title: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi, India.
authors.forename: Popy
authors.initials: P
authors.lastname: Dutta
authors.forename: Goutam Kumar
authors.initials: GK
authors.lastname: Tanti
authors.forename: Soni
authors.initials: S
authors.lastname: Sharma
authors.forename: Shyamal K
authors.initials: SK
authors.lastname: Goswami
authors.forename: Sneha Sudha
authors.initials: SS
authors.lastname: Komath
authors.forename: Marty W
authors.initials: MW
authors.lastname: Mayo
authors.forename: Joel W
authors.initials: JW
authors.lastname: Hockensmith
authors.forename: Rohini
authors.initials: R
authors.lastname: Muthuswami
mesh_terms: D020816:Amino Acid Motifs; D000617:Aminoglycosides / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004351:Drug Resistance / Q000235:genetics; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009355:Neomycin / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000617:Aminoglycosides; D009687:Nuclear Proteins; D014157:Transcription Factors; D019868:Kanamycin Kinase; D009355:Neomycin