Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse embyronic stem cells expressing the doxycycline-inducible Xist cDNA transgene inserted into chromosome 12 to study of Xist RNA mediated silencing

ABSTRACT: A doxycycline-inducible Xist cDNA transgene was introduced into mouse chromosome 12 at approximately 110Mb (NCBI build m36). Insertion of the transgene was heterozygous, so only one copy of chromosome 12 carried the transgene, and the other chromosome 12 was wild-type. Upon doxycycline induction, the transgene is expressed, producing Xist transgene RNA which localises to autosomal material in cis, leading to silencing of genes adjacent to the transgene. Due to the heterozygous nature of transgene insertion and the cis nature of silencing, for any silenced gene, the maximum possible downregulation in expression levels was 50%. To quantify the changes in the levels of gene expression in response to Xist transgene expression, total RNA was extracted from differentiated ES cells treated with doxycycline. As background control, total RNA was also prepared from uninduced, differentiated ES cells (cultured in parallel to doxycycline-treated cells). The total RNA was then used to make labelled cRNA, which was hybridised to an Affymetrix GeneChip Mouse Genome 430 2.0 array.

ORGANISM(S): Mus musculus

SUBMITTER: Amy Tang

PROVIDER: E-MEXP-2088 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Efficiency of Xist-mediated silencing on autosomes is linked to chromosomal domain organisation.

Tang Y Amy YA Huntley Derek D Montana Giovanni G Cerase Andrea A Nesterova Tatyana B TB Brockdorff Neil N

Epigenetics & chromatin 20100507 1

<h4>Background</h4>X chromosome inactivation, the mechanism used by mammals to equalise dosage of X-linked genes in XX females relative to XY males, is triggered by chromosome-wide localisation of a cis-acting non-coding RNA, Xist. The mechanism of Xist RNA spreading and Xist-dependent silencing is poorly understood. A large body of evidence indicates that silencing is more efficient on the X chromosome than on autosomes, leading to the idea that the X chromosome has acquired sequences that faci ...[more]

PMID: 20459652

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Project description:JARID2 ChIP-seq profile mediated by Xist was investigated using two separate approaches: In the first approach, we use the TX1072 female embryonic stem cell (ESC) line; this is a genetically polymorphic ESC line derived from a mouse with one X-chromosome (X-chr) from the Mus musculus castaneus (Cast) origin and the other of Mus musculus domesticus, C57BL/6 (BL6) strain origin, containing an inducible promoter on the BL6 Xist allele; differentiation and simultaneous induction of Xist for 4 days results in the inactivation of ONLY the BL6-derived X-chr and coating by JARID2 protein. We analysed this ESC line in the undifferentiated state, in which both X-chrs are active (and not coated by JARID2), and also after 4 days of differentiation under Xist inducible conditions, in which cells display one inactive X-chr from BL6 origin and one Xa of the Cast origin; the presence of SNPs allows for the comparison of the degree and localisation of JARID2 enrichment on the inactive X-chr versus the active X-chr; our results suggests that JARID2 is enriched on the inactive X-chr chromosome-wide and not in confined peaks as elsewhere in the genome; This pattern resembles patterns of Xist enrichment published before (Engreitz et al., 2013). In the second approach, we use a male ESC line that carries an inducible Xist transgene (TG) on chromosome 11 (chr11) (Wutz et al., 2000) in the presence (36:11 WT) or the absence of Eed (36:11 Eed-/-) Induction of Xist TG results in Xist coating and enrichment of JARID2 across the chr11, regardless of the presence of Eed when assess by IF/Xist RNA FISH experiments. We compare inducible and uninducible condition in both the 36:11 WT and Eed-/- ESCs; this way, we could assess the Xist-mediated JARID2 recruitment and the effect of the absence of Eed-/-; Our results shows that Xist induction result in a chromosome-wide enrichment of JARID2 on chr11, in a manner that resembles enrichment on the inactive X-chr; this pattern seem to be independent of EED, showing that in contrast to other regions in the genome where JARID2 occupancy is abrogated in the absence of EED, Xist-mediated JARID2 recruitment is not affected. JARID2 ChIP-seq analysis in differentiating female ESCs (TX1072) and in a male ESC harbouring a Xist transgene on chromosome 11 in WT and Eed-/- genetics settings

Dataset Information

Transcription profiling of mouse embyronic stem cells expressing the doxycycline-inducible Xist cDNA transgene inserted into chromosome 12 to study of Xist RNA mediated silencing

Publications

Efficiency of Xist-mediated silencing on autosomes is linked to chromosomal domain organisation.

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