ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells.
Bottom Line: Genome-wide expression analysis showed that ETS1 was required for activation of RAS-regulated cell migration genes, but also identified a surprising role for ETS1 in the repression of genes such as DUSP4, DUSP6 and SPRY4 that provide negative feedback to the RAS/ERK pathway.Consistently, ETS1 was required for robust RAS/ERK pathway activation.Therefore, ETS1 has dual roles in mediating epithelial-specific RAS/ERK transcriptional functions.
Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.Show MeSH
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Mentions: These results indicate that ETS1 mediates RAS/ERK activation through ETS/AP-1 sequences. However, previous mapping of ETS1 binding sites in T cells by ChIP-seq did not identify the ETS/AP-1 sequence as a common ETS1 binding motif (16,17). To test if this result was due to the differences in cell type, we measured genomic occupancy of four ETS proteins in DU145 cells by ChIP-seq (Bound regions available in Supplementary Table S2). These included three ETS proteins previously mapped in Jurkat T cells (ETS1, GABPA and ELF1) and an additional RAS/ERK regulated ETS protein, ELK4. As a control, two biologically independent replicates of ETS1 ChIP were sequenced, and these showed significant convergence (Supplementary Figure S4). In total, ETS1 bound 9994 regions and GABPA bound 2489 regions in DU145 cells (Figure 4A). These are similar to the number of bound regions reported for these factors in T cells (17,35). ELF1 only occupied 247 regions in DU145 cells, possibly due to a low expression level in this cell type. ELK4 also bound a small number of regions (338). Although ELK4 occupancy has not been published, this number is very similar to the 529 bound regions reported for the close ELK4 homolog, ELK1, in breast epithelia (36). ChIP/qPCR was used to confirm enrichment of five randomly selected regions in each dataset (Supplementary Figure S5). An overlap of ETS1, GABPA, ELK4 and ELF1 binding sites (Figure 4A) revealed a pattern similar to that identified in T cells (17), with GABPA binding primarily in promoter sequences (<300 bp to TSS) that were co-occupied by other ETS factors (redundant sites), and the other ETS proteins having a mixture of redundant promoter binding and specific distal enhancer binding sites (>300 bp to TSS). The GEM algorithm (27) was used to identify centered binding sites for each of the ETS tested. Following GEM, windows were extended 50 bp in either direction of the centered motif. The PeakMotifs algorithm (28) was used to conduct an unbiased search for the most over-represented sequence motifs in either promoter or enhancer binding sites. Motifs identified in promoter regions (Supplementary Figure S6) were similar to those previously reported in other systems (16,17), and none included AP-1. In enhancers, GABPA and ELF1 sites were enriched for ETS consensus sequences (Figure 3B). ELK4 sites were enriched for both an ETS site and a site for serum response factor (SRF), the factor known to cooperatively bind with ELK4 (Figure 4B). Strikingly, at ETS1 enhancer sites, the most enriched sequence was a composite ETS/AP-1 sequence (CCGGAAGTGACTCA). These data indicate that ETS1, and not GABPA, ELF1 or ELK4, binds to ETS/AP-1 sequences in the enhancers of DU145 cells.
Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.