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H3K9 methylation as regulator of epigenetic memory on bovine nuclear reprograming

Grant number: 15/08807-6
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): October 01, 2015
Effective date (End): August 31, 2019
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Flávio Vieira Meirelles
Grantee:Rafael Vilar Sampaio
Home Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil
Associated research grant:13/08135-2 - CTC - Center for Cell-Based Therapy, AP.CEPID
Associated scholarship(s):15/25111-5 - Analysis and modulation of H3K9 methylation in bovine somatic cells, BE.EP.PD


The persistence of somatic epigenetic memory is indicated as the main barrier for an efficient nuclear reprogramming. Reproductive technologies like somatic cell nuclear transfer (SCNT) are influenced by this memory. In mice H3K9 hypermethylation on somatic nuclei has been show as the major regulator of epigenetic memory. However, little is known in bovine. Based on that, we hypothesized that decreased H3K9 methylation in bovine fetal fibroblasts allows that important genes for development are transcript at embryonic genome activation of clones. In order to test this hypothesis fetal fibroblast (FFS) with H3K9 methyltransferases (G9a and SUV39h1) silenced will be produced trough siRNA. This study will be divided in four phases: On the first phase, we will identify genes related to H3K9 methylation using ChIP-seq for H3K9me2 and H3K9me3. On the second phase, fetal fibroblast (FFS) with H3K9 methyltransferases (G9a and SUV39h1) silenced will be produced trough siRNA. On the third phase, we will use RNA-seq to analyze the influence of decreased H3K9 methylation levels at maternal embryonic transition. To perform this experiment we will produce eight cells crossbreed embryos using FFS as nuclear donor (Bos taurus x Bos indicus) and analyzed by RNA-seq compared with IVF embryos crossbreed as well. This crossbreed approach will allow the analysis of allelic-specific expression genes, like imprinted genes. Lastly, embryos on blastocyst stage will be analyzed by differential staining: for inner cell mass and trophectoderm. Therefore, we believe this project will allow the understanding of epigenetic memory formation process during cellular reprograming and consequently improve future biotech applications. Thus, this proposal is groundbreaking and will serve as base to clarify important paradigms involving cloning and others assisted reproductive technologies.

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)
ROSS, PABLO J.; SAMPAIO, RAFAEL V. Epigenetic remodeling in preimplantation embryos: cows are not big mice. ANIMAL REPRODUCTION, v. 15, n. 3, p. 204-214, JUL-SEP 2018. Web of Science Citations: 1.

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