Egr1 regulates the coordinated expression of numerous EGF receptor target genes as identified by ChIP-on-chip

Abstract

Background: UV irradiation activates the epidermal growth factor receptor, induces Egr1 expression and promotes apoptosis in a variety of cell types. We examined the hypothesis that Egr1 regulates genes that mediate this process by use of a chip-on-chip protocol in human tumorigenic prostate M12 cells.

Results: UV irradiation led to significant binding of 288 gene promoters by Egr1. A major functional subgroup consisted of apoptosis related genes. The largest subgroup of 24 genes belongs to the epidermal growth factor receptor-signal transduction pathway. Egr1 promoter binding had a significant impact on gene expression of target genes. Conventional chromatin immunoprecipitation and quantitative real time PCR were used to validate promoter binding and expression changes. Small interfering RNA experiments were used to demonstrate the specific role of Egr1 in gene regulation. UV stimulation promotes growth arrest and apoptosis of M12 cells and our data clearly show that a downstream target of the epidermal growth factor receptor, namely Egr1, mediates this apoptotic response. Our study also identified numerous previously unknown targets of Egr1. These include FasL, MAX and RRAS2, which may play a role in the apoptotic response/growth arrest.

Conclusions: Our results indicate that M12 cells undergo Egr1-dependent apoptotic response upon UV stimulation and led to the identification of downstream targets of Egr1, which mediate epidermal growth factor receptor function.

Figure 1

Egr1 induction upon UV-C stimulation. (a) Time course and dose response of Egr1 induction upon UV-C stimulation was measured by qRT-PCR. RNA was collected at various time points and at different doses of UV-C in order to determine the optimal dose (40 J/m²) and time point (2 h) for Egr1 expression. The upper panel shows the time course and the lower panel shows the dose response of Egr1 expression upon UV-C stimulation. (b) Western analysis using anti-Egr1 to define time of maximum UV response. (c) Western analysis of M12 cells treated with UV-C or not (control) in the presence and absence of ERK1/2 inhibitor (ErkI). The results show that prior treatment with ErkI resulted in negligible induction of Egr1 upon UV stimulation. Hence, at least 90% of Egr1 induction in these cells was downstream of the ERK MAP kinase.

Figure 2

ChIP-on-chip hybridization results. (a) Western analysis of ChIP products for M12 cells treated with UV-C or not (control). (b) Yield and size of DNA precipitated with anti-Egr1 from M12 cells following treatment with UV-C. (c) Distribution of the consensus Egr1-binding sequence (5' GCGGGGGCG 3') in approximately 17,000 human genes. (d) M-A plots of promoter array hybridization intensities of ChIP products from control M12 cells and cells treated with 40 J/m² UV-C for 2 h. The lower panel shows significance (volcano) plots of the hybridization intensity data for ChIP products of M12 cells treated with UV-C for 2 h compared with control non-treated ChIP sample. The right arm of the lower plot shows significantly bound promoters.

Figure 3

Validation of ChIP-on-chip results. (a) ChIP products for M12 cells treated with UV-C or untreated controls (control) were analyzed by qRT-PCR. The data are expressed as relative fold change of real-time PCR results to untreated control ChIP sample. The experiments were performed in triplicates and the error bars represent the triplicate data. (b) Western blot analysis of Egr2 (an Egr1 target gene) to define time of maximum induction. (c) M12 cells were treated with siRNA against Egr1 (SiEgr1) or SiGenome control (SiControl) for 48 h followed by UV-C irradiation and western blot confirmed the suppression of Egr1 in SiEgr1 treated cells. (d) qRT-PCR analysis of Egr1 target genes using RNA extracted from SiEgr1- or SiGenome control-treated M12 cells with or without UV-C irradiation. All the results are expressed relative to GAPDH.

Figure 4

EGFR activation upon UV stimulation. (a) After 40 J/m² UV-C radiation, 100 μg of the protein lysate was used for immunoprecipitation with EGFR antibody and this sample was used for immublotting with pTyr antibody showing activated EGFR. pEGFR, phosphorylated EGFR. (b) Western blot analysis of M12 cells treated with suramin or PD153035 for 45 minutes followed by UV-C irradiation.

Figure 5

Role of Egr1 in apoptosis. (a) Growth curve of M12 cells over a period of 72 h, with or without UV-C irradiation. The experiments were performed in triplicates and the error bars represent the data from the triplicate experiments (b) Western blot analysis of M12 cells treated with UV-C, and collected at varying time points after UV irradiation. Anti-PARP (to demonstrate apoptosis) was used to identify PARP cleavage product (this antibody only detects the cleaved product and not the native protein). (c) M12 cells were treated with SiGenome control and SiEgr1 and followed by UV-C stimulation. Cells were collected after 24 h of UV treatment, and western blot analysis, using anti-PARP, showed that Egr1 is involved in apoptosis of M12 cells.

Figure 6

Schematic diagram of the activation of Egr1 and the identification of its downstream targets upon UV simulation.