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Functional dissection of the XIST non-coding RNA in X-chromosome inactivation in human ESCs
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Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation
Publication . Arez, Maria; Eckersley-Maslin, Melanie; Klobuar, Tajda; von Gilsa Lopes, João; Krueger, Felix; Mupo, Annalisa; Raposo, Ana Cláudia; Oxley, David; Mancino, Samantha; Gendrel, Anne-Valerie; Jesus, Bruno Bernardes De; da Rocha, Simão T.
Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.
H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensabe for inducing gene silencing
Publication . Tjalsma, Sjoerd J. D.; Hori, Mayako; Sato, Yuko; Bousard, Aurelie; Ohi, Akito; Raposo, Ana Cláudia; Roensch, Julia; Le Saux, Agnes; Nogami, Jumpei; Maehara, Kazumitsu; Kujirai, Tomoya; Handa, Tetsuya; Bagés-Arnal, Sandra; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi; da Rocha, Simão T.; Żylicz, Jan J.; Kimura, Hiroshi; Heard, Edith
During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non-coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3-specific intracellular antibody or H3K27me3-mintbody. By combining live-cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP-seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.
The tandem repeat modules of Xist lncRNA: a swiss army knife for the control of X-chromosome inactivation
Publication . Raposo, Ana Cláudia; Casanova, Miguel; Gendrel, Anne-Valerie; da Rocha, Simão T.
X-inactive-specific transcript (Xist) is a long non-coding RNA (lncRNA) essential for X-chromosome inactivation (XCI) in female placental mammals. Thirty years after its discovery, it is still puzzling how this lncRNA triggers major structural and transcriptional changes leading to the stable silencing of an entire chromosome. Recently, a series of studies in mouse cells have uncovered domains of functional specialization within Xist mapping to conserved tandem repeat regions, known as Repeats A-to-F. These functional domains interact with various RNA binding proteins (RBPs) and fold into distinct RNA structures to execute specific tasks in a synergistic and coordinated manner during the inactivation process. This modular organization of Xist is mostly conserved in humans, but recent data point towards differences regarding functional specialization of the tandem repeats between the two species. In this review, we summarize the recent progress on understanding the role of Xist repetitive blocks and their involvement in the molecular mechanisms underlying XCI. We also discuss these findings in the light of the similarities and differences between mouse and human Xist.
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Fundação para a Ciência e a Tecnologia
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SFRH/BD/137099/2018