Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse tet inducible NIH 3T3 cells expressing ATPase-defective BRG-1 to identify SWI-SNF-Regulated Genes

ABSTRACT: NIH 3T3 cells (mouse fibroblasts) were engineered such that the dominant negative ATPase-defective BRG-1 is expressed via the tet-off inducible expression system to inactivate the SWI-SNF chromatin remodeling complexes (Mol Cell Biol 20, 2839). These cell lines have been used for several studies to demonstrate the in vivo functions of the SWI-SNF complexes in transcription, differentiation and cell cycle control. We used one of those cell line designated as B05-1. B05-1 cells were cultured in the medium with tetracycline for routine maintenance, and cultured in the medium without tetracycline for four days to maximally induce the expression of the dominant negative BRG-1, which we designated as B05-1(+tet) and B05-1(-tet) cells, respectively. The aim of our microarray experiments was to determine the genes whose expression levels are changed by inactivation of the SWI-SNF complexes by analyzing the genes differentially expressed between B05-1(+tet) and B05-1(-tet) cells. It should be noted that, since the comparison was made between B05-1 cells cultured with and without tetracycline for four days, the genes differentially expressed between these two cells do not only represent the genes that are directly regulated by the SWI-SNF complexes but also include the genes that are secondarily affected by SWI-SNF inactivation as well as the genes whose expression is changed by tetracycline effect per se.

INSTRUMENT(S): GenePix 4200A

ORGANISM(S): Mus musculus

SUBMITTER: Anthony Imbalzano

PROVIDER: E-MEXP-434 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting gamma-H2AX induction.

Park Ji-Hye JH Park Eun-Jung EJ Lee Han-Sae HS Kim So Jung SJ Hur Shin-Kyoung SK Imbalzano Anthony N AN Kwon Jongbum J

The EMBO journal 20060824 17

Although mammalian SWI/SNF chromatin remodeling complexes have been well established to play important role in transcription, their role in DNA repair has remained largely unexplored. Here we show that inactivation of the SWI/SNF complexes and downregulation of the catalytic core subunits of the complexes both result in inefficient DNA double-strand break (DSB) repair and increased DNA damage sensitivity as well as a large defect in H2AX phosphorylation (gamma-H2AX) and nuclear focus formation a ...[more]

PMID: 16932743

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Project description:RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. Gene array analysis shows that RPF-1 transcription factor modify the transcriptome of HEK293 cells by regulating a genetic program endowed with developmental programs Comparison of HEK293 cells induced for RPF-1 expression (Tet-) over uninduced control (Tet+) and Mock cells. RPF-1 cDNA was inserted into the pTRE2puro vector to generate stable Tet-inducible KEK293 transfectants. Upon Tet removal from the medium, HEK/RPF-1 transfectants expressed the transcription factor RPF-1. Cells were then seeded in 10 cm dishes, cultured for 48 h either in complete medium supplemented with Tet (+) or in Tet-depleted medium (-), harvested, and spun down before RNA isolation. Comparative analysis between HEK/RPF-1 induced (Tet-) and uninduced (Tet+) transfectants allowed us to identify genes transcriptionally regulated by RPF-1. Residual effects on gene expression due to Tet supplementation into culture media was ruled out by comparison with the Mock transfectant.

Project description:Synthetic systems that use positive feedback have been developed to control human disease vectors and crop pests. The tTAV system, which has been deployed in several insect species, relies on a positive feedback circuit that can be inhibited via dietary tetracycline. Although insects carrying tTAV fail to survive until adulthood in the absence of tetracycline, the exact reason for its lethality, as well as the transcriptomic effects of an active positive feedback circuit, remain unknown. Understanding what factors contribute to the variance in tTAV-associated mortality is likely to inform the development of insect control systems. In the present study, the OXI513a tTAV feedback circuit was introduced and verified into D. melanogaster. The tight tetracycline regulation of the system affords a convenient method to conduct a strain-by-strain assessment of the tTAV system and determine the transcriptomic effect, if any, of a positive feedback circuit. Transcriptomic analysis of four independent D. melanogaster tTAV insertion lines, in both adults and larvae, was conducted to examine the transcriptomic influence of the tTAV system. The tTAV lines and non-tTAV control were maintained on TET-on media for 5 generations prior to commencing. Thirty flies, 15 male and 15 female, of the same age, from each of the strains were transferred to either TET-On or TET-Off media. Five days after transfer, adult flies were removed and 10 adult flies, 5 male & 5 female, were frozen at -80°C. Ten larvae from each of these matings were collected at the late second instar stage, the last life stage normally seen prior to lethality, and frozen at -80°C. Three biological replicates were produced for each combination of strain, life-stage, and media. RNA was prepared from frozen samples using Trizol and DNAse was treated using a PureLink RNA Mini Kit (Invitrogen). Microarray experiments employed Agilent Drosophila Gene Expression Microarrays 4x44K and were scanned on an Agilent G2505C scanner (Agilent Technologies). Data collection was divided into two separate experiments. A pilot experiment for strain 102D consisting of two life stages in two conditions each with three biological replicates – a total of 12 samples. The remaining four strains were run as a separate experiment with a total of 48 samples. For each experiment, sample chip position was randomized to avoid genotype- and treatment-specific batch effects.

Dataset Information

Transcription profiling of mouse tet inducible NIH 3T3 cells expressing ATPase-defective BRG-1 to identify SWI-SNF-Regulated Genes

Publications

Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting gamma-H2AX induction.

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