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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.


XIST RNA localizes and forms Cot-1 holes when expressed from nine different integration sites. Shown is XIST RNA FISH (green) upon expression of an inducible XIST transgene integrated into the indicated chromosomal locations in HT1080 cells. IMR90 cells (a female fibroblast line) are shown as a positive comparator. Cells were counter-stained with DAPI (blue) and co-hybridized with Cot-1 (labelled with spectrum-red, but shown in grayscale). Arrow indicates the location of the XIST signal and reduction in Cot-1 staining. Graphs to the right show the RGB intensities across the lines shown in the picture inserts drawn through the XIST clouds (XIST (green), Cot-1 (red), and DAPI (blue))
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Fig1: XIST RNA localizes and forms Cot-1 holes when expressed from nine different integration sites. Shown is XIST RNA FISH (green) upon expression of an inducible XIST transgene integrated into the indicated chromosomal locations in HT1080 cells. IMR90 cells (a female fibroblast line) are shown as a positive comparator. Cells were counter-stained with DAPI (blue) and co-hybridized with Cot-1 (labelled with spectrum-red, but shown in grayscale). Arrow indicates the location of the XIST signal and reduction in Cot-1 staining. Graphs to the right show the RGB intensities across the lines shown in the picture inserts drawn through the XIST clouds (XIST (green), Cot-1 (red), and DAPI (blue))

Mentions: For each clone, the inducible promoter was activated for 5 days and the localization of XIST RNA assessed by RNA FISH. At each integration site the XIST RNA was able to localize and formed an XIST cloud comparable to that observed in normal female cells (Fig. 1). The level of XIST RNA varied between cell lines and also within different cultures of the same cell line, showing from five- to 30-fold induction of XIST after 5 days of DOX (Additional file 1). The integrations into 3q, 7p, and 15q (all G-dark) showed lower expression by q-RT-PCR, which is consistent with a significantly smaller signal for XIST for the 3q integration clone relative to all other integrations except for 15q (P ≤0.01). Co-hybridization with fluorescently labelled Cot-1 also showed depletion of Cot-1 hybridization coincident with the XIST cloud at each integration site, observable in the line diagrams of signal intensity across the XIST cloud (Fig. 1). Recently stable Cot-1 repeat RNA has been shown to be associated with euchromatic chromosomes, yet excluded from the Xi resulting in the Cot-1 RNA hole [42]. We noted differences in the intensity of the Cot-1 holes; however, in attempting to quantify such differences it became apparent that the XIST RNA signal was often at the nuclear or nucleolar periphery in the HT1080 cells, and that measuring the intensity of the Cot-1 hole could be influenced by nuclear location. The Xi is generally located at the nuclear or nucleolar periphery; however, as these were autosomal integration sites, it seemed that expression of XIST might be altering nuclear location.Fig. 1


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)

XIST RNA localizes and forms Cot-1 holes when expressed from nine different integration sites. Shown is XIST RNA FISH (green) upon expression of an inducible XIST transgene integrated into the indicated chromosomal locations in HT1080 cells. IMR90 cells (a female fibroblast line) are shown as a positive comparator. Cells were counter-stained with DAPI (blue) and co-hybridized with Cot-1 (labelled with spectrum-red, but shown in grayscale). Arrow indicates the location of the XIST signal and reduction in Cot-1 staining. Graphs to the right show the RGB intensities across the lines shown in the picture inserts drawn through the XIST clouds (XIST (green), Cot-1 (red), and DAPI (blue))
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: XIST RNA localizes and forms Cot-1 holes when expressed from nine different integration sites. Shown is XIST RNA FISH (green) upon expression of an inducible XIST transgene integrated into the indicated chromosomal locations in HT1080 cells. IMR90 cells (a female fibroblast line) are shown as a positive comparator. Cells were counter-stained with DAPI (blue) and co-hybridized with Cot-1 (labelled with spectrum-red, but shown in grayscale). Arrow indicates the location of the XIST signal and reduction in Cot-1 staining. Graphs to the right show the RGB intensities across the lines shown in the picture inserts drawn through the XIST clouds (XIST (green), Cot-1 (red), and DAPI (blue))
Mentions: For each clone, the inducible promoter was activated for 5 days and the localization of XIST RNA assessed by RNA FISH. At each integration site the XIST RNA was able to localize and formed an XIST cloud comparable to that observed in normal female cells (Fig. 1). The level of XIST RNA varied between cell lines and also within different cultures of the same cell line, showing from five- to 30-fold induction of XIST after 5 days of DOX (Additional file 1). The integrations into 3q, 7p, and 15q (all G-dark) showed lower expression by q-RT-PCR, which is consistent with a significantly smaller signal for XIST for the 3q integration clone relative to all other integrations except for 15q (P ≤0.01). Co-hybridization with fluorescently labelled Cot-1 also showed depletion of Cot-1 hybridization coincident with the XIST cloud at each integration site, observable in the line diagrams of signal intensity across the XIST cloud (Fig. 1). Recently stable Cot-1 repeat RNA has been shown to be associated with euchromatic chromosomes, yet excluded from the Xi resulting in the Cot-1 RNA hole [42]. We noted differences in the intensity of the Cot-1 holes; however, in attempting to quantify such differences it became apparent that the XIST RNA signal was often at the nuclear or nucleolar periphery in the HT1080 cells, and that measuring the intensity of the Cot-1 hole could be influenced by nuclear location. The Xi is generally located at the nuclear or nucleolar periphery; however, as these were autosomal integration sites, it seemed that expression of XIST might be altering nuclear location.Fig. 1

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.