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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

Mutation of Spen Reduces Recruitment of Polycomb Proteins(A, C, E, and F) Combined Xist RNA FISH and Ezh2 (A), Ring1b (C), acetylated histone H4 (E), and RNA polymerase II (F) immunofluorescence analysis of Spen mutant and control ESCs after 24 hr of induction with doxycycline. Scale bar, 5 μm.(B and D) Quantification of focal recruitment of Ezh2 (B) and Ring1b (D) by Xist expression in 2i-cultured (2i) and 24-hr-differentiated (retinoic acid; RA) ESCs (n = 100). cl 2 and cl 3 represent clones 2 and 3, respectively.See also Figures S3 and S4.
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fig4: Mutation of Spen Reduces Recruitment of Polycomb Proteins(A, C, E, and F) Combined Xist RNA FISH and Ezh2 (A), Ring1b (C), acetylated histone H4 (E), and RNA polymerase II (F) immunofluorescence analysis of Spen mutant and control ESCs after 24 hr of induction with doxycycline. Scale bar, 5 μm.(B and D) Quantification of focal recruitment of Ezh2 (B) and Ring1b (D) by Xist expression in 2i-cultured (2i) and 24-hr-differentiated (retinoic acid; RA) ESCs (n = 100). cl 2 and cl 3 represent clones 2 and 3, respectively.See also Figures S3 and S4.

Mentions: To further explore whether loss of Spen affected Xist-mediated chromatin modifications, we performed Xist RNA fluorescence in situ hybridization (FISH) and immunofluorescence staining. Xist clusters in control and Spen mutant cells appeared with similar efficiency (Figures S2F, S3A, and S3B), suggesting that Xist localization was not affected by the deletion of Spen. The Polycomb proteins Ezh2 and Ring1b were also recruited by Xist, and focal enrichment of H3K27me3 could be observed in the absence of Spen (Figures 4A and 4C; Figures S3 and S4). Similarly, Xist clusters appeared in a volume that was characterized by low signals of acetylated histone H4 and RNA polymerase II, consistent with the presence of a repressive compartment (Figures 4E and 4F). However, Ezh2 and H3K27me3 foci were less prominent in Spen mutant cells compared to control cells. Combined immunofluorescence staining and RNA FISH showed that the efficiency of Ring1b and Ezh2 recruitment to the Xist cluster was reduced in Spen mutant cells (Figures 4B, 4D, and S4). Taken together, these data demonstrate that loss of Spen resulted in a decreased efficiency of recruitment of chromatin modifications by Xist in ESCs. These results are consistent with previous observations that a mutant Xist RNA lacking A repeat can localize and recruit chromatin modifications but does not initiate gene repression (Wutz et al., 2002). The A-repeat mutant Xist RNA also displays a strong deficit in Polycomb recruitment in ESCs, but recruitment increases upon entry of ESCs into differentiation (Kohlmaier et al., 2004). However, we did not see an increase in efficiency of recruitment of Ring1b and Ezh2 in Spen mutant cells upon entry in differentiation (Figures 4B and 4D).


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)

Mutation of Spen Reduces Recruitment of Polycomb Proteins(A, C, E, and F) Combined Xist RNA FISH and Ezh2 (A), Ring1b (C), acetylated histone H4 (E), and RNA polymerase II (F) immunofluorescence analysis of Spen mutant and control ESCs after 24 hr of induction with doxycycline. Scale bar, 5 μm.(B and D) Quantification of focal recruitment of Ezh2 (B) and Ring1b (D) by Xist expression in 2i-cultured (2i) and 24-hr-differentiated (retinoic acid; RA) ESCs (n = 100). cl 2 and cl 3 represent clones 2 and 3, respectively.See also Figures S3 and S4.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4530576&req=5

fig4: Mutation of Spen Reduces Recruitment of Polycomb Proteins(A, C, E, and F) Combined Xist RNA FISH and Ezh2 (A), Ring1b (C), acetylated histone H4 (E), and RNA polymerase II (F) immunofluorescence analysis of Spen mutant and control ESCs after 24 hr of induction with doxycycline. Scale bar, 5 μm.(B and D) Quantification of focal recruitment of Ezh2 (B) and Ring1b (D) by Xist expression in 2i-cultured (2i) and 24-hr-differentiated (retinoic acid; RA) ESCs (n = 100). cl 2 and cl 3 represent clones 2 and 3, respectively.See also Figures S3 and S4.
Mentions: To further explore whether loss of Spen affected Xist-mediated chromatin modifications, we performed Xist RNA fluorescence in situ hybridization (FISH) and immunofluorescence staining. Xist clusters in control and Spen mutant cells appeared with similar efficiency (Figures S2F, S3A, and S3B), suggesting that Xist localization was not affected by the deletion of Spen. The Polycomb proteins Ezh2 and Ring1b were also recruited by Xist, and focal enrichment of H3K27me3 could be observed in the absence of Spen (Figures 4A and 4C; Figures S3 and S4). Similarly, Xist clusters appeared in a volume that was characterized by low signals of acetylated histone H4 and RNA polymerase II, consistent with the presence of a repressive compartment (Figures 4E and 4F). However, Ezh2 and H3K27me3 foci were less prominent in Spen mutant cells compared to control cells. Combined immunofluorescence staining and RNA FISH showed that the efficiency of Ring1b and Ezh2 recruitment to the Xist cluster was reduced in Spen mutant cells (Figures 4B, 4D, and S4). Taken together, these data demonstrate that loss of Spen resulted in a decreased efficiency of recruitment of chromatin modifications by Xist in ESCs. These results are consistent with previous observations that a mutant Xist RNA lacking A repeat can localize and recruit chromatin modifications but does not initiate gene repression (Wutz et al., 2002). The A-repeat mutant Xist RNA also displays a strong deficit in Polycomb recruitment in ESCs, but recruitment increases upon entry of ESCs into differentiation (Kohlmaier et al., 2004). However, we did not see an increase in efficiency of recruitment of Ring1b and Ezh2 in Spen mutant cells upon entry in differentiation (Figures 4B and 4D).

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