These findings were corroborated using a myoblast cell line (C2C12) along with two matching cell lines derived by introducing either neomycin-resistance or puromycin-resistance to the parental cell line via the retroviral vector (pLNCX/pLPCX). The vectors are identical with the exception of the resistance cassette and neither of the derived cell lines (or vectors) carried any additional DNA sequences for expression and thus should be genetically identical with the exception of the differing mechanisms of antibiotic resistance. The C2C12 parental (Figure 4E) and C2C12 puromycin-resistant (Figure 4F) cell lines were sensitive to ADAADiK but the C2C12 neomycin-resistant cell line was ADAADiK-resistant (Figure 4G).
24 hours. This was not due to loss of APH transcript (Figure 5B) and therefore was attributed to cessation of ADAADi synthesis. Western blot analysis showed that there was a marginal reduction in the levels of SMARCAL1 (Figure 5C). In addition, the levels of Brg1, a chromatin remodeler required for active transcription [19] was also found to be slightly downregulated. However, the levels of Rad54B, a SWI2/SNF2 protein required for double-strand break repair [20,21], was found to be unaltered (Figure 5C). The subcellular localization of Brg1 as well as SMARCAL1, however, was found unaltered in the absence and presence of antibiotics (Figure S7A and B).
Alterations in epigenome and gene expression levels
Reduced levels of functional SWI2/SNF2 proteins such as ADAAD (SMARCAL1) lead to the hypothesis that there must be compensatory epigenetic changes occurring in order for stably transfected cells to survive the resultant production of ADAADi in the presence of aminoglycoside antibiotics. Consequently, we investigated the levels of H3K9 acetylation (H3K9Ac), associated with transcription initiation [22], and H3K9 dimethylation (H3K9Me2), associated with transcription repression [22], in aph transfected cells. H3K9Ac was found to be downregulated in aph (39)-IIa transfected cells when compared to the untransfected cells (Figure 5D).
Concomitantly, the transfected cells showed an increase in the level of H3K9Me2 as compared to the untransfected cells (Figure 5E), confirming alterations in histone modifications. As a corollary of the altered epigenetics, a microarray analysis using an Agilent platform showed that the transcription of many tissue-specific genes as well as metabolic enzymes was altered in vector transfected cell lines (Figure S8). Specifically, 2706 genes were upregulated and an equal number were downregulated (GEO accession number GSE36142). The microarray data corroborated a previous report that transfection of neomycinresistance gene into NIH3T3 cells results in decrease in expression of procollagen1a as well as fibronectin genes [23]. We further validated the microarray data for five genes, Nanog, ADH4, Runx2, Dicer1, and EP300, using quantitative RT-PCR.SWI2/SNF2 proteins are inactivated in aph (39)-IIa transfected mammalian cells Resistance to ADAADi connotes inactivation of SWI2/SNF2 proteins. As shown in Figure 5A, immunoprecipitated SMARCAL1, the mouse orthologue of ADAAD [18], from untransfected Neuro2A cells was active and responsive to ADAADiN and ADAADiG418, while the protein immunoprecipitated from transfected Neuro2A cells cultured post-selection either in the presence of both antibiotics or in the presence of pen-strep alone was inactive. The activity of SMARCAL1 was partially restored when the cells were grown in the absence of antibiotics for Figure 3. ADAADi formation is catalyzed by different isoforms of APH using different aminoglycoside substrates. (A). APH (39)-I, APH (39)-IIa, and APH (39)-IIIa catalyze ADAADi formation. ADAADi, synthesized by the three isozymes of APH, was purified and ATPase assays with 0.22 mM His-ADAAD and 68 mM kanamycin (Kan), 44 mM neomycin (Neo), 1.6 mM ADAADiK from APH(39)-I (I) and APH(39)-IIa (IIa), 1.2 mM ADAADiK from APH(39)-IIIa (IIIa), 1.6 mM ADAADiN from APH (39)-I (I), 2 mM ADAADiN from APH(39)-IIa (IIa) , and 1.4 mM ADAADiN from APH(39)-IIIa (IIIa) were done as described. (B). ADAADi is produced from G418 as well as streptomycin by APH (39)-IIIa. ATPase assays were done either in the absence or presence of 200 mM streptomycin, 2 mM G418, 4 mM ADAADiS, 4 mM ADAADiG418. (C). ADAADi produced using APH (39)-IIIa from commercially available aminoglycosides.
The microarray data showed Nanog and ADH4 were upregulated while Runx2, Dicer1, and EP300 were downregulated. These findings are consistent with the reported siRNA-mediated downregulation of Brg1 yields upregulated Nanog levels [24]. In stably transfected cells grown either in the presence of both the selective aminoglycoside (G418) and streptomycin or in the absence of G418 but presence of pen-strep alone, Nanog and ADH4 transcript levels were upregulated while Runx2 transcript levels were downregulated validating the microarray results. However, the regulation of Dicer1and EP300 levels could not be
verified (Figure 5F). These data confirms that ADAADi produced from the streptomycin in pen-strep is sufficient to maintain the epigenetic alterations. Further, the transcript levels of Nanog were restored to the untransfected levels while ADH4 were partially restored to untransfected levels when transfected cells were grown in the absence of antibiotics for 24 hours. However, the transcript levels of EP300, Runx2, and Dicer1 were further downregulated in these cells (Figure 5F).
Thus, epigenetic alterations and changes in gene expression pattern are prominent features of these aph transfected cells which are commonly labeled as neomycin-resistant.Brg1 recruitment to sg2na promoters is impaired
SG2NA, a member of the Striatin sub-family containing WD-40 repeats, plays a role cell signaling as well as vesicular trafficking [25,26] and is known to exist as multiple splice variants [27]. Microarray analysis corroborated our observation that SG2NA is repressed in aph transfected Neuro2A cells. Therefore, SG2NA was used as the test gene to understand the transcription repression mediated by ADAADi in stably transfected cells. As shown in Figure 6A, the transcript levels of endogenous SG2NA were downregulated in Neuro2A cells aph transfected cells, grown either in the absence or in the presence of antibiotics. Western blot analysis corroborated this observation (Figure 6B). Next, ChIP analysis showed that the RNAP II as well as Brg1 were present in the promoter region of sg2na in untransfected control cells but not in the stably transfected cells grown either in the presence or in the absence of both antibiotics (Figure 6C). However, the levels of H3K9Me2 and H3K9Ac were not found tobe statistically different between transfected cells and untransfected cells (Figure 6C). Thus, at the sg2na promoter, Brg1 levels correlate with the recruitment of RNAPII in untransfected cells. In stably transfected cells, lower Brg1 levels correlate with reduced RNAPII yielding apparent transcriptional repression.
