Dataset Information

Global epigenetic changes induced by SWI2/SNF2 inhibitors characterize neomycin-resistant mammalian cells.

ABSTRACT:

Background

Previously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes.

Methodology

Using fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase ADomain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized.

Significance

The use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.

SUBMITTER: Dutta P

PROVIDER: S-EPMC3509132 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Global epigenetic changes induced by SWI2/SNF2 inhibitors characterize neomycin-resistant mammalian cells.

Dutta Popy P Tanti Goutam Kumar GK Sharma Soni S Goswami Shyamal K SK Komath Sneha Sudha SS Mayo Marty W MW Hockensmith Joel W JW Muthuswami Rohini R

PloS one 20121128 11

<h4>Background</h4>Previously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes i ...[more]

PMID: 23209606

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Dataset Information

Global epigenetic changes induced by SWI2/SNF2 inhibitors characterize neomycin-resistant mammalian cells.

Background

Methodology

Significance

Publications

Global epigenetic changes induced by SWI2/SNF2 inhibitors characterize neomycin-resistant mammalian cells.

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