Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human amniotic fluid-derived stem cells (AFSC) treated with ethanol

ABSTRACT: The effects of ethanol may vary from induction of apoptosis to the inhibition of proliferation, differentiation, migration or other function. The complex and diverse response of fetal cells to ethanol has prompted us to use a bioinformatics approach to study the effect of ethanol on fetal stem cells derived from the amniotic fluid-derived (AFSC). To characterize the global response of human AFSC to ethanol, gene expression profiles of AFSC treated with or without 100mM ethanol for 48 hours were analyzed. Experiment Overall Design: Two microarrays were performed (AFSC sealed with parafilm for 48 hours treated with or without 100 mM of ethanol). Total RNA was isolated using RNA Bee according to manufacturer's instructions. Fragmented antisense cRNA was used for hybridizing with human U133 A arrays (Affymetrix, Inc. Santa Clara, CA, USA) at the Core Genomic Facility of Wake Forest University School of Medicine. Raw CEL files were provided by the Microarray Core Facility of the Wake Forest University School of Medicine and were then analyzed with a software package AffylmGUI (Affymetrix LIMMA, Linear Models for Microarray Data, Graphical User Interfaces). AffylmGUI reads the raw Affymetrix CEL files directly, summarizes the gene expression values using RMA, and then uses LIMMA to identify statistically significant differences in gene expression. LIMMA fits a linear model for every gene (like ANOVA or multiple regression analysis), and adjusts P values for multiple testings. Differentially expressed genes were identified with a fold change > 1.8.

ORGANISM(S): Homo sapiens

SUBMITTER: Jennifer Hipp

PROVIDER: E-GEOD-13569 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? Here we investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell lineage. We find that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global down-regulation of PRC2, recruitment of hypophosphorylated RNA Polymerase II (Pol II) to promoters, as well as expression and action of cell-type specific homologs of subunits of TFIID (tTAFs). In addition, action of tMAC, a tissue specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell-type specific localization of PRC1 components and tTAFs to the spermatocyte nucleolus. Together, action of the tMAC and tTAF cell-type specific chromatin and transcription machinery leads to loss of Polycomb and release of Pol II from the promoter of terminal differentiation genes to elongation, allowing robust transcription of the terminal differentiation genes. Total RNA from approximately 200 pairs of fly testes (bam1/bam-delta-86; aly2/aly5P; sa1/sa2; y,w) was extracted using TRIzol (Invitrogen, #15596-018, Carlsbad, CA, USA) following the manufacturer's instructions. The genomic DNA was degraded using 2 Units of DNase I (Fermentas, #EN0521, Glen Burnie, MD, USA) at 37 degreeC for 20 minutes. RNA integrity was checked by gel electrophoresis (1% agarose). Approximately 4 ≈í¬∫g of total RNA from each biological replicate were used to generate labeling probes to hybridize with the Affymetix GeneChip Drosophila Genome 2.0 Array according to the Affymetrix protocol. Three biological replicates were performed for each genotype. Microarray hybridization was processed at the Core Facility at the Stanford University School of Medicine and the raw data were exported from the Affymetrix Microarray Suite (MAS). The CEL files were used for signal normalization with RMA as part of the limma package from the Bioconductor R packages (http://www.bioconductor.org).

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Transcription profiling of human amniotic fluid-derived stem cells (AFSC) treated with ethanol

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