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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

Silencing of endogenous genes upon ectopic XIST expression. Allelic inactivation of chromosomes 1, 8, and 12 is shown as a heat map following DOX induction of XIST in cells containing integrations into the chromosome listed in green. Colors denote the allelic expression change as assessed by RNA-seq (red = downregulation; yellow = no change) for those genes with allelic reads with FPKM ≥5. The integration site is marked as a black arrow; confirmatory pyrosequencing assays are indicated by blue lines, and the centromere is shown as a black line. For chromosome 1, the chromosome is truncated at the site of a translocation as it is unknown whether the integration is on the translocated chromosome (boxed region)
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Fig4: Silencing of endogenous genes upon ectopic XIST expression. Allelic inactivation of chromosomes 1, 8, and 12 is shown as a heat map following DOX induction of XIST in cells containing integrations into the chromosome listed in green. Colors denote the allelic expression change as assessed by RNA-seq (red = downregulation; yellow = no change) for those genes with allelic reads with FPKM ≥5. The integration site is marked as a black arrow; confirmatory pyrosequencing assays are indicated by blue lines, and the centromere is shown as a black line. For chromosome 1, the chromosome is truncated at the site of a translocation as it is unknown whether the integration is on the translocated chromosome (boxed region)

Mentions: As our candidate genes examined by pyrosequencing had shown significant reductions in gene expression that ranged from as little as 6 % to approximately 80 % silencing of one allele, we did not expect complete silencing of one allele that would reduce expression levels overall by 50 %. We chose a stringent threshold of 30–60 % total reduction to classify genes as silencing, at which level a significantly greater proportion of genes were observed within 30 Mb of the 8p and 12q integration sites compared to the genome (Chi-square test, P <0.0001). Consistent with our candidate gene analysis, the increase in proportion of genes in this range was not significant for the 1p integration site. Examination of expression changes as an allelic change is more sensitive to the partial reduction of expression of one allele, and should also still detect changes in allelic expression when the total expression level of the gene is regulated in trans although the number of genes that can be examined is reduced by the requirement for an expressed polymorphism. We examined the allelic change in expression on chromosomes 1, 8, and 12 (Fig. 4), and again a significantly higher proportion of genes showing an allelic change of greater than 30 % was seen for genes flanking the 8p integration site. A Chi-square permutation test demonstrated that the number of genes silenced on 8p following XIST induction decreased with increasing distance (P = 0.008). The biological duplicate for RNA-seq was highly concordant for the percent allelic gene silencing for 8p (Spearman r = 0.5806; P <0.0001 for genes with FPKM ≥5). The proportion of genes showing allelic silencing for genes flanking the 1p integration site was significant (P = 0.0269); however no significant change was seen for the 12q integration site. We show only the distal end of chromosome 1 as the HT1080 cells carry a translocation of one chromosome 1 to chromosome 4 (approximately 55 Mb distal to the integration site). We validated several of the observed changes by pyrosequencing or q-PCR, and the assays are included in Additional file 3, and highlighted on Fig. 4. Percent allelic silencing was highly concordant between pyrosequencing and RNA-seq (r = 0.9341; P <0.0001). Of the 17 genes showing at least 50 % allelic silencing in both 8p RNA-seq replicates, two genes (DLC1 and STC1) did not show a decrease in total expression, suggesting that auto-regulation could be another source of discrepancy between total read and allelic read changes. The high rate of validation of the observed changes by pyrosequencing of biological triplicates substantiated that silencing spread to endogenous genes. Intriguingly, on chromosome 8 there appeared to be domains of silencing separated by areas that were more resistant to the action of XIST. In order to explore what features might lead to differential susceptibility to silencing we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) for induced and uninduced cells with XIST integrated at 8p.Fig. 4


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 endogenous genes upon ectopic XIST expression. Allelic inactivation of chromosomes 1, 8, and 12 is shown as a heat map following DOX induction of XIST in cells containing integrations into the chromosome listed in green. Colors denote the allelic expression change as assessed by RNA-seq (red = downregulation; yellow = no change) for those genes with allelic reads with FPKM ≥5. The integration site is marked as a black arrow; confirmatory pyrosequencing assays are indicated by blue lines, and the centromere is shown as a black line. For chromosome 1, the chromosome is truncated at the site of a translocation as it is unknown whether the integration is on the translocated chromosome (boxed region)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Silencing of endogenous genes upon ectopic XIST expression. Allelic inactivation of chromosomes 1, 8, and 12 is shown as a heat map following DOX induction of XIST in cells containing integrations into the chromosome listed in green. Colors denote the allelic expression change as assessed by RNA-seq (red = downregulation; yellow = no change) for those genes with allelic reads with FPKM ≥5. The integration site is marked as a black arrow; confirmatory pyrosequencing assays are indicated by blue lines, and the centromere is shown as a black line. For chromosome 1, the chromosome is truncated at the site of a translocation as it is unknown whether the integration is on the translocated chromosome (boxed region)
Mentions: As our candidate genes examined by pyrosequencing had shown significant reductions in gene expression that ranged from as little as 6 % to approximately 80 % silencing of one allele, we did not expect complete silencing of one allele that would reduce expression levels overall by 50 %. We chose a stringent threshold of 30–60 % total reduction to classify genes as silencing, at which level a significantly greater proportion of genes were observed within 30 Mb of the 8p and 12q integration sites compared to the genome (Chi-square test, P <0.0001). Consistent with our candidate gene analysis, the increase in proportion of genes in this range was not significant for the 1p integration site. Examination of expression changes as an allelic change is more sensitive to the partial reduction of expression of one allele, and should also still detect changes in allelic expression when the total expression level of the gene is regulated in trans although the number of genes that can be examined is reduced by the requirement for an expressed polymorphism. We examined the allelic change in expression on chromosomes 1, 8, and 12 (Fig. 4), and again a significantly higher proportion of genes showing an allelic change of greater than 30 % was seen for genes flanking the 8p integration site. A Chi-square permutation test demonstrated that the number of genes silenced on 8p following XIST induction decreased with increasing distance (P = 0.008). The biological duplicate for RNA-seq was highly concordant for the percent allelic gene silencing for 8p (Spearman r = 0.5806; P <0.0001 for genes with FPKM ≥5). The proportion of genes showing allelic silencing for genes flanking the 1p integration site was significant (P = 0.0269); however no significant change was seen for the 12q integration site. We show only the distal end of chromosome 1 as the HT1080 cells carry a translocation of one chromosome 1 to chromosome 4 (approximately 55 Mb distal to the integration site). We validated several of the observed changes by pyrosequencing or q-PCR, and the assays are included in Additional file 3, and highlighted on Fig. 4. Percent allelic silencing was highly concordant between pyrosequencing and RNA-seq (r = 0.9341; P <0.0001). Of the 17 genes showing at least 50 % allelic silencing in both 8p RNA-seq replicates, two genes (DLC1 and STC1) did not show a decrease in total expression, suggesting that auto-regulation could be another source of discrepancy between total read and allelic read changes. The high rate of validation of the observed changes by pyrosequencing of biological triplicates substantiated that silencing spread to endogenous genes. Intriguingly, on chromosome 8 there appeared to be domains of silencing separated by areas that were more resistant to the action of XIST. In order to explore what features might lead to differential susceptibility to silencing we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) for induced and uninduced cells with XIST integrated at 8p.Fig. 4

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