Journal: Nucleic Acids Research

Article Title: The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression

doi: 10.1093/nar/gkt563

Figure Lengend Snippet: Role of GCN2, ATF4 and CHOP in the transcriptional activation of a set of autophagy genes in response to leucine starvation. Wild-type, GCN2 −/− , ATF4 −/− and CHOP −/− MEFs were incubated either in control (+leu) or leucine-free medium (−leu). MEFs were harvested after 6 h, and total RNA was analyzed for autophagy gene mRNA contents. ( A ) In the first class of autophagy genes, Atg16l1 , Map1lc3b , Atg12 , Atg3 , Becn1 and Gabarapl2 were ATF4-dependent but CHOP-independent for amino acid-regulated transcription. ( B ) In the second class, the induction of Nbr1 and Atg7 in response to leucine starvation was dependent on ATF4 and CHOP . ( C ) The third class included Atg10, Gabarap and Atg5 , which were upregulated by amino acid starvation through ATF4 and CHOP. The graphs show means ± S.E.M. of three independent experiments. The asterisks indicate a P value of ≤ 0.05 relative to the control medium value. The promoter region of the different autophagy genes and the position of the putative AARE or CHOP-RE are represented. The promoter sequences were scanned with IUPAC patterns (Genomatix Software GmbH, Munich, Germany) ( Supplementary Figure S3 ). Numbers indicate the distance of the regulatory elements from the transcription start site (TSS). The symbols in brackets indicate the localization of regulatory elements in coding (+) or noncoding (−) strands. ChIP analysis was performed using antibodies specific for ATF4, and CHOP and primers to amplify a part of the corresponding promoter (see supplementary Table S2 for sequences) containing ATF4 or CHOP binding sites. Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin (% of Input). In CHOP-ATF4 double-ChIP assays, DNA fragments were first immunoprecipitated with CHOP antibody, eluted, and then subjected to the second immunoprecipitation with ATF4 antibody or normal rabbit IgG antibody. The enrichment of CHOP/ATF4 proteins was analyzed by qPCR using primer sets specific for Nbr1 and Atg7 promoters. In C/EBPβ–CHOP double-ChIP assays, DNA fragments were first immunoprecipitated with C/EBPβ antibody, eluted and then subjected to the second immunoprecipitation with CHOP antibody or normal rabbit IgG antibody. The enrichment of C/EBPβ–CHOP proteins was analyzed by qPCR using primer sets specific for Atg10 , Gabarap or Atg5 promoters. Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin (% of Input). The graphs show means ± S.E.M. of three independent experiments. t tests have been performed to compare the means. The asterisks indicate a P value of ≤ 0.05 relative to the +leu medium value.

Article Snippet: The promoter sequences were scanned with IUPAC patterns (Genomatix Software GmbH, Munich, Germany) ( Supplementary Figure S3 ).

Techniques: Activation Assay, Incubation, Control, Software, Binding Assay, Immunoprecipitation