H3K9 methylation as regulator of epigenetic memory on bovine nuclear reprograming.

• Grant number: 15/08807-6
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: October 01, 2015
• End date: August 31, 2019
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Flávio Vieira Meirelles
• Grantee: Rafael Vilar Sampaio
• Host 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

Abstract

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)

SAMPAIO, RAFAEL VILAR; SANGALLI, JULIANO RODRIGUES; CAMARA DE BEM, TIAGO HENRIQUE; AMBRIZI, DEWISON RICARDO; DEL COLLADO, MAITE; BRIDI, ALESSANDRA; CAVALCANTE MENDES DE AVILA, ANA CLARA FAQUINELI; MACABELLI, CAROLINA HABERMANN; OLIVEIRA, LILIAN DE JESUS; DA SILVEIRA, JULIANO COELHO; et al. Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming. SCIENTIFIC REPORTS, v. 10, n. 1, JUL 13 2020. (15/08807-6, 14/22887-0, 13/08135-2, 14/50947-7, 14/21034-3, 15/25111-5, 12/50533-2, 13/07160-3)

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. (13/08135-2, 15/08807-6)