Abstract
BackgroundPreviously, 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.MethodologyUsing fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase A Domain 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.SignificanceThe 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.
Highlights
The SWI2/SNF2 proteins play an important role in maintaining the cellular epigenome by harnessing the energy released by ATP to mobilize nucleosomes, and remodel chromatin architecture [1,2,3]
We show that Active DNA-dependent ATPase A Domain inhibitor (ADAADi) binds to a region known as Motif Ia in SWI2/SNF2 proteins and this interaction induces a conformation change in the protein that prevents ATP hydrolysis
ADAADi, a distinct product generated by APH Previously we have shown APH (39)-IIIa generates a product from aminoglycosides, kanamycin and neomycin, that inhibits the ATPase activity of the SWI2/SNF2 proteins [11]
Summary
The SWI2/SNF2 proteins play an important role in maintaining the cellular epigenome by harnessing the energy released by ATP to mobilize nucleosomes, and remodel chromatin architecture [1,2,3]. Prokaryotic resistance to aminoglycosides is mediated by many means, one of which includes a reaction catalyzed by prokaryotic APH, therein transfering a phosphate group from ATP to the 39 position of aminoglycosides to generate a phosphoaminoglycoside as the predominant product [7,8,9]. Mammalian cells transfected with aph acquire resistance to aminoglycosides and the gene has been used extensively as a selection marker [10] wherein transfected cells are selected using neomycin or G418. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. We hypothesized that the introduction of neomycinresistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
