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. 2011;6(10):e26848.
doi: 10.1371/journal.pone.0026848. Epub 2011 Oct 26.

Systems analysis of ATF3 in stress response and cancer reveals opposing effects on pro-apoptotic genes in p53 pathway

Yujiro Tanaka  1 , Aya Nakamura, Masaki Suimye Morioka, Shoko Inoue, Mimi Tamamori-Adachi, Kazuhiko Yamada, Kenji Taketani, Junya Kawauchi, Miki Tanaka-Okamoto, Jun Miyoshi, Hiroshi Tanaka, Shigetaka Kitajima
Affiliations

Systems analysis of ATF3 in stress response and cancer reveals opposing effects on pro-apoptotic genes in p53 pathway

Yujiro Tanaka et al. PLoS One. 2011.

Abstract

Stress-inducible transcription factors play a pivotal role in cellular adaptation to environment to maintain homeostasis and integrity of the genome. Activating transcription factor 3 (ATF3) is induced by a variety of stress and inflammatory conditions and is over-expressed in many kinds of cancer cells. However, molecular mechanisms underlying pleiotropic functions of ATF3 have remained elusive. Here we employed systems analysis to identify genome-wide targets of ATF3 that is either induced by an alkylating agent methyl methanesulfonate (MMS) or over-expressed in a prostate tumour cell line LNCaP. We show that stress-induced and cancer-associated ATF3 is recruited to 5,984 and 1,423 targets, respectively, in the human genome, 89% of which are common. Notably, ATF3 targets are highly enriched for not only ATF/CRE motifs but also binding sites of several other stress-inducible transcription factors indicating an extensive network of stress response factors in transcriptional regulation of target genes. Further analysis of effects of ATF3 knockdown on these targets revealed that stress-induced ATF3 regulates genes in metabolic pathways, cell cycle, apoptosis, cell adhesion, and signalling including insulin, p53, Wnt, and VEGF pathways. Cancer-associated ATF3 is involved in regulation of distinct sets of genes in processes such as calcium signalling, Wnt, p53 and diabetes pathways. Notably, stress-induced ATF3 binds to 40% of p53 targets and activates pro-apoptotic genes such as TNFRSF10B/DR5 and BBC3/PUMA. Cancer-associated ATF3, by contrast, represses these pro-apoptotic genes in addition to CDKN1A/p21. Taken together, our data reveal an extensive network of stress-inducible transcription factors and demonstrate that ATF3 has opposing, cell context-dependent effects on p53 target genes in DNA damage response and cancer development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ChIP-on-chip analysis of ATF3 targets.
(A) RT-PCR analysis of ATF3 in HCT116 cells treated with 50 ng/ml MMS. (B) Western blot analysis of ATF3 proteins in MMS-treated HCT116 cells. (C)Common and unique targets of ATF3 in HCT116 cells and LNCaP cells.
Figure 2
Figure 2. Physical distance between predicted motifs and ATF3 peaks.
Distribution of physical distance between ATF/CRE, BRCA1, and GATA1 motifs on ATF3 targets from ATF3 peaks identified by ChIP analysis. ATF3 peaks largely coincide with ATF/CRE motifs but show no association with unrelated BRCA1 and GATA1 motifs.
Figure 3
Figure 3. Impact of ATF3 knockdown on target gene expression.
(A) HCT116 cells were transfected with either siRNA for ATF3 (siATF3) or scrambled siRNA (siControl) and treated with MMS for 0, 3, and 6 hours. Amount of ATF3 proteins was analysed by Western blot with β-actin as a loading control. MMS induces ATF3 at 3 and 6 hours after stimulation, which is significantly blocked by siATF3. (B) Heat map representation of gene expression levels at 0, 6, 12, and 24 hours after MMS stimulation for known targets of ATF3 (upper panel) or transcription factors which binding motifs are over-represented in ATF3 targets (lower panel). Signals are normalized against those at time 0 of siControl and relative levels or ratio between siATF3/siControl are colour coded from 2−2.5 to 22.5.
Figure 4
Figure 4. Regulation of p53 target genes by ATF3 in DNA damage response.
(A) Heat map representation of gene expression levels at 0, 6, 12, and 24 hours after MMS stimulation for common targets of ATF3 and p53. Signals are normalized against those at time 0 of siControl and relative levels or ratio between siATF3/siControl are colour coded from 2-2.5 to 22.5. Genes are clustered according to expression patterns after MMS stimulation: a, strongly activated; b, strongly repressed, c, weakly activated; d, mixed; e, weakly repressed. Most genes are weakly down-regulated by ATF3 knockdown, while DNMT1 is an exception that is up-regulated by ATF3 knockdown. (B) Quantitative RT-PCR analysis of ATF3 and pro-apoptotic targets of p53. Expression levels in siATF3-transfected cells (hashed bars) are indicated relative to those in control (open bars). (C) Phase contrast micrograph of HCT116 cells after treatment with 50 ng/ml MMS for 12 hours in the presence of siControl or siATF3. (D) Number of live HCT116 cells after treatment with 50 ng/ml MMS for 0, 8, 12, and 24 hours in the presence of siControl (open circles) or siATF3 (closed circles). (E) Activation of Caspase 3 as measured by its cleaved products. HCT116 cells were transfected with siControl or siATF3 and treated with 50 ng/ml MMS for 0, 3, 6, and 12 hours. Whole cell extracts were subjected to Western blot analysis using antibodies against ATF3, β-actin, Caspase 3, and cleaved Caspase 3. Arrowhead indicates cleaved Caspase 3 of the predicted molecular weight.
Figure 5
Figure 5. Activation of p53 target genes by ATF3.
(A) The DR5-luciferase reporter construct containing a DR5 promoter fragment from −1,226 (PstI site) to +1 (AUG codon) which was subcloned into PicaGene PGV-B2. ATF/CRE motifs (ATF) and p53 motif (p53) are indicated. (B) Dual luciferase reporter assays for TNFRSF10B/DR5, GADD45A, BBC3/PUMA, DDIT4, and VIM. HCT116 cells were transfected with each reporter and stimulated with 50 ng/ml MMS for 12 hours. Firefly luciferase activity was normalized by CMV-renilla luciferase activity. (C) Dual luciferase assay using wildtype or ATF3-/- mouse embryonic fibroblasts (MEF).
Figure 6
Figure 6. Regulation of p53 target genes by ATF3 in cancer.
(A) Western blot analysis of ATF3 and p21 in LNCaP cells transfected with siControl or siATF3 with β-actin as a loading control. (B) Number of viable cells at 0, 3, and 5 days after transfection with siControl (open circles) or siATF3 (closed circles). (C) Quantitative RT-PCR analysis of pro-apoptotic genes in p53 pathway and p21 after transfection. Levels of expression in siATF3 (hashed bars) relative to those in siControl (open bars) are indicated.
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