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Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome.

Yamada N, Hasegawa Y, Yue M, Hamada T, Nakagawa S, Ogawa Y - PLoS Genet. (2015)

Bottom Line: Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells.Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells.Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.

ABSTRACT
To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

No MeSH data available.


Related in: MedlinePlus

Xist exon 7 is required for the chromosome-wide localization of Xist RNA and H3K27me3 during X-inactivation.(A) Immuno-FISH for Xist RNA (green) and H3K27me3 (red) before (day 0) and after (day 8) differentiation. Nuclei were counterstained with DAPI. The white arrowhead and arrow indicate representative “strong” and “weak” Xist clouds classified in Fig 2B, respectively. Scale bar, 10 μm. (B) Frequency of Xist cloud- and H3K27me3-positive cells upon differentiation from three independent experiments. More than 350 nuclei in each ES cell line at each time point from three independent experiments were counted and classified based on the Xist RNA and H3K27me3 signal. Robust and faint Xist RNA FISH signals were classified as “strong” and “weak” Xist RNA, respectively. (C) The graph shows the mean ± SD of nuclei with Xist RNA clouds from three independent experiments. P-values were derived from an unpaired t-test between control TsixTST6 and XistdelE7TsixTST6 cells on the same day upon differentiation (*p<0.05, **p<0.01, ***p<0.001).
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pgen.1005430.g002: Xist exon 7 is required for the chromosome-wide localization of Xist RNA and H3K27me3 during X-inactivation.(A) Immuno-FISH for Xist RNA (green) and H3K27me3 (red) before (day 0) and after (day 8) differentiation. Nuclei were counterstained with DAPI. The white arrowhead and arrow indicate representative “strong” and “weak” Xist clouds classified in Fig 2B, respectively. Scale bar, 10 μm. (B) Frequency of Xist cloud- and H3K27me3-positive cells upon differentiation from three independent experiments. More than 350 nuclei in each ES cell line at each time point from three independent experiments were counted and classified based on the Xist RNA and H3K27me3 signal. Robust and faint Xist RNA FISH signals were classified as “strong” and “weak” Xist RNA, respectively. (C) The graph shows the mean ± SD of nuclei with Xist RNA clouds from three independent experiments. P-values were derived from an unpaired t-test between control TsixTST6 and XistdelE7TsixTST6 cells on the same day upon differentiation (*p<0.05, **p<0.01, ***p<0.001).

Mentions: Having shown that neither the control TsixTST6 nor the XistdelEx7TsixTST6 mutation affected the expression of Xist or the stability of Xist RNA and that neither mutant exhibited severe growth defects in EB differentiation during X-inactivation, we next examined the chromosome-wide gene silencing induced by the mutant Xist RNA during X-inactivation. We performed RNA fluorescence in situ hybridization with immunofluorescence (immuno-RNA FISH) to observe the accumulation of Xist RNA and a hallmark of facultative heterochromatin marker, H3K27me3, on the Xi upon differentiation. In the TsixTST6 mutant cells, the number of robust (strong) Xist RNA cloud- and H3K27me3-positive cells gradually increased upon differentiation. This is greater than that in the wild-type 16.7 ES cells at each time point, likely due to faster Xist induction by the Tsix mutation in TsixTST6 mutant cells [48]. In contrast to the strong focal staining of the Xist RNA and H3K27me3 in the control TsixTST6 mutant cells, the immuno-RNA FISH results in the XistdelEx7TsixTST6 mutant cells indicated a significantly reduced number of Xist RNA cloud- and H3K27me3-positive cells, despite the comparable levels of Xist expression between the mutant and control TsixTST6 cells (Figs 1D and 2). Instead of strong focal Xist RNA clouds, the XistdelEx7TsixTST6 mutant cells often exhibited a faint (weak) Xist signal. While the TsixTST6 mutant cells exhibited strong Xist clouds associated with H3K27me3 staining in approximately 65~70% of the cells on both day 8 and day 12, the co-localization of strong Xist clouds and H3K27me3 in the XistdelEx7TsixTST6 mutant cells was less than 20% and 14% on days 8 and 12, respectively. The difference in terms of the percentages of the Xist cloud-positive nuclei between the TsixTST6 and XistdelEx7TsixTST6 mutant cells became more significant as differentiation progressed (Fig 2B and 2C). Notably, the total percentage of the Xist cloud-positive (strong and weak) cells in the XistdelEx7TsixTST6 mutant cells on days 8 and 12 were significantly decreased from 45% to 24% (clone #1) and from 37% to 19% (clone #2), respectively. In contrast, the TsixTST6 cells maintained a high percentage of Xist cloud-positive cells (over 75% Xist cloud-positive) during the transitional period from day 8 to day 12. Our results suggest that exon 7 of the Xist RNA is essential for stable Xist RNA localization, especially in the maintenance of Xist RNA localization and associated H3K27me3 modifications on the Xi.


Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome.

Yamada N, Hasegawa Y, Yue M, Hamada T, Nakagawa S, Ogawa Y - PLoS Genet. (2015)

Xist exon 7 is required for the chromosome-wide localization of Xist RNA and H3K27me3 during X-inactivation.(A) Immuno-FISH for Xist RNA (green) and H3K27me3 (red) before (day 0) and after (day 8) differentiation. Nuclei were counterstained with DAPI. The white arrowhead and arrow indicate representative “strong” and “weak” Xist clouds classified in Fig 2B, respectively. Scale bar, 10 μm. (B) Frequency of Xist cloud- and H3K27me3-positive cells upon differentiation from three independent experiments. More than 350 nuclei in each ES cell line at each time point from three independent experiments were counted and classified based on the Xist RNA and H3K27me3 signal. Robust and faint Xist RNA FISH signals were classified as “strong” and “weak” Xist RNA, respectively. (C) The graph shows the mean ± SD of nuclei with Xist RNA clouds from three independent experiments. P-values were derived from an unpaired t-test between control TsixTST6 and XistdelE7TsixTST6 cells on the same day upon differentiation (*p<0.05, **p<0.01, ***p<0.001).
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pgen.1005430.g002: Xist exon 7 is required for the chromosome-wide localization of Xist RNA and H3K27me3 during X-inactivation.(A) Immuno-FISH for Xist RNA (green) and H3K27me3 (red) before (day 0) and after (day 8) differentiation. Nuclei were counterstained with DAPI. The white arrowhead and arrow indicate representative “strong” and “weak” Xist clouds classified in Fig 2B, respectively. Scale bar, 10 μm. (B) Frequency of Xist cloud- and H3K27me3-positive cells upon differentiation from three independent experiments. More than 350 nuclei in each ES cell line at each time point from three independent experiments were counted and classified based on the Xist RNA and H3K27me3 signal. Robust and faint Xist RNA FISH signals were classified as “strong” and “weak” Xist RNA, respectively. (C) The graph shows the mean ± SD of nuclei with Xist RNA clouds from three independent experiments. P-values were derived from an unpaired t-test between control TsixTST6 and XistdelE7TsixTST6 cells on the same day upon differentiation (*p<0.05, **p<0.01, ***p<0.001).
Mentions: Having shown that neither the control TsixTST6 nor the XistdelEx7TsixTST6 mutation affected the expression of Xist or the stability of Xist RNA and that neither mutant exhibited severe growth defects in EB differentiation during X-inactivation, we next examined the chromosome-wide gene silencing induced by the mutant Xist RNA during X-inactivation. We performed RNA fluorescence in situ hybridization with immunofluorescence (immuno-RNA FISH) to observe the accumulation of Xist RNA and a hallmark of facultative heterochromatin marker, H3K27me3, on the Xi upon differentiation. In the TsixTST6 mutant cells, the number of robust (strong) Xist RNA cloud- and H3K27me3-positive cells gradually increased upon differentiation. This is greater than that in the wild-type 16.7 ES cells at each time point, likely due to faster Xist induction by the Tsix mutation in TsixTST6 mutant cells [48]. In contrast to the strong focal staining of the Xist RNA and H3K27me3 in the control TsixTST6 mutant cells, the immuno-RNA FISH results in the XistdelEx7TsixTST6 mutant cells indicated a significantly reduced number of Xist RNA cloud- and H3K27me3-positive cells, despite the comparable levels of Xist expression between the mutant and control TsixTST6 cells (Figs 1D and 2). Instead of strong focal Xist RNA clouds, the XistdelEx7TsixTST6 mutant cells often exhibited a faint (weak) Xist signal. While the TsixTST6 mutant cells exhibited strong Xist clouds associated with H3K27me3 staining in approximately 65~70% of the cells on both day 8 and day 12, the co-localization of strong Xist clouds and H3K27me3 in the XistdelEx7TsixTST6 mutant cells was less than 20% and 14% on days 8 and 12, respectively. The difference in terms of the percentages of the Xist cloud-positive nuclei between the TsixTST6 and XistdelEx7TsixTST6 mutant cells became more significant as differentiation progressed (Fig 2B and 2C). Notably, the total percentage of the Xist cloud-positive (strong and weak) cells in the XistdelEx7TsixTST6 mutant cells on days 8 and 12 were significantly decreased from 45% to 24% (clone #1) and from 37% to 19% (clone #2), respectively. In contrast, the TsixTST6 cells maintained a high percentage of Xist cloud-positive cells (over 75% Xist cloud-positive) during the transitional period from day 8 to day 12. Our results suggest that exon 7 of the Xist RNA is essential for stable Xist RNA localization, especially in the maintenance of Xist RNA localization and associated H3K27me3 modifications on the Xi.

Bottom Line: Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells.Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells.Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.

ABSTRACT
To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

No MeSH data available.


Related in: MedlinePlus