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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.

2022Journal article

Unknown

Generation and characterization of induced pluripotent stem cell line (IBBISTi004-A) from an Angelman syndrome patient carrying a class II deletion of the maternal chromosome 15q11.2-q13

Publication . Maranga, Carina; Pereira, Carolina; Raposo, Ana Cláudia; Vieira, Adriana A.; Duarte, Sofia; Bekman, Evguenia; Milagre, Inês; da Rocha, Simão T.

Angelman Syndrome is a rare neurodevelopmental disorder caused by several (epi)genetic alterations. The patients present strong neurological impairment due to the absence of a functional maternal UBE3A gene in neurons. Here, we generated and characterized a new induced pluripotent stem cell (iPSC) line from a female child with Angelman syndrome harbouring a class II deletion. iPSCs were reprogrammed from fibroblasts using Sendai viruses. The new iPSCs express pluripotency markers, are capable of trilineage in vitro differentiation and have the expected imprinting status of Angelman syndrome. These iPSCs are a valuable tool to elucidate the pathophysiological mechanisms associated with this disease.

2022Journal article

Unknown

The influence of DNA methylation on monoallelic expression

Publication . da Rocha, Simão T.; Gendrel, Anne-Valerie

Monoallelic gene expression occurs in diploid cells when only one of the two alleles of a gene is active. There are three main classes of genes that display monoallelic expression in mammalian genomes: (1) imprinted genes that are monoallelically expressed in a parent-of-origin dependent manner; (2) X-linked genes that undergo random X-chromosome inactivation in female cells; (3) random monoallelically expressed single and clustered genes located on autosomes. The heritability of monoallelic expression patterns during cell divisions implies that epigenetic mechanisms are involved in the cellular memory of these expression states. Among these, methylation of CpG sites on DNA is one of the best described modification to explain somatic inheritance. Here, we discuss the relevance of DNA methylation for the establishment and maintenance of monoallelic expression patterns among these three groups of genes, and how this is intrinsically linked to development and cellular states.

2019Journal article

Open access

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.

2021Journal article

Open access