Figure 6. Compound 36 is a mM inhibitor of PTPs and binds the PTPs active site in silico. (A) Compound 36 was tested for inhibition of PTPs. Compound 36 (at a final concentration from 0 to 100 mM) was pre-incubated with PTPs for 10 min at 37uC. Reactions were then performed for 10 minutes at 37uC following the addition of 225 mM pNPP substrate. Relative PTPs activity is plotted (percent of activity in DMSO). Dashed line indicates 50% inhibition (10 mM). Bars represent standard deviation from three experiments. (B) The structure of compound 36 is displayed. (C) Compound 36 was docked into the active site of PTPs (PDB ID: 2FH7) using the D1 apo crystal structure. Molecular surface is colored by electrostatic potential. Red corresponds to negative potential and blue to positive potential. Docking was ?performed using the Schrodinger suite of software as described in Methods and figures generated using ICM (MolSoft).
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 neomycinresistant 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 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. 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.
Citation: Dutta P, Tanti GK, Sharma S, Goswami SK, Komath SS, et al. (2012) Global Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize NeomycinResistant Mammalian Cells. PLoS ONE 7(11): e49822. doi:10.1371/journal.pone.0049822 Editor: Brian P. Chadwick, Florida State University, United States of America Received July 5, 2012; Accepted October 17, 2012; Published November 28, 2012 Copyright: ?2012 Dutta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: R.M. was supported by grants from Council for Scientific and Industrial Research, University Grants Commission, University Grants Commissioncapacity build up, and Department of Science and Technology-PURSE; J.W.H. was supported by grants from the University of Virginia School of Medicine, and the Jeffress Memorial Trust; and M.W.M was supported by grants from National Cancer Institute (CA132580 & CA104397). P.D. was supported by funds from Rajiv Gandhi Fellowship, G.K.T. and S.S. were supported by funds from Council for Scientific and Industrial Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
The SWI2/SNF2 proteins play an important role in maintaining the cellular epigenome by harnessing the energy released by ATP to mobilize nucleosomes, and thus remodel chromatin architecture [1?]. Consequently, inhibition of the SWI2/SNF2 proteins could alter the epigenome and we herein explore the effects of one such inhibitor derived from aminoglycosides. Aminoglycosides, including neomycin and G418, are a large family of bactericidal antibiotics that interact with nucleotide A1408 present in the A-site of 16S rRNA [4,5], thus inhibiting translocation. Aminoglycosides are also toxic to eukaryotic cells, however the mechanism of action is unclear, as the eukaryotic rRNA contains a guanine residue in lieu of adenine at position 1408 thus precluding aminoglycoside binding [4,6]. 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?]. Mammalian cells transfected with aph acquire resistance to aminoglycosides and therefore, the gene has been used extensively as a selection marker [10] wherein transfected cells are selected using neomycin or G418. We have identified a hitherto uncharacterized product of APH enzymatic action which we call ADAADi [11]. ADAADi specifically inhibits Active DNA-dependent ATPase A Domain (ADAAD) and other members of the SWI2/SNF2 protein family [11]. This has been confirmed by Felle et al. who showed that this product inhibits nucleosome translocation and thereby prevents translocation of RNA polymerase I on chromatin templates [12].
