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MiR-29a target two central components of active DNA demethylation, TET1 and TDG: potential mechanism during reprogramming

Grant number: 13/19545-7
Support type:Scholarships abroad - Research Internship - Doctorate (Direct)
Effective date (Start): January 01, 2014
Effective date (End): March 31, 2014
Field of knowledge:Biological Sciences - Genetics
Principal Investigator:Marco Antonio Zago
Grantee:Mariane Serra Fraguas
Supervisor abroad: Tobias Cantz
Home Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
Local de pesquisa : Hannover Medical School, Germany  
Associated to the scholarship:10/02616-0 - Manipulation of inhibitory pathways of pluripotency induction aiming efficiency increase in iPSs generation process, BP.DD

Abstract

Adult somatic cells can be reprogrammed into induced pluripotent stem cells (IPSCs) by the ectopic expression of pluripotency-related transcription factors (TF), such as, Oct4, Nanog, Sox2 and c-Myc. MicroRNAs (miRs) are non-coding RNAs involved in the post-transcriptional regulation of a wide range of targets, and may thus, influence cell fate by modulating distinct signaling pathways and biological processes. Thus, are considered promising tools to improve reprogramming. Interestingly, it was shown that, while miR-29a inhibits reprogramming; its depletion enhances reprogramming efficiency of MEFs (Mouse Embryonic Fibroblasts) by 2 - 3 fold. Moreover, the transduction of c-Myc, was capable to decrease miR29a levels, and subsequently regulate multiple signaling networks, boosting reprogramming. In our preliminary studies, we found that components of active DNA demethylation (Tet1 and TDG) are direct targets of miR29a in BJ fibroblasts. Importantly, Tet1 transcription is controlled by Oct4 and Sox2, and during reprogramming, CpG dinucleotides in the promoters of pluripotency factors bound by Tet1, are hydroxylated, becoming enriched for 5hmC, and consequently demethylated and reactivate. In order to explore the molecular mechanisms by which miR29a influences iPSC generation; we aim carry reprogramming experiments, based in the transfection of the miR-29 mimics and inhibitory molecules to evaluate if the transfection of an anti-miR-29 in MEFs OG2, recapitulates the positive effect during reprogramming. Also, we aim evaluate the negative effect of miR-29 on reprogramming. If this holds true, we can carry additional experiments to pinpoint if the positive effect of the anti-miR-29 is mediated by the consequent upregulation of miR-29 targets, Tet1 and TDG. This could be achieved by the concomitant transfection of anti-miR-29 with siRNAs directed against Tet1 and/or TDG. Additional experiments with vectors with luciferase sequence fused to the 3´UTR regions of Tet1 and TDG would confirm the specificity of miR-29 targeting. Additional experiments could further address the roles of DNMT3a/b in this experimental setting. Taken together, our findings could help to understand how endogenous fibroblasts miR29a may impairs IPSC generation by targeting Tet1 and TDG, and that its inhibition, through the use of anti-miR29a, could favor reprogramming by allowing Tet1 and TDG to accumulate. These findings will contribute to the understanding of how miRs modulation drives IPSC induction, and how miR-29a may modulate the epigenetic machinery involved in reprogramming. (AU)

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
FRAGUAS, MARIANE SERRA; EGGENSCHWILER, RETO; HOEPFNER, JEANNINE; DOS SANTOS SCHIAVINATO, JOSIANE LILIAN; HADDAD, RODRIGO; BOURGUIGNON OLIVEIRA, LUCILA HABIB; ARAUJO, AMELIA GOES; ZAGO, MARCO ANTONIO; PANEPUCCI, RODRIGO ALEXANDRE; CANTZ, TOBIAS. MicroRNA-29 impairs the early phase of reprogramming process by targeting active DNA demethylation enzymes and Wnt signaling. STEM CELL RESEARCH, v. 19, p. 21-30, MAR 2017. Web of Science Citations: 8.

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