Epigenetic status of buffalo fibroblasts treated with sodium butyrate a chromatin remodeling agent

Papori Sharma; A S Yadav; N L Selokar; Dharmendra Kumar; S S Dhaka; P S Yadav

doi:10.1016/j.tice.2017.12.006

Epigenetic status of buffalo fibroblasts treated with sodium butyrate a chromatin remodeling agent

Tissue Cell. 2018 Feb:50:51-58. doi: 10.1016/j.tice.2017.12.006. Epub 2017 Dec 15.

Authors

Papori Sharma¹, A S Yadav², N L Selokar³, Dharmendra Kumar³, S S Dhaka², P S Yadav⁴

Affiliations

¹ Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India; Department of Animal Genetics & Breeding, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar-125001, Haryana, India.
² Department of Animal Genetics & Breeding, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar-125001, Haryana, India.
³ Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.
⁴ Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India. Electronic address: psycirb@gmail.com.

PMID: 29429518
DOI: 10.1016/j.tice.2017.12.006

Abstract

The somatic cells having higher levels of DNA methylation and reducing it in donor cells before nuclear transfer (NT) by treating them with chemicals, may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Therefore, the present study was planned to understand mechanism of epigenetic changes in donor cells (buffalo fibroblasts) treated with different concentration of sodium butyrate (NaBu)-a chromatin remodeling agent. The cultured fibroblasts purity and lineage were confirmed by fibroblast specific protein and gene markers (Vimentin, Tubulin and Cytokeratin) at different passages using immuno-staining and qPCR respectively. The buffalo fibroblast cells were treated with 1, 3 and 5 mM of NaBu and observations were taken on their morphological changes, population doubling time and cell proliferation after 48 h of treatment. The epigenetic changes were observed using acetylation (H3K9ac) and methylation (H3K27me3) markers expression. The fibroblast cells derived from new born ear tissue started emerging and anchoring to cell culture flasks within 24 h and showed spindle-shaped morphology. The confluent culture was cryopreserved at different time interval. The post thaw culture behavior of the cryopreserved cells was also observed at different time interval and passages. The morphology of NaBu treated cells were changed with increase of dosages of NaBu treatment. The increase population doubling times and decreases the proliferation rate in the dose dependent manner. The NaBu treatment showed that the significantly increased the acetylation (H3K9ac) and methylation (H3K27me3) level over the control. Based on the observations of fibroblast characterization as well as epigenetic modifications of these cells after treatment with NaBu, results suggest that the cells may provide a useful approach for better epigenetic reprogramming in SCNT embryos.

Keywords: Buffalo; Cloning; Epigenetics; Histone acetylation; Methylation.

MeSH terms

Animals
Buffaloes
Butyric Acid / pharmacology
Chromatin / drug effects
Chromatin / ultrastructure*
Chromatin Assembly and Disassembly / drug effects*
DNA Methylation / drug effects
DNA Methylation / genetics
Epigenesis, Genetic*
Fibroblasts / drug effects
Fibroblasts / ultrastructure*
Gene Expression Regulation / drug effects
Nuclear Transfer Techniques

Substances

Chromatin
Butyric Acid