The common stress responsive transcription factor ATF3 binds genomic sites enriched with p300 and H3K27ac for transcriptional regulation

Abstract

Background: Dysregulation of the common stress responsive transcription factor ATF3 has been causally linked to many important human diseases such as cancer, atherosclerosis, infections, and hypospadias. Although it is believed that the ATF3 transcription activity is central to its cellular functions, how ATF3 regulates gene expression remains largely unknown. Here, we employed ATF3 wild-type and knockout isogenic cell lines to carry out the first comprehensive analysis of global ATF3-binding profiles in the human genome under basal and stressed (DNA damage) conditions.

Results: Although expressed at a low basal level, ATF3 was found to bind a large number of genomic sites that are often associated with genes involved in cellular stress responses. Interestingly, ATF3 appears to bind a large portion of genomic sites distal to transcription start sites and enriched with p300 and H3K27ac. Global gene expression profiling analysis indicates that genes proximal to these genomic sites were often regulated by ATF3. While DNA damage elicited by camptothecin dramatically altered the ATF3 binding profile, most of the genes regulated by ATF3 upon DNA damage were pre-bound by ATF3 before the stress. Moreover, we demonstrated that ATF3 was co-localized with the major stress responder p53 at genomic sites, thereby collaborating with p53 to regulate p53 target gene expression upon DNA damage.

Conclusions: These results suggest that ATF3 likely bookmarks genomic sites and interacts with other transcription regulators to control gene expression.

Keywords: ATF3; ChIP-seq; Enhancer; H3K27ac; p300; p53.

Fig. 1

ATF3 binding profiling using isogenic HCT116 cells. a rAAV-mediated genome editing was applied to generate ATF3-knocked out HCT116 cells. rAAV-mediated homologous recombination led to insertion of the AAV targeting vector into ATF3 exon 3. A deletion of 22 bp was generated in one ATF3 allele after Cre-mediated excision of the Neo selection gene. LA and RA, left and right homology arms; ITR, inverted terminal repeat; KO, knockout. b ATF3 expression was completely abolished in ATF3-KO cells. Indicated cells were treated with 1.5 μM of CPT and subjected to Western blotting. c Venn diagram showing ATF3-binding peaks in ATF3 wild-type (ATF3-WT) and knockout (ATF3-KO) cells. d Heatmap and intensity plots showing ATF3 peaks in ATF3 WT and KO cells. e Representative genome browser views of ATF3 peaks. ATF3 peaks near ATF3, STK40, HYI, SPRY1, and UTP23 were shown for both ATF3-WT and KO cells. f, g ChIP-qPCR was used to validate ATF3 binding to representative genome sites that were referred to as the names of their annotated genes. NR, no-binding control region. Error bars represent SD for three replicate measurements. h The binding intensity determined by independent ChIP-qPCR assays was correlated with ChIP-seq scores of peaks tested in (f) and (g)

Fig. 2

Global ATF3-binding profile under the basal condition. a A pie chart showing distribution of ATF3 binding sites relative to annotated genes. b 600 annotated genes with top peak scores were used for DAVID GO analysis. GO biological process (BP) terms and KEGG pathway terms are shown. c Top motifs identified in the ATF3 binding sites. d Schematic showing relative abundance and overlaps of the three known ATF3 binding motifs. The numbers are peak numbers. e The binding of ATF3 to the ATF/CRE motif appeared to be stronger than the AP-1 motif. Student t-test. f A table showing top motifs after combining motifs with same/similar sequences. g Venn diagram showing overlaps of CTCF, GATA3, and TEAD motifs with the ATF3 motif

