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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 in ESCs(A) Schematic representation of viral gene trap insertions in Xist and Spen locus (not to scale, gene size is indicated). Chr, chromosome.(B) Schematic representation of the Spen gene locus showing the location of CRISPR/Cas9 guide RNAs (gRNAs) used for engineering a deletion and genotyping PCR primers.(C) Genomic PCR confirming the absence of wild-type Spen fragment in Spen mutant ESCs.(D) Quantitative expression analysis of Spen using primer sets spanning exons as indicated. ΔSpen ESCs lack transcript from the deleted region. Error bars represent SD (n = 3).(E) Cell survival of control HATX3 ESCs and derived ΔSpen ESCs clone 2 (top; n = 2) and clone 3 (bottom; n = 3) after Xist induction (+ dox). Survival was calculated relative to uninduced cells. Error bars represent SD.See also Figure S1.
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fig2: Mutation of Spen in ESCs(A) Schematic representation of viral gene trap insertions in Xist and Spen locus (not to scale, gene size is indicated). Chr, chromosome.(B) Schematic representation of the Spen gene locus showing the location of CRISPR/Cas9 guide RNAs (gRNAs) used for engineering a deletion and genotyping PCR primers.(C) Genomic PCR confirming the absence of wild-type Spen fragment in Spen mutant ESCs.(D) Quantitative expression analysis of Spen using primer sets spanning exons as indicated. ΔSpen ESCs lack transcript from the deleted region. Error bars represent SD (n = 3).(E) Cell survival of control HATX3 ESCs and derived ΔSpen ESCs clone 2 (top; n = 2) and clone 3 (bottom; n = 3) after Xist induction (+ dox). Survival was calculated relative to uninduced cells. Error bars represent SD.See also Figure S1.

Mentions: For generating a genome-wide set of mutations, we adopted a viral gene trap mutagenesis strategy that has been previously devised for screening in haploid human tumor cells (Carette et al., 2009). We infected 80 million haploid HATX ESCs, which were sorted for a haploid genome content one passage earlier, with a high-titer gene trap vector retrovirus preparation. We confirmed efficient infection by analyzing the fluorescence of a promoter-less EGFP reporter that is encoded by the gene trap virus (Jae et al., 2014) (Figure 1G). The cell pools were subsequently divided into two samples that were either subjected to selection by inducing Xist expression or cultured without doxycycline for obtaining control samples. DNA from both control and selected samples was prepared and used for identification of viral insertion sites through next-generation sequencing (NGS) (Carette et al., 2009). We calculated the number of independent insertions for each gene in selected and control data sets and ranked candidates according to the fold increase in the number of insertions in selected samples over controls. As anticipated, Xist was found on top of the list of candidates (Tables S1 and S2). Insertions were preferentially observed within the 5′-most region of Xist and in an orientation that aligns the poly-adenylation sequences of the gene trap with the transcription unit consistent with the potential termination of Xist transcription (Figure 2A). The ROSA26 locus from which the tetracycline responsive transactivator nls-rtTA is expressed was also among the selected candidates. A large number of insertions further mapped within the nls-rtTA cDNA (Figure S1), demonstrating that our screen robustly recovered factors that were anticipated from the technical setup of the screen.


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 in ESCs(A) Schematic representation of viral gene trap insertions in Xist and Spen locus (not to scale, gene size is indicated). Chr, chromosome.(B) Schematic representation of the Spen gene locus showing the location of CRISPR/Cas9 guide RNAs (gRNAs) used for engineering a deletion and genotyping PCR primers.(C) Genomic PCR confirming the absence of wild-type Spen fragment in Spen mutant ESCs.(D) Quantitative expression analysis of Spen using primer sets spanning exons as indicated. ΔSpen ESCs lack transcript from the deleted region. Error bars represent SD (n = 3).(E) Cell survival of control HATX3 ESCs and derived ΔSpen ESCs clone 2 (top; n = 2) and clone 3 (bottom; n = 3) after Xist induction (+ dox). Survival was calculated relative to uninduced cells. Error bars represent SD.See also Figure S1.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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fig2: Mutation of Spen in ESCs(A) Schematic representation of viral gene trap insertions in Xist and Spen locus (not to scale, gene size is indicated). Chr, chromosome.(B) Schematic representation of the Spen gene locus showing the location of CRISPR/Cas9 guide RNAs (gRNAs) used for engineering a deletion and genotyping PCR primers.(C) Genomic PCR confirming the absence of wild-type Spen fragment in Spen mutant ESCs.(D) Quantitative expression analysis of Spen using primer sets spanning exons as indicated. ΔSpen ESCs lack transcript from the deleted region. Error bars represent SD (n = 3).(E) Cell survival of control HATX3 ESCs and derived ΔSpen ESCs clone 2 (top; n = 2) and clone 3 (bottom; n = 3) after Xist induction (+ dox). Survival was calculated relative to uninduced cells. Error bars represent SD.See also Figure S1.
Mentions: For generating a genome-wide set of mutations, we adopted a viral gene trap mutagenesis strategy that has been previously devised for screening in haploid human tumor cells (Carette et al., 2009). We infected 80 million haploid HATX ESCs, which were sorted for a haploid genome content one passage earlier, with a high-titer gene trap vector retrovirus preparation. We confirmed efficient infection by analyzing the fluorescence of a promoter-less EGFP reporter that is encoded by the gene trap virus (Jae et al., 2014) (Figure 1G). The cell pools were subsequently divided into two samples that were either subjected to selection by inducing Xist expression or cultured without doxycycline for obtaining control samples. DNA from both control and selected samples was prepared and used for identification of viral insertion sites through next-generation sequencing (NGS) (Carette et al., 2009). We calculated the number of independent insertions for each gene in selected and control data sets and ranked candidates according to the fold increase in the number of insertions in selected samples over controls. As anticipated, Xist was found on top of the list of candidates (Tables S1 and S2). Insertions were preferentially observed within the 5′-most region of Xist and in an orientation that aligns the poly-adenylation sequences of the gene trap with the transcription unit consistent with the potential termination of Xist transcription (Figure 2A). The ROSA26 locus from which the tetracycline responsive transactivator nls-rtTA is expressed was also among the selected candidates. A large number of insertions further mapped within the nls-rtTA cDNA (Figure S1), demonstrating that our screen robustly recovered factors that were anticipated from the technical setup of the screen.

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