Heterochromatin: study of different strategies to modulate the stability of epigenetic memory on bovine fibroblasts

• Grant number: 18/15089-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2018
• End date: December 31, 2019
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Flávio Vieira Meirelles
• Grantee: Dewison Ricardo Ambrizi
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil

Abstract

The mammalian adult organism contains approximately two hundred different cell types. Each of these cells types possesses different functions and morphologies, which is controlled by the gene expression pattern. The chemical modifications present in the chromatin regulate gene expression and are called epigenetic modifications. The main functions of these modifications are to choose which genes will be turn-on and turn-off according to the cell's necessity. These information’s are inherited throughout each cell division and this process is known as epigenetic memory. Techniques utilized for cellular reprogramming such as Somatic Cell Nuclear Transfer (SCNT) and induced pluripotent stem cells (iPSCs) are inefficient to remove this epigenetic memory from differentiated cells. This happens because the genes regulating pluripotency and embryonic development are silenced in the somatic nucleus by repressive histones modifications. These silenced regions of the chromatin are named heterochromatin, that can be permanently repressed (constitutive heterochromatin) or not (facultative heterochromatin). The main epigenetic modification of constitutive heterochromatin is H3K9me3, which is catalyzed by the lysine methyl-transferase proteins (KMTs), being the SUV39H1 the principal member. Several methodologies are currently being utilized to modulate the somatic cell epigenetic memory aiming to improve the epigenetic reprogramming. In murine, trichostatin (TSA) and sodium butyrate (NaBu) improved the efficiency of cellular reprogramming, because these molecules caused a relaxation in chromatin structure and decreased the HP1 levels, an essential protein for heterochromatin stabilization. Other methods such as knockdown of SUV39H1 mediated by siRNA resulted in an increased efficiency of SCNT embryonic development possibly due to the H3K9me3 reduction in important regions for pluripotency. Additionally, the chemical inhibition of the SUV39H1 using chaetocin, a specific inhibitor, has been shown to aid the reprogramming process. Thus, our hypothesis is that several molecules work in concert (i.e. HP1 and H3K9me3 catalized by SUV39H1) to confer the repressed chromatin state in bovine fibroblasts. Therefore, the goal of this study is to investigate the modulation of the main heterochromatin components combining chromatin modifiers agents that increase the global levels of acetylation and decrease HP1 (TSA and/or NaBu), as well as the chemical inhibition (chaetocin) or SUV39H1 knockdown through siRNA. In this way, we expect to better understand the mechanisms regulating the epigenetic memory formation and modulation in bovine fibroblasts.