X chromosome inactivation (XCI) is a crucial dosage compensation mechanism in mammalian XX females that renders one X chromosome transcriptionally and structurally inactive (Xi). XCI is initiated during embryonic development, by monoallelic upregulation of lncRNA Xist, which coats the chromosome of origin in cis, then recruits and coordinates other silencing machineries. XCI is therefore a valuable model to study how chromatin dynamics can regulate transcriptional silencing.
Currently, the initiation of XCI remains limited in understanding due to its intricate interplay with cell differentiation. In our previous work, we identified the E1A-binding protein p400 as a novel epigenetic candidate associated with XCI, positively influencing Xist expression based on bulk RNA-seq data. p400 typically operates within the TIP60-p400 complex, catalysing histone variant incorporation and activating target gene transcription through histone acetylation. Building on this, we hypothesise that p400 is a positive epigenetic regulator of XCI initiation, by activating Xist transcription in differentiating female mouse embryonic stem cells (mESCs).
To identify the window of p400’s action in XCI, we performed shRNA-based Ep400 knockdown in differentiating mESCs carrying fluorescently labelled X chromosomes. Confirming our hypothesis, we observed an increased proportion of cells retaining two active X chromosomes after Ep400 depletion, implying a potential delay or partial obstruction of XCI. Comparable XCI patterns were also seen in knockdown experiments involving Ruvbl1/2, both constituents of the TIP60-p400 complex. Our ongoing research, utilising RNA fluorescence in situ hybridization (FISH) and allele-specific genomics, seeks to delve into the consequences of Ep400 depletion on Xist activity at the single-cell level and explore the physical interaction between p400 and the silenced X chromosome.
Ultimately, our research endeavours to provide more insights into the initial phase of XCI. Understanding the complex epigenetic regulations involved could enhance therapeutic opportunities to treat X-linked diseases in heterozygous females, by targeted inactivation/reactivation of X-linked alleles.