RNA-sequence analysis of Burkholderia vietnamiensis strain G4 exposed to ethylzingerone 4-(3-ethoxy-4-hydroxyphenyl) butan-2-one)
ABSTRACT: Burkholderia vietnamiensis strain G4, representative of a species routinely encountered as a contaminant of industrial product, was exposed to a proprietary preservative agent for 24 hours and gene expression analysed by RNA-seq.
Project description:In this study we discover proteins that bind to G4 quadruplex DNA structure. We use modified c-myc quadruplex as a bait, and compare it to the control bait - T15 oligo. We use spectral counts and G-test to determine significant binders. T15 and G4 datafiles are uploaded
Project description:Telomere dysfunctional CMP/GMP have deregulated pathways that are associated with DNA damage signaling We compared differentially expressed genes in the G4/G5 CMP relative to the G0 control to identify pathways that may affect CMP differentiation. Bone marrow CMP and GMP cells were sorted from two paired pools of G0 TERTER/+ or G4/G5 TERTER/ER mice (5,000-20,000 cells per sample) using the Influx Cell Sorter. Every paired pool includes CMP or GMP sorted from 4 age and gender matched G0 or G4/G5 mice. RNA from the respective sorted cells was extracted using Trizol (Ambion) and profiled on 2100 Bioanalyzer (Agilent). Gene expression profiling was performed at the Sequencing and Non-coding RNA Program at MD Anderson Cancer Center. Briefly, the GeneChip® 3 IVT Express Kit (Affymetrix) was used to generate biotin-labeled cRNA, which were purified and fragmented, before target hybridization on the GeneChip® Mouse Genome 430 2.0 Array (Affymetrix) according to the manufacturer's instructions. Affymetrix raw data (CEL files) were normalized using Affymetrix Microarray Suite (MAS) version 5.0 using a TGT=100. Paired pools used in the study were: CMP pool 1: G0-1 and G4/5-1 CMP pool 2: G0-2 and G4/G5-2 GMP pool 1: G0-1 and G4/G5-1 GMP pool 2: G0-2 and G4/G5-2
Project description:Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. We developed a zinc finger nuclease translocation reporter (ZITR) and screened for factors that modulate rearrangements in human cells. We identified UBC9 and RAD50 as suppressors and 53BP1, DDB1, and PARP3 as promoters of chromosomal rearrangements across human cell types. We focused on poly(ADP)ribose polymerase 3 (PARP3) as it is dispensable for murine viability and has druggable catalytic activity. We found that PARP3 regulated G quadruplex (G4) DNA in response to DNA damage, which suppressed repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3-/- cells led to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways. Overall design: RNA-seq was performed on WT and PARP3-/- A549 cells with vehicle treatment or treatment with 1 micromolar pyridostatin. All conditions were performed in biological triplicate.
Project description:Differential hyper- and hypo-methylation regions in G0 versus G4/G5 CMP The goal of this study is to evaluate changes in CpG methylation profilings of telomere dysfunctional common myeloid progenitor cells (CMP) as compared to their wild type controls Genomic DNA was extracted from sorted CMP populations isolated from 3 pools of G0 or 2 pools of G5 mice using UltraPure Phenol:Chloroform:Isoamyl Alcohol according to manufacturer’s instructions (Life Technologies). 14,000 to 30,000 cells were available for each sample, resulting in a minimum of 45ng of DNA. Genome-wide DNA methylation profiling was performed by RRBS. Library preparation and sequencing were performed at the UT MD Anderson Cancer Center’s DNA Methylation Analysis Core and Sequencing and Microarray Facility, according to published protocols. RRBS sequencing data were aligned and methylation was called using Bismark v0.7.119. In brief, bisulphite-treated DNA was aligned to UCSC Genome Browser mm10 reference genome using Bowtie. In total 29-38 million reads were generated per sample with alignment rates around 63%. Next, MethylKit10 implemented with Fisher’s exact test was used to compare the cytosine methylation profiles of G0 and G5 CMP. Gene promoter regions were calculated based on RefSeq gene annotations with regions starting 1 kb upstream of the annotated transcription start site (TSS) and extending 500 base pairs downstream of TSS. Exons, introns, and CpG islands coordinates were collected from the UCSC Genome Browser mm10 version.
Project description:Telomere erosion causes cell mortality, suggesting that longer telomeres allow greater number of cell division. In telomerase-positive human cancer cells, however, telomeres are often kept shorter than the surrounding normal tissues. Recently, we have shown that telomere elongation in cancer cells represses innate immune genes and promotes their differentiation in vivo. This implies that short telomeres contribute to cancer malignancy, but it is unclear how such genetic repression is caused by long telomeres. Here we report that telomeric repeat-containing RNA (TERRA) induces genome-wide alteration of gene expression in telomere-elongated cancer cells in vivo. Using three different cell lines, we found that G4 forming oligonucleotide repressed innate immune genes in vivo 3D culture conditions. Most of the suppressed genes belonged to innate immune system categories and were upregulated in various cancers. We propose that TERRA G4 counteracts cancer malignancy through suppression of innate immune genes. Six samples are G4 oligo-transfected cells (PC-3/(uuaggg)^4, PC-3/AS1411, HBC4/(uuaggg)^4, HBC4/AS1411, MKN74/(uuaggg)^4 and MKN74/AS1411), and the other six samples are control oligo-transfected cells.
Project description:G quadruplex motifs are frequently located near TSS and in the first introns of transcribed genes. We investigated the role that G4 structures play in transcription by stabilizing G4 DNA with the selective G4 ligand PhenDC3 in the HT-1080 human fibrosarcoma cell line. After treatment of triplicate samples with PhenDC3 or a negative control (DMSO carrier only), we performed RNA-Seq after poly-dT selection to assess changes in gene and isoform expression, as well as changes to splicing patterns. Overall design: 6 samples total; 2 gene expression conditions (treated and untreated) in triplicate; 4 raw sequencing files per sample