Unknown,Transcriptomics,Genomics,Proteomics

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Modulation of NF-kB-Dependent Gene Transcription Using Programmable DNA Minor Groove Binders

ABSTRACT: Nuclear factor kappaB (NF-kB) is a transcription factor that regulates various aspects of immune response, cell death and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5' GGGWWW-3'. The compound is capable of binding to kB sites and reducing the expression of various NF-kB driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-kB inhibitor PS1145, identifying overlaps and differences in affected gene groups and showing that both affect comparable numbers of TNFa inducible genes. Inhibition of NF-kB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists. A549 cells were treated with either a Py-Im polyamide or PS1145, subsequently induced with TNFa and compared with the untreated TNFa induced and the basal state by RNAseq. The NF-kB binding motif was derived by ChIP-seq

ORGANISM(S): Homo sapiens

SUBMITTER: Jevgenij Raskatov

PROVIDER: E-GEOD-34329 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders.

Raskatov Jevgenij A JA Meier Jordan L JL Puckett James W JW Yang Fei F Ramakrishnan Parameswaran P Dervan Peter B PB

Proceedings of the National Academy of Sciences of the United States of America 20111227 4

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5'GGGWWW-3'. The compound is capable of binding to κB sites and reducing the expression of various NF-κB-driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy wi ...[more]

PMID: 22203967

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Project description:TNF time course series of HelA tet off cells cultured in presence or absence of Dox; TNF is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the Nuclear Factor-kB (NF-kB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kB dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kB inhibitor to systematically identify NF-kB dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kB signaling was analyzed. Methods: Cell culture, treatment and transfection-The human cervical epithelioid carcinoma cell line HeLa expressing tTA (HeLa Tet-Off) pBI-EGFP- IkBa Mut was constructed earlier (23). HeLa Tet-Off were grown in medium containing 90 % Dulbecco's Modified Eagle's Medium (DMEM), 10 % heat-inactivated fetal bovine serum (FBS), 4mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mg/ml G418, 100 units /ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5 % CO2. For TNF stimulation, freshly isolated cells were split into two cultures, one group maintained in Dox, the other without for 7 d, a time at which IkBa Mut expression was maximal. Thereafter, recombinant human (rh) TNFa (25 ng/ml, final concentration) was added directly to the culture medium for indicated times prior to harvest. Oligonucleotide probe based microarray-Hu95Av2 GeneChip (Affymetrix Inc, Santa Clara, CA) containing 12,626 sequenced human genes were hybridized according to the manufacturer's recommendations and washed using both non-stringent (1 M NaCl, 25Â°C) and stringent (1 M NaCl, 50Â°C) conditions prior to staining with phycoerythrin streptavidin (10 mg/ml final). Arrays were scanned using a Gene Array Scanner (Hewlett Packard) and analyzed using the Gene Chip Analysis Suite 5 software (Affymetrix Inc). For each gene, 16-20 probe pairs are immobilized as ~25-mer oligonucleotides that hybridize throughout the mRNA; each probe pair is represented as a perfect match (PM) oligonucleotide and a mismatch (MM) oligonucleotide as hybridization control. The the Absolute Call [e.g., the gene is detected (present) or not (absent)] and the Signal Intensity (measure of mRNA abundance) is determined. Four independent experiments were performed identically including control (0 h), 1, 3 and 6 h TNF stimulation (25 ng/ml) in the presence or absence of Doxycyline (2 mg/ml) in growth medium. These are indicated as 180, 181, 220 and 266 series, and are considered replicates of each other.

Dataset Information

Modulation of NF-kB-Dependent Gene Transcription Using Programmable DNA Minor Groove Binders

Publications

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders.

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