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. 2014 Oct 29;42(19):11928-40.
doi: 10.1093/nar/gku929. Epub 2014 Oct 7.

ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells

Joshua P Plotnik  1 , Justin A Budka  2 , Mary W Ferris  2 , Peter C Hollenhorst  3
Affiliations

ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells

Joshua P Plotnik et al. Nucleic Acids Res. .

Abstract

The RAS/ERK pathway is commonly activated in carcinomas and promotes oncogenesis by altering transcriptional programs. However, the array of cis-regulatory elements and trans-acting factors that mediate these transcriptional changes is still unclear. Our genome-wide analysis determined that a sequence consisting of neighboring ETS and AP-1 transcription factor binding sites is enriched near cell migration genes activated by RAS/ERK signaling in epithelial cells. In vivo screening of candidate ETS proteins revealed that ETS1 is specifically required for migration of RAS/ERK activated cells. Furthermore, both migration and transcriptional activation through ETS/AP-1 required ERK phosphorylation of ETS1. Genome-wide mapping of multiple ETS proteins demonstrated that ETS1 binds specifically to enhancer ETS/AP-1 sequences. ETS1 occupancy, and its role in cell migration, was conserved in epithelial cells derived from multiple tissues, consistent with a chromatin organization common to epithelial cell lines. 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.

