Compromised mitotic fidelity in human pluripotent stem cells

Human pluripotent stem cells (PSCs), which include both embryonic and induced pluripotent stem cells, are widely used in fundamental and applied biomedical research. They have been instrumental for better understanding development and cell differentiation processes, disease origin and progression and can aid in the discovery of new drugs. PSCs also hold great potential in regenerative medicine to treat or diminish the effects of certain debilitating diseases, such as degenerative disorders. However, some concerns have recently been raised over their safety for use in regenerative medicine. One of the major concerns is the fact that PSCs are prone to errors in passing the correct number of chromosomes to daughter cells, resulting in aneuploid cells. Aneuploidy, characterised by an imbalance in chromosome number, elicits the upregulation of different stress pathways that are deleterious to cell homeostasis, impair proper embryo development and potentiate cancer development. In this review, we will summarize known molecular mechanisms recently revealed to impair mitotic fidelity in human PSCs and the consequences of the decreased mitotic fidelity of these cells. We will finish with speculative views on how the physiological characteristics of PSCs can affect the mitotic machinery and how their suboptimal mitotic fidelity may be circumvented.info:eu-repo/semantics/publishedVersio

Identification of the regulatory mechanisms and factors involved in the brain-specific expression of CYP46A1

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2011Cholesterol has a crucial role in central nervous system physiology and cell signaling, and many studies correlate alterations in brain cholesterol homeostasis with neurodegenerative diseases. The CYP46A1 gene codes for the cholesterol 24-hydroxylase (CYP46A1), a cytochrome P450 specifically expressed in neurons, that is responsible for the majority of brain cholesterol turnover. However, despite its physiological importance, the molecular mechanisms underlying the human CYP46A1 expression had not been characterized. Therefore, our work aimed to identify regulatory elements and factors involved the CYP46A1 brain-specific expression, and further assess if CYP46A1 transcription is regulated by epigenetic modifications, such as DNA methylation. Moreover, we aimed to identify a human cell model that could be a valuable tool for the study of cholesterol homeostasis in human neurons. In our initial studies, we cloned and characterized the human CYP46A1 promoter. Functional deletion analysis, over-expression studies, site-directed mutagenesis and gel-shift assays identified that not only Sp transcription factors control CYP46A1 transcription, but are most probably responsible for cell type specificity. To test our hypothesis that an increase in the (Sp3+Sp4)/ Sp1 ratio would result in CYP46A1 transcriptional activation, we differentiated Ntera2/clone D1 (NT2) human teratocarcinoma cells into post-mitotic neurons (NT2N). We demonstrated for the first time a significant increase in CYP46A1 mRNA and protein levels in a human cell culture, and identified a concomitant decrease in the levels of Sp1 associated with the proximal promoter of this gene. Nevertheless, we did not observe any conserved pattern in Sp protein binding to other Sp-regulated gene promoters, suggesting that Sp-DNA binding and transcriptional activity is highly dependent on the neuronal chromatin context. Moreover, we showed that throughout NT2 differentiation, HMG-CoA Abstract xxiv synthase, HMG-CoA reductase, SREBP2 and LDLr, key players in brain cholesterol homeostasis, present expression profiles that mimic what is thought to occur in vivo. Our results also suggest that progenitor cells eliminate cholesterol in the form of 27-hydroxycholesterol while neurogenesis induces a shift to the 24-hydroxylase-dependent elimination pathway. Finally, we showed that the demethylating agent 5’-Aza-2’-deoxycytidine (DAC) is a CYP46A1 inducer. Surprisingly, bisulfite sequencing analysis revealed that the CYP46A1 core promoter is completely unmethylated in both human brain and non-neuronal human tissues where CYP46A1 is not expressed. We demonstrated that DAC induces CYP46A1 expression, in a DNA methylation- independent mechanism, by decreasing Sp3/HDAC binding to the proximal promoter. Collectively, our results provide new insights on the regulatory circuits that control CYP46A1 transcription, and contribute to the identification of potential therapeutic approaches that can modulate CYP46A1 expression.Fundação para a Ciência e a Tecnologia:SFRH/BD/27660/2006; PPCDT/SAU-MMO/55919/2004 and PTDC/SAU-GMG/64176/2006 (to Professor Elsa Margarida Teixeira Rodrigues) from FCT and FEDER.Fundação para a Ciência e Tecnologia (FCT),(SFRH/BD/27660/2006), Lisbon, Portugal; PPCDT/SAU-MMO/55919/2004; PTDC/SAU-GMG/64176/2006. FCT; FEDER

