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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.


Allelic silencing of flanking endogenous genes upon XIST induction. a Allele-discriminating RT-PCR pyrosequencing assay for genes closest to 5 Mb of integration site for each integration site, comparing triplicate cDNAs from untreated cells (No DOX) and following 5-day DOX induction of XIST in duplicate pyrosequencing reactions. cDNA from a different integration was also assessed (additional assays are shown in Additional file 3). P values of significantly silenced genes are listed. b Summary of the silencing observed for individual genes for each of the nine integration sites (color-coded as shown in the legend); plotted by distance from the integration site on the chromosome (from short to long arm). The allelic change is shown as percent silencing, which was calculated as: (allele frequency No DOX – allele frequency 5d DOX)/allele frequency No DOX × 100) for the pyrosequencing assays. For the Xq integration the silencing was determined by q-RT-PCR since the chromosome is hemizygous. Phase was determined for the 3q integration but for all integrations the allelic change is shown as silencing. Integrations on other chromosomes showed no silencing upon DOX induction. c Correlation between extent of silencing and level of XIST RNA. Five different clones (symbols) and cultures show variation in the level of XIST RNA after DOX induction (as measured by qRT-PCR for XIST relative to PGK1), and for 12q this correlates well with the extent of silencing of two genes assayed by allelic pyrosequencing after RT-PCR (OAS3, P <0.0001; POLR3B, P = 0.0004). A similar analysis (d) for the chromosome 8p integration site showed a similar variation in XIST levels, but no correlation with extent of silencing of two loci on 8p. e Removal of XIST expression after 5-day DOX induction resulted in substantial reactivation of endogenous genes in the 8p and Xq integration sites
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Fig3: Allelic silencing of flanking endogenous genes upon XIST induction. a Allele-discriminating RT-PCR pyrosequencing assay for genes closest to 5 Mb of integration site for each integration site, comparing triplicate cDNAs from untreated cells (No DOX) and following 5-day DOX induction of XIST in duplicate pyrosequencing reactions. cDNA from a different integration was also assessed (additional assays are shown in Additional file 3). P values of significantly silenced genes are listed. b Summary of the silencing observed for individual genes for each of the nine integration sites (color-coded as shown in the legend); plotted by distance from the integration site on the chromosome (from short to long arm). The allelic change is shown as percent silencing, which was calculated as: (allele frequency No DOX – allele frequency 5d DOX)/allele frequency No DOX × 100) for the pyrosequencing assays. For the Xq integration the silencing was determined by q-RT-PCR since the chromosome is hemizygous. Phase was determined for the 3q integration but for all integrations the allelic change is shown as silencing. Integrations on other chromosomes showed no silencing upon DOX induction. c Correlation between extent of silencing and level of XIST RNA. Five different clones (symbols) and cultures show variation in the level of XIST RNA after DOX induction (as measured by qRT-PCR for XIST relative to PGK1), and for 12q this correlates well with the extent of silencing of two genes assayed by allelic pyrosequencing after RT-PCR (OAS3, P <0.0001; POLR3B, P = 0.0004). A similar analysis (d) for the chromosome 8p integration site showed a similar variation in XIST levels, but no correlation with extent of silencing of two loci on 8p. e Removal of XIST expression after 5-day DOX induction resulted in substantial reactivation of endogenous genes in the 8p and Xq integration sites

Mentions: Given the capacity of the XIST RNA to silence in cis, and the apparent spread of the RNA along the chromosome based on our RNA FISH data, we questioned whether there would be silencing of endogenous genes at additional sites adjacent to the XIST transgenes. The HT1080 cells remain diploid although they carry several structural rearrangements (46,XY,del (1)(p21), i(3)(p10), i(3)(q10), der(4)t(1;4)(p21;p16), der(5)t(5;5)(p15;?), der(11)t(3;11)(q11;q25) see additional details in methods). We generated allele-discriminating pyrosequencing assays to examine silencing of candidate genes flanking the integration sites (Fig. 3). The phase of the polymorphisms relative to the integration was not known, but the allelic expression change upon DOX induction of XIST is presented as a cis-linked loss of expression as previously demonstrated for the 3q integration for which we were able to assign the allelic loss to the chromosome bearing the inducible XIST [40]. Individual pyrosequencing results for the locus closest to 5 Mb from the integration site are shown in Fig. 3a, with the silencing percentages for all genes examined shown in Fig. 3b as a function of distance from the integration site (all assays are shown in Additional file 3). While no significant changes were observed for the control integrations (clones with XIST integrated on different chromosomes), all XIST integration sites except 4q showed at least one gene with significant allelic silencing. There was much more variability between integration sites for endogenous gene silencing than was seen for the silencing of Hyg. The 8p-integrated XIST clone displayed the most silencing, with four out of the five genes tested showing 60–80 % silencing. The 4q clone, in contrast, showed no significant silencing for any of the three genes tested. Two different integration sites on chromosome 7 showed quite different results, with only one of seven genes assayed showing over 20 % silencing for the 7p integration site, while five of the seven genes showed over 20 % silencing with XIST expressed from the 7q integration site. There was also discontinuous spread of silencing. For example, in the 1p integration site clone, two genes located approximately 200 kb from the XIST transgene failed to silence (1 % silencing), whereas the RHBDL2 gene located approximately 400 kb from the XIST transgene silenced by approximately 70 %. In addition to variation between the integration sites in the number of genes that were silenced, there were also significant differences in the extent of gene silencing between genes that showed silencing. More than half of the significant changes demonstrated less than 50 % silencing of one allele, and a significant change as small as 6 % for the ZNF710 gene on 15q was observed, indicating that XIST can cause a continuum of silencing.Fig. 3


