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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

Characterization of Xist-Inducible Mouse Haploid ESCs(A) Structure of the Xist inducible system.(B) Chromosome spread of haploid HATX ESCs.(C) Xist induction (+ dox) leads to loss of cells compared to growth in absence of doxycycline (no dox).(D) Xist RNA FISH in HATX3 ESCs grown with and without doxycycline.(E) Immunofluorescence image showing focal Ezh2 and H3K27me3 staining in HATX3 ESCs after Xist induction.(F) qRT-PCR analysis showing induction of Xist and repression of X-linked genes in HATX3 ESCs after doxycycline addition. The autosomal β-actin gene is shown as a control. NT, nontreated.(G) Flow cytometry profile of HATX3 ESCs before (red) and after (green) infection with gene trap viruses. DNA content (left) and GFP fluorescence (right) of a gene trap encoded reporter are shown. A mixed haploid/diploid DNA content profile is shown for reference in gray.
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fig1: Characterization of Xist-Inducible Mouse Haploid ESCs(A) Structure of the Xist inducible system.(B) Chromosome spread of haploid HATX ESCs.(C) Xist induction (+ dox) leads to loss of cells compared to growth in absence of doxycycline (no dox).(D) Xist RNA FISH in HATX3 ESCs grown with and without doxycycline.(E) Immunofluorescence image showing focal Ezh2 and H3K27me3 staining in HATX3 ESCs after Xist induction.(F) qRT-PCR analysis showing induction of Xist and repression of X-linked genes in HATX3 ESCs after doxycycline addition. The autosomal β-actin gene is shown as a control. NT, nontreated.(G) Flow cytometry profile of HATX3 ESCs before (red) and after (green) infection with gene trap viruses. DNA content (left) and GFP fluorescence (right) of a gene trap encoded reporter are shown. A mixed haploid/diploid DNA content profile is shown for reference in gray.

Mentions: We derived haploid ESC lines from XistTX/TXR26nlsrtTA/nlsrtTA mice that carry an inducible promoter inserted within the Xist locus and a tetracycline-regulated transactivator targeted to the ROSA 26 locus on chromosome 6 (Savarese et al., 2006) (Figure 1A). We obtained 11 haploid ESC lines (HATX-1 to HATX-11) from a total of 170 activated oocytes (Figure 1B). Addition of doxycycline to HATX cultures induced Xist expression from the single X chromosome and caused cell loss (Figures 1C and 1D). After Xist induction, we observed focal recruitment of the Polycomb protein Ezh2 and the associated histone modification H3K27me3 (Figure 1E). qRT-PCR analysis further showed the repression of X-linked genes, but not of an autosomal control gene, after Xist induction in HATX ESCs (Figure 1F). These observations are consistent with earlier findings that Xist expression initiates X inactivation in ESCs (Wutz and Jaenisch, 2000; Wutz et al., 2002). In haploid cells, loss of gene expression from the single-X chromosome is not compatible with cell survival. The Xist-induced lethality then provides an efficient selection strategy for recovering mutations abrogating the silencing pathway of Xist and, therefore, preventing Xist-dependent cell death.


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)

Characterization of Xist-Inducible Mouse Haploid ESCs(A) Structure of the Xist inducible system.(B) Chromosome spread of haploid HATX ESCs.(C) Xist induction (+ dox) leads to loss of cells compared to growth in absence of doxycycline (no dox).(D) Xist RNA FISH in HATX3 ESCs grown with and without doxycycline.(E) Immunofluorescence image showing focal Ezh2 and H3K27me3 staining in HATX3 ESCs after Xist induction.(F) qRT-PCR analysis showing induction of Xist and repression of X-linked genes in HATX3 ESCs after doxycycline addition. The autosomal β-actin gene is shown as a control. NT, nontreated.(G) Flow cytometry profile of HATX3 ESCs before (red) and after (green) infection with gene trap viruses. DNA content (left) and GFP fluorescence (right) of a gene trap encoded reporter are shown. A mixed haploid/diploid DNA content profile is shown for reference in gray.
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Related In: Results  -  Collection

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fig1: Characterization of Xist-Inducible Mouse Haploid ESCs(A) Structure of the Xist inducible system.(B) Chromosome spread of haploid HATX ESCs.(C) Xist induction (+ dox) leads to loss of cells compared to growth in absence of doxycycline (no dox).(D) Xist RNA FISH in HATX3 ESCs grown with and without doxycycline.(E) Immunofluorescence image showing focal Ezh2 and H3K27me3 staining in HATX3 ESCs after Xist induction.(F) qRT-PCR analysis showing induction of Xist and repression of X-linked genes in HATX3 ESCs after doxycycline addition. The autosomal β-actin gene is shown as a control. NT, nontreated.(G) Flow cytometry profile of HATX3 ESCs before (red) and after (green) infection with gene trap viruses. DNA content (left) and GFP fluorescence (right) of a gene trap encoded reporter are shown. A mixed haploid/diploid DNA content profile is shown for reference in gray.
Mentions: We derived haploid ESC lines from XistTX/TXR26nlsrtTA/nlsrtTA mice that carry an inducible promoter inserted within the Xist locus and a tetracycline-regulated transactivator targeted to the ROSA 26 locus on chromosome 6 (Savarese et al., 2006) (Figure 1A). We obtained 11 haploid ESC lines (HATX-1 to HATX-11) from a total of 170 activated oocytes (Figure 1B). Addition of doxycycline to HATX cultures induced Xist expression from the single X chromosome and caused cell loss (Figures 1C and 1D). After Xist induction, we observed focal recruitment of the Polycomb protein Ezh2 and the associated histone modification H3K27me3 (Figure 1E). qRT-PCR analysis further showed the repression of X-linked genes, but not of an autosomal control gene, after Xist induction in HATX ESCs (Figure 1F). These observations are consistent with earlier findings that Xist expression initiates X inactivation in ESCs (Wutz and Jaenisch, 2000; Wutz et al., 2002). In haploid cells, loss of gene expression from the single-X chromosome is not compatible with cell survival. The Xist-induced lethality then provides an efficient selection strategy for recovering mutations abrogating the silencing pathway of Xist and, therefore, preventing Xist-dependent cell death.

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