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Effects of Firre knockdown on mouse gene expression

ABSTRACT: In mammals, genes located on the X chromosome are present in one copy in XY males and two in XX females. To balance the dosage of X-linked gene expression between the sexes one of the two X chromosomes in females is silenced by X inactivation initiated by up-regulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers for silencing. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus. We report a novel role for the X-linked lncRNA Firre in anchoring the inactive mouse X chromosome and preserving one of its main epigenetic features, trimethylation of histone H3 at lysine 27 (H3K27me3). Similar to Dxz4, Firre is expressed from a macrosatellite repeat locus associated with a cluster of CTCF and cohesin binding specifically on the inactive X. CTCF binding initially present in both male and female mouse embryonic stem cells was found to be lost from the active X during development. The Firre and Dxz4 loci on the inactive X were preferentially located adjacent to the nucleolus. Knockdown of Firre RNA disrupted perinucleolar targeting and H3K27me3 levels in mouse fibroblasts, demonstrating an important role for this lncRNA in maintenance of one of the main epigenetic features of the X chromosome. There was no X-linked gene reactivation after Firre knockdown; however, a compensatory increase in the expression of chromatin modifier genes implicated in X silencing was observed. In female ES cells Firre RNA knockdown did not disrupt Xist expression/coating nor silencing of G6pdx during differentiation, suggesting that Firre does not play a role in the onset of X inactivation. We conclude that the X-linked lncRNA Firre helps position the inactive X chromosome near the nucleolus and preserve one of its main epigenetic features.

ORGANISM(S): Mus musculus x Mus spretus

PROVIDER: GSE66172 | GEO | 2015/03/05

SECONDARY ACCESSION(S): PRJNA276013

REPOSITORIES: GEO

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Project description:One of the two X chromosomes in female somatic cells is transcriptionally silenced across cell generations, a classic paradigm of epigenetic regulation. Although most genes are stably silenced, certain X-linked genes escape X-chromosome inactivation (XCI), providing a fundamental dosage difference between females and males. A role for chromosome conformation has been proposed in XCI as the process is accompanied by a massive structural reorganisation. However a detailed molecular understanding of the three-dimensional architecture of the Xi has been lacking. Here we reveal an unusual three-dimensional configuration of the Xi, that provides novel insights into the relationship between gene expression and TAD organisation. Using allele-specific Hi-C, RNA-Seq and ATAC-seq, as well as DNA FISH, we show that the Xi is spatially segregated into two ‘mega-domains’ separated by a 200kb boundary including the DXZ4 macrosatellite, and is globally devoid of typical autosomal structural features such as active/inactive compartments and topologically associating domains (TADs). However, we find that a few regions along the Xi display TAD signatures corresponding to regions containing escape genes, and display DNA accessibility at promoter-proximal TF binding sites. Strikingly, when the DXZ4-containing boundary region is deleted prior to XCI, most facultative escapees are no longer expressed from the Xi, and the escape associated chromatin domains are no longer observed. Deletion of the DXZ4-containing boundary region from the Xi also results in fusion of the two mega-domains. We show that Xist A-repeat containing (gene silencing competent) RNA is sufficient to induce this spatial segregation of the X chromosome. Combined, our results point to a critical role for the DXZ4-boundary region, together with Xist RNA, in shaping the higher order structure of the Xi and in allowing facultative escape from XCI during differentiation. They also point to direct and uncover a close relationships between transcription and TAD formation organisation in the context of the Xi.

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Effects of Firre knockdown on mouse gene expression

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