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)

Allelic silencing of flanking endogenous genes upon XIST induction. a Allele-discriminating RT-PCR pyrosequencing assay for genes closest to 5 Mb of integration site for each integration site, comparing triplicate cDNAs from untreated cells (No DOX) and following 5-day DOX induction of XIST in duplicate pyrosequencing reactions. cDNA from a different integration was also assessed (additional assays are shown in Additional file 3). P values of significantly silenced genes are listed. b Summary of the silencing observed for individual genes for each of the nine integration sites (color-coded as shown in the legend); plotted by distance from the integration site on the chromosome (from short to long arm). The allelic change is shown as percent silencing, which was calculated as: (allele frequency No DOX – allele frequency 5d DOX)/allele frequency No DOX × 100) for the pyrosequencing assays. For the Xq integration the silencing was determined by q-RT-PCR since the chromosome is hemizygous. Phase was determined for the 3q integration but for all integrations the allelic change is shown as silencing. Integrations on other chromosomes showed no silencing upon DOX induction. c Correlation between extent of silencing and level of XIST RNA. Five different clones (symbols) and cultures show variation in the level of XIST RNA after DOX induction (as measured by qRT-PCR for XIST relative to PGK1), and for 12q this correlates well with the extent of silencing of two genes assayed by allelic pyrosequencing after RT-PCR (OAS3, P <0.0001; POLR3B, P = 0.0004). A similar analysis (d) for the chromosome 8p integration site showed a similar variation in XIST levels, but no correlation with extent of silencing of two loci on 8p. e Removal of XIST expression after 5-day DOX induction resulted in substantial reactivation of endogenous genes in the 8p and Xq integration sites
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Fig3: Allelic silencing of flanking endogenous genes upon XIST induction. a Allele-discriminating RT-PCR pyrosequencing assay for genes closest to 5 Mb of integration site for each integration site, comparing triplicate cDNAs from untreated cells (No DOX) and following 5-day DOX induction of XIST in duplicate pyrosequencing reactions. cDNA from a different integration was also assessed (additional assays are shown in Additional file 3). P values of significantly silenced genes are listed. b Summary of the silencing observed for individual genes for each of the nine integration sites (color-coded as shown in the legend); plotted by distance from the integration site on the chromosome (from short to long arm). The allelic change is shown as percent silencing, which was calculated as: (allele frequency No DOX – allele frequency 5d DOX)/allele frequency No DOX × 100) for the pyrosequencing assays. For the Xq integration the silencing was determined by q-RT-PCR since the chromosome is hemizygous. Phase was determined for the 3q integration but for all integrations the allelic change is shown as silencing. Integrations on other chromosomes showed no silencing upon DOX induction. c Correlation between extent of silencing and level of XIST RNA. Five different clones (symbols) and cultures show variation in the level of XIST RNA after DOX induction (as measured by qRT-PCR for XIST relative to PGK1), and for 12q this correlates well with the extent of silencing of two genes assayed by allelic pyrosequencing after RT-PCR (OAS3, P <0.0001; POLR3B, P = 0.0004). A similar analysis (d) for the chromosome 8p integration site showed a similar variation in XIST levels, but no correlation with extent of silencing of two loci on 8p. e Removal of XIST expression after 5-day DOX induction resulted in substantial reactivation of endogenous genes in the 8p and Xq integration sites
Mentions: Given the capacity of the XIST RNA to silence in cis, and the apparent spread of the RNA along the chromosome based on our RNA FISH data, we questioned whether there would be silencing of endogenous genes at additional sites adjacent to the XIST transgenes. The HT1080 cells remain diploid although they carry several structural rearrangements (46,XY,del (1)(p21), i(3)(p10), i(3)(q10), der(4)t(1;4)(p21;p16), der(5)t(5;5)(p15;?), der(11)t(3;11)(q11;q25) see additional details in methods). We generated allele-discriminating pyrosequencing assays to examine silencing of candidate genes flanking the integration sites (Fig. 3). The phase of the polymorphisms relative to the integration was not known, but the allelic expression change upon DOX induction of XIST is presented as a cis-linked loss of expression as previously demonstrated for the 3q integration for which we were able to assign the allelic loss to the chromosome bearing the inducible XIST [40]. Individual pyrosequencing results for the locus closest to 5 Mb from the integration site are shown in Fig. 3a, with the silencing percentages for all genes examined shown in Fig. 3b as a function of distance from the integration site (all assays are shown in Additional file 3). While no significant changes were observed for the control integrations (clones with XIST integrated on different chromosomes), all XIST integration sites except 4q showed at least one gene with significant allelic silencing. There was much more variability between integration sites for endogenous gene silencing than was seen for the silencing of Hyg. The 8p-integrated XIST clone displayed the most silencing, with four out of the five genes tested showing 60–80 % silencing. The 4q clone, in contrast, showed no significant silencing for any of the three genes tested. Two different integration sites on chromosome 7 showed quite different results, with only one of seven genes assayed showing over 20 % silencing for the 7p integration site, while five of the seven genes showed over 20 % silencing with XIST expressed from the 7q integration site. There was also discontinuous spread of silencing. For example, in the 1p integration site clone, two genes located approximately 200 kb from the XIST transgene failed to silence (1 % silencing), whereas the RHBDL2 gene located approximately 400 kb from the XIST transgene silenced by approximately 70 %. In addition to variation between the integration sites in the number of genes that were silenced, there were also significant differences in the extent of gene silencing between genes that showed silencing. More than half of the significant changes demonstrated less than 50 % silencing of one allele, and a significant change as small as 6 % for the ZNF710 gene on 15q was observed, indicating that XIST can cause a continuum of silencing.Fig. 3

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.