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

© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).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.This work is funded by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the projects UIDB/04565/2020 and UIDP/04565/2020 of Institute for Bioengineering and Biosciences – iBB, the project PTDC/BIA-MOL/29320/2017 of the Instituto de Medicina Molecular João Lobo Antunes and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB, as well by the Pedro Maria José de Mello Costa Duarte grant by Fundação Amélia de Mello. C. Maranga is supported by a PhD fellowship (PD/BD/135505/2018) and S.T. da Rocha is supported by an assistant research contract (CEECIND/01234/2017) from FCT.info:eu-repo/semantics/publishedVersio

MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs.

Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome

Demethylation of the Coding Region Triggers the Activation of the Human Testis-Specific PDHA2 Gene in Somatic Tissues

Human PDHA2 is a testis-specific gene that codes for the E1α subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the −122 to −6 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-2′-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos

Multi-omic rejuvenation of human cells by maturation phase transient reprogramming.

Funder: Biotechnology and Biological Sciences Research Council; FundRef: http://dx.doi.org/10.13039/501100000268Funder: Milky Way Research FoundationAgeing is the gradual decline in organismal fitness that occurs over time leading to tissue dysfunction and disease. At the cellular level, ageing is associated with reduced function, altered gene expression and a perturbed epigenome. Recent work has demonstrated that the epigenome is already rejuvenated by the maturation phase of somatic cell reprogramming, which suggests full reprogramming is not required to reverse ageing of somatic cells. Here we have developed the first "maturation phase transient reprogramming" (MPTR) method, where reprogramming factors are selectively expressed until this rejuvenation point then withdrawn. Applying MPTR to dermal fibroblasts from middle-aged donors, we found that cells temporarily lose and then reacquire their fibroblast identity, possibly as a result of epigenetic memory at enhancers and/or persistent expression of some fibroblast genes. Excitingly, our method substantially rejuvenated multiple cellular attributes including the transcriptome, which was rejuvenated by around 30 years as measured by a novel transcriptome clock. The epigenome was rejuvenated to a similar extent, including H3K9me3 levels and the DNA methylation ageing clock. The magnitude of rejuvenation instigated by MPTR appears substantially greater than that achieved in previous transient reprogramming protocols. In addition, MPTR fibroblasts produced youthful levels of collagen proteins, and showed partial functional rejuvenation of their migration speed. Finally, our work suggests that optimal time windows exist for rejuvenating the transcriptome and the epigenome. Overall, we demonstrate that it is possible to separate rejuvenation from complete pluripotency reprogramming, which should facilitate the discovery of novel anti-ageing genes and therapies

Recruitment of RNA pol II to the human <i>PDHA2</i> gene.

<p>Chromatin from SH-SY5Y was prepared at 72 hours after treatment with 5 µM DAC and precipitated with antibodies directed against IgG, Sp1 and RNA pol II. After DNA recovery, the precipitates were evaluated by real-time PCR as described in Experimental Procedures. Results are expressed as fold change over IgG and represent means of at least three independent experiments ± SEM (* p<0.001).</p

DAC increases <i>PDHA2</i> mRNA levels in SH-SY5Y cells.

<p>Real-time PCR analysis of <i>PDHA2</i> steady-state mRNA transcript levels in SH-SY5Y cells treated with 5 µM DAC for the indicated time points (A), and with 5 µM DAC for 96 h and/or 0.25 µM TSA for 24 h (B). Values were normalized to the internal standard β-actin. Data represent means ± SEM of at least three independent experiments and was expressed as pg of <i>PDHA2</i> mRNA per ng of β-actin mRNA (* p<0.001; § p<0.01).</p