Fig 1: ATF4 modulates association of positive and negative elongation factors within AA-responsive ARS genes. (A) Inhibition of CDK9 abolishes the induction of CARS and SARS during the AAR in HepG2 cells. HepG2 cells were pre-treated with or without flavopiridol (FVP) for 30 min, then incubated for 8 h in DMEM or DMEM + 5 mM HisOH, with (FVP) or without FVP (control). The steady state mRNA content for CARS and SARS was analyzed and the data plotted as the ratio to GAPDH. The results shown are the fold induction normalized relative to the DMEM value of control samples and presented as the averages ± standard deviations of three individual samples per experiment. The data are representative of multiple independent experiments. An asterisk (*) indicates a statistically significant change at P ≤ 0.05. (B) ATF4 interacts with both CDK9 and cyclin T1. FLAG-tagged ATF4 was expressed in HEK293T cells that bear a tetracycline-inducible construct encoding FLAG-ATF4 by incubating the cells in DMEM containing 0.1 μg/ml tetracycline for 6 h. Nuclear protein was isolated and subjected to immunoprecipitation with an antibody specific for the FLAG epitope. The immunoprecipitated protein was subjected to immunoblot analysis with antibodies against CDK9 or cyclinT1. Probing with anti-Atf3, a known Atf4 interacting protein, was used as a positive control. (C) ATF4 modulates changes in the gene association of NELF-A, and Spt5 during the AAR in MEFs. MEFs from WT or Atf4 KO mice were incubated for 8 h in DMEM or DMEM + 5 mM HisOH to activate the AAR. The Cars and Sars genes were subjected to ChIP analysis with antibodies specific for NELF-A or Spt5. Immunoprecipitation with a non-specific goat-anti rabbit antibody (IgG) or PCR at a distal site (+23 or +11 kb) were used as negative controls. The data are plotted as the ratio to input DNA and are the averages ± standard deviations for at least three samples per experiment. An asterisk (*) denotes a statistically significant difference of P ≤ 0.05 relative to the corresponding DMEM control value for that particular primer set. The arrows denote the annotated TSS of each gene and the boxes represent exons 1 and 2. The grey ovals represent the approximate location of CARE sites. The primer sequences used for RT-qPCR are listed in Supplementary Table S1.