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ETV4 and AP1 Transcription Factors Form Multivalent Interactions with Three Sites on the MED25 Activator-Interacting Domain

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ETV4 and AP1 Transcription Factors Form Multivalent Interactions with Three Sites on the MED25 Activator-Interacting Domain Featured Arcle KDC YJMBI-65461; No. of pages: 21; 4C: ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain Simon L. Currie 1,2, Jedediah J. Doane 1,2, Kathryn S. Evans 1,2, Niraja Bhachech 1,2, Bethany J. Madison 1,2, Desmond K.W. Lau 3, Lawrence P. McIntosh 3, Jack J. Skalicky 4, Kathleen A. Clark 1,2 and Barbara J. Graves 1,2,5 1 - Department of Oncological Sciences, University of Utah School of Medicine, Salt Lake City, UT, 84112-5500, USA 2 - Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 84112-5500, USA 3 - Departments of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada 4 - Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, 84112-5650, USA 5 - Howard Hughes Medical Institute, Chevy Chase, MD, 20815-6789, USA Correspondence to Barbara J. Graves: Department of Oncological Sciences, University of Utah School of Medicine, Salt Lake City, UT, 84112-5500, USA. [email protected]. http://dx.doi.org/10.1016/j.jmb.2017.06.024 Edited by Prof. M.F. Summers Simon L. Currie, Jedediah J. Doane, Kathryn S. Evans and Barbara J. Graves Abstract 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. © 2017 Elsevier Ltd. All rights reserved. Introduction and activity of RNA polymerase II (Pol II) [1]. These interactions are important for the foundation of The activation domains (ADs) of sequence spe- transcriptional programs that regulate development cific DNA-binding transcription factors interact with and establish cell-type identity [2]; as such, compo- general transcription factors, coactivators, and chro- nents of these interactions are commonly mutated in matin remodelers, in order to regulate the location human disease [3,4]. Acidic ADs, originally noted for 0022-2836/© 2017 Elsevier Ltd. All rights reserved. J Mol Biol (2017) xx, xxx–xxx Please cite this article as: S. L. Currie, et al., ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain, J. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jmb.2017.06.024 2 ETV4 and JUN/FOS bind to multiple sites on MED25 an enrichment of negatively-charged and non-polar scription factors [35,40,42,50–52]. For example, in residues [5,6], have an alternating pattern of addition to ETV5 the transcription factors ATF6α, negatively-charged/nonpolar and bulky hydropho- HNF4α, RARα, SOX9, and the viral protein VP16 bic/aromatic residues. Although usually disordered recruit the variable subunit MED25 to their respec- in isolation, ADs often become more helical when tive target genes [32,45,53–56]. The SOX9-MED25 interacting with cofactors [7–10]. These sequence interaction is implicated in chondrogenesis because and structural characteristics are presumably the reduced expression of either component results in foundation of the ability of a single AD to interact with similar palatal malformations in zebrafish [56]. Other multiple partners as a flexible hydrophobic/aromatic Mediator subunits, such as MED23 and MED1, interface that can be presented differently to diverse function with a distinct set of transcription factors proteins [11–20]. However, higher affinity for a (reviewed in Ref. [52]). Thereby, Mediator subunits particular factor, and thus specificity, can be accom- can have gene-specific and cell-specific functions. plished through the use of multiple ADs [21–24]. Here we investigate the biochemical basis and ETV1, ETV4, and ETV5 form a subgroup within functional implications of ETV4-MED25 interactions. the ETS family of transcription factors, sharing high High-affinity interaction with MED25 was specific to the sequence conservation both within and beyond the ETV1/4/5 subfamily of ETS factors. The ETV4-MED25 DNA-binding domain (DBD). This subgroup is interaction involves two domains, the N-terminal aberrantly overexpressed in a subset of prostate activation domain (AD) and the DNA-binding domain cancers [25–27], and promotes PI3-kinase and RAS (DBD), each binding the activator interacting domain signaling pathways resulting in an aggressive and (ACID) of