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To extend the value of biosensor-SPR in the characterization of DNA recognition by nucleoproteins, we report a comparative analysis of DNA-facilitated target search by two ETS-family transcription factors: Elk1 and ETV6. ETS domains represent an attractive system for developing biosensor-based techniques due to a broad range of physicochemical properties encoded within a highly conserved DNA-binding motif. Building on a biosensor approach in which the protein is quantitatively sequestered and presented to immobilized cognate DNA as nonspecific complexes, we assessed the impact of intrinsic cognate and nonspecific affinities on long-range (intersegmental) target search. The equilibrium constants of DNA-facilitated binding were sensitive to the intrinsic binding properties of the proteins such that their relative specificity for cognate DNA were reinforced when binding occurred by transfer vs. without nonspecific DNA. Direct measurement of association and dissociation kinetics revealed ionic features of the activated complex that evidenced DNA-facilitated dissociation, even though Elk1 and ETV6 harbor only a single DNA-binding surface. At salt concentrations that masked the effects of nonspecific pre-binding at equilibrium, the dissociation kinetics of cognate binding were nevertheless distinct from conditions under which nonspecific DNA was absent. These results further strengthen the significance of long-range DNA-facilitated translocation in the physiologic environment., (Copyright © 2021. Published by Elsevier Inc.)

ETS transcription factors play an important role in the specification and differentiation of endothelial cells during vascular development. Despite previous studies, the role of the founding member of the ETS family, Ets1, in vascular development in vivo is only partially understood. Here, we generated a zebrafish ets1 mutant by TALEN genome editing and tested functional redundancy between Ets1 and a related ETS factor Etv2/Etsrp/ER71. While zebrafish ets1 -/- mutants have a normal functional vascular system, etv2 -/- ;ets1 -/ embryos had more severe angiogenic defects and lower expression levels of kdr and kdrl, the two zebrafish homologs of the mammalian Vascular Endothelial Growth Factor Receptor 2 VEGFR2/Flk1, than etv2 -/- embryos. Expression of constitutively active Mitogen-Activated Protein Kinase1 (MAP2K1) within endothelial cells partially rescued this angiogenic defect. Interestingly, ets1 -/- embryos displayed extensive apoptosis within the trunk vasculature despite exhibiting normal vascular patterning. Loss of Ets1 combined with a partial knockdown of Etv2 function resulted in a decrease in endothelial cell numbers in the axial vasculature, which argues for a role of Ets1 in promoting vasculogenesis. We also demonstrate that although both Ets1 and Etv2 can induce ectopic vascular marker expression in zebrafish embryos, Ets1 activity is dependent on MAPK-mediated phosphorylation of its Thr30 and Ser33 residues, while Etv2 activity is not. Together, our results identify a novel function of Ets1 in regulating endothelial cell survival during vasculogenesis in vivo. Based on these findings, we propose a revised model of how Ets1 and Etv2 play unique and partially redundant roles to promote vascular development., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The molecular mechanisms initiating the formation of the lymphatic system, lymphangiogenesis, are still poorly understood. Here we have identified a novel role in lymphangiogenesis for an ETS transcription factor, Etv2/Etsrp, a known regulator of embryonic vascular development. Through the use of fully validated photoactivatable morpholinos we show that inducible Etv2 inhibition in zebrafish embryos at 1 day post-fertilization (dpf) results in significant inhibition of lymphangiogenesis, while development of blood vessels is unaffected. In Etv2-inhibited embryos and larvae, the number of lymphatic progenitors is greatly reduced, the major lymphatic vessel, the thoracic duct, is absent or severely fragmented, and lymphangiogenesis-associated marker expression, including lyve1b, prox1a, and vegfr3/flt4, is strongly downregulated. We also demonstrate that lymphatic progenitors in Etv2 deficient embryos fail to respond to Vegfc signaling. Chromatin immunoprecipitation and sequencing (ChIP-Seq) studies using differentiated mouse embryonic stem (ES) cells as well as luciferase reporter studies in the ES cells and in zebrafish embryos argue that Etv2 directly binds the promoter/enhancer regions of Vegfc receptor Vegfr3/Flt4 and lymphatic marker Lyve1, and promotes their expression. Together these data support a model where Etv2 initiates lymphangiogenesis by directly promoting the expression of flt4 within the posterior cardinal vein., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. High-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-motifs, which bind JUN and FOS transcription factor families, were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements., (Copyright © 2017 Elsevier Ltd. All rights reserved.)