DNA methylation analysis of bull semen according to peripubertal age (12 months vs. 16 months)
ABSTRACT: The DNA methylation pattern in spermatozoa of bulls at different age was investigated. Spermatozoa collected from 4 different bulls at 10, 12 and 16 month each. Genome-wide DNA methylation analysis was performed by micro array using EDMA platform. three condition experiment, 4 bulls, all collected at 10, 12 and 16 month
Project description:The DNA methylation pattern in spermatozoa of bulls at different age was investigated. Spermatozoa collected from 4 different bulls at 10, 12 and 16 month each. Genome-wide DNA methylation analysis was performed by micro array using EDMA platform. three condition experiment, 4 bulls, all collected at 10, 12 and 16 month
Project description:DNA from expanded bovine blastocysts and bovine sperm were extracted then subjected to methylation-sensitive enzymatic digestion and LM-PCR enrichment before being hybridized onto a microarray. Two-condition experiment, bovine blastocysts (pools of 10) vs bovine sperm. Four biological replicates of each tissue were hybridized to four two-color arrays in a dye-balanced design.
Project description:Transposable elements (TE) have been shown to contrain functional transcription factor (TF) binding sites for long, but the extent to which TEs contribute TF binding sites is not well know. Here, we comprehensively mapped binding sites for 26 pairs of orthologous TFs, in two pairs of human and mouse cell lines (i.e., leukemia, and lymphoblast), along with epigenomic profiles representing DNA methylation and six histone modifications. We found that on average, 20% of TF binding sites were embedded in TEs. We further identified 710 TF-TE relationships in which certain TE subfamilies enriched for TF binidng sites. TE-derived TF binding peaks were also strongly associated with decreased DNA methylation and increased enhancer-associated histone marks. Most of the TE-derived TF binding sites were species-specific, but we also identified conserved binding sites. Additionally, 66% of TE-derived TF binding events were cell-type specific, associated with cell-type specific epigenetic landscape. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf To evaluate the contribution of transposable elements (TE) to transcription factor (TF) binding landscapes, we profiled ChIP-seq datasets for 26 TFs in two cell lines in human and mouse, generated by the ENCODE and MouseENCODE consortia. The epigenomic profiles were evaluated from six histone modification in each of the cell lines, also generated by the consortia. We added DNA methylation to the epigenomic profiles, using two complementary techniques, MeDIP-seq and MRE-seq. The human data related to this study are available through GSE56774: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56774
Project description:Epigenetics (DNA methylation) profiling of sperm from Monozygotic (MZ) twin bull brothers comparing with each other Two-condition experiment, DNA methylation analysis of sperm from Monozygotic twin bull brothers which were hybridized as dye-swap in two-color arrays in a dye-balanced design.
Project description:DNA methylation and DNA replication timing were examined across a variety of human tissues and cell lines, applying microarray-based techniques. The analyses revealed that late-replicating DNA was demethylated compared to the methylation of early-replicating regions. DNA methylation: Epstein-Barr-Virus (EBV) transformed B-lymphocyte cell lines GM10849, GM12089, GM12092, GM12093, and GM08714 (ICF) (http://ccr.coriell.org/nigms) were cultured in RPMI-1640 supplemented with 15% FCS (Sigma) at 37oC and 5% CO2. Normal BJ foreskin fibroblasts (NHF cells) at different PDs (36)(34) were cultured in 4:1 DMEM : M-199 supplemented with 15% FCS (Sigma) at 37oC and 5% CO2. Replication timing: EBV-transformed female B-lymphocyte cell lines GM12092 and GM12093 were cultured as above, harvested from logarithmic growth cultures, washed once in ice-cold phosphate-buffered saline, immediately fixed in 70-85-95% ethanol at -20oC, stained with propidium iodide, and sorted into G1 and early-S fractions using a MoFlo cell sorter.
Project description:Background: It has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival has also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny. Methods: Hematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells. Results: In microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes have specific expression just at certain stages of ontogeny (STATs, GRB2, CREBB). Beside continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH). Conclusions: This broad overview of gene expression in erythropoiesis revealed transcription factors with a prevalence at certain stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway- coupled NFKBIA and YWHAH genes. Adult peripheral blood mononuclear cells were isolated from buffy coats of 3 healthy donors using Lymphocyte Separation Medium (BioWhittaker, Walkersville, MD). We washed mononuclear cells twice with Dulbecco's phosphate-buffered saline (PBS, Invitrogen Corporation, Carlsbad, CA), and CD34+ cells were purified by positive immunomagnetic selection using the MACS cell isolation system (Miltenyi Biotec, Auburn, CA). Fresh bone marrow CD34+ cells were collected (AllCells LLC, Berkeley, CA). Cord blood CD34+ cells (AllCells LLC) and fetal liver CD34+ cells (Cambrex Bio Science, Inc., Walkersville, MD) were collected and frozen. For analysis, CD34+ cells were resuspended in medium, which contained 30% FBS, 2 mmol/L glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% deionized BSA, 10 mmol/L beta-mercaptoethanol, 1 mmol/L dexamethasone, 33 µg/ml holo-transferrin, 10 ng/ml SCF, 1 ng/ml IL-3 and 1 ng/ml GM-CSF (Sigma, St. Louis, MO), and 1 U/ml human recombinant EPO (Amgen Inc, Thousand Oaks, CA) Erythroid progenitor cells differentiated from hematopoietic CD34+ progenitor cells of fetal liver, cord blood, bone marrow and peripheral blood origin Biological replicates: 2 fetal liver, 3 cord blood, 3 bone marrow, 3 peripheral blood origin
Project description:To understand functional roles of ncRNAs during exponential growth phase, we captured transcriptome changes by conducting strand-specific RNA-seq. Directional RNA-seq (dUTP method by TruSeq®) were generated in two biological replicates by using Illumina MiSeq
Project description:Transcripome of longissimus dorsi muscle was compared between Korean cattle bulls and steers by using a customized bovine Combimatrix microarray containing 10,199 genes. A customized bovine Combimatrix microarray containing 10,199 genes were constructed, and transcripome of longissimus dorsi muscle was compared between Korean cattle bulls (3 bulls) and steers (3 high-marbled and 3 low-marbled steers) by using the microarray hybridzation.
Project description:miRNA profiles of the MSC-MVs and EPO-MVs were analyzed with a quantitative PCR (qPCR)-based array of the whole mice genome. Further analysis revealed differences in the miRNAs of 212 EPO-MVs (fold change ≥ 1.5 compared to the MSC-MVs), which constituted approximately 22.64% of all of the evaluated mouse miRNAs. Of all of the differences, 70.28% of the changes in the EPO-MV group involved upregulation To study the differential miRNA expression in MV and EPO-MV which might contributed to the better treatment in chronic kidney disease, we performed miRNA expression profiling of the culture supernatant of MSC with or without EPO incubation using the miRCURY LNA Array (v.18.0) (Exiqon, Vedbaek, Denmark).