mark H3K27ac showed significant, but opposing changes on chromosome 8 upon DOX induction of XIST from the 8p integration site, and these changes were even more dramatic on the short arm where XIST is integrated (Fig. 5a, b). Surprisingly, DOX treatment significantly increased H3K27ac across the genome, with most chromosomes showing an increase, suggesting a widespread impact of the antibiotic (Fig. 5b). This unexpected change in chromatin accessibility did not extend to any significant change in H3K27me3 across the genome, and the anticipated enrichments in the imprinted regions of KCNQ1 and IGF2R were observed before and after DOX (Additional file 4). When examined across all genes on chromosome 8p, the loss of acetylation was most notable at the promoter, the site of most pre-existing acetylation, but loss was seen throughout the upstream and gene bodies (Fig. 5c). The genes from 8q showed changes similar to that of other autosomes, with an increase only detected at the promoter (Additional file 5). The gain of H3K27me3 on chromosome 8p was observed across both genic and intergenic regions (Fig. 5c), with no change observed for genes on 8q or autosomes (Additional file 5). In order to correlate the change in H3K27me3 and H3K27ac with the silencing observed, as well as other features of the chromosome, we plotted the initial levels (without DOX) as well as the total change in both marks along the chromosome (Fig. 5d). While the loss of H3K27ac could be anticipated to occur from locations where there was acetylation initially, the recruitment of H3K27me3 upon XIST induction also mirrored the pre-existing levels remarkably well (Spearman r = 0.8671; P <0.0001 for 8p). In Fig. 5e we show the average silencing determined from total reads, while in Fig. 5f we show allelic gene silencing. We assessed allelic changes in H3K27ac as well, and the limited informative genes showed a significant correlation between allelic silencing and loss of acetylation (correlation for 29 genes with data for both, r = 0.5904; P = 0.0007). Mouse Xist localizes to sequences that are in contact with the integration site as determined by chromatin conformation capture [43, 44], and therefore we extracted the Hi-C contacts anchored at the 1 Mb domain containing the 8p integration site from the published Hi-C data [45] (Fig. 5f). There are more contacts, as well as stronger silencing, closer to the integration site, confounding the ability to examine correlations. Interestingly, the Hi-C contacts correlated with the pre-existing (No DOX) H3K27me3 levels along the chromosome 8 short arm (Spearman r = 0.4996; P = 0.0006), although we did not identify an association of allelic silencing with domains designated as closed nor open [46] (Fisher’s exact test). Allelic silencing correlated with both the gain of H3K27me3 (Spearman r = 0.4599; P = 0.0003) and loss of acetylation (Spearman r = −0.4557, P = 0.0004). Given the proposed role for repetitive elements in the XCI process [47–49], and the tendency for mouse Xist to first localize to gene-rich regions during early expression in embryonic stem cells [43, 44] we show the LINE and ALU distribution (Fig. 5f) and the gene density (Fig. 5g) along chromosome 8; however, neither feature showed a significant correlation with silencing, although as would be expected, gene density is a major contributor to total levels of H3K27 acetylation. To further explore the relationship of the ability to modify chromatin and the silencing ability of the different chromosomal integration sites we performed immunofluorescence in conjunction with FISH for XIST.Fig. 5

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.