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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

Truncation of exon 7 of Xist RNA affects the X-linked gene silencing on the Xi during EB differentiation.(A) 129 mutant allele-specific RT-qPCR analysis of X-linked Mecp2 and Pgk1 genes upon differentiation; each was normalized to undifferentiated cells (set to 1) and Gapdh. The mean ± SD values from three independent experiments are shown with the unpaired t test P values (*p<0.05, **p<0.01). (B) Representative allele-specific RT-PCR analysis for Mecp2 and Pgk1. (C) Quantification of relative expression levels of Mecp2 and Pgk1 from the 129 allele. The mean ± SD values from the three independent experiments included in Fig 3B are shown. P-values were derived from an unpaired t-test (*p<0.05, **p<0.01).
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pgen.1005430.g003: Truncation of exon 7 of Xist RNA affects the X-linked gene silencing on the Xi during EB differentiation.(A) 129 mutant allele-specific RT-qPCR analysis of X-linked Mecp2 and Pgk1 genes upon differentiation; each was normalized to undifferentiated cells (set to 1) and Gapdh. The mean ± SD values from three independent experiments are shown with the unpaired t test P values (*p<0.05, **p<0.01). (B) Representative allele-specific RT-PCR analysis for Mecp2 and Pgk1. (C) Quantification of relative expression levels of Mecp2 and Pgk1 from the 129 allele. The mean ± SD values from the three independent experiments included in Fig 3B are shown. P-values were derived from an unpaired t-test (*p<0.05, **p<0.01).

Mentions: Intriguingly, the XistdelEx7TsixTST6 mutant clones exhibited impaired localization of the Xist RNA and H3K27me3 on the Xi. This observation prompted us to investigate whether the compromised Xist RNA and H3K27me3 localization on the Xi affects the expression status of X-linked genes on the Xi during EB differentiation using two different approaches (Fig 3): RT-qPCR analysis using allele-specific primers (Fig 3A) and allele-specific RT-PCR using a polymorphic restriction enzyme digestion (Fig 3B and 3C). The allele-specific RT-qPCR analysis using 129 allele-specific primer sets can discriminate the Pgk1 and Mecp2 expression on the mutant 129 X-chromosome from their expression on the Cast X-chromosome. Thus, we can only detect Pgk1 and Mecp2 expression on the mutant 129 X-chromosome. In the control TsixTST6 EB cells on days 8 and 12 of differentiation, the X-linked Mecp2 and Pgk1 expression from the 129 Xi gradually decreased upon differentiation and remained less than 13% and 12% compared to that of the undifferentiated ES cells (day 0), respectively. In contrast to the control cells, the mutant 129 allele-specific RT-qPCR for Mecp2 and Pgk1 in the XistdelEx7TsixTST6 mutant clones showed that the partial silencing of Mecp2 (40% and 25% in clones #1 and #2, respectively) and Pgk1 (55% and 47% in clones #1 and #2, respectively) occurred on day 8 instead of on day 0. The partial silencing of Mecp2 and Pgk1 in the XistdelEx7TsixTST6 mutant cells was then followed by the reactivation of Mecp2 (98% and 89% in clones #1 and #2, respectively) and Pgk1 (64% and 58% in clones #1 and #2, respectively) on day 12 upon differentiation, although the initial phase of silencing in Mecp2 and Pgk1 on day 4 was similar between the TsixTST6 and XistdelEx7TsixTST6 mutant cells. Additionally, to confirm the compromised Mecp2 and Pgk1 expression from the 129 Xi relative to that of the wild-type Cast Xa in the XistdelEx7TsixTST6 mutant cells, allele-specific RT-PCR analysis based on a polymorphic restriction enzyme digestion was performed (Fig 3B and 3C). The ratios of the X-linked gene expression from the 129 Xi were markedly reduced from differentiation day 8 in the TsixTST6 cells. However, in the mutant clones, the ratios of X-linked gene expression from the 129 Xi remained high during the late stages of X-inactivation. Combined with the data in Fig 2, these results suggest that the XistdelEx7TsixTST6 mutant Xist RNA can partially induce the silencing of X-linked genes, but cannot sustain transcriptional repression on the Xi. This is likely due to the loss of the Xist RNA clouds and the H3K27me3 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)

Truncation of exon 7 of Xist RNA affects the X-linked gene silencing on the Xi during EB differentiation.(A) 129 mutant allele-specific RT-qPCR analysis of X-linked Mecp2 and Pgk1 genes upon differentiation; each was normalized to undifferentiated cells (set to 1) and Gapdh. The mean ± SD values from three independent experiments are shown with the unpaired t test P values (*p<0.05, **p<0.01). (B) Representative allele-specific RT-PCR analysis for Mecp2 and Pgk1. (C) Quantification of relative expression levels of Mecp2 and Pgk1 from the 129 allele. The mean ± SD values from the three independent experiments included in Fig 3B are shown. P-values were derived from an unpaired t-test (*p<0.05, **p<0.01).
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pgen.1005430.g003: Truncation of exon 7 of Xist RNA affects the X-linked gene silencing on the Xi during EB differentiation.(A) 129 mutant allele-specific RT-qPCR analysis of X-linked Mecp2 and Pgk1 genes upon differentiation; each was normalized to undifferentiated cells (set to 1) and Gapdh. The mean ± SD values from three independent experiments are shown with the unpaired t test P values (*p<0.05, **p<0.01). (B) Representative allele-specific RT-PCR analysis for Mecp2 and Pgk1. (C) Quantification of relative expression levels of Mecp2 and Pgk1 from the 129 allele. The mean ± SD values from the three independent experiments included in Fig 3B are shown. P-values were derived from an unpaired t-test (*p<0.05, **p<0.01).
Mentions: Intriguingly, the XistdelEx7TsixTST6 mutant clones exhibited impaired localization of the Xist RNA and H3K27me3 on the Xi. This observation prompted us to investigate whether the compromised Xist RNA and H3K27me3 localization on the Xi affects the expression status of X-linked genes on the Xi during EB differentiation using two different approaches (Fig 3): RT-qPCR analysis using allele-specific primers (Fig 3A) and allele-specific RT-PCR using a polymorphic restriction enzyme digestion (Fig 3B and 3C). The allele-specific RT-qPCR analysis using 129 allele-specific primer sets can discriminate the Pgk1 and Mecp2 expression on the mutant 129 X-chromosome from their expression on the Cast X-chromosome. Thus, we can only detect Pgk1 and Mecp2 expression on the mutant 129 X-chromosome. In the control TsixTST6 EB cells on days 8 and 12 of differentiation, the X-linked Mecp2 and Pgk1 expression from the 129 Xi gradually decreased upon differentiation and remained less than 13% and 12% compared to that of the undifferentiated ES cells (day 0), respectively. In contrast to the control cells, the mutant 129 allele-specific RT-qPCR for Mecp2 and Pgk1 in the XistdelEx7TsixTST6 mutant clones showed that the partial silencing of Mecp2 (40% and 25% in clones #1 and #2, respectively) and Pgk1 (55% and 47% in clones #1 and #2, respectively) occurred on day 8 instead of on day 0. The partial silencing of Mecp2 and Pgk1 in the XistdelEx7TsixTST6 mutant cells was then followed by the reactivation of Mecp2 (98% and 89% in clones #1 and #2, respectively) and Pgk1 (64% and 58% in clones #1 and #2, respectively) on day 12 upon differentiation, although the initial phase of silencing in Mecp2 and Pgk1 on day 4 was similar between the TsixTST6 and XistdelEx7TsixTST6 mutant cells. Additionally, to confirm the compromised Mecp2 and Pgk1 expression from the 129 Xi relative to that of the wild-type Cast Xa in the XistdelEx7TsixTST6 mutant cells, allele-specific RT-PCR analysis based on a polymorphic restriction enzyme digestion was performed (Fig 3B and 3C). The ratios of the X-linked gene expression from the 129 Xi were markedly reduced from differentiation day 8 in the TsixTST6 cells. However, in the mutant clones, the ratios of X-linked gene expression from the 129 Xi remained high during the late stages of X-inactivation. Combined with the data in Fig 2, these results suggest that the XistdelEx7TsixTST6 mutant Xist RNA can partially induce the silencing of X-linked genes, but cannot sustain transcriptional repression on the Xi. This is likely due to the loss of the Xist RNA clouds and the H3K27me3 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