Selective reactivation of human X-linked genes in female fibroblasts reprogrammed by cell fusion
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ABSTRACT:
PROVIDER: PRJNA258003 | ENA |
REPOSITORIES: ENA
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Project description:In female mammals, one of the two X chromosomes is silenced as pluripotent cells differentiate. This process, termed X-inactivation, is random and results in mosaic expression of many X-linked genes in female tissues. Although X-inactivation can be reversed in vitro by reprogramming, the extent varies according to species, operator and conditions. Here, we have reprogrammed cloned female human fibroblasts by fusing them with mouse ESCs and examined the kinetics of X-linked gene re-expression. Allele-specific RNA sequencing analysis was used to determine the expression from the inactive X chromosome. This study revealed that a subset of loci within domains prone to escape X-inactivation, or that showed variable expression between somatic clones, were consistently reactivated upon pluripotent reprogramming. This suggests that stochastic events may be stabilised by reprogramming, and conversely, that variable expression is predictive for Xi genes susceptible to re-activation.
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Project description:Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we used cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show a rapid and widespread loss of Xi-associated H3K27me3 and XIST in fused cells that precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-Seq analysis confirmed that Xi reactivation remained partial and showed that induction of human pluripotency-specific XACT transcripts occurred, but was rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation, and suggest a hierarchy where early events such as XIST-delocalisation, are required but are insufficient to establish stable human X reactivation. We performed RNA-sequencing of human fibroblast clones derived from TERT-immortalised NHDF17914 (Lonza) before and at 5 days after fusion with mouse ESC (E14Tg2a:puroR). Two biological replicates were performed and sequenced.
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Project description:Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we used cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show a rapid and widespread loss of Xi-associated H3K27me3 and XIST in fused cells that precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-Seq analysis confirmed that Xi reactivation remained partial and showed that induction of human pluripotency-specific XACT transcripts occurred, but was rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation, and suggest a hierarchy where early events such as XIST-delocalisation, are required but are insufficient to establish stable human X reactivation.
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Project description:To investigate the dynamics of X-chromosome reactivation, we differentiated mouse embryonic stem cells (ESCs) into neural precursor cells (NPCs) and later reprogrammed them into induced pluripotent stem cells (iPSCs). During the conversions of NPCs into iPSC, we assessed the kinetics of X-chromosome reactivation. We generated allele-specific RNA-seq datasets to assess gene reactivation dynamics, employed ATAC-seq to investigate chromatin opening dynamics and performed in situ Hi-C to explore dynamics of chromosome structure.
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Project description:Female human induced pluripotent stem cell (hiPSC) lines exhibit considerable variability in X-inactivation status. Some lines maintain one transcriptionally active X chromosome (Xa) and one inactive X (Xi) from donor cells. However, hiPSC lines that have two Xas are infrequently produced. We show here Xinactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated using the Kyoto method, which employs leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Lines derived on other feeders maintained an Xi. In addition, there appears to be a window in which SNL feeders promote Xi-reactivation. Upon differentiation, Xa/Xa hiPSCs silenced one X. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and inactivation. Gene expression patterns were compared between several human embryonic stem cell (hESC) and hiPSC lines. Gene expression ratios between genes on X and those on autosomes were calculated from each cell lines.
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Project description:Viral infection leads to heterogeneous cellular outcomes ranging from resistance to virion amplification and cell death. In this study, we apply single-cell techniques to analyze diverse fate trajectories during Epstein-Barr virus (EBV) lytic reactivation in three B cell models. We expand upon the previous finding that c-Myc regulates lytic reactivation and determine that MYC downregulation during the lytic phase is correlated with a loss of germinal center (GC) B cell expression signatures. Consistent with prior work, lytic induction yields both abortive and complete reactivation. Abortive lytic cells, which express high STAT3 and initiate but do not complete the lytic cycle, also upregulate NF-kB and IRF3 pathway target genes. Cells that proceeded through the full lytic cycle exhibited unexpected and striking expression of genes associated with cellular reprogramming, developmental progenitor phenotypes, and cytokine upregulation. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.
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Project description:Female human induced pluripotent stem cell (hiPSC) lines exhibit considerable variability in X-inactivation status. Some lines maintain one transcriptionally active X chromosome (Xa) and one inactive X (Xi) from donor cells. However, hiPSC lines that have two Xas are infrequently produced. We show here Xinactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated using the Kyoto method, which employs leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Lines derived on other feeders maintained an Xi. In addition, there appears to be a window in which SNL feeders promote Xi-reactivation. Upon differentiation, Xa/Xa hiPSCs silenced one X. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and inactivation.
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Project description:HHV-6A is a human herpesvirus that integrates into human sub telomeric regions to acquire latency. This latent virus frequently reactivates causing numerous diseases. The project was aimed to understand changes in host cell prteomics upon virus reactivation, which might helpin understanding the pathophysiology of virus reactivation.
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