Fig. 3

ATF3 globally binds genomic sites enriched with p300 and H3K27ac. a Genome browser views showing co-localization of ATF3 with p300 and H3K27ac in several representative genomic sites. b Venn diagram showing overlaps between ATF3 peaks and p300 peaks. c Intensity plot showing co-localization of ATF3 with p300 and H3K27ac. d Venn diagram showing overlaps between p300 distal peaks (active enhancers) and ATF3 peaks. e ChIP-qPCR validation of p300 binding to the ATF3 binding sites. HCT116 cells were subjected to ChIP using a p300 antibody. Precipitated DNA was quantitated using qPCR. f H3K27ac was enriched in the ATF3-binding sites. ChIP-qPCR was carried out to determine H3K27ac levels in the ATF3-binding sites

Fig. 4

Binding of ATF3 to active enhancers correlates with ATF3-regulated gene expression. a Heatmaps showing ATF3-regulated genes, and their binding by ATF3 and p300. b qRT-PCR validation of genes differentially expressed between ATF3-wildtype and knockout cells identified by microarray. c, d Validation of ATF3 binding to differentially-expressed genes by ChIP-qPCR. e ATF3 was localized in regions distal to TSS (>2 kb) of differentially-expressed genes. f Representative genome browser views of co-localization of ATF3 and p300 in ATF3-regulatd genes. g ATF3 was localized in active enhancers of ATF3-regulated genes. Error bars represents SD

Fig. 5

DNA damage alters the ATF3-binding landscape for transcriptional regulation. a Venn diagram showing overlap of ATF3-binding peaks between the basal (ATF3-WT) and the CPT-treated (ATF3-CPT) conditions. b Heatmaps and intensity plots showing alterations in the ATF3-binding profile caused by DNA damage. c Intensity plots showing different enrichments of ATF3, p300, and histone markers between CPT-induced and decreased peaks. d Distribution of ATF3-binding sites under basal (ATF3-Ctrl) and stressed (ATF3-CPT) conditions. e Venn diagram showing overlaps of ATF3 peaks and p300 peaks under the CPT-treatment condition. f Heatmaps showing CPT-regulated genes. g Heatmaps of ATF3-binding sites associated with CPT-regulated genes showing that ATF3 was pre-loaded on most of these genes before stress. h Intensity plot showing that ATF3 binding to CPT-regulated genes was increased by the CPT treatment. i Heatmap showing ATF3-regulated genes under the DNA damage condition were pre-bound by ATF3. j Intensity plot showing that DNA damage increased ATF3 binding to ATF3-regulated genes

Fig. 6

Co-localization of ATF3 and p53 in genomic sites regulates gene expression in the DNA damage response. a Venn diagram showing the overlap between ATF3 peaks and p53 peaks under the DNA damage condition. b Genome browser views of p53 binding to several well-characterized p53 target genes. c Binding of p53 to indicated sites was validated by independent ChIP-qPCR assays. d Genome browser views of co-localization of ATF3 and p53 in representative genomic sites. e ATF3 and p53 were co-localized in genomic sites as demonstrated by re-ChIP assays. HCT116 cells treated with 1.5 μM of CPT for 4 h were first subjected to ChIP using the ATF3 antibody. The chromatin precipitated by the ATF3 antibody was then eluted from agarose beads, and subjected to the second round of ChIP using the p53 antibody. qPCR assays were used to quantitate re-ChIPed DNA. f Venn diagram showing the overlap of p53-binding sites containing the p53 motif or the ATF3 motif. g The ATF3 peak score correlated with the p53 peak score in the sites co-localized by ATF3 and p53. h ATF3 binding was often decreased in p53-knockout cells. p53-wildtype and knockout (p53-KO) HCT116 cells were subjected to ChIP-qPCR to measure binding of ATF3 to the indicated sites. i p53 binding was decreased in ATF3-knockout cells. ATF3-wildtype and knockout (ATF3-KO) HCT116 cells were subjected to ChIP-qPCR to measure binding of p53 to the indicated sites. j Expression of p53 target genes was repressed in ATF3-KO cells. Indicated cells were treated with 1.5 μM of CPT for qRT-PCR assays. ATF3 binding to these genes before and after CPT treatments in ATF3-WT cells were shown in Additional file 1: Figure S3