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Identification of Spen as a Crucial Factor for Xist Function through Forward Genetic Screening in Haploid Embryonic Stem Cells.

Monfort A, Di Minin G, Postlmayr A, Freimann R, Arieti F, Thore S, Wutz A - Cell Rep (2015)

Bottom Line: This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist.Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends.However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Hönggerberg, Otto-Stern-Weg 7, 8093 Zurich, Switzerland.

No MeSH data available.


Related in: MedlinePlus

Spen Is Required for Xist Function in ESCs and Binds Xist A-Repeat Sequences In Vitro(A and B) qRT-PCR analysis of the X-linked genes (A) and the autosomal control genes (B) as indicated in HATX and Spen mutant ESCs. Error bars represent SD (n = 3). The locations of the genes tested on the X chromosome (chr) is shown at the left. q represents the region of the chromosome. +dox, Xist induction; NT, nontreated.(C) Electrophoretic mobility shift analysis of Spen RRM domains using a 32P-labeled synthetic XCR RNA probe and cold XCR, SRA, and tRNA competitors as indicated. Asterisk and black triangle indicate position of the RNA-protein complex and free probe, respectively.(D) Competition with cold XS1 and XNX competitors as in (C).See also Figure S2.
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fig3: Spen Is Required for Xist Function in ESCs and Binds Xist A-Repeat Sequences In Vitro(A and B) qRT-PCR analysis of the X-linked genes (A) and the autosomal control genes (B) as indicated in HATX and Spen mutant ESCs. Error bars represent SD (n = 3). The locations of the genes tested on the X chromosome (chr) is shown at the left. q represents the region of the chromosome. +dox, Xist induction; NT, nontreated.(C) Electrophoretic mobility shift analysis of Spen RRM domains using a 32P-labeled synthetic XCR RNA probe and cold XCR, SRA, and tRNA competitors as indicated. Asterisk and black triangle indicate position of the RNA-protein complex and free probe, respectively.(D) Competition with cold XS1 and XNX competitors as in (C).See also Figure S2.

Mentions: Next, we investigated whether mutation of Spen abrogates the ability of Xist to induce X inactivation. For this, we measured cell survival after Xist induction. Whereas induction of Xist caused cell loss in parental HATX3 ESCs, cells with a deletion of Spen appeared unaffected (Figure 2E). To further confirm that Xist was induced and repression of X-linked genes was abrogated, we performed qRT-PCR analysis. Xist induction was observed 24 and 48 hr after doxycycline addition in Spen mutant and control cells (Figure 3A). Repression of X-linked genes was observed in HATX3 control cells but not in cells carrying a Spen deletion. Autosomal control genes remained unaffected by Xist in both Spen mutant and wild-type cells (Figure 3B). Taken together, these observations showed that a mutation in Spen abrogated the ability of Xist for initiating gene repression. We further analyzed the effect of the Spen deletion in clone 36 ESCs, in which Xist induction represses a puromycin-resistance gene that was co-integrated with the Xist transgene into chromosome 11 and the imprinted Meg1 gene. Xist induction caused efficient repression of the puromycin marker after 48 hr in control cells but less efficient repression in cells lacking Spen (Figure S2C). Consistent with this observation, Spen mutant cells showed an increased survival in the presence of puromycin and less efficient silencing of Meg1 when Xist was induced (Figures S2D–S2F), indicating that loss of Spen also led to a reduced efficiency of Xist-mediated repression in 36 ESCs. Taken together, our results demonstrate that Spen is required for gene repression by Xist in ESCs.


Identification of Spen as a Crucial Factor for Xist Function through Forward Genetic Screening in Haploid Embryonic Stem Cells.

Monfort A, Di Minin G, Postlmayr A, Freimann R, Arieti F, Thore S, Wutz A - Cell Rep (2015)

Spen Is Required for Xist Function in ESCs and Binds Xist A-Repeat Sequences In Vitro(A and B) qRT-PCR analysis of the X-linked genes (A) and the autosomal control genes (B) as indicated in HATX and Spen mutant ESCs. Error bars represent SD (n = 3). The locations of the genes tested on the X chromosome (chr) is shown at the left. q represents the region of the chromosome. +dox, Xist induction; NT, nontreated.(C) Electrophoretic mobility shift analysis of Spen RRM domains using a 32P-labeled synthetic XCR RNA probe and cold XCR, SRA, and tRNA competitors as indicated. Asterisk and black triangle indicate position of the RNA-protein complex and free probe, respectively.(D) Competition with cold XS1 and XNX competitors as in (C).See also Figure S2.
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fig3: Spen Is Required for Xist Function in ESCs and Binds Xist A-Repeat Sequences In Vitro(A and B) qRT-PCR analysis of the X-linked genes (A) and the autosomal control genes (B) as indicated in HATX and Spen mutant ESCs. Error bars represent SD (n = 3). The locations of the genes tested on the X chromosome (chr) is shown at the left. q represents the region of the chromosome. +dox, Xist induction; NT, nontreated.(C) Electrophoretic mobility shift analysis of Spen RRM domains using a 32P-labeled synthetic XCR RNA probe and cold XCR, SRA, and tRNA competitors as indicated. Asterisk and black triangle indicate position of the RNA-protein complex and free probe, respectively.(D) Competition with cold XS1 and XNX competitors as in (C).See also Figure S2.
Mentions: Next, we investigated whether mutation of Spen abrogates the ability of Xist to induce X inactivation. For this, we measured cell survival after Xist induction. Whereas induction of Xist caused cell loss in parental HATX3 ESCs, cells with a deletion of Spen appeared unaffected (Figure 2E). To further confirm that Xist was induced and repression of X-linked genes was abrogated, we performed qRT-PCR analysis. Xist induction was observed 24 and 48 hr after doxycycline addition in Spen mutant and control cells (Figure 3A). Repression of X-linked genes was observed in HATX3 control cells but not in cells carrying a Spen deletion. Autosomal control genes remained unaffected by Xist in both Spen mutant and wild-type cells (Figure 3B). Taken together, these observations showed that a mutation in Spen abrogated the ability of Xist for initiating gene repression. We further analyzed the effect of the Spen deletion in clone 36 ESCs, in which Xist induction represses a puromycin-resistance gene that was co-integrated with the Xist transgene into chromosome 11 and the imprinted Meg1 gene. Xist induction caused efficient repression of the puromycin marker after 48 hr in control cells but less efficient repression in cells lacking Spen (Figure S2C). Consistent with this observation, Spen mutant cells showed an increased survival in the presence of puromycin and less efficient silencing of Meg1 when Xist was induced (Figures S2D–S2F), indicating that loss of Spen also led to a reduced efficiency of Xist-mediated repression in 36 ESCs. Taken together, our results demonstrate that Spen is required for gene repression by Xist in ESCs.

Bottom Line: This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist.Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends.However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Hönggerberg, Otto-Stern-Weg 7, 8093 Zurich, Switzerland.

No MeSH data available.


Related in: MedlinePlus