36 EPIGENETIC MODIFICATIONS OF CULTURED BOVINE FIBROBLASTS FOR NUCLEAR TRANSFER

Abstract

Evidence indicates that a significant portion of the failure of nuclear transfer embryos to develop normally can be attributed to the use of a differentiated cell nucleus as the donor karyoplast. Studies suggest that epigenetic modifications, and the resulting chromatin compaction of in vitro-harvested cells, may change during culture. It has been hypothesized that blastocyst production and development to term of cloned embryos may differ between population doublings (PDs) of the same cell line as a consequence of changes in DNA methylation and histone acetylation patterns. The objective of this study was to determine levels of DNA methylation, histone acetylation, and gene expression patterns of the chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), histone deacetyltransferse-1 (HDAC1), and methyl CpG binding protein-2 (MeCP2) in bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were established from four 50-day fetuses. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO₂ at 37°C and passaged at 100% of confluence. Relative levels of methylated DNA, acetylated histone, Dnmt1, HDAC1, and MeCP2 were analyzed at PDs 2, 7, 15, 30, 45, and 70 in 3 replicates per PD. Global levels of methylated DNA and acetylated histone were determined by incubation of fixed cells with an anti-5-methylcytidine and an anti-acetyl-histone H3 antibody, respectively. Cells were labeled with a second antibody, contra-stained with propidium iodide, and analyzed by flow cytometry. The expression patterns of Dnmt1, HDAC1, and MeCP2 were characterized using Q-PCR. Relative levels of gene expression at different PDs were analyzed by the ” ”C_T method using Poly A as the reference gene. Differences in fluorescence and gene expression patterns between PDs were analyzed by one-way ANOVA (P < 0.05). The relative fluorescence levels of acetylated histone H3 were constant, whereas methylated DNA increased progressively from PD 2 to PD 45 [159 vs. 242 arbitrary units (AU)] in 3 of the cell lines analyzed. The remaining cell line showed higher fluorescence levels of acetylated histone at PD 2 than at PD 45 (196 vs. 72 AU, respectively) and methylated DNA peaks at PDs 2, 30, and 70 (240, 271, and 175 AU, respectively). When compared with cells at PD 2, Dnmt1 gene expression levels decreased by <50% at PD 7 and remained low during culture in all cell lines. HDAC1 and MeCP2 gene expression levels decreased significantly after PD 2 by 70% and 80%, respectively, in the cell lines analyzed. These data demonstrate that methylated DNA and acetylated histone patterns of in vitro cells change with time in culture. Chromatin remodeling proteins involved in such epigenetic modifications are also altered during in vitro culture. Additionally, these results indicate that cell lines respond differently to in vitro conditions. Subsequent use of these cells for NT will provide insight as to how these epigenetic modifications affect reprogramming.

This study was supported by Grants from Louisiana State University Board of Regents to K. R. Bondioli.