Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ChIP-chip from HEK293-RPF-1 stable transfectants expressing the HA-tagged RPF-1 transcription factor

ABSTRACT: RPF-1 binds promoters of genes modulated by its induction in HEK/RPF-1 transfectant Chromatin IP (ChIP) combined to chromatin array analysis (ChIP-chip) shows that RPF-1 transcription factor bind to promoter elements of genes repressed or activated by RPF-1 expression Comparison of cells induced for RPF-1 expression (Tet-) over uninduced control cells (Tet+)

ORGANISM(S): Homo sapiens

SUBMITTER: gabriele bucci

PROVIDER: E-GEOD-55270 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Retina-derived POU domain factor 1 coordinates expression of genes relevant to renal and neuronal development.

Fiorino Antonio A Manenti Giacomo G Gamba Beatrice B Bucci Gabriele G De Cecco Loris L Sardella Michele M Buscemi Giacomo G Ciceri Sara S Radice Maria T MT Radice Paolo P Perotti Daniela D

The international journal of biochemistry & cell biology 20160715

Retina-derived POU domain Factor 1 (RPF-1), a member of POU transcription factor family, is encoded by POU6F2 gene, addressed by interstitial deletions at chromosome 7p14 in Wilms tumor (WT). Its expression has been detected in developing kidney and nervous system, suggesting an early role for this gene in regulating development of these organs. To investigate into its functions and determine its role in transcriptional regulation, we generated an inducible stable transfectant from HEK293 cells. ...[more]

PMID: 27425396

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Project description:The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and spike-ins comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. This SuperSeries is composed of the following subset Series: GSE9732: Spike-in Experiment for ChIP-chip Simulation GSE9842: Systematic evaluation of variability in simulated ChIP-chip experiments GSE9848: Systematic evaluation of variability in simulated ChIP-chip experiments Kevin_Encode GSE9849: Systematic evaluation of variability in simulated ChIP-chip experiments Myles_Encode GSE10004: ENCODE Spike-In, Yale Group GSE10076: ENCODE spikein, amplified DNA samples, NimbleGen arrays GSE10090: ENCODE spikein, nonamplified DNA samples, NimbleGen arrays GSE10112: Systematic evaluation of variability in simulated ChIP-chip experiments Keywords: SuperSeries For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Refer to individual Series

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.

Dataset Information

ChIP-chip from HEK293-RPF-1 stable transfectants expressing the HA-tagged RPF-1 transcription factor

Publications

Retina-derived POU domain factor 1 coordinates expression of genes relevant to renal and neuronal development.

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