The SAM Domain of Human TEL2 Can Abrogate Transcriptional Output from TEL1 (ETV-6) and ETS1/ETS2

Regulation of gene expression downstream of the Receptor Tyrosine Kinase signaling pathway in Drosophila relies on a transcriptional effector network featuring two conserved Ets family proteins, Yan and Pointed, known as TEL1 (ETV6) and ETS1/ETS2, respectively, in mammals. As in Drosophila, both TEL1 and ETS1/ETS2 operate as Ras pathway transcriptional effectors and misregulated activity of either factor has been implicated in many human leukemias and solid tumors. Providing essential regulation to the Drosophila network, direct interactions with the SAM domain protein Mae attenuate both Yan-mediated repression and PointedP2-mediated transcriptional activation. Given the critical contributions of Mae to the Drosophila circuitry, we investigated whether the human Ets factors TEL1 and ETS1/ETS2 could be subject to analogous regulation. Here we demonstrate that the SAM domain of human TEL2 can inhibit the transcriptional activities of ETS1/2 and TEL1. Drosophila Mae can also attenuate human ETS1/ETS2 function, suggesting there could be cross-species conservation of underlying mechanism. In contrast, Mae is not an effective inhibitor of TEL1, suggesting the mode of TEL2SAM-mediated inhibition of TEL1 may be distinct from how Drosophila Mae antagonizes Yan. Together our results reveal both further similarities and new differences between the mammalian and Drosophila networks and more broadly suggest that SAM domain-mediated interactions could provide an effective mechanism for modulating output from the TEL1 and ETS1/2 oncogenes

Conservation of the mammalian and Drosophila Ets networks.

<p>(A) Schematic representation of the <i>Drosophila</i> and Mammalian Ets Networks. Activated MAPK (dpERK) phosphorylates Yan (TEL1) and PntP2 (ETS1/2) to inhibit transcriptional repression of target genes by Yan and to potentiate transcriptional activation by PntP2 respectively. Mae negatively regulates Yan and PntP2 to modulate signaling by the RTK network. Similarly, TEL2SAM negatively regulates the transcriptional activity of the vertebrate orthologs TEL1 and ETS1/2. (B) Sequence alignment of the SAM domains of Yan, TEL1, TEL2, Mae, PntP2, ETS1 and ETS2. Amino acids that are identical in at least four of the seven proteins are in bold, grey boxes highlight critical residues that mediate EH-ML surface interactions, and the asterisks indicate the specific residues mutated in the TEL2SAM^EHmut and TEL2SAM^MLmut constructs. (C) Dendrogram analysis using the sequences in (B) shows the phylogenetic relationships of the SAM domains.</p

Mae suppresses transcriptional activation by ETS1/2 in <i>Drosophila</i> S2 cells.

<p>(A) Activation of the argos-luciferase reporter by ETS1/2 is enhanced by expression of Ras^V12 and inhibited by Mae. (B) Myc-Mae coimmunoprecipitated with Flag-ETS1/2 from lysates of cotransfected <i>Drosophila</i> S2 cells cotransfected (lanes 2 and 3) but not from lysates of cells transfected with Myc-Mae alone (lane 1). Myc-Mae runs below the IgG light chain (strong band marked with asterisk). Flag-ETS1/2 run as doublets.</p

Inhibition of TEL1 repression by TEL2SAM is alleviated by mutations that prevent SAM domain-polymerization.

<p>(A) Communoprecipitation of myc-TEL2SAM with HA-TEL1 from cotransfected HeLa cells (lane 2) but not from cells transfected with HA-TEL1 alone (lane 1). Top and bottom panels were from the same gel, as were the middle two panels. (B) TEL2SAM can inhibit transcriptional repression by Yan, although not as effectively as Mae. (C) Repression of the E74tkluciferase reporter by TEL1 is suppressed by TEL2SAM but not by TEL2SAM^EHmut or TEL2SAM^MLmut.</p

TEL2SAM inhibits transcriptional repression by TEL1 of the E74tkluciferase reporter.

<p>(A) Repression by TEL1 is suppressed by TEL2SAM but <i>Drosophila</i> Mae has only a weak effect. The full-length TEL2 alone lane derives from an independent experiment in which repression by TEL1 was almost identical to that shown here. Using the TEL1 alone values to normalize between experiments, the % transcriptional activity for TEL2 was adjusted by a factor of 0.82. (B) TEL2SAM effectively inhibits repression by TEL1 at decreasing TEL2SAM concentrations. The ratio of TEL2SAM DNA to TEL1 DNA that was used for titration ranged from 2∶1 to 0.25∶1.</p

TEL2SAM inhibits transcriptional activation by ETS1/2 in HeLa cells.

<p>Ras^V12 enhanced and Mae or TEL2SAM suppressed activation of (A) the MMP9-luciferase reporter and (B) the dEts-luciferase reporter. (C) Mutations in the EH or ML surfaces of TEL2SAM do not alter its ability to suppress ETS2.</p

Synthesis and Evaluation of Apoptotic Induction of Human Cancer Cells by Ester Derivatives of Thujone

Thujone (1), thujol (2), and aromatic thujol esters (3–9) were evaluated for their ability to induce cell death in human cervical (HeLa), melanoma (A375), and colon (HCT-116) cancer cell lines, using etoposide as a positive control. The compounds showed dose-dependent activity at concentrations ranging from 50–400 μg/mL. Etoposide exhibited an IC50 value of 116 μg/ mLin HeLa cells, and α-thujone, α/β-thujone (7:1), and thujol showed comparable activity with IC50 values of 191, 198, and 1234567890();,: 1234567890();,: 136 μg/mL, respectively. All seven ester derivatives were cytotoxic to HeLa and HCT-116 cells, while a subset was cytotoxic to A375 cells. In HeLa cells, t-cinnamate (4), t-isonicotinate (5), t-nicotinate (6), and t-furoate (8) were more potent than either α-thujone or α/β-thujone. Similarly, t-furoate (8) was more potent than thujone in A375 cells, and t-isonicotinate (5) and t-nicotinate (6) were more potent against HCT-116 cells. Based on cell morphology, PARP cleavage and an increase in the caspase-3/7 levels, the esters exert their cytotoxic effects by induction of apoptosis

Sterile Alpha Motif Domain-Mediated Self-Association Plays an Essential Role in Modulating the Activity of the Drosophila ETS Family Transcriptional Repressor Yan▿

The ETS family transcriptional repressor Yan is an important downstream target and effector of the receptor tyrosine kinase (RTK) signaling pathway in Drosophila melanogaster. Structural and biochemical studies have shown that the N-terminal sterile alpha motif (SAM) of Yan is able to self associate to form a helical polymeric structure in vitro, although the extent and functional significance of self-association of full-length Yan remain unclear. In this study, we demonstrated that full-length Yan self associates via its SAM domain to form higher-order complexes in living cells. Introduction of SAM domain missense mutations that restrict Yan to a monomeric state reduces Yan's transcriptional repression activity and impairs its function during embryonic and retinal development. Coexpression of combinations of SAM domain mutations that permit the formation of Yan dimers, but not higher-order oligomers, increases activity relative to that of monomeric Yan, but not to the level obtained with wild-type Yan. Mechanistically, self-association directly promotes transcriptional repression of target genes independent of its role in limiting mitogen-activated protein kinase (MAPK)-mediated phosphorylation and nuclear export of Yan. Thus, we propose that the formation of higher-order Yan oligomers contributes to proper repression of target gene expression and RTK signaling output in developing tissues