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Figures

Figure 1.
Figure 1.
ETS/AP-1 sequences predict increased activation by RAS signaling. Box plots show all genes or genes with the indicated neighboring sequence, activated by expression of Ha-RASV12 or Ki-RASV12 in Caco2 or HMEC cells. Genes are only included if the indicated sequence is in open chromatin in the cell line where expression changed, and has the nearest transcription start site within 25 kb. Plotted gene numbers are All: 24826, ETS/AP-1: 3849, mutETS/AP-1: 1691, ETS/CRE: 334, ETS/ETS: 1037, ETS/SP1: 2331. P-values calculated by a Welch Two Sample t-test: ***<0.0001, unmarked are >0.05.
Figure 2.
Figure 2.
Phospho-ETS1 is required for the migration of the RAS-active prostate cancer line, DU145. (A) Immunoblot with antibodies shown (left) of DU145 cells with shRNA mediated knockdown of five ETS factors (top). An shRNA targeting luciferase is a negative control. Tubulin is a loading control. (B) A transwell assay measured relative cell migration of DU145 cells with indicated knockdown. Mean and SEM of ≥ 3 biological replicates shown. P-values (*<0.05, **<0.01, ***<0.001) are calculated by two-tailed T-test. (C) An MTT assay compared proliferation of DU145 cells with ETS1 and control shRNA knockdowns. Three biological replicates shown. Proliferation rates are not significantly different (P = 0.73). (D) A reporter assay compares relative luciferase units (firefly/renilla) from DU145 cells expressing control luciferase (luc) shRNAs or ETS1 shRNAs and treated with the MEK inhibitor, U0126 50 μM, as indicated. The firefly luciferase vector has three copies of the ETS/AP1 element (WT) 5′ to the minimal promoter or the same vector with point mutations in each ETS binding site (MUT). Values are shown as a ratio to the first column and are the mean and SEM of three biological replicates. P-values are as in (B). (E) Anchorage-independent growth by soft agar assay for selected ETS knockdown in DU145 cells. Number of colonies calculated relative to luciferase shRNA. Mean and SEM of three biological replicates reported. (F) Immunoblots show levels of E-cadherin and Vimentin in DU145 cells with the indicated shRNA knockdown. (G) Immunoblot with antibodies shown (left) for DU145 cells transduced with a retrovirus to stably overexpress empty vector, Flag-ETS1, Flag-T38A/S41A ETS1 and Flag-ETS2. (H) A transwell assay measured relative cell migration of DU145 cells with indicated ETS overexpression constructs as in (G). Mean and SEM of ≥3 biological replicates each containing two technical replicates are shown. P-value of ETS1 compared to T38A/S41A ETS1 by T-test. (I) Relative luciferase expression as in (D) for a 3xETS/AP-1 reporter in DU145 cells overexpressing indicated ETS construct.
Figure 3.
Figure 3.
ETS1 only regulates cell migration in cells with an activated RAS/ERK pathway. (A) Immunoblot of PC3 cells stably expressing either luciferase, ETS1 or ETV4 shRNAs (top) demonstrates knockdown of each ETS factor. (B) Transwell assay of PC3 cells with indicated knockdowns. Replicates and significance as in Figure 1B. Transwell assays of RWPE1 (C) or KiRAS expressing RWPE2 (D) cells stably overexpressing retroviral ETS1. Replicates and significance as in Figure 1B. (E) Transwell assay of RWPE2 cells transfected with stable lentiviral expression of indicated shRNA. Replicates and significance as in Figure 1B.
Figure 4.
Figure 4.
ETS1 binds ETS/AP1 sequences in vivo. (A) Venn diagram demonstrates overlap of all ChIP-seq bound regions in DU145 cells for ETS factors tested using Useq platform. (B) Most enriched motifs for bound enhancer regions (>300 bp from a TSS) as determined by RSAT peak-motifs algorithm after GEM analysis. For GABPA and ELF1, only one motif was identified. (C) Overlap of ETS1 bound enhancer regions with JUND bound regions in DU145 as determined by ChIP-seq analysis. Number in parenthesis indicates the number of expected overlaps using the assumption that any region of open chromatin is potential binding site (determined by RWPE1 DNase-seq). (D) ETS1 and JUND occupancy is plotted by -log binary P-value near the ETS1 gene.
Figure 5.
Figure 5.
ETS1 is required for migration and demonstrates genome-wide enrichment for ETS/AP1 across multiple RAS-active cancer cell types. (A) Overlap of ETS1-bound regions in RAS-active prostate (DU145), lung (A549) and pancreatic (PANC1) cell lines. A549 data is from ENCODE (Richard Myers Group, GSM1010829). (B) ETS1, JUND and GABPA occupancy near the gene encoding vimentin (VIM) is plotted by -log binary P-value from ChIP-seq data in the indicated cell lines. (C) Immunoblots compare ETS1 and tubulin (loading control) levels in PANC1 cells after treatment with shRNAs targeting luciferase (negative control) or two independent shRNAs targeting ETS1. (D) A Boyden chamber transwell assay measures relative cell migration of PANC1 cells expressing each lentiviral shRNA of ETS1 or luciferase shRNA control. Mean and SEM of four biological replicates shown for ETS1 shRNA #1 and two replicates for shRNA #2. P-value (*<0.05) by two-tailed T-test.
Figure 6.
Figure 6.
ETS1 regulates two positive feedback loops through two distinct mechanisms. (A) Venn diagram displaying overlap between genes downregulated by the MEK inhibitor U0126 and genes neighboring an ETS1-bound enhancer as determined by ChIP-seq. Downregulated genes are those with significant (P-value < 0.05) decreases after 6 h of 50 μM U0126 compared to mock treated cells as measured by RNA-seq of three biological replicates. Parenthesis indicates randomly predicted overlap. (B) The 272 genes overlapping in (A) were compared to genes with expression changes of greater than log2 = 0.2 or −0.2 in ETS1 shRNA compared to control shRNA by RNA-seq. (C) Ontology analysis of ETS1 enhancer targets from overlaps in (B) using g:Profiler. The top three informative, non-redundant, categories are listed along with their representative P-values. (D) Immunoblot of ETS1, ERK, pERK or tubulin in DU145 cells treated with ETS1 shRNA or U0126, as indicated. (E) Representative immunoblot of DU145 cells expressing luciferase (control) or ETS1 shRNAs and serum starved (12 h) and treated with 50 mg/ml PMA for 5 min as indicated. (F) Quantification of immunoblots as in (E), by ImageJ. P-value (*<0.05) by two-way T-test after three independent biological replicates. (G) Model demonstrating ETS1 regulating two distinct positive feedback loops perpetuating RAS/ERK signaling and cell migration.
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