Project description:ERYTHROID CELL-TYPE SPECIFIC GENE EXPRESSION Post-translational histone modifications, acting alone or in a context-dependent manner, influence numerous cellular processes via their regulation of gene expression. Monomethylation of histone H3 lysine 27 (K27me1) is a poorly understood histone modification. Some reports describe depletion of K27Me1 at promoters and transcription start sites (TSS), implying its depletion at TSS is necessary for active transcription, while others have associated enrichment of H3K27me1 at TSS with increased levels of mRNA expression. Tissue- and gene-specific patterns of H3K27me1 enrichment and their correlation with gene expression were determined via chromatin immunoprecipitation on chip microarray (ChIP-chip) and human mRNA expression array analyses. Results from erythroid cells were compared to those in neural and muscle cells. H3K27me1 enrichment varied depending on levels of cell-type specific gene expression, with highest enrichment over transcriptionally active genes. Over individual genes, the highest levels of H3K27me1 enrichment were found over the gene bodies of highly expressed genes. In contrast to H3K4me3, which was highly enriched at the TSS of actively transcribing genes, H3K27me1 was selectively depleted at the TSS of actively transcribed genes. There was markedly decreased to no H3K27me1 enrichment in genes with low expression. At some locations, H3K27 monomethylation was also found to be associated with chromatin signatures of gene enhancers. The gene expression profiles of three replicates of 4 human cell lines were obtained using Illumina microarrays.
Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-alpha tagged with TAP-tag at the C-term (C-TAP-ER-alpha). All cells were grown in Dulbecco's modified Eagle's medium (DMEM), starved by using DMEM w/o phenol red and 5% Dextran Coated Charcoal stripped serum (DCC-FBS) for 5 days. Cells were stimulated with 17 beta Estradiol (E2), 4-idrossi-tamoxifen (Tam), raloxifene (Ral) and Fulvestrant (ICI) at the concentration of 10-8M for 12 hours or with vehicle Ethanol. Biological replicate were lysed and RNA extracted were pooled. For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 19 hours on Illumina HumanHT-12 v4.0 BeadChips and after scanning, data analysis was performed.
Project description:Pantethine, an anti-cholesterol drug, was shown to inhibit the Ab (amyloid beta) peptide-induced expression of IL-1b in primary culture astrocytes derived from either untreated Ab-treated and untreated 5XFAD or Ab-treated WT. These observations prompted us to investigate the effects of pantethine at the transcriptomic level in the mouse AD model.
Project description:Analysis of gene expression levels of whole transcripts obtained from two WA09 ES cell cultures, two dermal fibroblast cultures, two lines each for L-iPSCs, F-iPSCs and LF-iPSCs. Total RNA obtained from WA09 ESCs, the dermal fibroblasts from which all iPSCs in the microarray study were derived, two F-iPSC clones, two L-iPSC clones and two LF-iPSC clones on day 100-120 were compared.
Project description:The goal of this experiment was to compare the gene expression programs mediated by androgen/AR vs. constitutively active, truncated AR variants in castration-resistant CWR-R1 prostate cancer cells. Because constitutive activity of truncated AR variants can mask androgen/AR target genes, the androgen/AR transcriptional program was assessed by silencing the trucnated AR 1/2/3/CE3 variant with siRNA targeting AR exon CE3 and treating cells with vehicle (ethanol) or 1nM DHT. Similarly, because full-length AR activity can mask truncated AR variant target genes, the AR variant transcriptional program was assessed under castrate conditions by selectively silencing full-length AR with siRNA targeting AR exon 7, and comparing this profile with CWR-R1 cells transfected wtih siRNA targeting AR exon 1, which silences all AR expression (full-length and truncated AR variants). CWR-R1 cells were maintained under castrate conditions in long term culture in order to enrich for the population of cells harboring a 48kb intragenic deletion in AR intron 1. These late-passage CWR-R1 cells were electroporated with siRNAs targeting AR exon 1, AR exon 7, or AR exon CE3. Electroporated cells were seeded in RPMI 1640 medium containing antibiotics and 5% charcoal-stripped, steroid-depleted medium and allowed to recover for 48h. After this 48h recovery, electroporated cells were switched to serum-free RPMI 1640 with 1nM DHT or 0.1% ethanol (vehicle control) for an additional 24h prior to extraction of total RNA. Three independent biological replicates were performed.
Project description:MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21M-bM-^@M-^S23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3M-bM-^@M-2untranslated region (3M-bM-^@M-2UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity. Control (no drug treatment): 1 hour and 4 hour samples, 3 replicates each; Drug-treated: 1 hour and 4 hours samples, 3 replicates each
Project description:Transcriptional profiling of E14.5 XY germ cells (marked with Oct4-EGFP transgene) from two strain backgrounds (C57BL/6J and 129S1/SvImJ) that differ in their susceptibility to testicular teratomas. Two condition experiment. For each sample, Oct4-EGFP+ germ cells from all XY embyos within a litter were pooled. For the C57BL/6J background, n=3 pooled biological replicates were profiled, and n=2 replicates were obtained and profiled for 129S1/SvImJ.
Project description:To compare gene expression in human myometrium after TSA treatment to gene expression after treatment with a specific HDAC8 inhibitor Four Control, four TSA and four Compound 2 samples (2 patients, replicated) derived from non-pregnant myometrial strips