title: DNA methylation and histone acetylation patterns in cultured bovine fibroblasts for nuclear transfer.
abstract: Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of a differentiated cell nucleus as the donor karyoplast. 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 during in vitro culture. The objective of this study was to determine gene expression patterns of the chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), methyl CpG binding protein-2 (MeCP2), and histone deacetyltransferse-1 (HDAC1), in addition, to measuring levels of DNA methylation and histone acetylation of bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were established from four 50-day fetuses. Relative levels of Dnmt1, MeCP2, HDAC1, methylated DNA, and acetylated histone were analyzed at PDs 2, 7, 15, 30, 45, and 70. RNA levels of Dnmt1, HDAC1, and MeCP2 were examined using Q-PCR. Global levels of methylated DNA and acetylated histone were determined by incubation of fixed cells with an anti-5-methylcytidine and anti-acetyl-histone H3 antibody, respectively. Cells were labeled with a second antibody, counter-stained with propidium iodide and analyzed by flow cytometry. These data demonstrate that chromatin remodeling protein mRNAs involved in epigenetic modifications are altered during in vitro culture. Methylated DNA and acetylated histone patterns of in vitro cells change with time in culture. Subsequent use of these cells for NT will provide insight as to how these epigenetic modifications affect reprogramming.
authors: Department of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA.
authors: Angelica M
authors: AM
authors: Giraldo
authors: John W
authors: JW
authors: Lynn
authors: Megan N
authors: MN
authors: Purpera
authors: Robert A
authors: RA
authors: Godke
authors: Kenneth R
authors: KR
authors: Bondioli
mesh_terms: D000107:Acetylation; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000201:enzymology*; D015870:Gene Expression; D006655:Histone Deacetylases / Q000235:genetics; D006657:Histones / Q000378:metabolism*; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics; D053652:Nuclear Transfer Techniques; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism
chemical_list: D006657:Histones; D051783:Methyl-CpG-Binding Protein 2; D012333:RNA, Messenger; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D006655:Histone Deacetylases