MED25 via a similar motif (ΩxxxΩΦ or metastatic disease phenotype [28,29]. Upregulation ΦΩxxxΩ,whereΩ is an aromatic residue, Φ is a of the ETV1/4/5 subgroup mimics RAS/MAPK hydrophobic residue, and x is any residue). Full-length signaling to gene expression changes in prostate ETV4, bearing both regions, had higher affinity for cell lines ultimately resulting in increased cellular MED25 than either domain alone. Furthermore, the migration [30]. ETS binding motifs, in association kinetics of association and dissociation by each domain with motifs recognized by JUN and FOS transcrip- differed, suggesting a complex binding reaction when tion factors (AP1 factors), are a hallmark of RAS/ both are present. NMR spectroscopy, mutational MAPK-responsive gene expression [31]. Previously studies, and protein-docking modeling provided evi- it was demonstrated that the N-terminal AD of ETV5 dence that the AD and DBD bound to the same site on binds to the activator interacting domain (ACID) of MED25 ACID. However, the DBD also interacted with Mediator subunit 25 (MED25) [7,32]. However, ~ two additional, distinct sites on MED25, such that 10% of prostate cancers frequently harbor trunca- simultaneous occupancy was possible in spite of tions of ETV1, ETV4, or ETV5 that lack this AD due overlapping contact surfaces. MED25 activated the to chromosomal rearrangements [33,34]. This sug- DNA binding of ETV4 by relieving a previously gests that the AD is dispensable for the function of described autoinhibition mechanism. Additional func- these factors in prostate cancer. Therefore, we tional implications were provided by genome-wide hypothesized that ETV1/4/5 subfamily factors could studies of MED25 and ETV4 occupancies. There was use an additional MED25-binding site, outside of the a significant overlap in physical location and in genes N-terminal AD, to interact with MED25. Such a whose expression was affected by depletion of either domain, if it functioned in the absence of the AD, factor. MED25-occupied regions were enriched for might explain the retained transcriptional activity of ETS binding sequences. In addition, recognition the oncogenic ETV1/ETV4/ETV5 truncations. sequences for members of the JUN and FOS family The Mediator complex is a critical transcriptional of transcription factors (AP1 sites) were enriched in coactivator that serves as a primary conduit for MED25-occupied regions. We report that JUN/FOS transmitting regulatory signals from specific tran- heterodimers also contacted MED25 through a similar scription factors to Pol II [35]. The 26 subunits of mechanism as ETV4. In conclusion, we propose that Mediator (not including the CDK8 kinase module) both ETV1/4/5 and AP1 transcription factors use form distinct modules termed the head, middle, and multivalent interactions to recruit MED25 to gene tail. A reconstituted complex comprised of 15 regulatory regions and promote the stable assembly subunits from the head and middle modules repre- of transcriptional machinery. sents the minimal functional, or “core”, complex required for the general coactivator function of Results Mediator [36–38]. In contrast, the presence of, and requirement for, other subunits of Mediator is more variable and gene-specific [39–49]. The simplest High-affinity interaction with MED25 is specific model to explain gene-specificity is that non-core to the ETV1/4/5 subfamily of ETS factors Mediator subunits are required only for the transcrip- tion of the genes to which they are directly recruited The interactions between MED25 ACID (residues via interactions with distinct sequence-specific tran- 391–553) and several ETS transcription factors were Please cite this article as: S. L. Currie, et al., ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain, J. Mol. Biol. (2017), http://dx.doi.org/10.1016/j.jmb.2017.06.024 ETV4 and JUN/FOS bind to multiple sites on MED25 3 measured by biolayer interferometry in which one tion (Fig. S1c). As other experimental approaches, species is attached to a substrate and solution discussed below, also support a multivalent bindingofananalyteismonitored(Fig. 1a,b). ETV4-MED25 interaction, we report values calculat- Testing single concentrations of these full-length ed using the two-to-one model.
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