Fig. 4.

ATF1 trans-activates core pluripotent and mitochondrial factors. a Representative GSEA enrichment plots for gene sets enriched in control and shATF1 (shATF1#1 and shATF1#4) groups. b QRT-PCR (QPCR) analysis of stemness genes in iDox-shATF1 MDA-MB-231 cells. Fold changes (presented as Log2) in gene expression were compared to cells without Dox treatment (n = 3; mean ± SD). c Top biological processes enriched in ATF1 co-expressed genes based on the TCGA pan-cancer datasets. P values were EASE scores (modified Fisher Exact P value) provided in the DAVID database. d QPCR analysis of mitochondrial biogenesis genes in iDox-shATF1 MDA-MB-231 cells. Fold changes (presented as Log2) in gene expression were compared to cells without Dox treatment (n = 3; mean ± SD). e Top motifs enriched in ATF1 binding DNA element in MDA-MB-231 cells determined by de novo motif analysis of ATF1-CutTag peaks. f Biological processes enriched in ATF1 target genes, as determined by genes with transcription start sites 2k bp from in ATF1 binding peaks. g Left panel, representative tracks of normalized ATF1 CUT&TAG-seq signals at MYC, NAONG, NRF1 and TFAM loci. Right panel, PCR quantification of ATF1 ChIP products in the regulatory regions of stemness and mitochondrial regulators. ATF1 ChIP signal was normalized over control IgG (n = 3; mean ± SD). h Luciferase reporter assay for ATF1 mediated gene transactivation. 293 T cells were co-transfected with a promoter-Firefly-luciferase reporter construct (MYC, NANOG, NRF1 and TFAM) in combination with empty vector, wild-type or mutant ATF1. Results were expressed as ratio of Firefly/ Renilla and normalized to vector construct (n = 4; mean ± SD; (n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA). i The ENCODE ChIP-seq datasets (https://www.genome.ucsc.edu/index.html) showing ATF1 and CREB1 peaks in the regions of nuclear and mitochondrial regulators according to the data from the UCSC Genome Browser. Red boxes represent region near the transcription start site. Blue arrows represent transcription orientation. j Pearson correlations among ATF1, MYC, NRF1 and stemness ssGSEA scores based on the transcription profiles of the CCLE cancer cell lines (P values, Pearson correlation). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant