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

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

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 transcription 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

(a) 2.0 + MED25 + ETV4 - ETV4 [ETV4]

1.5

1.0

0.5 Response (nm) Baseline Association Dissociation 0.0 Load 250 500 750 1000 time (s) -0.5 * (b) (c) 0.15 Association Dissociation 10,000 * 50 nM ETV4 1,000 * 0.10 * 500 nM SPDEF 100 (nM)

500 nM ETS1 D 0.05 K 10 ETV4 Response (nm) 500 nM EHF ETV1 SPDEF 0.00 1 0200400 K (1) K (2) D D Time (s) Interaction with MED25

Fig. 1. High-affinity interaction with MED25 is specific to the ETV1/4/5 subfamily of ETS factors. (a) Representative ETV4-MED25 sensorgrams using biolayer interferometry. Streptavidin sensors were loaded with the biotinylated ACID domain of MED25391–553 (+ MED25), followed by the addition (+ ETV4), then removal (− ETV4) of six different concentrations of ETV4 in order to measure the association and dissociation, respectively, between ETV4 and MED25 ACID. The bottom purple line is a control sensor with no MED25 loaded and the highest concentration of ETV4 added during the association step. (b) Representative sensorgrams of binding assays with a single concentration of ETV4 (50 nM), SPDEF (500 nM), ETS1 (500 nM), and EHF (500 nM). Only the association and dissociation phases of the sensorgram are shown. ERG (500 nM) and ETV6 (500 nM) were also tested but showed no detectable association; each ETS factor was tested twice. (c) Equilibrium dissociation constants (KD) for the interaction between MED25 ACID and SPDEF, ETV1, and ETV4. Two KD values, denoted KD (1) and KD (2) with KD (1) being the higher-affinity interaction, are reported as these interactions better fit a 2:1 (ETS:MED25) binding model (Fig. S1c). KD values were determined using a group fit with six different concentrations of each ETS factor (Figs.1a and S1a,b). Circles and squares represent the KD determined from a single, six-concentration experiment. Horizontal lines represent the mean and standard deviation for three to five replicate experiments. KD, ka, and kd values for these interactions are summarized in Table S1. “*” Indicates p b 0.05 in a Mann–Whitney U test.

ETV1/4/5 subfamily and MED25 ACID. Using ETV4 ETV443–84, and a broader N-terminal fragment, as a model for this subfamily, we interrogated the ETV41–164, were equivalent in binding to MED25 – interaction between different fragments of ETV4 and (Fig. 2d). Likewise, ETV4337 436, which corresponds MED25 with biolayer interferometry (Fig. 2a). The to the ETS domain and an additional α-helix H4 that N-terminal AD, ETV443–84, bound to MED25 in a is specific to the ETV1/4/5 subfamily, interacted with 165–484 one-to-one manner with a KD of 700 ± 100 nM (Fig. MED25 with similar affinity to that of ETV4 . 2b,d and Table S1). This value is comparable to Therefore, we conclude that the N-terminal AD and previous measurements of the interaction between the C-terminal DBD contribute to the high-affinity the conserved AD of ETV5 and MED25 by fluores- binding of full-length ETV4 with MED25. cence polarization (580 ± 20 nM) and by isothermal Interestingly, the kinetics of the ETV4 AD and DBD calorimetry (540 ± 40 nM) (Fig. S2) [7,32]. As the interactions with MED25 were noticeably different. AD bound MED25 with an approximately hundred-- The AD-MED25 interaction had relatively high fold weaker affinity than full-length ETV4, we association and dissociation rate constants (ka and surmised that additional regions within ETV4 also kd, respectively), reflecting the faster association and contribute to the interaction with MED25. Indeed, dissociation for this interaction (Fig. 2b and Table ETV4165–484, which lacks the N-terminal AD, also S1). In contrast, the DBD-MED25 interaction had bound to MED25 and used a two-to-one interaction relatively low ka and kd values indicating slower mode reminiscent of full-length ETV4 (350 ± 80 and association and dissociation (Fig. 2c and Table S1). 2200 ± 500 nM) (Fig. 2c,d). Testing of additional These data suggest that the high affinity interaction ETV4 fragments revealed that the minimal AD, between ETV4 and MED25 could be due to

0.15 (a) (b) + ETV443-84 - ETV443-84 ETV4 1 - 484 (FL) AD DBDED 0.10 165 - 484 DBDED 0.05 337 - 436 (DBD) DBDED

1 - 164 AD 0.00 Response (nm) Response 43 - 84 (AD) AD 10 20 30 40 50 -0.05 Time (s) (d) (c) 2.0 100,000 + ETV4165-484 - ETV4165-484 * 10,000 * * 1.5 * 1,000 1 - 484 1.0 (nM)

D 100 165 - 484 K 337 - 436 0.5 10 1 - 164

43 - 84 (nm) Response 0.0 1 500 1000 K (1) K (2) D D -0.5 Time (s) Interaction with MED25

Fig. 2. Two regions of ETV4 can independently bind to MED25. (a) Schematic of ETV4 truncations used for binding studies with MED25 ACID. AD, DBD, and FL are abbreviations for activation domain, DNA-binding domain, and full length, respectively. (b) Representative ETV443–84-MED25 sensorgrams with the association and dissociation phases shown. 30 μM ETV443–84 was used for the top sensorgram with serial 1.5-fold dilutions for the next five sensorgrams, and the bottom gray sensorgram corresponds to a control with no ETV443–84. (c) Representative ETV4165–484-MED25 – sensorgrams displayed as in (b). 3 μM ETV4165 484 was used for the top sensorgram. The difference in x- and y-axis scales between (b) and (c) reflects the different kinetic constants for these interactions (Table S1). (d) KD values for the interactions between fragments of ETV4 and MED25 ACID. Filled circles and squares represent the KD value from a single experiment with six different concentrations of ETV4, and the horizontal lines represent the mean and standard deviation 165–484 for three to five replicate experiments. “*” Indicates p b 0.05. Note that there are two KD values for ETV4 and 337–436 ETV4 as these sensorgrams were better fit by a 2:1 (ETV4:MED25) model. In contrast, there is a single KD value for ETV41–164 and ETV443–84 as these sensorgrams were better fit by a 1:1 model. ETV41–484 data are included from Fig. 1c for reference.

combining the fast association mechanism and the (a) 15 H1 N-DBD + MED25 slow dissociation mechanism used by the AD and A349 DBD, respectively. V348 H4 Interaction between MED25 and ETV4 DBD: N N435 Molecular interface and influence on DNA bind- H2 Y402 Y418 S429 E425 C ing G406 H3 Previous studies provided structural characteriza- ∆δ(ppm) tion of the interaction between the ETV5 AD and > 0.02 MED25. Notably, the predominantly disordered AD 0.02 > > 0.0075 becomes more helical in the MED25-bound state 0.0075 > and phenylalanine and tryptophan residues in the (b) AD are critical for this interaction [7,32]. Based on the H1 DBD mutations robust sequence conservation between the ADs of T363 I372 ETV1, ETV4, and ETV5 (Fig. S2), we surmised that the ETV4 AD would interact with MED25 in a E373 H4 K370 F432 conserved fashion. Indeed, the introduction of H2 F428 Phe54Ala, Phe60Ala, or Trp64Ala mutations into S429 ETV4 significantly disrupted interaction with MED25 (Fig. S3a,b). Therefore, we focused on characteriz- H3 ing residues that are important for the newly Fold-difference K discovered interaction between the DBD of ETV4 D and MED25. 30 > > 10 NMR spectroscopy was used to investigate the 10 > > 4 MED25-interface of the ETV4 DBD. We compared the 15N–HSQC spectra for 15N–labeled ETV4 DBD (c) H4 AD (model) [57] with or without unlabeled MED25 ACID (Fig. L427 F432 S4a). MED25 most strongly perturbed the signals W64 α from residues (Glu425 and Ser429) within -helix H4 L65 of ETV4 (Figs. 3a and S4b,c). Additionally, signals F428 F60 from residues within H1, H3, and the β-sheet were perturbed. Based on the specificity of the ETV1/4/5 Fig. 3. A similar motif in ETV4 AD and DBD is critical for subfamily for high-affinity interactions with MED25, 15 – β binding to MED25. (a) Changes to the N HSQC of ETV4 we focused on residues near the -sheet and in H4. DBD upon addition of unlabeled MED25 ACID at a 1:1.2 M Surface-exposed residues on the β-strands, as well ratio are colored onto the structure of ETV4 DBD bound to as residues in loops flanking the β-strands, are DNA (PDB: 4UUV) [59]. Amide chemical shift perturba- 2 2 ½ poorly conserved among all ETS factors (Fig. S2). tions (Δδ =[(ΔδH) + (0.2ΔδN) ] ) that were greater than Likewise, the sequence and secondary structure of the mean are colored orange and those that were in the H4 is not conserved in any ETS factors outside of the highest 10 % are colored in red. The peak for the amide of ETV1/4/5 subfamily (Fig. S2). To test the functional Glu425 is also colored red as this peak was broadened to importance of individual residues we analyzed the baseline. DNA (light blue) is shown for illustrative influence of single site alanine substitutions on the purposes, although it was not included in the NMR experiment. See Fig. S4 for NMR spectra and quantifica- ETV4-MED25 interaction. Mutations near the β tion. (b) Fold-differences of KD values for interaction with -sheet (Thr363Ala, Lys370Ala, Ile372Ala, MED25 are mapped onto the DBD of ETV4. Indicated Glu373Ala) had a ~ four- to ninefold disruption on residues were mutated to alanine in full-length ETV4, and MED25 binding (Figs. 3b and S3a). Mutation of interaction with MED25 was compared to wild type ETV4. Ser429 within H4 disrupted MED25 binding greater See Fig. S3 for example sensorgrams and quantification of than tenfold. Ser429 occurs within a motif (LFSLAF) ETV4 mutants. (c) Structure of H4, left, and model of AD, on H4 that is reminiscent of the N-terminal AD right, in cartoon representation with side chains shown in (FQETWL). This portion of the AD is critical for stick representation. Note that the AD is intrinsically interaction with MED25 (Fig. S3a) and the con- disordered, but takes on partial helical character when served sequence in ETV5 becomes more helical interacting with MED25 [7]; this helical model for the AD was generated by swapping AD amino acids onto the when interacting with MED25 (Fig. S2) [7].By structure of H4. analogy to the AD, we reasoned that the surface-exposed phenylalanines in H4 might also be important for interaction with MED25. Indeed, mutation of both ing (Fig. S3a,c). These results suggest that a broad Phe428 and Phe432 to alanine resulted in an interface, including critical phenylalanines from approximately thirty-fold disruption of MED25 bind- α-helix H4 as well as residues from the β-strand of

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 6 ETV4 and JUN/FOS bind to multiple sites on MED25 the ETS domain, contributes to the interaction of the (a) +MED2 DBD with MED25. Helix H4, by itself, resembles the [MED25] AD suggesting that the AD and DBD of ETV4 bind 5 ETS MED25:ETV4:DNA MED25 through a similar motif (Fig. 3c). However, ETV4:DNA the contribution of additional residues outside of H4 suggests that the DBD uses a broader interface for interaction with MED25. We previously demonstrated that α-helix H4 inhibits the DNA binding of the ETS domain of ETV4 [57]. Leu430 on the interior face of H4 is critical for this autoinhibition and interacts with conserved hydrophobic residues of the ETS domain. Hence, we Free DNA questioned whether MED25 binding to the exterior face of H4 would affect DNA binding. Using an EHF:DNA electrophoretic mobility shift assay (EMSA), we observed that the addition of MED25 to the equilibrium DNA binding reaction resulted in a slower migrating band on the EMSA gel, which we propose to be a MED25-ETV4-DNA ternary complex (Fig. 4a). As a control, equivalent amounts of MED25 Free DNA did not interact with the EHF-DNA complex. The (b) presence of MED25 increased the affinity (K )of 10 D + MED25 ETV4 for DNA approximately two-fold (Fig. 4b). This matches the two-fold magnitude of DNA-binding * - MED25 autoinhibition that was previously attributed to H4 [57]. Mutation of the MED25-interaction site on H4 (Phe428Ala/Phe428Ala) or of the ETS domain-interaction site on H4 (Leu430Ala) both abrogated the activation of DNA binding by MED25 (Fig. 4b). We [ETV4:DNA (nM)] conclude that the interaction between MED25 and D α-helix H4 activates the DNA binding of ETV4. K 1 WT F428A/F432A L430A ETV4 AD and DBD interactions with MED25: ETV4 Single-site versus multisite binding Fig. 4. Interaction with MED25 relieves the DNA- Having determined that the AD and DBD use binding autoinhibition from α-helix H4 of ETV4. (a) similar motifs to interact with MED25, we next Electrophoretic mobility shift assay (EMSA) titrating wanted to explore the AD and DBD binding sites MED25 ACID (500–1 nM) with a single concentration on MED25. We utilized assigned 15N–HSQC spec- (50 nM) of ETV4 (top) or EHF (bottom). EMSA gels are tra and the tertiary structure of the ACID domain of representative examples of at least three replicates for MED25 that were previously reported [9,10]. The each protein. (b) Using a single concentration of MED25 ACID domain is a seven-stranded β-barrel with three ACID (500 nM), ETV4 WT or indicated mutants were – peripheral α-helices (Fig. 5a). 15N–labeled MED25 titrated (100 0.01 nM) to determine the KD of the ACID was titrated with either the AD or the DBD of ETV4-DNA interaction in the presence or absence of MED25. Filled circles represent a single experiment and ETV4 (Fig. S5a,b). The addition of the AD resulted in the horizontal lines represent the mean and the standard robust and widespread changes in the 15N–HSQC deviation. “*” Indicates p b 0.05. The KD values for the spectra of MED25 ACID (Fig. S5c,e). Several amide ETV4-DNA interaction were (mean ± standard deviation): 1 H 15 N - N peaks displayed progressive chemical shift WT (+ MED25) = 2.1 ± 0.5 nM; WT (− MED25) = 3.4 ± changes indicative of fast exchange behavior. We 0.8 nM; Phe428Ala/Phe432Ala (+MED25) = 3.4 ± observed substantial line broadening, even at the 1.0 nM; Phe428Ala/Phe432Ala (− MED25) = 3.7 ± lowest titration point, as has also been observed in 0.8 nM; Leu430Ala (+ MED25) = 1.9 ± 0.5 nM; MED25 ACID interactions with other activation Leu430Ala (− MED25) = 1.9 ± 0.4 nM. domains [7,9,10]. Comparatively, the addition of ETV4 DBD resulted in more subtle changes in the spectra of MED25 ACID (Fig. S5d,f). Amide signals titration, we interpreted the changes in the MED25 showed relatively smaller chemical shift changes ACID 15N–HSQC spectra as evidence for interaction and line broadening was more gradual as DBD was with both of these ETV4 fragments. added at 0.2:1, 0.5:1, and 1.2:1 M ratios. Although We mapped the MED25 residues that exhibited exhibiting different effects upon AD and DBD amide peak intensity changes from both ETV4

(a) MED25 ACID (b)MED25 ACID (c) MED25 ACID + ETV4 AD + ETV4 DBD

C Site 1 Site 1 H2 H3 S3 S2 S5

180° 180° 180° Site 3 Site 3 C H2 H3 S5 S2 S6 S7 H1 S4 S1 Site 2 Site 2

Fig. 5. NMR spectroscopy suggests a single MED25 binding site for ETV4 AD and multiple binding sites for ETV4 DBD. (a) Cartoon representation of MED25 ACID structure (PDB: 2KY6) [9]. Bottom view is rotated 180° relative to the top view of MED25 ACID. α-helices and β-strands are abbreviated H and S, respectively, and numbered according to progression from N- to C-termini of the domain. (b) MED25 ACID oriented as in (a) but with surface representation and changes to the 15N–HSQC of MED25 ACID upon addition of unlabeled ETV4 AD indicated by color. Upon addition of 0.2 M equivalents of ETV4 AD, MED25 amide relative peak intensities that were less than the mean are colored teal and those that were in the lowest 10 % are colored blue. (c) MED25 ACID as in (b) with changes to the 15N–HSQC upon addition of unlabeled ETV4 DBD indicated by color. Upon addition of 0.2 M equivalents of ETV4 DBD, MED25 amide relative peak intensities that were less than the mean are colored orange and those that were in the lowest 10 % are colored in red. Sites of clustered changes upon titration of AD and/or DBD are indicated by dotted lines and arbitrarily named site one, two, or three. See Fig. S5 for 15N–HSQC spectra of MED25 ACID alone and with unlabeled ETV4 AD or ETV4 DBD.

titrations onto the structure of MED25 ACID (Figs. 5 grooves with positively charged residues lining the and S5e,f). The AD most strongly perturbed residues perimeter. While the AD appears to most strongly that clustered in a single site formed by β-strands S3, interact with Site 1 on MED25, we propose two S5, and α-helix H3, which we will term Site 1. The possibilities to explain the absence of clustering for DBD influenced residues within Site 1 as well, but amide signal changes upon DBD titration to a single also at two different sites on additional faces of MED25 site. One possibility is that the DBD may MED25 formed by S4, S7, and H1 (Site 2), and by alternately interact with multiple sites on different S2, S4, and H2 (Site 3), respectively. These multiple faces of MED25 ACID, thereby forming a “fuzzy” sites all have concave grooves that are suitable for binding interface [8,12]. Alternatively, the DBD may interaction with an α-helix, such as that formed by interact with a single site on MED25 and have the AD or H4 of ETV4 (Fig. S6). Hydrophobic and significant through-molecule effects that influence uncharged polar residues form the floor of these other surfaces of MED25 without direct interaction.

We next pursued further biochemical and structural Lys545Glu) were located at Site 1, suggesting that characterization in an attempt to distinguish between this may be the preferred binding site for either of these two models. these ETV4 fragments in isolation (Figs. 6, S7, and Mutagenesis was used to further investigate the Table S2). Interestingly, the mutations that disrupt binding of MED25 and ETV4. Using MED25 resi- AD binding cluster near the center of the groove in dues implicated from the NMR experiments as a Site 1, whereas, the mutations that preferentially starting point, we focused on those that had affect DBD binding are more spread out on Site 1. surface-exposed side chains. Residues from the This suggests a broader surface at Site 1 is used for concave groove floors, and surrounding positively DBD binding, mirroring the broad surface on the charged residues, of all three MED25 ACID sites DBD that is used for MED25 binding (Fig. 3a,b). were mutated. Based on the importance of aromatic Although some mutations in MED25 Site 2 and Site 3 residues in the AD and DBD for binding to MED25 also disrupted AD binding, all MED25 mutations at (Fig. 3a), most residues were mutated to glutamate these sites (Gln430Glu, Arg466Glu, His474Glu, as we surmised that a negative charge would Tyr487Ser, Leu514Glu, Lys518Ala/Lys519Ala/ successfully disrupt any π-cation or hydrophobic Lys520Ala, Ile521Glu, and Met523Glu) more strong- interactions. The MED25 mutants that most strongly ly, or only, affected DBD binding (Fig. 6 and Table disrupted binding for both the AD (Gln451Glu) and S2). The spacing of the DBD-specific mutations at DBD (Lys422Glu, Arg509Glu, Arg538Glu, and MED25 Sites 2 and 3 were also suggestive of a

(a)AD (b) DBD Site 1 Site 1

K545E K545E K422E Q451E R538EQ451E R538E

R509E

180° 180°

K518A/ Site 3 K518A/ Site 3 K519A/ K519A/ Y487S K520A K520A L514E L514E I521E I521E

M523E H474E H474E R466E M523E Q430E Site 2 Site 2

> 8 fold > 8 fold 4 - 8 fold 4 - 8 fold 2 - 4 fold 2 - 4 fold

Fig. 6. Broad surfaces on multiple MED25 sites contribute to DBD binding. MED25 ACID represented as in Fig. 5b and with point mutants that perturb ETV4 binding colored onto the structure. Blue scale (a) and red scale (b) indicate mutations that disrupted the interaction with ETV4 AD and DBD, respectively. Fold inhibition of binding was calculated by comparing to a wild type MED25 control with AD or DBD. See Table S2 for summary of all KD values, and Fig. S7 for examples of sensorgrams with MED25 mutants.

(a)MED25 ACID (b) MED25 ACID + ETV4 AD + ETV4 DBD

Site 1 Site 1

180° 180°

Site 3

Site 2

Fig. 7. Modeling suggests multiple potential MED25 binding sites for ETV4 DBD. (a) The top ten predictions for the interaction between MED25 ACID and ETV4 AD using the ZDOCK protein-docking program [58]. MED25 ACID is represented as in Fig. 5b, ETV4 AD is displayed as a blue α-helix in cartoon representation with the side chains for Phe60 and Trp64 shown in green. AD residues Phe60 and Trp64 and MED25 residue Gln451 were selected as residues involved in the binding site for the prediction. (b) The top-ten predictions for the interaction between MED25 ACID and ETV4 DBD. The DBD was binding to MED25 Site 1 in two predictions, Site 2 in seven predictions, and Site 3 in one prediction. Modeling and representation were performed as in (a). The entire DBD is shown in cartoon format in red, and Phe428 and Phe432 side chains are displayed in green. DBD residues Phe428 and Phe432 were selected as residues involved in the binding site for the prediction.

(a) (c) Shared Peaks C(A/C)GGAA Shared Peaks ETV4 211 636 406 MED25 ≥ 1 36% (7%) ≥ 2 22% (5%) ETV4 MED25 Input 25 (d) MED25 ETV4 20 Input

ChIP Enrichment ChIP 5

ANK2 SDC1 HEY1 CDK14 IGF2BP1 Regions (e) MED25a ETV4a MED25b ETV4b 1.0

0.8

0.6

(b) 0.4 ETV4 0.2

MED25 level Relative mRNA Input 0.0

IGF2BP ANK2 SDC1 HEY1 CDK14 IGF2BP

Fig. 8. ETV4 and MED25 occupancy at regulatory regions genome-wide and effects on expression of associated genes. (a) Overlap of ETV4 and FLAG-MED25 bound regions from PC3 cells displayed as heatmaps of the RPKM from MED25, ETV4, and input control data across the set of shared regions. Heat map ranges from 0 (blue) to 72 (red) RPKM. (b) Graphical display of enriched reads for ETV4 and MED25 ChIP DNA and input DNA in IGF2BP intron (hg19 chr10:54,218,829–54,242,010). Red bar, region assayed by qPCR. (c) ETS binding motif is most over-represented sequence in shared regions as determined by MEME [86]. The MEME, expect-value, E, is 1 × 10−21. Percentages of peaks with match to motif are shown to right with comparison to randomly generated size-matched peak set in parentheses. (d) qPCR quantification of MED25-FLAG and ETV4 enrichment at putative regulatory elements for genes shown; input values displayed for comparison. Two to three independent biological replicates provided similar patterns, but different maximum levels of enrichment. A representative experiment is shown. (e) Relative expression values for indicated gene as determined by qPCR of total cDNA derived from ETV4 and MED25 knockdown PC3 cells. Two different shRNA constructs were used for both ETV4 and MED25 and denoted as a or b. Average values for biological triplicates are graphed with standard deviation. Relative expression of control knockdown cells is set at 1, and experimental sample values are graphed relative to control.

ETV4 and MED25 share transcriptional targets in these common genes, 70% were associated with prostate cancer cells potential regulatory elements occupied by both ETV4 and MED25 (Fig. S8b). The occupancies of Given the strong interaction between MED25 and putative regulatory elements for five genes (ANK2, ETV4,aswellastheestablishedroleofMED25asa CDK14, HEY1, IGF2BP1, and SDC1)were transcriptional co-regulator [32,45,53,54],wehypoth- esized that MED25 and ETV4 would share transcriptional targets. To test this, we assayed ETV4 and MED25 DNA occupancy genome-wide in PC3 cells, a prostate cancer tumor cell line that overexpresses ETV4. Chromatin immunoprecipitation (ChIP) of a FLAG-tagged version of MED25 detected 1042 MED25 bound regions while the parallel ETV4 ChIP detected 847 ETV4 bound regions (Table S3). The vast majority of the enriched regions for each dataset mapped greater than 5000 base pairs (bp) from defined transcriptional start sites, with almost half lying greater than 50,000 bp away, suggesting robust binding to distal enhancer elements (Fig. S8a,b). Intersection of the two datasets demonstrated a striking high degree of overlap; ~75% of the ETV4 peaks were in the MED25 dataset (Fig. 8a,b and Table S3). The ~50% of MED25 sites that do not overlap with ETV4 sites could reflect recruitment of MED25 via other transcription factors. Also, MED25 is bound to fewer genomic sites compared to the over 10,000 sites that other Mediator subunits occupy in various cell types [60,61]. These data support the role of MED25 as a variable and loosely associated Mediator subunit [36,49,62],andsuggestthatMED25is present in only a subset of total Mediator complexes. We observed high enrichment of ETS binding motifs at the overlapping sites; the ETS binding site (CAGGAA) was the top overrepresented motif in the shared peaks and second top hit for all MED25 peaks after the AP1 binding sequence (Figs. 8c and S8c). We compared the frequency of the ETS binding motif between the shared peaks and a size-equivalent, randomly generated set of genomic sequences. Of the 611 ETV4-MED25 shared peaks, 223 had at least one C(C/A)GGAA sequence in their central 100 bp core; whereas, only 44 regions of a control dataset had the motif (Fig. 8c and Table S3). Furthermore, only two of the 44 regions in the control dataset had multiple occurrences of the motif, while Fig. 9. JUN/FOS heterodimers bind to multiple sites on 50 of the 223 shared peaks had two or more ETS MED25. (a) Representative sensorgrams of binding motifs. Several targets were confirmed by qPCR to assays between MED25 ACID and 500 nM JUN (red), validate the ChIPseq data and verify that these 500 nM FOS (blue), or 250 nM JUN/FOS (purple). (b) represent overlapping binding regions (Figs. 8d and Representative sensorgrams of binding assays between S8b). MED25 ACID wildtype (WT) and point mutants as labeled, To test the functional relevance of this genomic and 500 nM FOS. (c) Representative sensorgrams of occupancy, we sought to identify genes whose binding assays between MED25 ACID wildtype (WT) and transcription was dependent on both MED25 and point mutants as labeled, and 250 nM JUN/FOS. Note that sensorgrams corresponding to Tyr487Ser and Arg466Glu ETV4. We performed shRNA-mediated knockdown are shown as dotted and dashed lines, respectively, to aid of endogenous MED25 and ETV4 in the PC3 cell line in visualization because they are almost completely followed by RNAseq. Of the 153 and 57 genes down overlapping. See Fig. S10 and Table S6 for further regulated by MED25 and ETV4 knockdown, respec- quantification of JUN/FOS-MED25 and FOS-MED25 tively, 18% were in common (Table S5). Focusing on interactions.

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 12 ETV4 and JUN/FOS bind to multiple sites on MED25 confirmed by qPCR of DNA in the peak region (Figs. of MED25. Three distinct sites on MED25 bind the 8d and S8b). Consistent with RNA-seq results, all DBD; whereas, only one site binds the AD, resulting five of these genes were down-regulated in PC3 in a multivalent interaction between MED25 and cells expressing either MED25 or ETV4 shRNA ETV4. MED25 activates the DNA binding of ETV4 by construct as assessed by RT-qPCR (Fig. 8e). From relieving autoinhibition, and we propose that the these findings we propose that MED25 and ETV4 ETV4-MED25 interaction is important for the tran- interact at genomic sites and work together to scriptional output of ETV4 target genes. JUN/FOS regulate the transcription of ETV4 target genes. heterodimers also interact with multiple sites on MED25, indicating that multivalent interactions may JUN/FOS also binds to multiple sites on MED25 be a common mechanism of MED25 recruitment by diverse transcription factors. The AP1 binding sequence (TGAGTCA) was also overrepresented at MED25 peaks, thus implicating a Model for ETV4- and JUN/FOS-MED25 interac- possible involvement of JUN and FOS families of tions transcription factors (Fig. S8c). As a MED25-AP1 interaction had not been previously reported, we Biochemical results from biolayer interferometry explored this possibility. Using a previously reported binding studies as well as structural insight from JUND ChIP-seq dataset from PC3 cells [30],we NMR spectroscopy and protein-docking predictions found a 29% overlap in MED25- and JUND-occupied provide a detailed description of the ETV4-MED25 regions (Table S3). Furthermore, JUN and FOS also interaction. The AD and DBD of ETV4 can each bind Ω ΩΦ ΦΩ Ω had one and two of the MED25-interacting motifs MED25 independently. Similar xxx or xxx (ΩxxxΩΦ), respectively, occurring within previously motifs are found in both domains and are critical for annotated activation domains (Fig. S9) [63,64]. MED25 binding. However, this motif in the flexible To investigate a potential interaction between AD is at the core of a single interaction surface; MED25 and AP1 factors, we used full-length JUN Phe60Ala and Trp64Ala mutations almost and FOS to prepare JUN/FOS heterodimers, JUN completely abrogate binding to MED25. By com- homodimers, and FOS homodimers. MED25 strong- parison, additional residues assist the interaction motif in the DBD and form a broader interface with ly bound FOS (KD = 9 ± 3 nM) (Figs. 9a, S10a, and Table S6). Although JUN did not measurably interact MED25. with MED25 at a concentration of 500 nM, the On the Mediator side, the DBD interacts with three presence of JUN in JUN/FOS heterodimers resulted distinct sites on MED25, and the AD interacts with only one of these sites. These three MED25 sites are in a slightly higher affinity for MED25 (KD =5± 3 nM) compared to FOS by itself (Fig. S10b). The relatively similar in overall appearance; hydrophobic apparent complex binding of JUN/FOS to MED25 and non-polar residues form the bottom of concave was reminiscent of the AD and DBD of ETV4 grooves with positively charged residues lining the combining to interact with MED25; the fast initial perimeter. We speculate that the difference in association of JUN/FOS was similar to FOS by itself, single-site versus multisite binding for the AD- and and the addition of JUN resulted in a second, slower DBD-MED25 interactions, respectively, reflects the association not observed for either JUN or FOS different binding interfaces. For the AD, the exact alone. This complex interaction behavior suggested spatial and chemical nature of the binding groove on that JUN/FOS also binds to multiple sites on MED25. MED25 Site 1 is critical for binding because this is We used MED25 mutants to further characterize the only point of contact. In contrast, the specifics of JUN- and FOS-binding sites. Mutation of MED25 Site 1 the MED25 sites for binding to H4 are less critical for (Gln451Glu) strongly disrupted the binding of both the overall interaction because other parts of the JUN/FOS and FOS alone (Fig. 9b,c and Table S6). In DBD also contact MED25 to form a broader contrast, MED25 Site 2 (Arg466Glu) and Site 3 interface. Based on the spacing of the sites on (Tyr487Ser) mutations had essentially no impact on MED25 ACID, one DBD molecule could only interact FOS binding but disrupted JUN/FOS binding by two to with one MED25 site at a time. Yet, the presence of threefold.Basedonthesemutationaldatawepropose additional binding sites suggests that all three that FOS within JUN/FOS heterodimers strongly binds MED25 sites collectively contribute to the macro- to MED25 Site 1, and JUN weakly enhances the overall scopic DBD-MED25 interaction. interaction by contacting Sites 2 and 3. We propose a multi-step kinetic model for the ETV4-MED25 interaction (Fig. 10). An important assumption supported by our findings is that the AD Discussion and DBD cannot co-occupy the same MED25 site due to steric clash. The predominant pathway for Here we report that both the DNA-binding domain complex formation would involve the AD of ETV4 (DBD) and the N-terminal activation domain (AD) of first binding to Site 1 on MED25 due to its fast ETV4 bind to the activator interacting domain (ACID) association. The DBD of the bound ETV4 with its

Fig. 10. Model for the multivalent ETV4-MED25 interaction. In the predominant pathway (top) the first step of interaction involves the activation domain (AD) binding to MED25 Site 1 due to its drastically faster association compared to the interaction between MED25 and the DNA-binding domain (DBD, bottom). The association and dissociation rate constants, ka (AD), kd (AD), ka (DBD), and kd (DBD) for the first binding steps are assumed equivalent to those measured for the isolated AD-MED25 and DBD-MED25 interactions, respectively. In the second step of the predominant pathway, the DBD of the bound ETV4 can only bind to the two MED25 sites that are not occupied by the AD (right). The ETV4-MED25 interaction would then be in equilibrium between two states with the DBD alternately occupying Sites 2 and 3 on MED25. The predominant pathway for dissociation of ETV4-MED25 will be limited by the slower release of the DBD. ETV4 DBD binding to MED25 is not predicted to be the predominant first step for the interaction for full-length ETV4 and MED25 (bottom), due to the drastically slower association rate (bottom). However, in circumstances where the AD is bound to another protein, or in oncogenic translocations of ETV4 that do not have the N-terminal AD, binding of the DBD to MED25 would still be a viable mode of interaction. The size of the arrows indicating association and dissociation between the unbound and AD-MED25 or DBD-MED25 complexes, and dissociation between the ETV4-MED25 and AD-MED25 or DBD-MED25 complexes, is representative of the measured rate constants for these steps. Other arrows are qualitatively sized based on mutational data and interpretations, and should not be considered as measured values. slower association would then interact with MED25 Interestingly, JUN/FOS factors also use a multi- at either Site 2 or Site 3 and could toggle between valent mechanism for interacting with MED25. FOS alternately binding to either of these two MED25 strongly interacts with MED25 by binding to Site 1, sites. The predominant pathway for dissociation will like ETV4 AD. JUN weakly interacts with MED25 by be limited by the slower dissociation of the DBD. In itself, but binds to MED25 Sites 2 and 3 when FOS is this scheme, the AD and DBD of ETV4 and three also present. Therefore, the multiple binding sites on MED25 ACID sites combine to form a high-affinity MED25 provide a route for combinatorial interaction and multivalent interaction. from a single transcription factor and may allow for

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 14 ETV4 and JUN/FOS bind to multiple sites on MED25 cooperative recruitment of MED25 by multiple interaction could also explain the retained function of factors, an idea further explored below. ETV1/4/5 truncations in prostate cancer that lack the N-terminal AD [33,34]; the tighter binding FOS would Autoinhibition and regulation of ETV4 outcompete ETV4 AD for interaction with MED25 Site 1, making the AD of ETV4 dispensable for the The conserved ETS domain results in similar recruitment of MED25 to composite ETS-AP1 DNA DNA-binding properties for most ETS transcription sequences. factors [65]. However, evidence clearly indicates that In addition to the ETS factors and JUN/FOS individual ETS factors have distinct biological roles described here, ATF6α, CBP, HNF4α, RARα, and [66]; how is such specificity achieved? Part of this SOX9 also interact with MED25 [45,53,54,56]. specificity can be ascribed to the diverse flanking ATF6α, CBP, and SOX9, bind to the ACID domain, inhibitory regions that are specific to individual though detailed structural and biochemical studies subfamilies of ETS factors. In the ETS1 and ETV1/ describing their binding sites have not been per- 4/5 subfamilies, both inhibitory α-helices that pack formed. Our genomic investigation did not suggest a against the ETS domain and intrinsically disordered broad role for these factors in recruiting MED25 in sequences contribute to autoinhibition [57,67]. The PC3 cells. However, given the plasticity of MED25 subfamily-specific α-helices also provide unique ACID binding sites used by ETV4, other ACID-bind- scaffolds for intermolecular interactions. In addition ing factors may also be able to recruit MED25, along to MED25, USF1 relieves the DNA-binding auto- with ETV4, at select regions or in different cell types. inhibition of ETV4 [68], whereas, RUNX1 and PAX5 The nuclear receptors (NRs), HNF4α and RARα, counter ETS1 autoinhibition [69,70]. This DNA-bind- bind to MED25 through a NR box located outside of ing activation in ETS1 is likely the reason that ETS1 the ACID domain, which is thus distinct from Sites 1– specifically regulates ETS-RUNX composite DNA 3 described here. Therefore, cooperative recruit- recognition sites in T-cells [71]. Here, we have ment of MED25 could be accomplished through demonstrated that MED25 interacts with the ETS simultaneous interaction with a nuclear receptor and domain and with H4, an inhibitory helix specific to the any one of the ACID-interacting transcription factors. ETV1/4/5 subfamily. MED25 interaction with H4 In particular, cooperative MED25 recruitment by relieves autoinhibition from H4 but does not appear ETV1/4/5 and androgen receptor (AR) could be an to disturb inhibition from the disordered N-terminal important mechanism for the oncogenic role of these inhibitory domain. Interestingly, acetylation of lysines transcription factors in prostate cancer; transcrip- relieves the inhibition from this disordered domain tional co-regulation by ETS and AR has been [57]. MED25 also interacts with CBP, a protein previously described [29,72–74], although the po- acetyltransferase [53]. Therefore, a hypothetical tential importance of MED25 recruitment was not ETV4-MED25-CBP complex could maximally acti- investigated. More broadly, interaction between vate ETV4 DNA binding by relieving autoinhibition multiple transcription factors and different Mediator from both inhibitory domains and warrants further subunits could be an avenue for cooperative investigation. In conclusion, the ETV4-MED25 inter- recruitment of the entire Mediator complex [52]. action is an example of how distinct flanking MED25 ACID interacting with several different inhibitory sequences can set up specific protein transcription factors is broadly illustrative of AD-co- partnerships that regulate the DNA-binding activity factor interactions in general. Many cofactors are of a particular ETS factor. capable of interacting with ADs from multiple transcription factors. Conversely, most ADs can Multivalent AD-cofactor interactions bind to multiple cofactors. Several examples, including our findings with ETV4, suggest that multivalency The multivalency of MED25 ACID with at least is one route for forming specific, higher-affinity three distinct ETV4 binding sites could enable AD-cofactor interactions. For example, two ADs cooperative recruitment of MED25 to target genes. from p53 form bivalent interactions with both the In PC3 cells, MED25 occupies genomic regions that TAZ1 and TAZ2 domains of CBP by binding to were enriched for ETS and AP1 DNA-binding opposite faces of these domains [21]. The p53 ADs sequences. Thus, cooperative MED25 recruitment also interact with at least two other CBP domains could be accomplished through interactions with [24], so the tetrameric form of p53 has been AP1 and ETV4. We propose that FOS and ETV4 predicted to form a highly multivalent interaction could simultaneously contact MED25 based on with full-length CBP. RAD4, RAD34, and TFIIE differences in binding modes; the strong interaction utilize bivalency to strongly bind to the PHD domain with FOS would occupy MED25 Site 1 while ETV4 of the general transcription factor TFIIH [22,23]. Two DBD would bind to Sites 2 or 3 (Fig. S11). Many GCN4 ADs interact with three different activator-- genomic regions have ETS and AP1 DNA-binding binding domains in MED15/Gal11 [20].Thus, sites in close proximity that would allow for this ETV4-MED25 interactions described here extend concurrent interaction [30]. Such a combinatorial the emerging picture of multivalent interactions

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 15 between transcription factors and transcriptional buffer with 15 mM glutathione (Sigma) added. coactivators in the assemblage of transcriptional Elutions were then dialyzed overnight into 25 mM machinery. In the case of oncogenic proteins, such Tris pH 7.9, 10% glycerol (v:v), 1 mM EDTA, 50 mM as ETV4 as well as related factors ETV1 and ETV5, KCl and 1 mM dithiothreitol (DTT; Sigma). After this understanding of multivalent interactions may ultracentrifugation for 30 min at 40 k rpm in a enable a multipronged approach for therapeutic Beckman Ti45 rotor, proteins were purified over a strategies. SP-sepharose cation exchange column (GE) (ETV4337–436 and MED25 ACID) or a Q-sepharose anion exchange column (GE) (ETV41–164 and 43–84 – Materials and methods ETV4 )usinga501000 mM KCl gradient. Proteins were then further purified over a Superdex 75 gel filtration column (GE) in 25 mM Tris pH 7.9, Protein Expression & Purification 10% glycerol (v:v), 1 mM EDTA, 300 mM KCl, and 1 mM DTT. Purified proteins were concentrated with The genes encoding full-length ETS factors and a 3, 10, or 30 kDa molecular weight cutoff (MWCO) truncated ETV4 fragments were cloned into the centricon device (Sartorius), snap-frozen in liquid pET28 (Novagen) bacterial expression vector using nitrogen and stored in single-use aliquots at −80 °C sequence and ligation independent cloning (SLIC) for subsequent NMR, EMSA, or biolayer interferom- [75]. ETV4337–436 that was used as a ligand in etry studies. biolayer interferometry was cloned into a custom-- Full-length ETV4 (ETV41–484) and ETV4165–484 made vector, described below. Plasmid of human expressed into the insoluble fraction and were MED25 cDNA was ordered from GE Dharmacon. readily induced by autoinduction [76]. Briefly, bacte- The gene encoding MED25 ACID (residues 391– ria in 250 mL of autoinduction media were grown in 553) was cloned into pET28 to enable protein 4 L flasks at 37 °CtoanOD600 ~0.6–1. The production for NMR spectroscopy, and into a temperature was then reduced to 30 °C and cultures custom-made vector to express ligand protein for were grown for another ~12–24 h. Final OD600 biolayer interferometry. This vector was based on a values were typically ~6–12, indicating robust pGEX backbone with N-terminal GST, Avitag, and autoinduction. Harvested cells were resuspended HIS6 tags separated by thrombin and TEV cleavage as described above, sonicated and centrifuged at sites, and followed by a Precission protease site 15 k rpm in a JA-17 rotor (Beckman) for 15 min at before MED25 ACID. Plasmids of JUN and FOS in 4 °C. The soluble fraction was discarded and this the pET28 bacterial expression vector were a kind procedure was repeated with the pellet/insoluble gift from Dr. Peter Hollenhorst. fraction twice more to rinse the inclusion bodies. The All proteins, except JUN and FOS, were final insoluble fraction was resuspended with 25 mM expressed in Escherichia coli BL21 (λDE3) cells. Tris pH 7.9, 1 M NaCl, 0.1 mM EDTA, 5 mM – – MED25 ACID, ETV41 164,ETV443 84,and imidazole, 2 mM βME, 1 mM PMSF, and 6 M urea. ETV4337–436 expressed into the soluble fraction of After sonication and incubation for ~1 h at 4 °C, the cells, and were grown in 1 L cultures of Luria broth sample was centrifuged for 30 min at 40 k rpm and 2+ (LB) at 37 °CtoOD600 ~ 0.7–0.9, induced with 4 °C. The soluble fraction was loaded onto a Ni 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; affinity column and refolded by immediately switch- Gold Biotechnology), and grown at 30 °C for ~2– ing to the same buffer except without urea. After 3 h. For MED25 ACID or ETV4337–436 protein used elution with 5–500 mM imidazole, the remaining as the ligand in biolayer interferometry, 1 mL of purification steps (anion-exchange and size-exclu- 50 mM D-biotin (Fisher Scientific) was added at the sion chromatography) were performed as described induction point. Cells were centrifuged at 6000 rpm above. in a JLA 8.1 rotor (Beckmann), resuspended in a Full-length JUN and FOS proteins expressed into buffer containing 25 mM Tris, pH 7.9, 200 mM NaCl, the insoluble fraction using IPTG induction and were 0.1 mM ethylenediaminetetraacetic acid (EDTA), expressed and purified as for insoluble ETV4 2 mM 2-mercaptoethanol (βME), and 1 mM phenyl- proteins, described above, with the following excep- methanesulfonylfluoride (PMSF), and snap-frozen in tions. FOS was expressed in Rosetta 2 cells for liquid nitrogen. After 3 to 5 freeze–thaw cycles, the supplementation of rare Arg tRNAs. Inclusion bodies cells were lysed by sonication and ultracentrifuged were purified and solubilized as described above, for 30 min at 40 k rpm in a Beckman Ti45 rotor. The then JUN and FOS were combined for JUN/FOS soluble fraction was then loaded onto a Ni2+ affinity heterodimers (or kept separate for JUN homodimers column (GE Biosciences) and eluted over 20 column or FOS homodimers), diluted to 200 ng/μL (total volumes of a 5–500 mM imidazole (Sigma) gradient. protein), then dialyzed for at least 3 h each against For MED25 ACID used in biolayer interferometry, the following three buffers (in sequential order): (1) protein eluted from the Ni2+ column was loaded onto 25 mM Tris pH 6.7, 0.1 mM EDTA, 10% glycerol, a GST-affinity column (GE) and eluted with the same 5 mM BME, 1 M NaCl, 1 M Urea; (2) same as (1) but

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 16 ETV4 and JUN/FOS bind to multiple sites on MED25 without urea; (3) same as (2) but with NaCl reduced JUN homodimers and FOS homodimers were to 100 mM. Refolded samples were then purified purified separately and tested for interaction with using Ni2+ affinity column and gel-filtration column MED25 ACID. Purified JUN and FOS were com- as described above. bined at a 1:1 ratio for the “JUN/FOS” sample. Due to varying dimerization affinities [77], the majority of the Biolayer interferometry sample will be JUN/FOS heterodimers, and a very small minority of the sample will be either JUN/JUN Data were collected with an Octet Red96 instrument or FOS/FOS homodimers. JUN/JUN homodimers (ForteBio) and processed with Octet Data Analysis did not interact with MED25 ACID at the concentra- software (v8.1, ForteBio). MED25 ACID (residues 391– tions tested. The composite JUN/FOS sample had 553) was used as the ligand for all interactions except an obviously different sensorgram appearance (Fig. for with ETV4337–436 as this truncation of ETV4 9a) and a quantifiably higher-affinity interaction with displayed too much non-specific interaction with the MED25 (Fig. S10a,b), as compared to FOS/FOS. biosensor tip to be used as the analyte. Biotinylated Therefore, we interpret that JUN/FOS heterodimers ligand (500 nM) was immobilized using high precision have a higher affinity for MED25 than FOS/FOS streptavidin sensors (ForteBio). Protein concentrations homodimers. were determined after thawing each aliquot of protein, using the Protein Assay Dye Reagent (Bio-Rad). NMR spectroscopy Interaction experiments were conducted using 25 mM Tris pH 7.9, 150 mM NaCl, 10 mM DTT, 5 μg/mL 1H-15 N HSQC measurements were recorded on bovine serum albumin (Sigma) and 0.05% (v:v) a 500 MHz Varian Inova spectrometer at 25 °C with Tween20 (Sigma). Biosensors were dipped in various protein in the following sample buffer: 20 mM sodium concentrations of the analyte of interest to measure phosphate, pH 6.5, 200 mM NaCl, 2 mM DTT, association, and transferred back to buffer wells for 1 mM EDTA, and 10% D2O. Proteins were either monitoring dissociation. purified into NMR sample buffer using a size-exclu- For a more quantitative analysis, six concentra- sion column, or buffer-exchanged using centricon tions of analyte, with exact concentrations varying spin columns. Labeled protein was concentrated to dependent on the affinity of the specific analyte-li- ~0.2–0.25 mM and titrated with 0.2, 0.5, and 1.2 M gand combination, were fit using a global (full) equivalents of unlabeled protein. Upon addition of analysis (simultaneous, constrained fit of all six each titration equivalent, the entire sample was sensorgrams). The reported kinetic rate constants removed and concentrated back down to approxi- (ka and kd) were determined from the fit of a 1:1 mately the same starting volume. Significant loss of binding model for ETV443–84 and ETV41–164, and a 15N–MED25 ACID was not observed upon progres- 2:1 (ETV4:MED25) binding model for all full-length sive titration of either ETV4 AD or DBD. Assignments ETS factors, ETV4165–484, and ETV337–436. The were transferred from previous work for MED25 decision to use either of these binding models was ACID [9,78] and ETV4 337–436 [57]. NMR data were determined empirically based on chi-squared processed and analyzed using Vnmr (Varian) and values, as evidence from NMR spectroscopy and Sparky (UCSF) [79]. Chemical shift perturbations 2 2 ½ protein-docking experiments also suggested that (Δδ =[(ΔδH) + (0.2ΔδN) ] ) and relative peak in- ETV4337–436 interacts with MED25 using a multiva- tensities for ETV4 and MED25, respectively, that lent mode. The equilibrium dissociation constant were above the median and in the top 10 % for (KD) was derived from the kinetic constants. Use of chemical shift perturbations, or below the median either the 1:1 or 2:1 binding models resulted in and in the lowest 10 % for relative peak intensities, relatively similar KD values for all interactions tested. were colored onto the structures of ETV4 (PDB: For example, the full-length ETV4-MED25 interac- 4UUV) [59] or MED25 (PDB: 2KY6) [9] using Pymol tion results in a KD of 5 ± 1 nM using the 1:1 binding (v1.7.0.5 enhanced for Mac, Schrödinger). model and KD values of 7 ± 3 and 28 ± 7 using the 2:1 binding models. Agreement was similar for other Electrophoretic mobility shift assays (EMSAs) ETV4 fragments and ETS factors. Therefore, the use of either model supports the conclusions that ETV1/ DNA-binding assays of ETS factors utilized a 4/5 subfamily factors bind MED25 more tightly than duplexed 27-bp oligonucleotide with a consensus other ETS factors and that full-length ETV4 binds ETS binding site: 5′-TCGACGGCCAAGCCGGAAGT- more tightly to MED25 than the AD or DBD alone. All GAGTGCC-3′ and 5′ -TCGAGGCACT- constants were averaged separately from replicate CACTTCCGGCTTGGCCG-3′. Boldface GGAA experiments, therefore the reported KD value may indicates the consensus ETS binding site motif. Each not exactly equal kd / ka. Mean and standard of these oligonucleotides, at 2 μMasmeasuredby deviation of KD, ka, and kd values from at least absorbance at 260 nm on a NanoDrop 1000 (Thermo three independent experimental replicates are dis- Scientific), were labeled with [γ-32P] ATP using T4 played in figures and tables. polynucleotide kinase at 37 °C for 30 min. After

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 17 purification over a Bio-Spin 6 chromatography column and are indicated by “*”; whereas, values greater (Bio-Rad), the oligonucleotides were incubated at than 0.05 were not considered significant and are 100 °C for 5 min, and then cooled to room temperature indicated by brackets without an asterisk. Replicate over 2 h. The DNA for EMSAs was diluted to 1 × 10− numbers are indicated by the number of dots in each 12 M and held constant. ETV4 and EHF concentrations bar graph, and are included in Tables S1, S2, and ranged was diluted to 5 × 10−7 and held constant while S6. MED25 was diluted from 5 × 10−8 to ~1 × 10−9 M. For measuring ETS-DNA KD values, MED25 was held Cell culture and viral expression constant at 5 × 10−7 and ETS factors were diluted from 1×10−7 to ~1 × 10−11 M. Protein concentrations PC3 cells were obtained from American Type were determined after thawing each aliquot of protein, Culture Collection and cultured accordingly. Full-- using the Protein Assay Dye Reagent (Bio-Rad). The length MED25 cDNA was cloned into pQCXIH binding reactions were incubated for 45 min at room (Clontech) with an added C-terminal 3× FLAG tag. temperature in a buffer containing 25 mM Tris pH 7.9, Oligonucleotide sequences for MED25 shRNA 0.1 mM EDTA, 60 mM KCl, 6 mM MgCl2,200μg/mL hairpin design are as follows, with targeting se- BSA, 10 mM DTT, 100 ng/μL poly(dIdC), and 10% quences in lower case: (v:v) glycerol, and then resolved on an 8% (w:v) native MED25a_fwd-CCGGtgattgagggtacggccaaTT- polyacrylamide gel at room temperature. The 32P– CAAGAGAttggccgtaccctcaatcacaTTTTTG labeled DNA was quantified on dried gels by MED25a_rev-AATTCAAAAAtgtgattgagggtacggc- phosphorimaging on a Typhoon Trio Variable Mode caaTCTCTTGAAttggccgtaccctcaatca Imager (Amersham Biosciences). Equilibrium disso- MED25b_fwd- CCGGtcaaaggcctctaccgcatTTCAA- ciation constants (KD) were determined by nonlinear GAGAatgcggtagaggcctttgagaTTTTTG MED25- least squares fitting of the total protein concentration b_rev- AATTCAAAAAtctcaaaggcctctaccg- [P]t versus the fraction of DNA bound ([PD]/[D]t)tothe catTCTCTTGAAatgcggtagaggcctttga. MED25 eq. [PD]/[D]t =1/[1+KD/[P]t)] using Kaleidagraph (v. shRNA hairpins were cloned into the pLKO.1 3.51; Synergy Software). Due to the low concentration lentiviral expression vector [80], and expression of total DNA, [D]t, in all reactions, the total protein and infection of lentivirus performed following concentration is a valid approximation of the free, standard protocol. ETV4 shRNA retroviral constructs unbound protein concentration. Reported KD values were previously described, and retrovirus production represent the mean and the standard deviation from at and infections were carried out following previously least four independent experiments. published methodology [81]. Whole-cell extracts from cells expressing control shRNA constructs, Protein docking ETV4 shRNA or MED25 shRNA were run on SDS-PAGE gels and blotted to nitrocellulose mem- Modeling for ETV4 AD- and DBD-MED25 ACID branes following standard procedures. Antibodies interactions were performed using the ZDOCK server used for immunodetection were ETV4, ARP32262 (http://zdock.umassmed.edu) [58]. PDB files 4UUV [59] (Aviva Systems Biology); MED25, ARP50699_P050 and 2KY6 [9] were used as inputs for ETV4 DBD and (Aviva Systems Biology); and beta-Tubulin, MED25 ACID, respectively. An α-helix with the sc-55,529 (Santa Cruz). sequence “LSHFQETWLAEA” was generated using Pymol and used as the input for ETV4 AD based on the Chromatin Immunoprecipitation (ChIP) and conserved sequence in ETV5 becoming more helical ChIPseq analysis when interacting with MED25 ACID [7].Fromour mutational data, we selected ETV4 residues Phe60 ChIPs were performed as described previously and Trp64, and Phe428 and Trp432 to be involved in [71], with the following modifications. Cross-linked the interactions between AD-MED25 ACID and chromatin was sheared with a Branson sonifier and DBD-MED25 ACID, respectively. We did not select magnetic beads were washed with buffer containing any ETV4 residues to be blocked from the interface. On 500 mM LiCl. Antibodies used for ChIP were: the basis of NMR and mutational analysis we selected MED25, anti-Flag M2, (Sigma Life Sciences); MED25residueGlu451tobeinvolvedintheAD ETV4, ARP32262 (Aviva Systems Biology). ChIP- interaction but did not select any MED25 residues to be seq libraries were prepared using the NEBNext® involved in the DBD interaction. No MED25 residues ChIP-Seq Library Prep Master Mix Set for Illumina were blocked from either interaction. (NEB, E6240) and run on a Hiseq2000 sequencer. Two ETV4 and two MED25 ChIPseq libraries were Statistical analysis generated from biological replicates and analyzed as replicates with one input control library. Sequence An unpaired Mann–Whitney test was used to reads were aligned with Bowtie [82] to human calculate p values using Graph Pad Prism (v.6). genome HG19 and enriched regions (peaks) deter- Values less than 0.05 were considered significant mined using the Useq analysis package [83],atan

FDR of 1% and Log2ratio of 2 with input DNA library expression of a gene had to be significantly affected used as control sample and either ETV4 or MED25 by all four shRNA constructs (ETV4a, ETV4b, aligned reads as treatment sample. Shared regions MED25a, MED25b). between ETV4 and MED25 enriched regions were defined using IntersectRegion in Useq package with qPCR analysis of ChIP DNA and RNA no gap. Heatmaps of shared regions were generated with DeepTools [84] using a bedfile corresponding to Primer-BLAST [88] was used to generate primer coordinates from the MED25-ETV4 shared regions sets for amplification of enriched regions; primer from MED25 dataset, and bigwig files generated sequences and their coordinates are provided in from the aligned ETV4, MED25 and input ChIPseq Supplemental Table S7, and regions are also reads for one replicate. Data were aligned using the denoted as red bars in IGB tracks (Figs. 8b and center point of this shared peak bedfile for all three S8b). qPCR of ChIP DNA was performed using maps. Enriched regions were also visualized graph- Roche FastStart Essential DNA Green Master and ically using IGV [85]. run on a Roche Lightcycler 96. Serially diluted input Overrepresented DNA sequences present in the was used to create a standard curve for absolute ETV4 and MED25 enriched regions were deter- quantitation of amplified regions from ChIP DNA. mined using the MEME-ChIP program [86] (http:// PCRs for each sample and primer pair were run as meme-suite.org) using default settings except for triplicates and signal averaged over the three values. following parameters for MEME- 1) any number of Data are displayed in graphical form as a ratio of the repetitions for site distribution; and 2) maximum site signal of the target region over the signal of a width of 13. The central 100 bp of the ETV4 and negative control genomic region. An input sample MED25 enriched regions were interrogated by was also subject to same qPCR reactions graphed MEME-ChIP to find centrally located binding sites to confirm validity of negative control region. For all within the regions. We used these tighter peak primer pairs, the input enrichment value was coordinates with IntersectRegions to generate a approximately one. more stringent set of shared ETV4-MED25 regions For qPCR analysis of RNA samples, total cDNA resulting in 611 shared regions to interrogate with was generated from RNA using iScript Reverse MEME-ChIP. A set of size-matched random regions Transcription Supermix (Bio-Rad) and used as a for comparison to the 611 ETV4 and MED25 shared DNA template for qPCR with Roche FastStart regions was generated using the shuffle command in Essential DNA Green Master. Primer pairs for each bedtools (http://bedtools.readthedocs.io/en/latest/). gene assayed were designed with Primer-BLAST The 611 shared regions and 611 randomly generat- against the corresponding mRNA. Relative gene ed regions were searched for the occurrences of expression was calculated using the ΔΔCq method, C(A/C)GGAA using the FIMO program from MEME with GAPDH as an internal reference. Reactions suite [87]. were performed in triplicate for each sample and ΔΔCq values averaged for each knockdown RNA RNAseq sample preparation and analysis sample relative to the average ΔCq value for the control with standard deviation. Expression levels of RNA samples for each shRNA construct (ETV4a, individual genes in samples from ETV4 and MED25 ETV4b, MED25a, MED25b, lenti-luciferase shRNA) shRNA knockdown samples are reported as values were analyzed as biological triplicates except for relative to control shRNA. ETV4 shRNA retroviral control, which had two biological replicates. RNA was prepared from cells at day 7 post-infection using the Qiagen RNAeasy Mini Kit. Samples were treated on column with DNase to remove genomic DNA. Samples were Acknowledgments depleted of ribosomal DNA using a bead-based method (Illumina TruSeq Stranded RNA Kit with We thank Peter Hollenhorst for providing JUN and Ribo-Zero Gold) prior to library construction. Library FOS plasmids and for critical review of the manu- construction was performed using the Illumina script. This study was funded by the National TruSeq Stranded RNA Kit and sequenced using Institutes of Health (R01GM38663 to B.J.G.), the the Illumina Hiseq2000 sequencer. Sequence reads Canadian Cancer Society Research Institute were aligned to hg19 using novoalign (Novocraft) (CCSRI 2011-700772 to L.P.M.), and the Canadian and differentially expressed transcripts determined Institutes of Health Research (CIHR MOP-136834 to with drds analysis package in Useq. Expression L.P.M.). Instrument support was provided by CIHR, changes of an absolute log 2 fold change N0.585 the Canada Foundation for Innovation (CFI), the and FDR b1%, relative to control shRNA, were British Columbia Knowledge Development Fund counted as significant. In order to be considered (BCKDF), the UBC Blusson Fund, and the Michael affected by both ETV4 and MED25 shRNA, the Smith Foundation for Health Research (MSFHR).

Support to B.J.G. from the Huntsman Cancer [8] P.S. Brzovic, C.C. Heikaus, L. Kisselev, R. Vernon, E. Institute/Huntsman Cancer Foundation and the Herbig, D. Pacheco, et al., The acidic transcription activator Howard Hughes Medical Institute is acknowledged. Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex, Mol. Cell 44 Shared resources at the University of Utah, including – the NMR facility, were supported by the National (2011) 942 953. [9] A.G. Milbradt, M. Kulkarni, T. Yi, K. Takeuchi, Z.Y. Sun, R.E. Institutes of Health (P30CA042014 to the Huntsman Luna, et al., Structure of the VP16 transactivator target in the Cancer Institute). The content of this publication is mediator, Nat. Struct. Mol. Biol. 18 (2011) 410–415. solely the responsibility of the authors and does not [10] E. Vojnic, A. Mourao, M. Seizl, B. Simon, L. Wenzeck, L. necessarily represent the official views of the Lariviere, et al., Structure and VP16 binding of the mediator National Institutes of Health or other funding Med25 activator interaction domain, Nat. Struct. Mol. Biol. 18 agencies. (2011) 404–409. Supplementary data to this article can be found [11] P.E. Wright, H.J. Dyson, Intrinsically disordered proteins in online at http://dx.doi.org/10.1016/j.jmb.2017.06.024. cellular signalling and regulation, Nat. Rev. Mol. Cell Biol. 16 (2015) 18–29. [12] P. Tompa, M. Fuxreiter, Fuzzy complexes: polymorphism Received 11 April 2017; and structural disorder in protein-protein interactions, Trends Received in revised form 4 June 2017; Biochem. Sci. 33 (2008) 2–8. Accepted 5 June 2017 [13] P. Di Lello, L.M. Jenkins, T.N. Jones, B.D. Nguyen, T. Hara, Available online xxxx H. 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Simon L. Currie1,2, Jedediah J. Doane1,2, Kathryn S. Evans1,2, Niraja Bhachech1,2,

Bethany J. Madison1,2, Desmond K. W. Lau3, Lawrence P. McIntosh3, Jack J. Skalicky4,

Kathleen A. Clark1,2, Barbara J. Graves1,2,5

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: Tel: 1-301-215-8718; Fax: 1-301-215-8828;

Email: [email protected]

SUPPLEMENTARY DATA

Supplementary Tables

Table S1. Equilibrium dissociation constants (KD), association rate constants (ka), and dissociation rate constants (kd) for interactions between ETV4 truncations and MED25 ACID.

-9 a,b 3 -1 -1 a -3 -1 a b ETS KD (x 10 M) ka (x 10 M s ) kd (x 10 s ) n p SPDEF1-335 Interaction 1 320 ± 90 3.5 ± 0.9 1.1 ± 0.3 3 0.04 Interaction 2 5,000 ± 2,000 1.6 ± 0.7 20 ± 10 3 0.04 ETV11-477 Interaction 1 16 ± 5 2,600 ± 300 40 ± 20 3 0.04 Interaction 2 21 ± 2 70 ± 10 1.5 ± 0.2 3 0.25 ETV41-484 (FL) Interaction 1 7 ± 3 120 ± 40 0.9 ± 0.6 5 - Interaction 2 28 ± 7 1,600 ± 700 40 ± 10 5 - ETV4337-436 (DBD) Interaction 1 230 ± 80 2 ± 1 0.6 ± 0.2 4 0.02 Interaction 2 6,000 ± 3,000 0.3 ± 0.2 2 ± 1 4 0.02 ETV4165-484 Interaction 1 350 ± 80 1.0 ± 0.2 0.4 ± 0.1 4 0.02 Interaction 2 2,200 ± 500 6.0 ± 0.4 1.5 ± 0.2 4 0.02 ETV41-164 700 ± 100 330 ± 20 210 ± 20 3 0.04 ETV443-84 (AD) 700 ± 100 400 ± 100 290 ± 30 3 0.04 a Six concentrations of ETS factor, or ETV4 fragment, were used to calculate KD, ka, and kd for each experimental replicate. Mean and standard deviation are reported for the indicated number of replicates. KD may not exactly equal kd / ka as each of these values was averaged separately. Values were calculated using a model assuming 1:1 interaction for ETV443-84 and ETV41-164, and using a model assuming a 2:1 (ETS:MED25) interaction for SPDEF, ETV1, ETV41-484, ETV4165-484, and ETV4337-436. See methods and Fig. S1 for details. b The p-values were calculated using a Mann-Whitney U test with ETV41-484 as the reference; ETV443-84 and ETV41-164 were compared to ETV41-484 interaction 1.

Table S2. 43-84 165-484 Equilibrium dissociation constants (KD) for interactions between ETV4 or ETV4 and MED25 ACID mutants.

ETV443-84 ETV4165-484 Fold MED25 K (x 10-9 M)a,b K (x 10-9 M)a,b,c Fold Differencea n D Differencea D WT 700 ± 100 - 350 ± 80 - 3 K422E ~ 1,200 ~ 2 N.B. > 80 2 Q430E ~790 ~ 1 ~ 1,600 ~ 5 2 Q451E N.B. > 8 ~ 5,800 ~ 20 2 R466E 700 ± 30 1.0 ± 0.1 3,000 ± 1,000 8 ± 4 3 M470E ~ 900 ~ 1 ~ 280 ~ 1 2 H474E ~ 1,700 ~ 2 ~ 1,800 ~ 5 2 Y487S ~ 770 ~ 1 ~ 6,100 ~ 17 2 R509E ~1,100 ~ 2 N.B. > 80 2 L514E ~ 1,500 ~ 2 ~ 1,500 ~ 4 2 K518Ad ~ 2,700 ~ 4 ~ 3,900 ~ 11 2 I521E 4,000 ± 1,000 6 ± 2 5,000 ± 1,000 13 ± 4 3 M523E 3,000 ± 900 4 ± 1 8,000 ± 2,000 19 ± 8 3 R538E ~ 2,400 ~ 4 N.B. > 80 2 K545E ~ 3,100 ~ 5 N.B. > 80 2 a Values with error indicate that six concentrations of ETV4 were used in each experimental replicate; the mean and standard deviation are reported from three replicates for KD and fold-difference values. Estimated values (~) are averaged from two replicates using a single concentration of the indicated ETV4 fragment, and are reported without error to convey the more approximate nature of these values. b “N.B.” indicates no detectable binding with 40 μM of ETV443-84 or with 35 μM of ETV4165- 484. Fold difference is estimated by comparison to sensorgrams with wildtype MED25 ACID (Fig. 2b,c). c ETV4165-484-MED25 interaction data resembled a 2:1 (ETV4:MED25) binding model for all MED25 species; only the higher-affinity interaction is reported. d “K518A” is an abbreviation for the K518A/K519A/K520A triple mutant.

Table S3. ETV4 and MED25 occupy overlapping genomic regions.

See excel sheet online for table of ETV4- and MED25-occupied regions.

Table S4. ETS DNA binding sequences are enriched in regions occupied by ETV4 and MED25.

See excel sheet online for regions with ETS DNA binding sequence that were occupied by ETV4 and MED25, as compared to a randomly generated control.

Table S5.

Significant overlap in genes regulated by ETV4 and MED25.

See excel sheet online for RNA-seq data upon ETV4 or MED25 shRNA knockdown.

Table S6. Equilibrium dissociation constants (KD) for interactions between FOS or JUN/FOS and MED25 ACID mutants.

FOS JUN/FOS Fold MED25 K (x 10-9 M)a,b K (x 10-9 M)a,b,c Fold Differencea n D Differencea D WT 9 ± 3 - 5 ± 3 - 3 Q451E ~ 160 ~ 18 ~ 74 ~ 15 2 R466E ~ 12 ~ 1 ~ 13 ~ 3 2 Y487S ~ 11 ~ 1 ~ 12 ~ 2 2 a Values with error indicate that six concentrations of FOS or JUN-FOS were used in each experimental replicate; the mean and standard deviation are reported from two replicates for KD and fold-difference values. Estimated values (~) are averaged from two replicates using a single concentration of FOS or JUN/FOS, and are reported without error to convey the more approximate nature of these values. b “N.B.” indicates no detectable binding with 500 nM of FOS. Fold difference is estimated by comparison to sensorgrams with wildtype MED25 ACID (Fig. S9a). c JUN/FOS-MED25 interaction data resembled a 2:1 (JUN/FOS-MED25) binding model for all MED25 species; only the higher-affinity interaction is reported.

Table S7. Oligonucleotides used in this study ChIP qPCR Forward primer Reverse Primer ANK2 GGCAGAGCACGTACTCACATTT ACATAGTACTTGATGAGTCTCCCT SDC1 TACGCACCGCTGAGTAATGG CCAGCCTATGGCTCACTTCC CDK14 GCCCTCACGCCAGTTTAAGT AACCCAATCATCATCAAATGCAAG HEY1 GGAGCAGGCAGTGAAGAACT GCCAATAGCAGAGAGCGGTA IGF2BP1 TTGCAAGGGTCTTGGGACTC AGCAGGCTGTCCAGTACCTA RT qPCR Forward Primer Reverse Primer ANK2 CGAGCAGTCAGAGGACAACA GGAGGTCGTCCTGGTCTAGT SDC1 TGACAACTTCTCCGGCTCAG GACGTGGGAATAGCCGTCA CDK14 GATGTGTGACCTCATTGAGCC TTCCCTGGCAGTTCCGTGTA HEY1 GTCTGAGCTGAGAAGGCTGG TCCCGAAATCCCAAACTCCG IGF2BP1 AGCTCCTTTATGCAGGCTCC CCGGGAGAGCTGTTTGATGT

Supplementary Figures

(a) (b) 0.4 + ETV1 - ETV1 0.15 + SPDEF - SPDEF 500 nM 50 nM ) 0.3 ) m m 0.10 n n ( (

0.2 se se 0.05 pon 0.1 pon es es R 0.0 R 0.00 200 400 200 400 -0.1 Time (s) -0.05 Time (s) (c) 1:1 fit (ETV4:MED25) 2:1 fit (ETV4:MED25) 0.4 + ETV4 - ETV4 0.4 + ETV4 - ETV4 50 nM 50 nM ) 0.3 ) 0.3 m m n n ( (

0.2 0.2 se se pon 0.1 pon 0.1 es es R 0.0 R 0.0 200 400 200 400 -0.1 -0.1 Time (s) Time (s)

Fig. S1. ETV1/4/5 subfamily factors bind to MED25 with higher affinity than other ETS factors. Representative titrations of ETV1 (a) and SPDEF (b) with MED25 ACID. Only the association and dissociation portions of the sensorgram are shown. The highest concentration was 50 nM for ETV1 and 500 nM for SPDEF; both were diluted 1.5-fold for each subsequent titration point. ETV1 binds to MED25 with a similar affinity as ETV4, and both ETV1 and ETV4 bind to MED25 with a 20-50 fold higher affinity compared to SPDEF. See Fig. 1c and Table S1 for quantification of replicate experiments for each of these proteins. (c) Comparison of binding models for ETV4-MED25 interaction data. The black lines are the actual data and are identical in the left and right panels. The red lines are the fit of the data assuming a one-to-one interaction between ETV4 and MED25, left, or a two-to-one interaction with two molecules of ETV4 binding to each MED25 molecule, right. The better fit of the two-to-one binding model suggests that ETV4 interacts with MED25 using a complex mechanism. However, both models are in reasonable agreement about the affinity of the ETV4-MED25 interaction as the one-to- one model results in a KD of 5 ± 1 nM versus KD values of 7 ± 3 and 28 ± 7 for the two- to-one model (two ETV4 molecules binding to one MED25 molecule). See methods for details.

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ETV6 ------MSETPAQCSIKQERISYTPPESPVPSYAS-ST EHF ------MILEGG------G--VMNLNPGNNLLH------QPP-AWTDSYST-C- ETS1 --IIKTEKVDLELFPSPDMECADVPLL------TPSSKEM----MSQ ERG QDWLSQPP----ARVTIKMECNPSQVNGSRNSPDECSVAKGGKMVGSPDTVGMNYGSYME SPDEF ------ETV4 AGYLDQQVPYTFSSKSPGNGSLREALIGPL------GKLMDPGSLPPLD----SE ETV1 DGFYDQQVPYMVTNSQRGRNCNEKPTNVRK------RKFINRD--LAHD----SE ETV5 DGFYDQQVPFMVPGKSRSEECRGRPVIDRK------RKFLDTD--LAHD----SE

ETV6 ------MSETPAQCSIKQERISYTPPESPETV6VPSY A S - S T PLHVPVPRALRMEEDSIRLPAHLRLQPIYWSRDDVAQWLKWAE--NEFSLRPIDSNTF-E EHF ------MILEGG------G--VMNLNPGNNLLH------QPP-AWEHFETV6TDS Y S T - C ------N-VSSGFFGGQWHEIHPQYMWSTEKTYPQAVQWCESWILKQHELLRIDSTYNTQPPLDEASNPCVIPPSFYQAESF--SDT ETS1 --IIKTEKVDLELFPSPDMECADVPLL------TPSSKETS1EHFEM---- M S Q A------LKATFSG-FTMKIELQQEGGR------LGIPKGD--PRVQMWNTLENTPHGVNNRDLLWVHM------WAV--NQEPPFS-LAKWGTVDDSFYQSKTF--CC- ERG QDWLSQPP----ARVTIKMECNPSQVNGSRNSPDECSVAKGGKMVGSPDTVERGETS1GMN Y G S Y M E E--KHIIMPPPKTENK-VMDTTLENLEFRRPS----PDMEVCIAVDPVAPDLLPT------LWSTDHVRQWLEWAV--KETYPGSSLPKDEVMN----ILLFMQSNQ SPDEF ------SPDEFERG ------QDWLSQPP----ARVTIKMECNPSQVNGSRNSPDECSVAKGGKMVGSPDTVGMNYGSYME ETV4 AGYLDQQVPYTFSSKSPGNGSLREALIGPL------GKLMDPGSLPPETV4SPDEFLD---- S E D------LFQDLS------H----FQETWLA------EAQVPD------ETV1 DGFYDQQVPYMVTNSQRGRNCNEKPTNVRK------RKFINRD--LETV1ETV4AHD---- S E EALGFYQLDLQQS------VPYTFSSKSPGNGSLREALIGQP----L------LQETWLA------GKLMDEPAGQSVLPPPD------LD----SE ETV5 DGFYDQQVPFMVPGKSRSEECRGRPVIDRK------RKFLDTD--LETV5ETV1AHD---- S E EDLGFQYDLQQS------VPYMVTNSQRGRNCNEKPTNVQR----K------LQEAWLA------RKFINERADQ--VPLDA------HD----SE Clustal Omega ETV5 D G F Y D QQ V P F M V P G K S R S EE C R G R P V I D R K ------R K F L D T D -- L A H D ---- S E

ETV6 PLHVPVPRALRMEEDSIRLPAHLRLQPIYWSRDDVAQWLKWAE--NEFSLRETV6PIDS N T F - E MNGKALLLLTKEDFRYRSPH-SGDVLYELLQHILK-QRKPRILFSPFFHPGNSIHTQPEV EHFETV6 ------N-VSSGFFGGQWHEIHPQYMWSTEKTYPQAVQWCESWILKQHELLRIDSTYNTQPPLDEASNPEHFETV6CVIPPS FY QA ES F- -S DT IPNLGHEVHPLVCPSRMASLLRQMEEEFTDRSAAIRGLTP-AAHGLQRLLLQYPSINYLWQSHRLDDKWV-ANQGWQLCKSW---AE--SDNLEFQSSLTRHP------IDSNTFN-VE ToolsETS1EHF > Multiple A------SequenceLKATFSG-F AlignmentTMKIELQQEGGR------>LG ClustalIPKGD--PRV QMOmegaWNTLENTPHGVNNRDLLWVHM------WAV--NQEPPFS-LAKWETV6ETS1EHFGTVDDS FY QS KT F- -C C------M------NGAALCALGKND-CVFSSLEGLFFAPGGDFQVWGHDEILHWPEQMHYSLWETTIKP---YAQQVCLWSQEIKWKELQDEVHR---LLISDYKTTPNPPYQQLEVDS----APNVCPISNPYGFAVQSNE-PFS---TD ERGETS1 E--KHIIMPPPKTENK-VMDTTLENLEFRRPS----PDMEVCIAVDPVAPDLLPT------LWSTDHVRQWLEWAV--KETYPGSSLPKEHFERGETS1DEVMN---- I LL FM QS NQ ------IADLGKKAETLFCSKGM-TFMKTIDDKLEEFQQGGQR------L----TPSYLNGAIDPGIK--LLDPVSRMHQNLWLHTNYEP---TGHNNVLRLLRDEWHTV------PMLW---AV--PQHNPPLETF-SA----LWKTGDVSDDDYFSVQTDK-KFC----C ResultsSPDEFERG for job ------QDW Lclustalo-I20170117-172757-0553-82261980-oySQPP----ARVTIKMECNPSQVNGSRNSPDECSVAKGGKMVGSPDTVETS1SPDEFERGGMN Y G S Y M E ------EKIIHMKPPPTEKNV-DMLTTELNFEPRRSP----DMECVAIDVVPPALLDP------TLWSTDHVRQWLEWAV--KTEPYSSGLKPEDMV----NILLMFSQQN ETV4SPDEF D------LFQDLS------H----FQETWLA------EAQVPERGETV4SPDEFD------Q------DWLSQPP----ASRDVETQIFKVMPEDCFNHPSSEQNVLNAGFSHRSNPSTP-DTERCISKKVAEKPGGQ---KMVSGPSRPTDTPVAGLMSNCYSGRSKYPM--E ETV1ETV4 EALGFYQLDLQQS------VPYTFSSKSPGNGSLREALIGQP----L------LQETWLA------GKLMDEPAGQSVLPPPSPDEFETV1ETV4D------LD---- S E ------DLFQDLS------NDEQFVPDYQAESLAFHG----LP-LFKQIEKKTWELPAH------SPCESAEQIVSSPDA------CSQEQ-- ETV5ETV1 EDLGFQYDLQQS------VPYMVTNSQRGRNCNEKPTNVQR----K------LQEAWLA------RKFINERADQ--VPLETV4ETV5ETV1DA------HD---- S E A------EGLYFLQDQQLSV------PYTFSSDKESQPFGVNPGDSFLQRSEDANLIVGLPHQLA------PPPTLKQIEKTRWELLAH------G---KLMSDPPSSEGASEQLVPP-PSSDL------DC----SHEQS--E ETV5 D G F Y D QQ V P F M V P G K S R S EE C R G R P V I D R K ------R K F L D T D -- L ETV1 ETV5A H D ---- S E D EG LF Y QD QQ L SV ------P Y M V T N S Q R G R N C N E K P T N V R QK ------L Q E A W L A ------R K F I N R ED A-- Q VL PA DH ------D ---- S E ETV5 D G F Y D QQ V P F M V P G K S R S EE C R G R P V I D R K ------R K F L D T D -- L A H D ---- S E CLUSTAL O(1.2.4) multiple sequence alignment ETV6 MNGKALLLLTKEDFRYRSPH-SGDVLYELLQHILK-QRKPRILFSPFFHPG ETV6N S I H T Q P E V I L H Q N H EE D N C V Q R ---- T P R P S V D N V HH N PP T I E LL H ------R S R S P I TT --- EHFETV6 IPNLGHEVHPLVCPSRMASLLRQMEEEFTDRSAAIRGLTP-AAHGLQRLLLQYPSINYLWQSHRLDDKWV-ANQGWQLCKSW---AE--SDNLEFQSSLTREHFETV6HP------ID S N T F N- VE IMVNKGTKEAQLLLL------TKEDFRYRSPTHE-PSSGIDMVNLTYWEKLLDEQ------HILK-QRKPRILFSPFFHPGNYSLI------HTQPEV ETS1EHFETV6 M------NGAALCALGKND-CVFSSLEGLFFAPGGDFQVWGHDEILHWPEQHYMLWSETIKT---YPQAVQLWCQESKWIELKDQVHE---LLRIDSKTYPNTYQPPQLVDE----ASNPETV6ETS1EHFCVIPNPS GFY VQA NES PF- ---S DT PAIL------NHGVEPHVLPCRSAMLSRLMQEEYEPFDETSRIRAARYLTGPSTAD-HYALFGRIQLSLLQYPGYISYENWHLSAQRQHDDCLVKVPPWA-QSNWEGLFQKSCWESAP---ES--FISNTDELFSSYQLQSRTPLHIH------DPSINSSTFEE-NE-V ETV6 ------ETV6 ------MSETPAQCSIKQERISYTPPESPVPSYAS-ST ERGETS1EHF IA------DLGKKAETLFCSKGM-TFKTMDDKIELFQQEQGGRL------TPSYLNGAIDPIKGLLD--PSRVHQMLWNHTLYEN---TPHGVNNLRRDLLEWTVHPM------LW---AV--PHNQLEPPTFS-----LAKWEHFERGETS1GTVDDDDS FY VQS DKT KF- ---C C------AM------NGAALCALGNK-DVLCSSQFNLGSEFFPLRAGG------PDQFWVHGEDIHLPWQLEYMHWHLTAEKRIYN---QTVGGWLE-----QWKLEQDHVLLAA---DFTIKNFPQPYL------QDVA----NCIPNFGQVENFP---D EHF ------EHF ------MILEGG------G--VMNLNPGNNLLH------QPP-AWTDSYST-C- SPDEFERGETS1 ------E--KHIIMPPPKTENK-VMDTTLENLEFRRPS----PDMEVCIAVDPVAPDLLPT------LWSTDHVRQWLEWAV--KETYPGSSLPKETS1SPDEFERGDEVMN---- I LL FM QS NQ A------ILDKGAKTEFLSCGK-MFTTKKDDEQQFQR----LTPSLYGNIAPDKIDMLLPGRSQAHWSLTPHE---YT---HVGRLDSSRWEVTM-PWSLAP---VS--HLLLPNHELFPPTSLD----KTGVVSDRDDFTQGVKLDFEK-K--CA ETS1 ------MKA------AETS1VDLK P T L T - --IIKTEKVDLELFPSPDMECADVPLL------TPSSKEM----MSQ ETV4SPDEFERG ------QDWLSQPP----SADREVQTFIVKPMDEFCHNSPESNQLVANFGHSSRPNTS-PTDREICKKSVEAPKQGG---KMSVPGRSTPDPTAVERGETV4SPDEFLGSMCN SY RG KS PY --M E EP------KLHPMYPPPHH-NG-EMQTTCLNYESSRR-----AYDPPVIRVQPIADIPKTSLPWASPTGDAHLVGRQQSWPLE-WQAPVF--P-----KEYGLRP------DVNILLFQN ERG MIQTVPDPAAHIKEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTERGSKM S P R V P Q QDWLSQPP----ARVTIKMECNPSQVNGSRNSPDECSVAKGGKMVGSPDTVGMNYGSYME ETV1ETV4SPDEF ------D------LFQDLS------NDEQFVPDYQAESLAFHG----LP-LFKQIEKKTWELPAH------SPCESAEQIVSSPSPDEFETV1ETV4DA------CS Q E Q ------P------FKFSY-GEKCLYSNDVESQAFYVDPQDKFPHQSVEGNMLRAPFSHNSPPPTT-PTSSRITKKPVE-PSQP---L------SPRTDHHPAALSPCNSSRTKHPT--P SPDEF ------SPDEF ------ETV5ETV1ETV4 ------EALGFYQLDLQQS------VPYTFSSDEKQSFPVGPNDGFSQLSRDENALVILGHQPA----LPPP------TLKQIEKTRWELLAH------GKLSMPDSSEPAGEQSLV-PPPETV4ETV5ETV1SSD------LCDS---- H E Q --S E DA------LFGQADNLYS------GEKCLYNNDYECQAFYVDPRDKYPPQASEGSFLKAPFHL----TGPPLPTT-FLQPKELITSKKWPLTEA-P------HQ---NPLSFPEPPPCASQEVQIPASSDT------LAPCTSSQGEHQA--P ETV4 ------ETV4ME RR M K - AGYLDQQVPYTFSSKSPGNGSLREALIGPL------GKLMDPGSLPPLD----SE ETV5ETV1 E DL GF Q YD L QQS ------V P Y M V T N S Q R G R N C N E K P T N VQ R---- K ------L Q E A W L A ------R K F I NE RA DQ --V P LETV1 ETV5D A ------H D ---- S E E ------L F Q D L S ------D E Q F V P D F Q S D N L V LQ H---- A PPP L TQ KE IT KW RL EA L------H --- S PE SSA Q EV LP -D SS------C S H E Q -- ETV1ETV5 ------D G F Y D QQ V P F M V P G K S R S EE C R G R P V I D R K ------R K F L D T D -- LETV5ETV1 A H D ---- MS -E ED LG F QY D LQQ S ------V P Y M V T N S Q R G R N C N E K P T N V QR ----K ------L Q E A W L A ------R K F I N ER AD Q-- V PL DA ------H D ---- S E ETV5 ------ETV5 M - DGFYDQQVPFMVPGKSRSEECRGRPVIDRK------RKFLDTD--LAHD----SE ETV6 I L H Q N H EE D N C V Q R ---- T P R P S V D N V HH N PP T I E LL H ------R S ETV6R S P I TT ------N H R P S P D P E Q R P L R S P L ------

EHFETV6 IMVNKGTKEAQLLLL------TKEDFN-terminalRYRSPTHE-PSSGIDMVNL ActivationTYWEKLLDEQ------HILK-QR KDomainPRILFSPFF (ETV4HPGEHFETV6NYSLI ------H residuesT Q P E V ------ILH Q43-84)NHEEDNC VQR----TPRPSVDNVHHNPPTIELLH------RSRSPITT--- ETS1EHFETV6 AIP------NLGHEVHPLVCPSRMASLLRQMYEEEPFETDSRRAAIYRTGLSTPD-AYAHFGLIQRSLLLYQGYPISENYHLWAQSQHRCLDDVKPPWV-ASNQEGWFQLSCKESWP---ASEF--ISTDNELSFYQSQSLTRETV6ETS1EHFLHPH------IPD IS SSN T EEF N- -VE M------INVGKKTAELLLLQ------TKEDFRYRSPHT-ESPGSDIVMLNYTEWLLKDQEH------ILK-QRKLLPRSILLKFYSEPNFFDYHPPSGVNISLYIRLH------TQPEV ETV6 PLHVPVPRALRMEEDSIRLPAHLRLQPIYWSRDDVAQWLKWAE--NEFSLRPIDSNTF-E ERGETS1EHF AM------NGAALCALGKND-LCVQFSSNLSEGPLFFRA------PGGDFQVWGHDEILHWPLEQMHYHLWAETRIKN---YTQGGVLW-----QEKWELDQVHAA---LLFDIKTFPNPYQ------QLVD----ANEHFERGETS1CINP GF VQ NE PF --- D I------AN------GEHLCSMSLQEYFPTERSAARYGTTS-DAYGFQILLSYYGSINELHQAHQLCKVWPP-NSGEQFCSSE---PSNFSTIDSTLVEFYSQPYSEQTAHTL------HPISSEENV- EHF ------N-VSSGFFGGQWHEIHPQYWTKYQVWEWLQHLLDTNQLDANCIPFQEF-D SPDEFERGETS1 ------IADLGKKAETLFCSKGM-TFKTDDKEFQQQRL----TPSYLNGAIDPIKMLLDGPSRAHQSLWPHT---YE---THGVLRSSRDEWVT-PMSLWP---ASVH--LLLPHNLEPPTFSD----LTKETS1SPDEFERGETV4VGSVRDDD TF GVQ LDK EKF K--- AC MAAAVN------DGQAAAGG------LVCNAGLHGRKYDPCLGFQALNGESVVLPARIP------KDQFEVQGTDDIFL-WAELYHMDLHSEADIR------NTGGLQ-----KVETDGVC---AAASFMKIYPFLYPHQ------TVE----GFSGNPGSVPNGPD--GA ETV4 VDQGGVNGHRYPGAGVVIKQEQTDF-AYDSD------VTGCASMYLHTEGFETS1SGPS P G D G A ALKATFSG-FTKEQQR----LGIPKDPRQWTETHVRDWVMWAV--NEFSLKGVDFQKF-C ETV4SPDEFERG P------ELKPHYMHHPPP-GNE-QMCTTLYNSSERR-A----YDPPVRIQVIPAIDKPSTPLAWPSGTADLHGVQRSQPWL-EQWPAFVP------KEYGRL------PERGETV4SPDEFETV1DVN I LL F Q N I------DGKGESLAACKSMQTSKFDDP-FPPQRLLMTIPKSQYENAPAERDQDIRFLLNM-FGASLYHRADLSSSSHPEYG------TSQGLPR------SSEVTVPP-SLSC---PHHSSPHIPGLLLYHMLGRTYPPQSLE----GDGETFHVLSSSADDRHVTPVFGSDQQL--KE--PKRLAT ETV1 ---GSAASQSFP-PPLMIKQEPRDF-AYDSE------VPSCHSIYMRQEGFERGLAH P S -- R T EKHMPPPN-MTTNERR----VIVPADPTLWSTDHVRQWLEWAV--KEYGLPDVNILLFQN ETV1ETV4SPDEF P------FKFSY-GEKCLYSNDVESQAFYVDPQDKFPHQSVEGNMLRAPFSHNSPPPTT-PTSSRITKKPVE-PSQP---L------SPRTDHHPASPDEFETV1ETV4ETV5ALSPCNS SR TK HP T-- P ------P--LPMYPHHGPP-GAEHQGCFLQY-SSSP-MAGYIDKPPQKEPRPDQR-IDRAYAI-FKCPSVAPDHAS------PEG------ALGQLSPPPVLPS-NQCSPQIFSSPD-----YSSMRYGGPMRYD------FHS----RFRRS--QLSSH ETV5 --MPGPPAHGFQ-SPMGIKQEPRDY-CVDSE------VPNCQSSYMRGGYFSPDEFS---S -- S H ------ETV5ETV1ETV4 A------DLGFAQNDYL-SG------EKCLYNNDYECQAFYVDPRDKYPPQASEGSFLKAPFLHTG----PPLPTT-LFPKQLIESKKTPWTEL-PAH------Q---NPLSFPPPPCESAEQQIVASSPETV4ETV5ETV1 TD LA------PC TS SQ GE HQ A-- P ------AAP F GK PF VS QY G- VG GE PK AC PL SAY DPN EHV QS FLA VPY PED DPQ FGK HP SQQQQ EV NG LTM AFR FAP HVS SPN PRPP TPP -T THP RQSS IP KKLT QP EMV P- QKS ---MMP L P------SE PN RQ TY DP PSHH AE LQA S- CRP SFN RQS KRT PQH --LT SP ETV4 DLFQDLS------H----FQETWLA------EAQVPD------ETV5ETV1 ------E L F Q D L S ------D E Q F V P D F Q S D N L V L H QA ----PPP T LK QI EK TR WE LL AH ------S P SS E AE QL V- PETV1 ETV5SS D ------C S H E Q ------A L G A N Y - G E K C LN YD NE YQ CF AV YP DD RY KQ PPA E SS GL FA KF PH LG TL PPP - TTL K PI LKK S PE TP -H --- Q N PS LP FC PPPS E I QSS A TA LC PS TQ SE GQ H-- A P ETV5 E L F Q D L S ------Q ---- L Q E A W L A ------E A Q V PETV5ETV1 ETV6 D ------E ------L F Q D L S D------G L H R E GD KE PQ IF NV LP SD HF -Q RS -D EN DL LV AL YHQ MA------PPP TL KQ NIE HKT IRW MEL VLA SH------V--- S PP S EEP SSE HA AEQ MLV P-P ISSD G------RC IS AH DE CQ R-- LL ETV6 ------DGLHREGKPINLSH-R-EDLAYM-----NHIMVSVSPPEEHAMPIETV5GRIA D C R LL ELFQDLS------Q----LQEAWLA------EAQVPD------ETV6 ------N H R P S P D P E Q R P L R S P L ------ETV6EHF ------KK E ------D N M I - RR L S P A E R A Q G P R P H QQ ED NNPP HA Q- EK SC YH PT LKK S VH SN PP MR EG -T NNH L EHF ------KKE------QDPPA-KCHTKK H N P R G T H L EHFETV6ETS1 ------IQLRHVQPNSHYEEDSDFNDCSVEQDRY----PAALTPPNRHPKSPVKDGNTVFHHKDNYPPVRTDIREALLDLHN------KDKPVIPAAALRASGEHFETV6ETS1RYSTPG IS TTG P ---I Q L ------QRVPSYDSFDSEDYNPHAARPLSPPNDHPKEPQKRGPTLFRKSDPYLV------RDRADLNKDKPVIYPDAAATNYLGASGTYVTDGLLSGDPSIKQTL ETS1EHFETV6 ------IMVNKGTKEAQLLLL------TKEDFRYRSPTHE-PSSGIDMVNLTYWEKLLDEQ------HILK-QRLLKPSRLIKLYFESNPDFFYPHSPVGETV6ETS1EHFERGINLYSRLI ------H T Q P E V I------LHQNHPEEQSDKNAACVQQPRS----PSTVTPPKR------PSVDNDV------HHNTPPEDTPQILRE-PLLQQTLHDD------TPLYQQNIDLYGFPATISSKQRRELSVVARNSTPPGIDSTT----GQ---IQNL ERG -----PQSKAAQPSPSTVPK------TEDQRPQLDPYQILGPTSSRLAETV6NPGS G Q I Q L MNGKALLLLTKEDFRYRSPH-SGDVLYELLQHILK-QRKPRILFSPFFHPGNSIHTQPEV ERGETS1EHF ------AI------NGEHLCSMSLQYEPFETSRRAAYTGSTD-YAFGIQSLLYGYISENHLAQQHCLVKPPW-SNEGFQSCESP---SNFTISSTDVELYSFPYQEQSATEHFERGETS1SPDEFTLH------H------P I SS EE N -V I------EV------KTEQ------EHSLEQV------TEPSIMQNSTMWVVKDGEE------VL-KDIELLTASCLKKLLYENNIDTYAQPDRSPIVMNTTIDYLWLRS------PP----SNVQDK SPDEF E------EHSLEQV------QSMVVGEVL-KDIETACKLLNITADPMEHFDWS P S N V Q K INGEHLCSMSLQEFTRAAGT-AGQLLYSNLQHLKW-NGQCS---SDLFQSTH------NV SPDEFERGETS1ETV4 AAAMV------NDGQAAAGG------LVCNAGLHGRKYDPLCGQFANLGSEVVPLRAI------PKDQFEVQGTDIFL-WALEYMHDHLSAEDRI------N---TGGL-----QKVETDGVCAA---ASFMIKYFPLPYH------QTVE----GFETS1SPDEFERGETV4ETV4SGNP GS VP NG PD --G A A------VM------D------QGGVNGGHYRGYYPEYGKPAPEGLSVVRRPYVIFTGKPSLQ----DEYRRQFTIDDDSWFYS-VGPACISYVVEPPDHSPADEQT------KCPFVEEPPQGGDSLIESVKFATQSFGEEYCGPLA-SVFYNMGIFTYATDSLFEMVHRSLYTEYPEGQPEGPPTEAFLDTSYHSS------GQPRRIWSSS----PGGAEEDLGQ-AL ETV4 M------GYGYEKPLRPFP----DDVCVVPEKFEGDIKQEG-VGAFREGPPETS1YQRR G A L Q L MNGAALCALGKDCFLELAPDFVGDILWEHLEI---LQKEDV---KPYQV----NGVNP-- ETV4SPDEFERGETV1 ------I---DGKGESLAACKSMQTSKFDDP-FPPQRLLMTIPKSQYEANPEARQDRIFNMLL-FGALSYRAHDSSSLPHEG---Y------T---SQGPL------SSREVVTP-PSSLCP---HHSPHIGLLLPYHMGLRYPPTQLSEGD----ETFERGETV4SPDEFETV1ETV1HVLSSSARDDH VTP FGVS QQLD-- EK PK--R LAT ADAA---E------I------CGHAGS------FAATSGQCGGGSMFFPELR-KQEPPGNPPSLRPMQRAIF------PKYYQ----QEHPQRLDDDSFE-TPACLCYVVMPDH---SPAEER------KNPFTYDGG-GPD-----QQIVKSPQFSEKC--AAQHESPFYIGIHYMFDMYPRR------LQEYEGEGPQFTALYGAQQHRRP----SG--SLRQATL ETV1 E------GCMFEKGPRQFY----DDTCVVPEKFDGDIKQE--PGMYREGPTERGYQRR G S L Q L IDGKELCKMTKDDFQRLTPSYNADILLSHLHY---LRETPL---PHLTS----DDVDK-- ETV1ETV4SPDEFETV5 ------P------LPMYPHHGPP-GAEHQGCFLQY-SSSP-MAGYIDKPPQKEPRDQR-IDRAYAI-FKCPSVAPDHAS------PEG------ALGQLSPPPVLPS-NQCSPQIFSSPD-----YSSMRYGGPMRYD------FSPDEFETV1ETV4ETV5ETV5HS----RF RRS -- Q LS SH ------E------EP------CMNPSGFPPPPAGLHFPGSTFYMQEP-KRSDEPPGMRRGLPIYMKFYQ----QERAPQERMQDDDSRYMEN-TGPFCCSNLVVVAIRDS---SSPPEE---G------RPTLFSEG-QGLPPPKSS------VQVKVGPQ-FNESKC--PQSEHSSPHYPTLLLHGYMDHMYPGRRPPVYGGEYLGDEGYPPTHEFVNHSYSTSS---QRMRRTVG-----FSGLQQ--SELKPSQALHL ETV5 E------GFSYEKDPRLYF----DDTCVVPERLEGKVKQE--PTMYREGPPSPDEFYQRRG S L Q L ------ETV5ETV1ETV4 AAP------F GK PF VS QY G- VG GE PK AC PL AYS PND HVE SQ LAF PYV EDP PQD GKF PH QQQQS VE GN TML FRA APF VSH PNS RPPP PPT T- HPT QSSR PI LTKK QP MVE P- KSQ MMP--- L P------ES NP QR YT PD SHHP EA ETV4ETV5ETV1 QAL -S RPC FNS QSR RTK QHP LT-- SP PE------LP PC YH HHP F -PP G EQ QP CG LV :YP SSG D -N AR YP DS PPY HK RP QD IM- ASR IEA KPF SIP PVA APH PAA------G APP L G- QPP SL PQPP LG -FS QK PQS FEI PY -----HD DSS P LY YP REM ------HD GH V- PR GF -----RR Q L S : ETV4 ------SDEQFVPDFHSENLAFHSPT-TRIKKEPQ---SPRTDPALSCSRKP-- ETV5ETV1 A ------L G A N Y - G E K C L Y NN DY EC QA FY VD PR DK YPP Q AS EG SF LK AP FL HT GPP L PTT - LP KL IS KKP T E- PH Q ---N P L SF PPPP C S EQ IA SSETV1 ETV5T L A P C T S S Q G E H Q A -- P P AAF K GF PS VY Q- GG VE GK PC AL PY AN PV HS A LY PD EQ PK GP Q QQQV G M TR FP AS VN PPP R PPT P HSS Q PT LP QV M- PS KP MML ------P E N Q Y PHH S EA QS -P RN FS QT RH QT LP S ETV5ETV6 ------D G L H R E G DK EP QI FN VL PS DH F- QR S- DE ND L VA LY HM A----- PPP T KN IH KI RM EV LS HV ---S PP SEE P SSH A EM LP -IETV5ETV1 ETV6ETV6 SSG R CI SA HD EC QR --LL A------WL DG YA VN YY --- DG QE LLLK HC R-L ESYN GDND KSYE PRCQ IYAF NEYV LNDP SFRD HIKY -RPPQ R-A -WSE EGS DDFL KKA EPF YSLH MKTG -----IPPL FP RTT- IL VPK NDLI HPSKK INP MGTE VL-P SAH VRQ--- SLN PPWP GLS EENFP HPPPC HKS ANE MRQI PTASS INT GMLA RTPC IYTS AESQ DKGE CMHQ RSA-- LLRP A ETV6 WDYVY--QLL-SDSRYENFIR-WEDKESKIFRIVDPNGLARLWGNHKNRTNETV5MTYE K M S R A ------DEQFVPDFQSDNLVLHAPPPTKIKRELH---SPSSEL-SSCSHEQ-- ETV6 EHF ------KK E ------D N M I - RR L S P A E R A Q G P R P H Q E DNN PP H AQ -E KS CY HP TL KK ETV6EHFEHFS V H S N P M R E G - T NN H L H------W CE PF AI SSR -- E SD HI PKKLL K PEN SS------P D PK RN --P G QL EI SK T- RW VE ID QR LS ME PG SV PF IR MF HL PK LS IE LA NV PA RQQ HDL SPPW VG DAKKK F- K QCNN SH RTSS LKK SM EHT ------NY PE RK GL TS HR LA EHF WEFIR--DILLNPDKNPGLIK-WEDRSEGVFRFLKSEAVAQLWGKKKNNSS M T Y E K L S R A EHFETV6ETS1 ------WQQRFVLLPS---YDSEFLLDSTEDKYNSPHCAARQPSLSFPPINDSHP-KEWPQTKR-GPGTLDFRGKSWDPEYLFV------KRLDSRDAPDDLENVKADRRKPWVGIYKPDRAAATKNYKLGPASKGEHFETV6ETS1ETS1TMYVNTDYG LLES KG DLP SI KRQ TGL F------QWCRQRVFAPLLQSIY---SDMSTTTFEDLLSSETHDDLYKPS------AACQLSPFNIDHSNK-MPWIKT-G-RRTGVFDALKGESDWSPYEPAVF------ERKRDLARSQADGDPPLDRNEPKVHDAQKRREPNNVWIGDHPKMQAAAR------EKSNYLKPAPLGKSYMVTNSGYPSEMGKEPL-ISNNQRLG ETS1EHFETV6ERG ------I-----LHQNHPEEQSDKNAACVQQPRS----PSTVTPPKR------PSVDDN------VHHNTPPEDPTQLIR-EPQLLTLDHT------PLYQNIDLYGFPATISSKQRERLVVSAETV6ETS1EHFERGERGRNTSPPG DIS ----TTG Q ---I Q NL ------M------WCQMF-LLGRP---TQSRKEGAALLKLQSGGPDSSSNQPH----SNRTSSPVSPCPDKIDS------TPF-EWQSERI-PEGLSTRYNSDGPSTEL------EF------DKQMRTPDQPLDDEPVYACQRR---ILWGGDYPERDTRLTSSKTNSQYRKSGLPWSANSSTNMVPNQDGYSSLLSDGKFDQLNSISSKQRLTA ERG WQFLL---ELLSDSSNSSCIT-WE-GTNGEFKMTDPDEVARRWGERKSKPNETV6MNYD K L S R A ILHQNHEEDNCVQR----TPRPSVDNVHHNPPTIELLH------RSRSPITT--- ERGETS1EHFSPDEF ------IEV------KTEQ------EHSLEQV------TEPSIMQNSTMWVVKDGE------VL-KDIELLTASCLKKLLYENNIDTYAQPDRSPIVMEHFERGETS1SPDEFSPDEFTTINDLYWRLS P------S N V Q DK ------L------EWP------LLY-WETPPEH------EQHYSRLLE-QPPV------MGKAFQELAGQ-DSK------MEVVGMGEEFVKPLLL-I-KEQDPTIEEDETVLAAQCRRNKDLLWYGFNIAIQRRITKKANSQDRAEPPWVVMATDMGTWNHPSYGDPDH----SKPNLTVS--QRNKS SPDEF WLLWTEHQYRL-PPMGKAFQELAG-KEGMEFKLIEPEEVARRWGIQKNRPAEHFMNY D K L S R S IVKTEQ------TEPSIMNTWKDE------NYL------SPDEFERGETS1ETV4 ------AMV------DQGGVNGYHGRYEPYKGPAELGSRVVRPVYFIGTPKLS----QEDRRYQFTDIDDWSFSYV-PGCASIVVYPPEDHPSAEDTQK------PCFEVQPPGGDLSISEKVAFQTFSEGYEGCLP-ASVYNFGMFTIAYDSTFLMVERHLYSETYPGEPEQPPGEATFETS1SPDEFERGETV4ETV4DTLYSSS------HQGPRR WIS ----SSGP AG EELD QG A-L ------A------MWK------QAFPLGVA---SSGRYAEEGLLYPEDD--KPPPLT----RNPAFHPFEE----IAP-EWQDDTC-PVGVCRIVVGDMSPEQEF------KKFLEIGEDPIEEAKLLPQVAEASGRL-LKSVWYLGGEDAINLFQDVRKYPENPGGQGRSRPPPVLIAITTTYML------QNRRRY------PD----GKALLSQRLDS ETV4 WQFLV---ALLDDPTNAHFIA-WT-GRGMEFKLIEPEEVARLWGIQKNRPAETS1MNYD K L S R S A------YPESRYTSDYFISYGIEHAQCVPPSEFSEPSFITESYQTLHPISSEE- ETV4SPDEFERGETV1 DAAAWE---I------Q------CGFHALGS------VF---AATSGQACGGGLLMSFDDEPRLK-EQPGPPPNSLSNRPSQMARHFIP------FYKYI----QQAEH-PQWRLTDDDS-FEGT-PRCACLGVVYPMD---HEPSAFERK------PNLFYTID-GGEGPDQQ-----EEIKVSVQPFAESKRR--CQAAHEWPSYFGIHIMYDFQYMPKRL------NEQYRGEEPGQATFERGETV4SPDEFETV1ETV1AMYLGNQAQYRRH PD ----KGS LS-- SL RQ ASLT ------V------EWD------QQFGGLVV---NGHCARMLLYFPEDDGKAGPGPSVVRNQSIVFHKGYFQL----IEAQRR-TWDDDTFW--STGAPCRYSVVGDPPMSPEDAEF------TKKPFLEDIQGEGDPLIEEVSKTAQVGFEACY--RRALSPWNMYGGTYFMISLDYQVHMRKYTLENPEYGREGPPAFETATSDYM------GSSQNPRRYSWDP----GKGSLDLSGQRALS ETV1ETV4SPDEFETV5 E------P------CMNPSGFPPPPGALFHPSGTYFMEQPK-RDSEPGRMRLGPYIMFKY----QQERAPQERMQDDDSRYENMT-PFGCNLSVVVIRAD---SSSPPEG---R------PTLFSE-QGPPPLK------SSVQKVGVQPF-ENKS--CQPEHSPSSYPHTHGLLLMYDHYMPGRVYPPEGGYLGEGDPPYHETFSPDEFERGETV1ETV4ETV5ETV5NHVYSTSSSQ---MRRR VT -----FGS QQLS-- EL PKQS LAH AAA---DEW------I------QGCFAGHL------SVAAF---TSGQFTSGGGLLFYPEDDL-RKQPPEDPNPASLRNPMLARIAYH------KPFFQY----IEQAPH-RQWDLDDTFS--ETGAPCRLYCVVGMDPMHS---PEAEFR------KNPLLTYEIGG-GEPKP-----QQVEEVKPSQVSFEACK--RRAAHQSEPWFIYTGIYHMIFMDYQPRK------QLENEYGRGEPPPFQALAYMAGQNHQRRYPDS----GK--SLLSRQRTALS ETV5 WQFLV---TLLDDPANAHFIA-WT-GRGMEFKLIEPEEVARRWGIQKNRPASPDEFMNYDK L S R S ------MGSASP---GLSSV-SPSHLLLPPDTVSRTGLEKA ETV5ETV1ETV4 E------P* PL CP HY PHH F PP- G QE PQ GC VL :PY GSS D N- RA PY SD YPP HK RP QD M-I SRA EAI PFK IPS VAP PHA AA------P*:: G PPA L -G. PPQ LS: QPPP GL:*: FS- KQ QSP EIF** YP HD----- DSS:*.. P LY YP EMR HD------ETV4ETV5ETV1 GH*.*:*:**. V- PR GF -----RR Q L S ------E *P MC PN GS PPF PP A HL GP FT QM: -P SR PE MG GR IP KM QY EQ APR ERQ QDM RYS N-E FCP LVN RDI SSS--- E *::G------TP SF Q- .PPP ------:VQ PG :*:NF CK Q HSSE **PY GYH HMD :*..GRP YGGV LY GYE EFH HSN *.*:*:**.SS---T M V FS----- QQ-- PS LH ETV5ETV1 AA P FG KP FV SQ YG -V GG EP KA CP LA YP NH VS L AP YE DP QG KP QQQ Q V GT MF RA PV SP NR PPPP TH PQ SSP L TQ PM VP -K SMM P LP ------E N Q Y P S HHE ETV1ETV4 ETV5Q A - S R P F N Q S R T Q H L T S P ------P EL PP CYDNA-Binding HHH P F- PPG E QQ PC GL VY PSS G D- NA RY P D SDomainPP YK HPR DQ -I MRA SAI EFK P PSI(ETV4AP VHA P------P AAG A PPL G -residuesQ PPLS PPP QL GS- FQ KSP QIF EP YD----- H SS D339-436) PY LP YMR ED------HH G- VR PF GRR ----- Q L S ETV1 PFKFSY-GEKCLYNVSAYDQKPQVGMRPSNPPTPSSTPV-SPL------HHASPNSTHTP ETV5ETV6 ALW------LRD GHY AYYV NY YK-- -LD GNQ EIILLL KH CR- LKSE YEDG NPSK YGRP CQYI AREN YLLNL DFS RFIH KR- PPF-R MW- SKE GTD FPKL KDEA PESY LIKM TMI----- PPSF GR TTRI TV PDN L-PH SRNI P-GM TLV -EAS HRV QLS NEWPP PSG LQNEE FEH PPPLKH DNA ERM QTP AINETV5 ETV6ETV6 TYMG L------TR PYI TEA SKD GMC HSR ARLL PA AA WL DRG YHP VYYV YQ --KG LV QNG LLIIP A -RP SKA DEP SPH RGS YQL ERP NLLE FP IFG RRP -FQQQ WM EK DTT KPF EDA SEV KIP IMR FSPP RG IRH VTQ DDP -L NRQ G-M LLP AEK RHMM LL WEP GSE NQN HEQ KLY NDP RES TQE NIQ MY- T------R YF EQ KR MQ SL RS A ETV6 EHF H W------C EP FA ISS R --E S DH IP LLKKK P NESS P------DP KR N-- P GQ LE IS KT -R WV EI DQ RL SM EP GS VP FI RM FH LP KL SI EL AN VP AR QH LDS WPPV GD KKKAF -K Q NNCS HR SSTL KK ETV5ETV6EHFEHFS M E TH ------YN EP KR LG ST RH AL A ------WML ERG FYYYA IN RY --K- DRG DEE LIIK HLLLC REL ENRY GPVN KDDY PKGC INRRA NPY LGLD SLVR HIYK -KPP R-F -WGS EKG DNF LRAK ASRP YEGL MGWT -----VRPP FE RNTT FE LNP NK------L HSS IEP MAT V- SAH VQQ SLN PPWP GL EEKKKF PPP H ANN MQ PSSA IT GML RTP IYT AES DKG CLH RSA LLRP A EHF MRYYYKREILERVDGRRLVYKFGKNARGWRENEN------EHFETV6ETS1 F------LWQCRQRYYYFVALLPQSID---YSKDMNSTTTIIEFLLDHSSKTEHTDLAKYPGS------KCAARQYSLVFPYINDRSHNF-KMVWPICTK-DGRRLGTVQDFALSGKES--WDSPEYPALLFV------EKRRGLDAYSRQTDAGPDPEDLR------ENPVKHADQRRKEPNNWVGIDHKPMQERAAA------ELKSHNYAKLPMALKGEHFETV6ETS1ETS1SDMYVNTS------YG PES MKG ELP -SI NNRQ GL ------HWLCQRPFYYYALLSS---DEKSNHEIIKKPLLKHEPTK------SSDTKAPSGRCK--QRSYQFVEIYSRT-FRWVVTCI-DQGLLDQMGSPW--SEPFLLIKMLGHSYPDTLPPIDELE------NVPAQRRRDHPPSWVGADK-FREKLCQNHHSKATRPMKKLKLSMDHENVN------Y------PERKGLTSHRLG ETS1EHFETV6ERG M------W-----CQMF-LLGR---PTQSREKGLLAAKLSQGGDPSSSQNP----HNSRSSTPVSCPDPIKSDT------FP-EWSQEIR-EPGSLTYRNDSGSPET------LFE------KDMQTRDPPQDLEDVPAYCRRQ---IWLGDYEPRDTLTKSSTNSQYKRSGPLWSNAETV6ETS1EHFERGERGSSTMNVNPQDYG SSLLDS KG FDLQ NSI SKRQ LTA ------QFWLRCQRVRFYYYPALLSQYI---DDSKSMNFTTTEIDLLMSTESKDHVYLSSHPGAA------NKSSRLYPCANIYDHTKNK-FMPWDIKEF-GHRRTGGFVTILKANASDEGQPYSEAAVPFLER------KQRDMPARTHQADPPGDPPLDERNESSPKVHDALQKRRYEPKNNVWYIGPHPDESQAAAMRDE------KLSPYLKYPAMLGNGSYMSVTNYSGYHPSDAMGKHEPLP-ISQNNQRKLAM ERG LRYYYDKNIMTKVHGKRYAYKFDFHGIAQALQPHPPESSLYKYPSDLPYMGETV6SYHA H P Q K M ------NHRPSPDPEQRPLRSPL------ERGETS1EHFSPDEF L------WEPRLL------YYYY-WETPPEKH------GQEIYHMRSQLK-EVPPQAVGM------EGRKYAVFYQKEFLVACGD-QDPKS------EMLGVVCMAEGMFESKVPEELLI--QEKQFPDTREEIDQETRVLSAQPRRCNLKDGGWLLYGFDINAVQIRRILKTKHNASQARDAEHPWVVLAMEHFERGETS1SPDEFSPDEFTDMGT------NWHPYS GDP H----KS PLN TSV --RQ NSK ------MWLCLLRMYYY-WGTPREEQTHKSQGKRYIAAGRMKLQQL-KPGGPPVSAPQMGS----GETKRVAYPFVKDQY------SEKFLFEAVSGCI-DEKPSEEYGLDMCTSEAE------FMDKSQLEERIPEQQPFLEERDQPVRYASQCRRPI---LLWGGGYPDIDDTRQVTSSLKLNTHYRQAGLSPHSAWLTNSSMDVPN------DGQYLLSSSDGKDQFLSINKQSRTLS SPDEFERGETS1ETV4 A------WMKQ------AFPLGVA---SSGRAYEELLGYPDDE--KPPTL----NRAPHFFPEEI----AP-EWQTDDC-PGVVRCIGVVDMSEPQFE------KLFIEEGPDEEIAKPLLVQAEGSR-LSKWVLYGDEIALNQFVDKRPYNEGGQPRGRSPPPLIVAETS1EHFSPDEFERGETV4ETV4TTTIMY------LNQRYRR P------D ----KG LA SL RQ LDS ------ELWL------PQRYFYYY-LEVPP---EKE------GHAISLLMLQEDDKQVVPA------TGNEARHYFVIYAK-FWVTCQ-EDSGP------MREVVGAMLGEFEFSVKLPLAL-IF-KEPQDPDTIEENDEQTVRLAPQCRANKLLDLLWKYGAFNIEAIQFITRRK-KANDQDSRREPAPVVMWAVDTMSTWGN------PSHYDPG----SHKNPLVTSQ--RNKS ETV4 LRYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDNQRPALKAEF-DRPVETS1S------LLSLKYENDYPSVILR------SPDEFERGETV1 ------LWERQ------YYYFLVE---KGIACMLLQFKDDEVKAPGGSPENRRSQVYHFGVFYLYI----EKARRF-VWDCTDDWD-SPGTPERCSAGVVPPLMFEPASFETMKPALFEFIDQPEGGDPLNEEISQKARVQFPAEYLLRR--LSKWPYNTGFTDIMDSMQYMV-KRLYENYPRGPEHEAITERGETV4SPDEFETV1ETV1DTNMYSS------NQYRR WD ----KG LS SL RQ ASL ------MAWL------KQRAFYYYPLGVA---ESSKGGYRAIGEELLMYQEPDDKK--VPALPSGR----NEPSRFHYPFV----EEIYAKP-FEWVDDQTCC-DVPGPCVREVVIGADMLPSEFEQFSK------MFLAEIFGEPDPDIEENKAQQPVREAPGRRLL-VSWKGLTADIDFLQMRVK-EPNEQGGGRRRPPPHILAITTYTMNQ------N------RRRYPD----GKALLSQRDLS ETV4SPDEFETV5 DAAWEIQ------CGFHALS------VF---TSGQTFGGGLLSYDDERKEPDPALNRPALAHYPFYI----QAH-QWLTDDS-EGTPRCCGVVPM---EPFEKRPLYIE-EGPKQQEEVKSVQFAEKRR--QEWPYGTHIMDQYPKRLNEYRGEPPPQASPDEFERGETV1ETV5ETV5AMYGNQQYRR PD ----KG LS SL RQ ASL ------EWL------QRFYYYLV---EKGGCTIMLLMFQEDDKKVGPAPAGRNEQARVFHYGYFVL----IYEAKRR-FWVDDDTCW-DSTGPPCRDSVVGAPPMLPEFAEFSTKMPFLAEDIFQGEPGDPDLIEENSKQAQVRFEAPY--RRFLLSPWKYNGAFTMIEDSYQSMVRK-LYENEYPGRCPEHEATLDTYMSSS------QN------RRYWD----GKSLLSQRALS ETV5 LRYYYEKGIMQKVAGERYVYKFVCDPDALFSMAFPDNQRPFLKAES-ECHLSPDEFS------AAGA------ETV1ETV4 E------:*:**.* P C N S F PP L*: P T M: P R E*.* G R P M Y::* Q RA QE MQ SR EN PF NL IR ---SS *::G PT FS -Q .PPP ------:Q G :*:F K Q EH **YP HG DH :*..PG VY YL EG HE ETV4ETV5 NH *.*:*:**. TSS M V -----F QQ P L DE*:*:**.I------C H S F T SG QF *:GGGS Y E :RK ED P *.*LR PL AY PF Y---- ::*Q H Q LDD S ET PC CVV P ---P E*:: R PL YE -G. PK QQV: K SQ:*: FE K-- Q EP** YT HM DY:*.. PR LE YG EPP Q AY*.*:*:**. GQ QRR P ----G S L Q AL ETV5ETV1 E------PCHPFPPQPGVPGDNRPSYHKRPQDM-SREAPFIPVAPHAA------PP-PPLQPPGFSKQQSEIYPHDDSSPLYYPEMHDETV1ETV4 GH V- PR GF -----RR Q L S E------P C N S F PP L P T M:P R E G R P M Y Q RA QE MQ SR EN PF NL IR ---SS G PT FS -Q PPP ------Q G F K Q EH YP HG DH PG VY YL EG HE NH TSS M V -----F QQ P L ETV1 ------H1 KPD-RAFPAH------LPPSH2QSI PDSSYPMDH-RFRRH3QL S ETV5ETV6 AA-----LW RD GHY PYYV VY QK-- GEL VDNQ GEIILL PC A------R- PKS AED PS HGR SQY LRE PLLN EF PFI GR PF- QQQMW KE TD TPK FDE AES VIK PMI RSF PPGR RI HTV QD P- LRN Q-G ML PEA KHR MML EW PSG EQN NEH QLK YDN PER SQT EINETV5ETV6ETV6 QYM ------T RY FE QK RM QS LR SA EL-----PRCHHYYPFKQPPLENDQIIEPCGR------VKPEGPDGNQRPLLSYFHRRFQMMKSTEPPDIEVIPMAASGPPRT-DPP-RQ-GLFEKHQLEYSHQDEPLLDYEEQHIGYV------PG----- EHF MW RE YYYF I R K-- R ED I LLL E RN VP D GK RRN P LG VL YI K F- GW KE ND AR RS GE WG RV EF NR EF NL ------K S E A V A Q L W G KKK NN SS ETV5ETV6EHFEHF M T Y E K L S R A AA WM------DRG YYYYP VV YQ --KG RV QEG LLIP LA -EP SRA DVP SDH RGS YRRL EP NLE FVP IYG RKP -FQQQ WG EK DNT KAF ERA SGV KWP IRR FEPP RN IEH VNQ D------P L NQ GM LP AK RMM L WP GE NN HQ KY NP RS TE NQ M- TR YF EQ KR MQ SL RS A EHF ------ETV6ETS1 H---LWCRQPYYYFAKLLSSPD---EAKSDNHEIIP------LLKHPKTSSTDAKPGSRKC--RQYSQVFEYISRTF-RVWVCTID-QLGLQDMSGP--WSEPLLFIKMGLHYSPTDLPIEDL------ENVPARRRHSWVGDKFERKLQHNSAKRMPLKEHFETS1ETS1SDMEVN------Y E K L S R G WL---ERFYYYIKRP--DDKADNDIIIELL------HNKPTDAKGNKPRGYLVIYKR-FWVECDRLSQESG--VFLLRFGLYKTSPEA------VAQLWGKKKELNNHASSMLMDTVY------EKLSRA EHFETV6ERG F------LWCRQRYYYFALLQID---SKMNTTTIEMLLTSKHVDLHSSPG------KNRSSYACYIDKTNF-MDWIFE-HRRGVITALNESQGSPAEPALF------EQKRPMAHTQPPDGPPEDRSSEPVHLAQYRREKNNYWPGDHSEMQDR------ELKSPYKPMLGNETV6ETS1ERGERGSMVYNSHY PAD MHK EPL -QS NNKR MA HWLNCQRFPFYYYVALLASSP---DHEKPPSNHEIAPLLMLKTPPTKVSSDVTKHSSSPSGRCK--QR------SYQFAEIYSKT-FRWDVTFI-HQGLDIMGAPWQSEAPFLIKQMLPHSPDPPLPIDELESSNVPALRRRYHKSWYVGPDKSFRDKLQNPSKYRPMLKGFFSMENYAA------YHEAPKHNLPPSQYRKWGMN ERG NFVAPHPPALPVTSSS------FFETV6AAP N P Y W N ------DNMI-RRLSPAERAQGPRPHQENNHQESYPLSVSPME-NN ETS1EHFSPDEF M------LWCRLLMYYY-WGTRETKHSGQRIYGMRKQLLK-GGVPPAQIGM----WEGKRSYAAAVFDYQSWKEFMLEKVASECGIRD-ETPKSEYPLGDGCMSAE------IMFHSKYEELCIAQESFPTREESQEERVSACSPRR---WLTGGWDGDYSIRDEVQLTVKTNDHQYSSARSGHPWSCLAEHFERGSPDEFSPDEFSSTSDMVG------NQDY SSLLPD IK FDHL NSL SKWR LTQS FWL------CQRRFYYYALLQI---ESKMGTTTEILLMQSKHDVLSSAPGI------NEWSSRKYSCVAAIYTKW-FMWVKECE-DRGPTVTESANLPEGCGSEAAPFMI------KSHMEEYTCDQAPFSDRTEQSVREEASRRPSLWWGGTGDDESMRVE------KLVSHDKASSPHNLCMDSN------GYQDPKILHSLRWAQ ERGETS1ETV4 L------WPRQYYYYF-LEVPPE---K------GIAMLLQKDDVAPGTENRAYHVFYIKAF-VWCTE-DPG------ERAGLMFESFLKPALLFI-PEQDPTNEEDQTRVLPAQARNLDKWYAGFEIAFQRRI-KKDNSQRAEPWVVVAETS1EHFSPDEFETV4ETV4TSMGT------NHPY GD H----K PL TS --R NS M------WL-----CLLRMYYY-WGTREEETHKSQGDRYITGRMVKLQPL-KLGGPPVSAHQMGL----GEDKREAYSFVPDQYASEKYFLLEAVPSGCEI-ELEKPASEGYGAPDML------SEF------FSKLLAIFEPPDEENQVRAPCRRA---LWKGADYIERDQFLTK-TNDQYRSGPWSAVASSTMSQVN------PQDYFSSLLDGKPFDLKNS---SKRLTS ETV4 -----EEDTVPLSHLDESPAYLPELAGP------AETS1QPFG P K ------D------PL-QTDTLQNDYFAIKQEVVTPD----N SPDEFERGETV1 A------LWKRQAYYYFPLGVAEEE---SSKDGRTIAEEVMLLPQPLKDD--SVHAPPFGS----DENERSSYHMVFEEAYIYKAPMF-EPVWQECTCGGD-PGVCERI------AGDLMSFEQSF------MKALFIPEDPNEEAQPRVAPGLLRR-SKWLTGDILMQV-KPENGGQRRHPLICAERGETV4ETV1ETV1TTTNM------P------NRHY PD ----YK NL ---S R LDS LW-----PQRYFYYY-LEVPP---EEEK------GDAITLLMVQPDDKLVSPAHTGFNEDAREHYSFVMIYAAKY-FMWVPTCE-DGGGPRECGA------MLEFFSKMLAIFEPPDEENQVRAPRLLLWKGTIDQMRRK-NESRAPHWAICTMNNGN------PHYHGDPHKYPLNTS-----RS SPDEFETV5 ------LWRQYYYFLVE---KGITMLLQKDDVAPGAENRAVYHGVFLYIEKARRF-VWDCTWD-SPGPDRSAGPPLMFEASFTMKPALEFIQPEGDPLNEESQARVFPAYFRRLSKWYAGFEIDSQM-KLENYCRPHELASPDEFERGETV1ETV5ETV5DSMSS------NY WD ----K L S R AS A------WL-----KQRAFYYYPLGVA---EEESSKGDRAITEELLMLQPPDDKL--VTPAHPSGF----NEESRDHYSFVPEEIYAAKYP-FLLEWVQTCDC-DMPGDVRDRIGA------DMLSEFQFS------KMLAIFEPPDEENAQPVRAPGRRF-LSWKLGADIELQSVK-PNEGGQRCRPHLIACTTTMSSS------N------RYLPDP----KYLAS---RLDS ETV5 -----EEDTLPLTHFEDSPAYLLDMDR------CSPDEFSSLPY A ------VGLERRDWSPSPPATPEQGLSAFYLSYFDMLYPEDSSW----A ETV4 D :*:**.*I C H S F T S Q *:GGG :R E P *.*L P A P Y ::*Q H Q L S E P C P --- *:: P Y - .P QQ : S :*:F K Q E **Y H D :*..P L Y E Q ETV5 A *.*:*:**. G Q P ---- A W:*:**. Q F L V --- T*: LL DD: P A*.* N A H F I::* A - W T - G R G M E F K L I E P EE V A RR W G I Q K N R P A M N Y D K L S R S ETV1 EPCNSFPPLPTMPREGRPMYQRQMSEPNI---PF-PPQGFKQEYHDPVYEHETV4 N T M V ----- D*I C H S F T S Q GGG R E P L P A P Y Q H Q L S E P C P --- *::PY -.P QQ: S:*:FKQ E**YH D:*..PLYE Q A*.*:*:**.GQP----A H3 H4 ETV1 EPCNSFPPLPTMPREGRPMYQRQMSEPNI---PF-PPQGFKQEYHDPVYEHNTMV----- ETV5ETV6 E------LPRCHHYYPFQKPPELDNQEIIPCG------RVKPEGPDGNQRPLLSYFHRRFQMMKSTEPPDIEVIPMAASGPPRT-DPP-RQ-GLFEKHQLEYSHQDEPLLDYEEQHIETV6ETV6GYV------PG ------QEDEC------EHF ------M R YYY K R E I L E R V D G RR L V Y K F G K N A R G W R E N E N ------ETV5ETV6EHFEHF EL------PRCHHYYPFKPPLNQIIPGRVKPEGPDGNQRPLLSYFHRRFQMMKSTEPPDIEVIPMAASGPPRT-DPP-RQ-GLFEKHQLEYSHQDEPLLDYEEQHIGYV------PG----- EHF ------ETS1 ------LRYYYKPDAKDNEII------HKTAGKRYVYRFVCDLQS--LLGYTPE------ELHAMLEHFETS1ETS1DV------M------RYYYKPKDRAEDIEL------ERVDGRRLVYKFGKNARGWRENEN------ETV6ERG HNLCFRPVYYYASSPHDEPPKSNHAIPLMKPTPVKSSTVSSSHPGRK------RYQAEYSKTFRDVFIHQGLIMAPQSAPLIQMPHPPPLIELSSNPLRYHKSYVPDSFDKLQPSYRMLFFGETS1ERGERGSEAAY------H PA NH P YQ WK NM LN------RFYYYVAPDHKPPNIIALHPKVTASSSGKR------YVYSRPFTVGGCDILYQPSN--TRLL------GYTPE------ELLHPATMSLFFHDMVAAP------SPHNLPGYTWYYN ERG ------SPTGGIYPNTR------LPTSHETV6MPSH L G T YY HCPASSESHPKPSSPR--QESTRVIQLMPSPIMHPLILNPRHSVDFKQSRLSE------EHFSPDEF F------LCLRRKYYYAEQLLLIESKMKGTTTPIHMSQSYKHGVLRAPFIG------IWERKWSYLAAVNYKWEMFKVGECIRDFTPVKSEAIPLEGCSDAPSIM------AHSQYEEVCAQRSFLTRWSQGEERISRSPKWLNTGGRDDPSMAEV------MVLNDHYSSADHKCLERGSPDEFSPDEFLSDSG------RQ SP I RH QL YYW Q L------FRLYYYKELLLDKNKIPMHTSKYVGHRGIFKWIRKRYSWAAALYNKWKFMEDKKFEGHRIGTFISKAPIQGEADLISQHAPYQHCVPPAASRETLSSSWEEGLIYSRKWKYTNPDRSPDEALVMPDNYSSYMDGCKSLYGSHQRAPSHIIPHRQLQKWYYMQ ETS1ETV4 M-----LCRMYYY-GREEETKSDGRTIGVMKPQLKGGSVHAQLG----DEERSYPVDAYSYKFLEPVSECILEEAPSGEYPAD------LSF------SLAFPDNQRPCA---LKADERFL-TDQRSPWAVEHFSPDEFETV4ETV4SSQSP------QF SSG P FK N--- S L FL------CRRYYYAQIEEESKMGDTTTITMVQPSKLHVSLAHGGPGL------EDYRESYSYVP------YAKYFLVPCEDLPAVEGALPEC------SAPM------SEEQFRQRSPLGGDMV------LHAHLADQ------PFGPK--- ETV4 ------GGYSY------ETS1 MCM-GRTSRGKLGGQ----DSFESIESYDS------C---DRLTQSWSSQSSFNSL ERGETV1 L------PRYYYY-EPPEEEK------DGTIVMPQLKSVEHAGFYDEVERYS------MVAYYKMFPVECGGDPCE------ALFSMAFPDNQRPLLKTDMRR-ESRAHWCIETV4ETV1ETV1TNGP------HGP HY PN T--- -- L------RYYYEEEKGDITMVQPKLVSAHEGFGEDYREVYSYVM------YAKYFMVPCEEGGPECA------LFSLAFPDNQRPALKAEF-DRPVCSN------PHPYN--- SPDEFETV5 A-----LKRAYYYPGAEEESSKDGRTIEELMPQPLK--TVHAPFG----EDRSYPVEEAYYKPLLFEVQDCCMDPDVRDI------ADLSFQS------MAFPDNAQPRAPGF-LSKLADELSV-PEGGCHLCERGETV1ETV5ETV5TSSS------L P Y A --- L L------PRYYYY-EPPEEEK------GDITMLQPKLVTAHEGFGEFRDAYSYVP------YAKYFLLVCDDMPDERA------LFSMAFPDNQRPLLKTDMRR-ESRAHWICTNSSG------HLGPHYPAT----- ETV5 ------EGFAY------SPDEF AKAPGASSREEP--P----EEPEQCPVIDSQ------APAG-SLDLVPGGLT------L :*:**. *: : *.* ::* ETV5 L R YYY E K G I M Q K V A G E R Y V Y K F V C D P D A L F S M A F P D N Q R P F L K A E S - E C H L S ------:*:**. *: : *.* ::* ETV6 ------QEDEC------ETV6ETV6 ------EHF ------ETV6EHFEHF ------QEDEC------ETS1 ------KPDADE------EHFETS1ETS1 ------ERG ------NFVAPHPPALPVTSSS------SPTGGIYPNTR------LPTSHFFETS1ERGERGMPAAS HP LN GP TY YYW N ------KPDADE------SPTGGIYPNTR------LPTSHMPSHLGTYY Fig.SPDEF S 2 .KKF------LAlignmentGKIIELLLRKPKDPIHSQYRGLRVFIYIWQRK FWSofVLAAHNPKW IEMETSKGEIRFTKSIP EGfactorDASIAHQYVCARSLTWS GEEsIRSequences.KWNTRDPSAEMVNDYSSDKCERGSPDEFSPDEFLSSGRQ SP I RH TheQL YYW Q NFKKFL VKGentireAEIIPLLLHRPPKKPAPDLHIPSSV YQTsequencesGRSSSRLFVIY------RQWFLVNHKPEIKGIFKIE DofSAQ VETSARLWGIR KfactorsNRPAMNYDFFKL AASthatRPSNIPRYQWYY N ETV4 ------EEDTVPLSGGHLYDSEYS------PAYLPELAGP------ASPDEFETV4ETV4QPFG P K ------GGIWYKSSYAA------WMKERTSPGAIHYCASTSEESWTDSEVDSSCSGQPIHLWQ wereETV1 investigated ------EEDTVPLSEHGF YDfoVEYS------MrA YinteractionMPEGGC------with MED25CETV4ETV1ETV1NPHP Y N (Fig.------1)EE DwereTVPLSHELG DYalignedEVSYP------AYLPELAG Pwith------Clustal OmegaAQPFGPK--- ETV5 ------EEDTLPLTEHGFFEADYS------PAYLLDMDR------CETV1ETV5ETV5SSLP Y A ------EEDTVPLSHEFGDFEASYM------AYMPEGGC------CNPHPYN--- [1] . Blue and red boxes highlight the N - terminal ETV5 activation ----- EE D T L P L T H F domainE D S P A Y LL D M D R (AD)------and the DNA C SS L P -Y A ---

bindingETV6 domain,------respectively, and sequenceETV6 numbers ------refer to ETV4. The secondary EHF ------ETV6EHF ------structureETS1 ------of the DNA-binding domain is illustratedEHFETS1 ------below the sequence with rectangles ERG ------SPTGGIYPNTR------LPTSHETS1ERGMPS H L G T YY ------andSPDEF arrows KKFLGKIIELLL RindicatingKPKDPIHSQYRGLRVFYIQRFWVLHNPK IEαKG-IhelicesFKIEDSAQVAR LandWGIRK NβRP-AMstrands,NYDKERGSPDEFLSR S I R Q YY respectively. ------KKGIIRKPDISQRLVYQ FNoteVSHPTIGGIY PthatNTR------sequencesLPTS HoutsideMPSHLGTYY ETV4 ------GGYSY------SPDEFETV4 F------LKELLLKPHSYGRFIRWLNKEKGIFKIEDSAQVARLWGIRKNRPAMNYDKLSRSIRQYY ofETV1 the conserved ------E GETSYVY------domain (α-helix 1 throughETV4ETV1 ------β-strandGG 4)YSY ------are not conserved in these ETV5 ------EGFAY------ETV1ETV5 ------EGYVY------ETS factors. However, regions a re conserved ETV5 in ------a subfamily E G F A Y ------specific manner. The N-

terminalETV6 ------AD and α-helix H4 are two regions in the ETV1/4/5 subfamily that display this EHF ------ETV6 ------subfamilyETS1 ------specific conservation. Likewise,EHF regions ------that are only highlighted by red boxes ERG ------ETS1 ------inSPDEF one, orKKG IIaR Kfew,PDISQR LETSVYQFVHP Ifactors indicate subfamilyERG - specific------α-helices that flank the ETS ETV4 ------SPDEF KKGIIRKPDISQRLVYQFVHPI domain.ETV1 ------ETV4 residues that were mutatedETV4 (Phe54, ------Phe60, Trp64, Thr363, Ile372, ETV5 ------ETV1 ------Glu373, Phe428, Ser429, and Phe432) areETV5 indicated ------by black arrows underneath the

sequences; See Fig. S3a for quantification of interactions between MED25 and ETV4 with mutations to these residues.

(a) * 1000 * * * * 100 ) * * * (nM D K 10

WT AD ETV4: DBDF60A F54A W64A T363AI372AE373AK370AS429A

F428A/F432A (b) (c) 0.15 0.15 ) ) 0.10 0.10 50 nM ETV4 WT 300 nM ETV4 50 nM ETV4 WT F428A/F432A 1,000 nM ETV4 0.05 W64A 0.05 Response (nm Response (nm

0.00 0.00 0 200 400 0 200 400 Time (s) Time (s)

Fig. S3. ETV4 mutations disrupt binding with MED25. (a) Summary of KD values for interactions between MED25 and ETV41-484 wild type (WT) or with indicated point mutations. Circles represent the mean from a single experiment using six concentrations of ETV4, and the horizontal lines represent the mean and standard deviation from three to five replicate experiments. “*” Indicates p < 0.05 in a Mann-Whitney U test. Data from ETV4 DBD, WT (full length, 1-484), and AD are included from Fig. 3d for reference. Although the interaction data for MED25 and ETV4 with all point mutants was better fit using a 2:1 (ETV4:MED25) interaction model, only the higher-affinity KD is reported here for brevity. The KD values (mean ± standard deviation) are: DBD, 230 ± 80; F60A, 380 ± 60; W64A, 320 ± 50; F54A, 42 ± 6; WT, 7 ± 3; T363A, 27 ± 3; I372A, 40 ± 6; E373A, 44 ± 7; K370A, 58 ± 9; S429A, 90 ± 10; F428A/F432A, 190 ± 30; AD, 700 ± 100. Representative titrations of three concentrations of ETV4 wild type (WT) and ETV4 W64A (b) or ETV4 F428A/F432A (c). The highest concentration was 50 nM for WT, 1,000 nM for W64A, and 300 nM for F428A/F432A, subsequent sensorgrams were diluted twofold.

(a) 105 15N-ETV4 DBD 15N-ETV4 DBD H358 110 + MED25 S429 T363 115 E425 V348 120

N (ppm) N391 15 125

130 I372

10 9 8 7 1H (ppm)

(b) 0.06 (c) 1.0 y 0.8

) 0.04

m 0.6

(pp 0.4

∆δ 0.02 0.2 Relative Intensit 0.00 0.0

337 347 357 367 377 387 397 407 417 427 337 347 357 367 377 387 397 407 417 427 Residue Residue

Fig. S4. MED25 interface of ETV4 investigated by NMR. (a) 15N-HSQC spectra of ETV4 DBD alone (black) and with 1:1.2 molar excess of unlabeled MED25 ACID (red). Select ETV4 peaks are labeled from this previously assigned spectra [2]. (b) Amide chemical 2 2 ½ shift perturbations (Δδ = [(ΔδH) + (0.2ΔδN) ] ) were calculated with the addition of MED25 ACID. The orange and red dashed lines indicate cutoffs that correspond to the top ten percent of all signals and the median of all signals, respectively. Residues with amide chemical shift perturbations above these lines are colored onto the structure of ETV4 DBD (Fig. 3b). (c) ETV4 NMR peak relative intensities were calculated with the addition of MED25 ACID.

(a) (b) 105 AD : 15N-MED25 105 DBD : 15N-MED25 G462 0.0 : 1.0 G532 0.0 : 1.0 0.2 : 1.0 0.2 : 1.0 G462 110 0.5 : 1.0 110 0.5 : 1.0 1.2 : 1.0 1.2 : 1.0 115 Q409 115

V471 N (ppm) N (ppm) 120 120 15 15 R538 R538 125 125

130 130 11 10 9 8 7 11 10 9 8 7 1H (ppm) 1H (ppm)

0.15 0.03 (ppm) (ppm) 0.10 0.02 ∆δ ∆δ

0.05 0.01

0.00 0.00

397 407 417 427 437 447 457 467 477 487 497 507 517 527 537 547 397 407 417 427 437 447 457 467 477 487 497 507 517 527 537 547 Residue Residue

(e) (f) 15 15 1.0 N-MED25 + AD 0.2 : 1 1.0 N-MED25 + DBD 0.2 : 1 0.5 : 1 0.5 : 1 0.8 1.2 : 1 0.8 1.2 : 1 y y

0.6 0.6

0.4 0.4

Relative Intensit 0.2 Relative Intensit 0.2

0.0 0.0

397 407 417 427 437 447 457 467 477 487 497 507 517 527 537 547 397 407 417 427 437 447 457 467 477 487 497 507 517 527 537 547 Residue Residue Fig. S5. ETV4 interface of MED25 investigated by NMR. (a) Aligned15N-HSQC spectra of MED25 ACID alone (black) and with 0.2 (yellow), 0.5 (green), and 1.2 (blue) molar equivalents of unlabeled ETV4 AD. Select assignments are displayed. (b) Aligned15N- HSQC spectra of MED25 ACID alone (black) and with 0.2 (yellow), 0.5 (orange), and 1.2 (red) molar equivalents of unlabeled ETV4 DBD. Amide chemical shift perturbations (Δδ 2 2 ½ = [(ΔδH) + (0.2ΔδN) ] ) were calculated with the addition of ETV AD (c) or of ETV4 DBD (d). The relative intensities of amide peaks were calculated with the addition of ETV4 AD (e) or of ETV4 DBD (f). The upper lines indicate the median peak intensity for the 0.2:1 (ETV4:MED25) titration point (yellow data set), and the lower lines indicate the lowest ten percent of peak intensities for the same titration point. Residues with relative peak intensities below these lines were colored onto the structure of MED25 in Fig. 5b,c. Using chemical shift perturbations instead of relative peak intensities resulted in very similar data, overall. However, in the AD titration many MED25 NMR peaks were broadened to baseline at the first titration point, and therefore using chemical shift perturbations underrepresented the contribution of these residues.

(a) (b) K422 Site 1 Q451 H2 K545 H3 R538

R509

180° 180° C K519

H2 Y487 H1 I521 R466 H474 Site 3 L514 Q430 M523 Site 2 M470

Fig. S6. Structure of MED25 ACID. (a) MED25 ACID (PDB: 2KY6) [3] oriented as in Fig. 6 with surface representation and colored according to electrostatic potential; blue is positively charged and red is negatively charged. The ACID domain has concave features on multiple faces that could serve as interaction sites with an α-helix from ETV4. These surfaces have hydrophobic and uncharged polar residues on the floor of the concave features and are primarily surrounded by positively charged residues. MED25 sites 1, 2, and 3 are labeled as in Fig.5. (b) MED25 ACID as in (a) with cartoon representation and with side chains shown. Side chains that were mutated are labeled (Fig. 6, Fig. S7, and Table S2). N- and C-termini and α-helices of MED25 ACID are also labeled.

(a) 43-84 (b) 165-484 ETV4 (AD) 4 ETV4 43-84 43-84 43-84 43-84 + ETV4 - ETV4 0.10 + ETV4 - ETV4 ) 3 MED25 WT ) MED25 WT 2 0.05 K422E, R509E 1 K518A, R538E, K545E 0.00 K422E, R509E, R538E, K545E Response (nm 0 100 200 300 400

Response (nm 10 20 30 Time (s) -1 Time (s) -0.05

(c) MED25 M523E + ETV443-84 (d) MED25 M523E + ETV4165-484 0.4 + ETV443-84 - ETV443-84 0.10 + ETV443-84 - ETV443-84 ) 0.3 ) 0.2 0.05 0.1

0.00 Response (nm 0.0 500 1000

Response (nm 2 4 6 Time (s) -0.1 Time (s) -0.05

Fig. S7. MED25 mutations disrupt ETV4 AD and DBD binding. Representative examples of association and dissociation phases for 40 μM ETV443-84 (a) (AD), or 35 μM ETV4165- 484 (b), and sensors loaded with wild type MED25 (WT) or indicated point mutants. The kinetic rate constants (ka and kd) and equilibrium dissociation constant (KD) for the mutants that displayed detectable binding were calculated as an average of two single- concentration experiments and are reported as approximate values (~) in Table S2. Representative examples of association and dissociation phases for six different concentrations of ETV443-84 (c) or ETV4165-484 (d) and sensors loaded with MED25 Met523Glu. The highest concentration was 40 μM for ETV443-84 and 5 μM for ETV4165-484, followed by 1.5-fold serial dilution. Both experiments were replicated three times, and the mean and standard deviation for KD, ka, and kd values for the MED25 Met523Glu interactions with ETV443-84 and ETV4165-484 are reported in Table S2.

Fig. S8. ETV4 and MED25 share transcriptional targets. (a) Histogram showing distribution of distances of ETV4 (black) and MED25 (gray) bound regions to closest transcriptional start site (TSS) from respective ChIP-seq data. (b) Graphical display of enriched reads for ETV4 and MED25 ChIP DNA and input DNA in putative regulatory elements for SDC1 (upper panel; hg 19 chr2 chr2:20,412,282-20,438,273) and CDK14 (lower panel; hg19 chr7:90,245,870- 90,393,286). Red bar, regions assayed by qPCR in Fig. 8c. (c) ETS and AP1 DNA binding sequences are overrepresented in ETV4, MED25, and both (shared) ChIP-seq datasets, as determined by MEME [4]. (d) Western blot confirming knockdown of MED25 (top) or ETV4 (bottom) using two different shRNA constructs (a,b) for each gene. Three biological replicates are shown for each, as well as two biological replicates for a control knockdown.

(//www.ebi.ac.uk/)

(//www.ebi.ac.uk/) Clustal Omega Clustal Omega

(//www.ebi.ac.uk/)

Clustal Tools (/Tools/) > Multiple Sequence Alignment (/Tools/msa) > Clustal Omega (//www.ebi.ac.uk/) OmegaResults for job clustalo-I20170307-220716-0910-62109583-es

ToolsClustal (/Tools/) ::::.*::> Multiple Sequence * Alignment (/Tools/msa) . * >. Clustal***** ******: Omega ::::.*:: * . * . ***** ******: JUNB_Hs PNSNGVITTTPTPPGQYFYPRGGGSGGGAGGAGGGVTEEQEGFADGFVCLUSTALKALDDLH KO(1.2.4)MNHV multiple sequence alignment JUNB_MmResults forPNSN jobGVITTT clustalo-I20170307-220716-0910-62109583-esPTPPGQYFYPRGGGSGGG---TGGGVTEEQEGFADGFVJUNB_HsKALDDLH K M N H V PNSNGVITTTPTPPGQYFYPRGGGSGGGAGGAGGGVTEEQEGFADGFVKALDDLHKMNHV cJUN_Hs QSSNGHITTTPTP-TQFLCPKN------VTDEQEGFAEGFVJUNB_MmRALAELH S Q N T L PNSNGVITTTPTPPGQYFYPRGGGSGGG---TGGGVTEEQEGFADGFVKALDDLHKMNHV OmegacJUN_Mm QSSNGHITTTPTP-TQFLCPKN------VTDEQEGFAEGFVcJUN_HsRALAELH S Q N T L QSSNGHITTTPTP-TQFLCPKN------VTDEQEGFAEGFVRALAELHSQNTL JUNB_Hs MCTKMEQPFYHDDSYTATGYGR------APGGLSL------HDYKLLKP JUND_Hs QS-NGLVTTTPTS-SQFLYPKV------AASEEQEFAEGFVJUNB_MmcJUN_MmKALEDLH K Q N Q L MQCSSTKNMGEHQIPTTTFYHPDDTPS-YTAAAQFLGCYPGKRN------SPGSLSVLT------DEQEGFAEGFVRALAHEDLYHKSLLQNKTPL JUND_Mm QS-NGLVTTTPTS-TQFLYPKV------AASEEQEFAEGFVcJUN_HsJUND_HsKALEDLH K Q S Q L MQTSA-KNMGELTTVTTTFYDDPTASL-NSAQSFFLLYPSKEVS------GPY------GAAYSNEE------QEFAEGFVKALEDLPHKIQLNKQQL . ** :***** *:: *: .:.*:: **:***:**cJUN_MmJUND_Mm :**. . : MQTSA-KNMGELTTVTTTFYDDPTASL-NTAQSFFLLYQPSKEVS------GAY------GAAYSNEE------QEFAEGFVKALEDLPHKIQLSKQQL ::::.*:: * . * . *****JUND_Hs ******: ---- . **ME T:*****PFYGDE A L S*::GLGGG *:A S G ------S GG S.:.*::FASPGR L**:***:**FPGAPPTAAA :**.GSMM KK. D: CLUSTALJUNB_Hs O(1.2.4) TPPN VmultipleSLG------sequence AalignmentTGGPPAGPGGVYAGPEPPPVYTNL SSJUND_Mm Y S P A S A SS GG A::::.*::----METP F Y G EE A L*S G L AA G A SS V A G A T G A P GGGG F.A PP G*R A F P.G A*****PPTS--- ******:SMLKKD JUNB_HsJUNB_Mm PTNPPSNGVVSILTTTG------PTPPGQYFYPRGGGASSGGGGGPQAGGGPAGGGGGVYVATGEEPEQPPPEGFVAYDTGNFLVSS JUNB_HsK AY LS DDP A LS HA KP MS NGG H VS T PP N**VS L G**------: :. . A T GG PP A G P GG V Y.A G P E PPP V Y T N L SS Y S P A S A SS: GG* A (a)JUNB_MmcJUN_Hs PNSSVNTGSVAAITTTQPVPNTGPPAGGMQVYAFPYAPVRAGGGSVASGGGGGSG---S-GGTGGGFSAVSTLEEHSQEEPPGFVAYDAGNFLJUNB_HsVSJUNB_MmKNAFLNDDPGLA HL KSS M NGGG H VPTNPPSNGVVSILTTTG------PTPPGQYFYPRGGGASSGGGGGPQAGGGPAGGGGGVYVATGEEPEQPPPEGFVAYDTGNFLVSSKAYLSDDPALSHAKPMSNGGHVS JUNB_HscJUN_HscJUN_Mm MQPCSSSTVKNTMGSEHAAQIPTTTQFPYVHPSDDTGPAS-GYTMTQVAFTLPGCAYPVGKARNS------VAGAGGG-AGGPGGYSLASVLT------DHESQEEPPGFVAYEAGNFLJUNB_MmVSJUNB_HscJUN_HsRNAFLNAPHEGDLA YHL KSSS LLQ NGGG KT PLPSNLSSAVNVTGNSVLAAIATTTDQPYVPRNTSGPPLAKGGAMQPVYGAFAPYRAPGVR-AGGGPSGVPASEGGGGGGGGGGSG---S-SGGTYGGGF----SAVSTLEESHGSQEEGPPGSFDVATYDGAGANFSLVLSKNALFLANDDSSPGLEAHLKESSMRNLGGGHIV JUNB_MmcJUN_MmJUND_Hs MQGCSSATGKNAAAAAAAAAAMGEHQIPTTTFYHPDDTPS-GGYTAAAQPFSL---GCYPGKRNT------ATGSAPPGSEPLGASPLAAAASVLT------DEPQEEAGPFVAYEAGNFLcJUN_HsVSSJUNB_MmcJUN_MmRAYLAAGGHEDLA YHGG KS LLQA NGG KT PL-QTPSSLSAVNLTGNSHLAAIATTTDQPYVPRSTGPLA-KGTGMQPVFGALAPCRAPGVK-ANPS------GVPAEGGASGGGGAG-SGGYFY----SAVSTLDSHEGSQEEGPPGSFDVATYEGAGANFSLVLSRKNALFLANASPETGLEAHLSESSQRNLGGGTILV cJUN_HsJUND_HsJUND_Mm MQGTSAAA-KNTMGAAELTTVTTTTSFGYADDPTAAS------L-NSAQSFFLLYPSKEVS------GPYPP------APADLAATGAAPYGSANEET------EQTEPFVAYEAGNFLcJUN_MmVSScJUN_HsJUND_HsKAFLAEGGDLA PHG KPP IQ LNGG KQ QL-QSGSSMATGNLAAAAAAAAAAGNHLIATTTDPVPGT-PS-LGGTKQPF------SL---CPKGNT------ATGSAPPGHELRAAPKAAAAVNTSDELLPQEETAGSPFPVADYEVAGNFLLLVSSRKALYLAASGGEPLEAHLGGSEQRANLGGTIIL- cJUN_MmJUND_Mm MQ TS A- KN MG EL:TTV TTT F Y DDP T AS L- NT AQ SF FL LY QP SK EV S------G A Y ------GAA Y S NEE ------Q E***:***.:FAEGFJUND_HsVcJUN_MmJUND_MmKAL E D.:L PH K IQ LS** KQ QLQSGSMAA-TNLTGNAALVATTTTDSPGVAPGT-AS------LSKQPF------LYPKV------PPAPAHDLRAAAKTAANPSGSDAEELLTEQTESPFPVADYEVAGNFLLLVSSKALFLAESGGDPLEAHLGKEPPQRNLGGQIIL- JUND_Hs ---- . **ME T:*****PFYGDE A L S*::GLGGG *:A S G ------S GG S.:.*::FASPGR L**:***:**FPGAJUND_MmPPJUND_Hs T AAA :**. G S MM KK. D:QA SL -T NL GS L:VS TTTE Q V PAAA T S -L TK QP FAAA L Y PPPP K V ------T P L R A D G A P S AA PP AA D SG EELL QA ES***:***.:FPADELGFLLVKALLA ES DP.:LEHL KE QR SL**QIIL JUND_MmJUNB_Hs ::::.*::----GAAVMGETTGP- FSS Y GY EEP TTT A L*SI GS LY AAL P GH A SSP - VP AF GA AGG T GH AP PA GGGGQ L G L FG.AR PPG A GS*RT AF FK PEE.G AP *****PPQJUND_Mm T TV SP ---E A ******:R S MR LD KKA T DP A.L T**LS L:*****AEQGAA G L K*::PGS A*:TA P S A L R P D G A ----- .:.*::PDGLLA S**:***:**PDLGLLKLA S:**.PELE R.L II: JUNB_HsJUNB_Mm TGPPTANVVGSTLGG------SSYPTATISYLPHAAPT-GGPFPPAGGAGHPGGAQVLYGALGSPREGPPPASAVFYKTEENLPSSQJUNB_HsTYVSPPEASRAS SSR D GGA T AP GAAVGTG-SSYPTTTISYLPHAP-PFAGGHPAQLGLGRGASTFKEEPQTVPEARSRDATP ** ** : :. . . Tools (/Tools/) : * ::::.*:: > Multiple Sequence * Alignment (/Tools/msa) . * .> *****Clustal ******: Omega JUNB_HsJUNB_MmcJUN_Hs PTANPPPSNYGVVSAAILTTTG------LAPFTPPPAQGPQQQQQQYFYPR---GGGASPPSGGGGGHHPQLAPGGGQQPAGGMGGGPVYQVA-TGHEEPERQPPPLEQGAFVLAYKDTEEGNFLPJUNB_HsVSSQJUNB_MmKTAYVLSPDDPEAMLSP HA-- KP MSG NGGE HT VSPTGPPTANVVGSTLGG------SSYPTATISYLPHAAPT-GGPFPPAGGAGHPGGAQVLYGALGSPREGPPPASAVFYKTEENLPSSQTYVSPPEASRASSSRDGGATAP JUNB_MmJUNB_HscJUN_HscJUN_Mm PSANLSPSAVNVTYGNSVLAAIATTTDQGPLYVAPRNFTSGPPPLASKGQGAMPQPVQQQQQYGAFAPYRAPGVR-APPGGGPSGVQPAPPSEGGGGGGGGGGHHSGL---SP-QQSGGTYIGGGFP----SVAQVS-TLHEESHPGSRQELEGPPQGSAFDVLATYKDGAEEGANFSLPJUNB_HsJUNB_MmVLSQcJUN_HsKNTALFVLANPDDSSPEGMLEAP HL-- KESS MRG NLGGGE HIT VPPTANPPPSNYGVVSAAILTTTG------LAPFTPPPAQGPQQQQQQYFYPR---GGGASPPSGGGGGHHPQLAPGGGQQPAGGMGGGPVYQVA-TGHEEPERQPPPLEQGAFVLAYKDTEEGNFLPVSSQKTAYVLSPDDPEAMLSPHA--KPMSGNGGEHTVSP cJUN_HsJUNB_MmcJUN_MmJUND_Hs QTP-----SSLSAVNLTGNSHLAAIATTTDQTPVYVAPRSFTGAAPLA-KGETGMPQPVFGAPLAPFCRA---PGVK-ANPSPPPPP------GVPAEGGASGGG----GAG-SGGPYGFYA----SLAGVS-TLPPDSHEGSRQELEGPPAAGSFDVLATYKEGADGANEFSLPJUNB_MmcJUN_HsVLSQcJUN_MmResultsRKNTALFVLANPASPDETGVLEAP HLS SESSF QRG NLGGGE TIS LVPforPANSPSVNTY GSjobVAAITTTQGPL VAclustalo-I20170307-220625-0735-57317599-pgPNFTGPPPASGQGMPQVQQQQQYAFPYAPVRAPPGGGSVQAPPSGGGGGHHSGL---SP-QQGGTIGGGFPSVAQVS-TLHEEHPSRQELEPPQGAFVLAYKDAEEGNFLPVSQKNTAFVLNPDDPEGMLAPHL--KSSMGNGGGEHTVP cJUN_MmFOSL2_HscJUN_HsJUND_HsJUND_Mm Q SG-----SSIMAPTGNTLAAAAAAAAAAGINHNLAAIATTTIDTTTPVVAPSGFTQ-AAPDS-LGGETQKPQWPVFM------SPLV---FCQ---PKTGNVTPPPP------IATSGMSGSANPPLPGYGPPPPPPHERLSRA------APKAAAAHVN-TSPPDELLPRQELEHTAAAGPSFYPVLASDYKEPVADGLNEFPLLLcJUN_HscJUN_MmVGSSQJUND_HsRLKTALYVL-APASGGDEVPLPEA HGLGGS SHEF QMRAG NALGGD TLIIS LP-QPR-----SSSVNTGSHAAITTTQTPVVAPSFTGAAPA-GETMPQVFAPLPFCA---PVKANSPPPPP------VAGAGGG-----GGPGYASLAGVS-TLPPDHESRQELEPPAAGFVLAYKEADGNEFLPVSQRNTAFVLNPAPDEGVLAPHLSSSSFQGNGGGETSLP :. :: : * ::*:******: . :* JUND_HsFOSL2_MmcJUN_MmJUND_Mm Q SGSIMAA-PTNTLGINAALNVATTTTIDSTTPGVAPSGTQ-ASD------LSQKQWPFM------LVYQPKTV------ITSMPPSNAPYAPHDRLSRAA------AKTAANPSGSDAEELLTEQHTEPSFYPVASDYEPVAGLNFPLLLcJUN_MmJUND_HsVGFOSL2_HsSSJUND_MmKLALFL-AESGGDVPLPEA HGL KHEPP QMR NALGG QLII LP-QGR -----SSAIGPNAAAAAAAAAATGIHNAAIATTTITTTVAPSFTQAAPD-GGLETQPQPWVFSMPL---VFCQ---PKGTNTVPPPP------AITGSSMGASPPNLPGGYPPPPPEPLRASP------AAAAHV-TPPDEPRQELEAHAAGPFVYLAYSKEAPDGNLEFLPVSSGQRLTAYVLA-PAGGSDEVLAPHGGGSSHFQAMGNGGADTLSL-PR JUND_MmFOSL1_HsJUND_Hs Q ASVL -PT NSL GIS LN:VTS TTTME SQ GV PSAAA TQ SE -L TQK QWP FMAAA LV YQ PPPP KH VF ------LT GP PL SSR A YD PG RA P LS TAA ------PP AA D SG EELL QYA EPS***:***.:FQPAYDE------LGFLLJUND_HsJUND_MmVFOSL2_Mm K AL LA ES DP.: LE HL KSE QPPR SL** QII LPQGR SAAI -P NT GAAI LN:.VTATTTSI GTT A::PSTAQ S------D:-L SQ QW FM LV YQ P KT V ------I T S*PPM S AN P AY DP LR AAS ------T AAP G SA EET E QTH EP::*:******:FVYAYSEAPGNLFLPVSSGKLAFLA-EGGSDV LAP HG KPPH QM.NGGA QL:*L-PR JUNB_Hs PVSPINMEDQERIKVERKRLRNRLAATKCRKRKLERIARLEDKVKTLKAENAGLSSTAGL FOSL1_MmJUND_Mm A.VL PT**SLIS DL:*****SA--EQSSGAAQ EG L HK*::WPMGVS QA*:PTHA FP LS GA PL TR GP YD PG RA P----- L A ------.:.*::PDGLLYA PS**:***:**QPYD------LGLLJUND_Mm FOSL1_Hs K L A S :**.P E L SE PPR .L QII :PQ RS V- PN SG IL:NV TTTT M S GP ST QS E- LT QQ WF ML VY QP K HV F------L G P SS Y P R P L T ------AA S EE Q YE***:***.:PFQAYE------GFVKAL E D.:LH K SQ PPS**QL PR JUNB_Mm PVSPINMEDQERIKVERKRLRNRLAATKCRKRKLERIARLEDKVKTLKAENAGLSSAAGL JUNB_Hs G:*::AAVG T:G - SS *Y P:*:*:***TTTISYLP H.:AP - P F.A GG H P A Q L G L G R G A S T F K EE P FOSL1_MmQJUNB_Hs T V P E A R S R D A T P P.V PS**SPI DN:*****SM--EDSSQEQR EI LK HV*::WEMRVK QR*:PLHR FN LR GL PAA T GT YK PC R PK LR AK ------L E.:.*::RIARLYE PD**:***:**QKYV------KTLKAEN A:**.GLSSS PPT.A QG:PLR cJUN_Hs PLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANM JUNB_HsJUNB_Mm TGPPTANVVGSTLGG------SSYPTATISYLPHAAPT-GGPFPPAGGAGHPGGAQVLYGALGSPREGPPPASAVFYKTEENLPJUNB_HsSS QJUNB_Mm TY VS PP EA S R AS SSR D GGA T APG PAA:*::VSVPGIT NG:M- ESS D QY*EP RTTT:*:*:***IKVIESRYKLRPLH RA.:NPR- LP AAF A.TGG K CH RP KA RQ KL G EL RG IR AG RA LS ET DF KK VEE K TP LQ KT AV EP NE AA GR LS SSR D AAA T GP L JUNB_MmFOSB_HscJUN_HscJUN_Mm T APPPGGPLSNAYPVSGISGAADLSMGGGE------LSPAQSFETPRSAIGQKTTPAQQQQQESRGKPRGMP---RANARSPPPIGGAAARHHPASQRLKAPCGRRQQRPKGGPMRRPKVEELYQEAT-RGLHIPTAEPRPPPEEEELQEEAVLKYKVTRREEKNTLVPJUNB_HsJUNB_MmSSRRQKcJUN_HsTAYEVQSRPNPNESAKMESLP AAAA-- PS SGT GGKEA CTN SRPMTGNPPPTLASNVPVGISTDLGMG-E------SSSQYEPRTIAKTAIESRYKLRPMHRANAPRT-IGGPAAFPPASGGKACGHRPKGGARQKVLYGEALRGSIPRAEGRPPPALSEEAVFKYKVTEEKNTLPSSQKTAYVQSPNPESAESRLASSSRSDTGGATNAPM ToolsJUND_Hs (/Tools/) P>L SMultiplePIDMDTQE SequenceRIKAERKRLR NAlignmentRIAASKCRKR (/Tools/msa)KLERISRLEEKV K>T LClustalKCLUSTALSQNTEL AOmega SO(1.2.4)TASL multiple sequence alignment cJUN_HsFOSB_MmcJUN_Mm P ASGGPVSTAYSGAAGSQGGGPLVPANSFGTPASGTTTQMPVQQQQQASPGAPVASPPSAVRQAPPPGGARHHSAGRLSP-RRQQGGPIFRPSEEVAQST-LHHTPSPREEEEELPPQAVLYKARREENLVPJUNB_MmcJUN_HsSRRFOSB_HsQcJUN_MmNTFEVNRPPNEGKMALP LAAA-- SS G GGGKE CT RPTAN PPPPGGLSNYAPVGSISAAGDLSMGGGE------LSAPQFSEPTRASIQGKPTTAQQQQQESRGKPRGM---PRANARSPPPIGGAAAHHRPASQLRKAPCGQQRRRPKGGMPRPRKVEELYQEA-TRGHLIPTAERPPPPLEEEEQEEAVLKYKVTEERRKNTLPVSSQRRKTAYVEQSPRNPENSAMKESPLA--AAAPSSGTGGEKATCNSPRMN JUND_Mm PLSPIDMDTQERIKAERKRLRNRIAASKCRKRKLERISRLEEKVKTLKSQNTELASTASL cJUN_MmFOS_HsJUND_Hs P -----SAVGT------SAAQTPVVASFGAAAGEMVVPVAKPPTFAM---VTAGGSPPPPPVRA-GAAQGGGS----IG-RRGGPGYKASVLAEGSQ-LPPHSSPREEEEELPPAAVLYKARRDNELIPcJUN_HscJUN_MmSRRFOSB_MmQJUND_HsNTFEVNRPPNDGKVAMP LAAAS SSF G GGGKE CS RPPAN SPGGLVSTYAPSGIAAGDSMQGGGDPLTVAPQNFSEGPTRASIGQTTTKMPAVQQQQQEASRPGKAPRVLAPPSRSANVQRAPPPIGGAAAHHRSAGLRKSPC-QQRRRGGKIPRFPRKSVEELAQES-TRLHIHPTSSRPELEEEEPPQEEAVLKYKVAEERRKNTLPVSQRRKNTSFVEQNPRPENTGMKEAPLL--AAAASSSGTGGGEKATCSPRLN JUND_HsFOS_MmJUND_Mm G -----*:***:*:AGAAAAAAAAAA------AATV A*****.****:***:**:**********:***:******::*:FAAGGEMPPVSKP---TFV---SGGGTPPPPART-GASQGASPPILGGRRPPPPPELGAKPVAAAAEHQ-LPPSPREEEEELAAAPVLYKARRDNELIPcJUN_MmJUND_HsSSRRQJUND_MmTYEVARPGGNDKV AMP*:*:*.: GGAAAS F AG GGKD CS -RPP-----NSLVSTPSIAADMQTDPVTVAQSFEGAARAIGEKMPAVEAPRPFKA---RVLARSPPPPPNVRAIGAAAGGG----SKC-RGGPKGRYAKSLAGES-RLPPIHSSRELPPAAEEVLKYKVADKNETLPSQKNTSFVQNPPDTGVEAPLLSASSFSGTGGGEASPL Results for job clustalo-I20170307-220625-0735-57317599-pgFOS_HsFOSB_Hs A-GM------FQAFPGDYDSGVVSRKCTSSSMTGGPSRA-EASQQS----IGRRYGLKSSVEVQDLSSFPGEEEESPP-KTRRAAAIRRSQEERCNAKGMLAAAGEMKPCGRSNF JUND_MmFOSL2_Hs G AAP G T------AA :.TS G A::PA ------: V I K T I G TT ---- *PP V AG PRRR A D L- AAR D TE PQ GL AS TP EEEEE T P::*:******:VYKARRNLIJUND_HsJUND_MmSSRR FE AR GGN K AL GAAA PP . GGK C:* -RG-----N*:***:*:AGAAAAAAAAAAAATV A*****.****:***:**:**********:***:******::*:FAAGGEPPVSP---F---GTPPPPATGSGAPPLGGPPPPPELAPAAAAH-PPPRELAAAPVLYKADNELPSSQTYVAPGGDV AP*:*:*.:GGSFAGGGDS-P FOS_MmFOSB_Mm A-GM------FQAFPGDYDSGMSVRKCTSSSVSGGPSRA-EASQQS----IGRRYGLKSSVEVQDLSSFPGEEEESPP-KTRRAAAIRRSQEERCNAKGMLAAAGEMKPCGRSNF FOSL2_MmJUNB_Hs L PR GE ------Q V:A Q L K Q K V M T VH IV KS TN IG GC TTQ LLL ---- G V VK G RRRH A F - R D E Q L S P EEEE ***:***.:KRRIJUND_Mm RR E R N .:K L AAA **K C RG NAA T AA :.TS G A::PA ------: *PP A P A D L AA T P G A T E T P::*:******:VYANLSSFAGG A G PP .GG :*- FOSL2_Hs IPTINAITTSQDLQWMVQPTVITSMSNPYPRS------HPYSPLPGFOSL2_HsFOS_HsLA-SVP G H M A L P R PMMG------FSGFNADYEASSSVIKRTCISSGTTAS----PAGDVSGLRRRSYY-HRSDPEAQDLSSFPSSEEEEMGSKPRRVNIARRQDEFRCNTKDLLAAAAVSSKCARNNF FOSL1_HsJUNB_HsJUNB_Mm PLPVRGSE------PQIVNAMQELDKQEKRVIMKTVHIEVRSAKNLRGGLCPPRQNLLL----RLAAGVPTKGKVCHRRRRAKFRPKCLEQRISAPREEEELEDKRRRVKTVL RRKJUNB_Hs A E RN NA KG L AAASS T KA CG RL NL R E Q V:A Q L K Q K V M T H V S N G C Q LLL G V K G H A F ***:***.: .: ** FOSL2_Mm IPTINAITTSQDLQWMVQPTVITSMSNPYPRS------HPYSPLPGFOSL2_MmFOSL2_HsFOS_MmLA-SVP G H M A L P R PIMMGP------TFISNGAFINTTADSYQEDALSSSVQIWKMRTVCIQSSGPTTTAVS----IPTASGMDVSGNLRRRPSYYYP-RHRSD------PEAQDLSSFPSSEEEEHMPGYSKSPRRPVLNIPTRRGQLDEAFR-CNSAKVDLPLAAAGSHVMSSKACLARPNNRF JUNB_HsFOSL1_MmJUNB_MmcJUN_Hs G PLAAPVRGSEV------PQGIVTNAGMQ-ELSSDKQYEKPRVTTTIMKNVHIEVSRNYAKSLRGPGLCHPPRQANLP----RM-LPAATFQQAPTGGGKLVCQHRRRRTPKFARQPKLCGELQRGIRSAGPRAEEEELSETDFKKRRRVEEKTPVLJUNB_HsQRRKJUNB_MmTAVEPRNENAAKGRL SAAASS R DAA AK TCG PRLPNLVRSEPQIVNAMQELDKQEKRVIMKTVHEVRSKNRGLCRQNLLLRLAAGVTKKGCHRAKFRKLERIARLEDKVKTLKAENAGLSSTAGL FOSL1_Hs VPSINTMSGSQELQWMVQPHFLGPSSYPRPLT------YPQY------FOSL1_HsFOSL2_MmFOSL2_HsS PP Q P R PI-GPM------TYIQNDAYIPTTGNSFQDDTLSSVQIWRRMGAV-LQSSGPPPTGV------ITSMPSGNVPRRRYPRPSC------PEAQHIASEPSEEEEYHSSPYGGGGRRRSPLVPQQRRGLKEAFR-RNSVKVDLPMAAAGPHGMSKAGCLSRPANRF JUNB_Mm cJUN_HscJUN_Mm G PLT LRA*SEV PQG IVT DAG MQ- ELSS SK QY EKP RVT IMA KNT AHI EVS RNY KSL RGP MCH RQA NLP RM- ILP AATF QQA SGG KL CQH**RTPKFA RQ KL G*EL RS::*****:**:******:*******IRAGRALSEEAFKKVEEKTPLJUNB_HsJUNB_MmQKcJUN_HsTAVQPNESAERL SA RS DT A TN PMGPLAAVRSEVPQGIVTNAGMQ-ELSSDKQYEKPRVTTTIMKNVHIEVSRNYKSLRGPLCHRQANLPRM-LPAATFQQATGGKLCQHRTPKFARQKLGELRGIRAGRALSETDFKKVEEKTPLQKTAVEPNEAAGRLSSSRDAAATGPL FOSL1_Mm VPSIDS--SSQELHWMVQPHFLGPTGYPRPLA------YPQY------FOSL1_MmFOSL1_HsFOSL2_MmS PP Q P R PV-GPM------SYIQNDTYMPSGGNSFQDETLSSVQIWRRMGAV-LQSSGPPPHGF------LGPSSPGYVPRRRRPLPTSC------PEAQHIASEPSEEEEYYSSPQGGGGRRRY------VQQRRKEFRRNVKDLMAAAPSGPPSKGCQSRPANRF cJUN_HscJUN_MmJUND_Hs APLPLRSEYPQGIVAADAMQGELLSKAQFEKPRVAILQKSPAHQQQQQEVRNKSRGMC---RQNLLRPPIPAAQHHHSQLKVPCQQRAKYMRPKVLQE-RHIPARLQEEALKKVEEKTPLJUNB_MmcJUN_HsQKcJUN_MmTAVQPNESMEPL --A S GT EA TN PMGPLTLRASEVPQGIVTDAGMQ-ELSSSKQYEKPRVTIMAKNTAHIEVSRNYKSLRGPMCHRQANLPRM-ILPAATFQQASGGKLCQHRTPKFARQKLGELRSIRAGRALSEEAFKKVEEKTPLQKTAVQPNESAERLSARSDTATNPM :*:: : * :*:*:*** .: . FOSL1_MmFOSL1_Hs V-*PM SF IR D SF --G E SSP G QP ESS L HG WN MGGG V Q PY HG F------L G P T G Y P**RP L AG ------P*A Q::*****:**:******:*******PPAAAYP--QYQ------AAQQ------SPPKQFPHRL cJUN_MmFOSB_HsJUND_HsJUND_Mm A PLPRRRLRSEYPQGEIVAALDATMQGDLLRTKALQFQEKPARVSEILQTKSPDAHQQQQQQEVLRNEEEKSRGLCPPKRQANLLQERPPLIPEAAQHHSHESQLIKVPACQQERALKYIQRPKVELQKE-ERHRIPLSRELFQVEEALLVKKAVEEHKKTPLcJUN_HscJUN_MmQGKJUND_HsTCSVKQPINEPTMYEPEEL --A GS GPT EGA TPS PGLAPLPLRSEYPQGIVAADAMQGELLSKAQFEKPRVAILQKSPAHQQQQQEVRNKSRGMC---RQNLLRPPIPAAQHHHSQLKVPCQQRAKYMRPKVLQE-RHIPARLQEEALKKVEEKTPLQKTAVQPNESMEPL--ASGTEATNPM CLUSTAL O(1.2.4) multiple sequence alignment FOSL1_Mm :*::-MYR D:Y G E P*G P:*:*:***SSGAGSPY G.:------. R P A Q PP Q A Q A Q T A QQQ ------K F H L JUND_HsFOSB_MmJUND_Mm ----- P************:.**..****:RRRLSPEIAALDTMTDVRTALQFQEAAAREIETKPDAVQEPLRFEEEK---RLKRPPPPPANERLI EAA S ----ES:IK AC ER::PLKGQRAKLEGKE-ERPPRILSRELFAAVEELLVKKAVDHKEKTPLcJUN_MmJUND_HsQGKJUND_MmTCSVKQPINDPTVYEPEEL SA FGS GPT EGA SP PGLAPLPLRSEYPQGIVAADAMQGDLLTKAQFEKPRVSILQKSPAHQQQQQEVRNKSRGLCPPRQNLLQRPPIPAAQHHHSQLKVPCQQRAKYIRPKVLQE-RHIPSRLQEEALKKVEEKTPLQKTSVQPNETMEPL--ASGTEATSPL FOSB_Hs GGASGSGGPSTSGTTSGPGPARPARARPRRPREETLTPEEEEKRRVRRFOSB_Hs E R N K L AAA K C R N RRR *:E L T:D R L Q A E:..TDQ L EEE K A E L E S E I A E L Q K E K..ER L E F V L V A H K P G.C K I P Y EE G P G P G P: JUND_MmFOS_Hs ----- *:***:*:RRREAALTTDVT AL*****.****:***:**:**********:***:******::*:FQAAAEETPDVQPLFE---DEKPPPPSALQGTAELIGAPPPPPNLLKEHK-EPPKLRELFAAILLAAKDHERPJUND_HsJUND_MmQA TC VK PI DP VDD P*:*:*.: SL FG GF DP SEE P-----P-************:.**..****:LSPIAADMTDVTAQFEAARIEKPAVEPRFK---RLRPPPPPNRI AA ----S:K C R::PKGRAKLGE-RPPISRLAAEELKKVDKETPLQKTSVQPNDTVEPLSAFSGTEASPL (b)FOSB_Mm GGASGSGGPSTSTTTSGPVSARPARARPRRPREETLTPEEEEKRRVRRFOSB_MmFOSB_HsERNKLAAA K C R N RRRGGAESLGTSDGGRLPQSATESTGDTTQLSEEEGPGKPAERLPEASREAIRAPERRLQPKREEEKETRLLTEPFEEEEVLVAKHRRKPVGRRCKEIRPNYKEELAAAGPGKPCGRPN FOS_Mm RRR E:.LT D T::LQ A:E T D Q L E D E K S A L*Q T E I A N LL K E K E K L E F I::*:******:LAAHRPJUND_Mm A C K I P DD L G .F P :*EE -----*:***:*:- AATV A*****.****:***:**:**********:***:******::*:FAAEPVPF---PPPPGALGPPPPPH-PPRLAALKDEPQTVPDV P*:*:*.:SFGDSP FOSB_HsFOS_Hs -AMGF------QAFPGDYDSGSVVRCKSSSTMTPGGSARE-SAQ----SIGRRYLGSSKVVEDQSLFSGPSEEEEPP-TKAAARRISRRFOS_HsFOSB_MmFOSB_HsQECRANGKLM GAAA E M PK GC SR FN RRRGGVPATESVLGTTSADGGITTTLPQSATQESDTTTTLDQQWLSLEGVDPQEVPKSTSALARILPSSQATRMEAIRQAPSNRRQLLGPQKRPEEELKAETSKLQLTPPEPFEEEEVVILDAAPKYHRRDRMPVPARRGCTKESIRYPNSDDKTLPLAAAGGMFSPKGEECYRSS-N FOSL2_HsJUNB_Hs LRRRREQEVLATQELKLQQKAVEMTEEHVLSEEENGCKQSLLLGLQGKVEKIGAHEALFQKEKEKLEFMLVAHGP V C K I S P EE RR S PP A :. :: : * ::*:******: . :* FOSB_MmFOS_Mm -AMGF------QAFPGDYDSGSMRVCKSSSTVSPGGSARE-SAQ----SIGRRYLGSSKVVEDQSLFSGPSEEEEPP-TKAAARRISRRFOS_MmFOS_HsFOSB_MmQECRANGKLM GAAA E M PK GC SR FN RRRAVGP------TEVLTTADITTTLQSAQEDTLDVVQQWLKLETVDMQETPKGGTSLARIL-SSQATQMESAIQAGSNRRQLLGGQKPEVLKEAEQSKLQLSPPEPFEEEEAIVLDAAPKYHRRDRMPIPARRGCTKESIRYPNSDDKTMPLAAAGGLFSPKAEECYRS-NT JUNB_HsFOSL2_MmJUNB_Mm P LVRRRRSEPQIEVNLAMTQELDKQLEQKRAVIEMKTVEEHEVRLSKEEENRGLCRKQNSLLLRGLAAQGKVTEKKIGCAHREAKLFRQKLEEKREIKALRELFEMDLKVVAKHTGLPJUNB_HsKVACEKNIASGPLEE SS RR T AS GPP LLTREQVAQLKQKVMTHVSNGCQLLLGVKGHAF FOS_HsFOSL2_Hs MMPGF------SGFNADYEASSSVIRKCTSSIGATTSP----AGDSVLGSRRRYYH-SRPDAEDQSLFSSSPEEEEMGSPKVRRNAIQRRFOSL2_HsFOS_MmFOS_HsDFECRTNDKL AAAA V SS K AC NR FN RRRAIGP------TEVLTTAEIKSLTQSAPEDTLEEMQVWLKLEEETVVQSPKGGASLGRVL-SSQAKQVESAIPAGSERRQLTQGRKAEV------KEEQKLLSEPFEEEEMPLHVPAKFHRRGGVPI-----VRRCKEIRSNPPKAEEMPAAASRRAGSKAPPCYRSANR JUNB_MmFOSL1_HscJUN_Hs P LVRRRSEPKQIEVNLAMTQEDLDFKQLEQKRAVIEMKTNVDHEKVRLNKESRDGLECRKQNSLRGMLAAQTRQQTEKILCEEQRTKLFRQKLQEKREIRALRELEVDLKEVAKHTRLPJUNB_HsJUNB_MmKIACEKNIAPGELG SSA K AAE -- G LGPLDVRSEPQIVNAMQELDKQEKRVIMKTVHEVRSKNRGLCRQNLLLRLAAGVTKKGCHRAKFRKLERIARLEDKVKTLKAENAGLSSTAGL FOS_MmFOSL2_MmFOSL2_HsPLEASE NOTE: MMPIGP F------TShowingSIGNFANIATTDYSEQ ADcolorsSSSLVQIWRKMCTVSSIQ GPonATTTSV P----IlargeATGSDMSV LGNalignmentsSRRRPYYYPH-RSRPD------AEDQSLFSSSP isEEEEMH GPslow.SYPKSVRRPNLTIPQRRFOSL2_MmGFOSL2_HsFOS_MmDLFEACR-ANSDKVLP SAAAG V H SS M K A AC L NR P FN R RRRPIGP------TEVLTTAEIKSLTQSAPEDTLEEVQIWLKLEEETVIQGPKTTTSLG----VLSSQKVEVAIGPARRRSEQLTQ-RKRAED------KEEQKLLSEPFEEEEMPLHVPAKYHRRGGLPI-----VRRCKEIRSNPPKTEELQAAASRRAGSKAPPCYRATNR cJUN_HsFOSL1_HsFOSL1_MmcJUN_Mm P LLPRRRSGEP------KQIEVDLAMTQEDLSFKQLEQKRAVIEMKTNAVDHEIKVRLNKAESRLDGMGECRPPKQNSLR----GMILAAQTRQQSPEKGILCVEEQRRRRTKLFRQKPLCQEKRQEIRASLRPELEEEEEEVLKEVRRRAKHTRLVPJUNB_MmcJUN_HsKRRIFOSL1_HsACQEKNRISNPEKLG AAAAD SKK T AKD NCP MRGGPLN VRRRSEPQKIVENALMQTELDDKFQLEKQRVAIMEKNTVHDEVKRNLKSERGDLCERQKNLSRMGLAATQQQRTEKLICQEERTKFLRQKLQEKREIRALRELEVDLKEVAKHTRLPKIACEKNIAPGELGSSAKAAE--GLGD FOSL2_HsFOSL2_Mm -IMPYTQIDNYAPIGTTNFSDQTDSSLQRWGM-VSSQPGT------VITSMSNPYPRSP------AHAESYSSHPGGGGYSPLQQPGPLEASEFOSL2_MmFOSL2_HsKLFAR-VSDVMPPGGH SMNOTE: GA SL AP FR PIGP ------ShowingTINAITTSQ DcolorsLVQIWKMTVIQ onGPTTTV ----largeITSMVS GNalignmentsRRRPYP-RRSD------EQLSP isEEEEH slow.PYKSRRPLIPRRGLEAR-NSKVLPAAAGHMKACLRPNR cJUN_MmFOSL1_Mm JUND_Hs P LLP**:***:RSGEP------QIVDAMQELS KQ*****::**:**:EKRVILKSAVHEIVRNKASRLGMGCRPPQNLLR---- I*:PAAQ HSP**QKGVCV PRRRR:*AKYR KP*:**:**::*LCERQIASRPLEEEEEEK VRRR**KT LV*cJUN_HscJUN_MmKRR FOSL1_MmA***QENRSN EK L AAAA S T AK NC MRPLN LRRRSEPQKIVEDALMQTELDSKFQLEKQRVAIMEKNTAHDEVKRNLKSERGDMCERQKNLSRMGILAATQQQRSEKLICQEERTKFLRQKLQEKREIRALRELEEVLKEVAKHTRLPKIACQKNISPELGADSKKTADNPMGG FOSL2_MmFOSL1_Hs -VMPYSQIDNYTPMGSNGFSDQTESSLQRWGM-VSSQPGH------FLGPSSYPRPLSTP------AHAESYSSYPGGGGQY------QQFOSL1_HsFOSL2_MmKFRVDMPGS SPP G SQ AP FR PIGP------TINAITTSQDLVQIWRMAVLQGPPPTV----ITSMPSGNVPRRRYPRPSC------EQISPEEEEHPYRRRSPLVPRRGLEAR-NSKVLPAAAGHMKACLRPNR JUND_Hs JUND_Mm P LL RS*EP QI VD AM QD LT KQ E KR VI LK SA HE VR NK SR GL CR QN LLR I PAA Q HS QK VC PR**AKYR K L E*R I::*****:**:******:*******SRLEEKVKTLcJUN_MmJUND_HsK S Q N T E L A S T A S LPL LR**:***:SEPQIVDAMQELSK Q*****::**:**:EKRVILKSAHEVRNKSRGMCRQNLLR IP*:AAQH SQ**KVCP RA:*KYR K*:**:**::*LERIARLEEK V**KT L*K A***QNS E L A S T A N M FOSL1_HsFOSL1_Mm -VMPFSRIDFSG--EPSSGPQSSELGHNWGGGMVQYPGH------FLGPTGYPRPLGAP------AQPPAAAY--PQQYAA------QQFOSL1_MmFOSL1_Hs------S PP K FQ HP LR PVGP------SINTMSGSQELVQIWRMAVLQGPPPHF----LGPSSPGYVPRRRRPLPTC------EQISPEEEEYPQRRRY------VRRERNKLAAASPPKCQRPNR JUND_MmFOSB_Hs P ************:.**..****:LSAPEI-DVMRDTLQPEGRSIAKPA-----ERKRLARKNERDIG AAF S SW:KLL C RPPPPPPP::KRKLERI------SRLEEKVKTLJUND_HsJUND_MmKSQNTEL A S T AL SP LFPLQLRSEPQIVDAMQDLTKQEKRVILKSAHEVRNKSRGLCRQNLLRIPAAQHSQKVCPRAKYRKLERISRLEEKVKTLKSQNTELASTASL FOSL1_Mm -:*::MYRD Y:G E P G*P SS:*:*:***GAGSPYG ------.: . R P A Q PP Q A Q A Q T A QQQ FOSL1_Mm------K F H L V*P S I D S -- SS Q E L H W M V Q P H F L G P T G Y P**RP L A ------* ::*****:**:******:*******YPQY------SPPQPR FOSB_HsFOSB_Mm *:***:*: RRRLAEE-LVTRD RL*****.****:***:**:**********:***:******::*:LPQGASETTSD-----QLEEEAKAEEDLGEFSGEWILLAEPPPPPPPLQKEKER------LEFVLVAHKPJUND_Mm GFOSB_Hs C K I P Y *:*:*.:EE G P GL P GFP************:.**..****:Q LSAPEI-DVMRDTLQPEGRSIAKPA-----ERKRLARKNERDI GAA F SS:WK LLC R::PPPPPPPKRKLERI------SRLEEKVKTLKSQNTELASTALSPLFQ *: : :.. .. . : :*:: : * :*:*:*** .: . FOS_Hs MSVASLDLTGGLPEVATPESEEAFTLPLLNDPEPKPSVEPVKSISS M E L K T E P F DD F L F*:***:*:P *****.****:***:**:**********:***:******::*: *:*:*.: FOSB_MmFOSB_Hs RRRGGAESLGTSDGGRLPQSATESTGDTTQLSEEEGPGKPAERLPEASREAIRAPERRLQPKREEEKETRLLTEPFEEEEVLVAKHRRKPVGFOSB_MmRRFOSB_HsCKEIRPNYKEEL AAAG P G KP CG RP NLRRRAE-EVLRTDLRPLGQSATEST-----DQLEEEAKEADEGLFEGSWELLIAPPPPPPPELQKEKE------RLEFVLVAHKPGCKIPYEEGPLGPFGQP JUNB_HsFOS_HsFOS_Mm L RRRRMESQVVEAALSQTLLDKTLQLTKQGGVAMELTPHDEVQASLNETGDPCEQKSLLLSEEALAGQFVTKELGIPHALLANFLLNDKPEKPEKKPLSELFEIPLVAAKSHIRSPNAVFOS_HsCEKLIKPADDEP LF GDD F PF EEL F -P MSVASLDLTGGLPEVATPESEEAFTLPLLNDPEPKPSVEPVKSISSMELKTEPFDDFLFP FOSB_HsFOSB_Mm VGGPTAVSTGASIGGTTPSQTDSLTTTQWLSVGQPPVTSLAIRSSPAMRAQRSPQRRGQPPRLEEASTQLPPTPVVEEEEDPYKDRRMPVGRRFOSB_MmFOSB_HsTSEYRSNTKPLGAAA M S GK YC SSR N RRRGGAESLGTSDGGRLPQSATESTGDTTQLSEEEGPGKPAERLPEASREAIRAPERRLQPKREEEKETRLLTEPFEEEEVLVAKHRRKPVGRRCKEIRPNYKEELAAAGPGKPCGRPN JUNB_MmFOS_MmFOSL2_Hs L RRRRPEGQLVEQALPQTMLDRKTSQLGGGKQVAMEST------HDVQSLNEGDCEQKLLLSALGQVTKEGIHAANFLLKEKEKLEFILAAHVRGPAJUNB_HsAVVVFOS_MmCKIKPQDDEP L GEE F PD EES PL-S RMESQVVAASQLLDKLQTKGGVMLTPHEVASNTGPCEQSLLLEEAGFVTKLGPHLLAFNDPEPKPSLEPVKSISNVELKAEPFDDFLFP FOSB_MmFOS_Hs VAPGT------VTAITTSQDLQVVWLKVTQMPTTGGLIRSS-AMQASQISGQRRGQGPKLVAESQQLPPSPAEEEEVDPYKDRRMPIGRRFOS_HsFOSB_MmTSEYRSNTKPM GAAA L S AK YC SR TN RRRGGAESLGTSDGGTLPQSATESTTTTDQLSEGDPEVKSSAARLPQATREAIRAPNRRLLPKREEEKETKLLTEPFEEEEILAAKHRRRPVARRCKEIRPNDDKLLAAAGFPKEECR-N cJUN_HsFOSL2_HsFOSL2_Mm L RRRRSEGQLVEQALSQTLLERKGQLTKQGVASMEANT------HEEVNLSEEEGCQKLSMGLTQQQKELIQATEFLQKEKEKLEFMLVAHVGPJUNB_MmVVVVFOSL2_HsCKIKSQPEEEPP RREE SD PPS PLAS RPEGQLVQAPQMLRKSQGGGKVMST------HVSNGCQLLLGVKGHAF VGAVVVKQEPLEEDSPS FOS_HsFOS_MmPLEASE NOTE: IAPG T------ShowingVTAISTSP DcolorsLQMWVLKVTQV PSonAGGL VRlargeSS-AVQAS PIalignmentsSGQRRTRGAK------VEQLSP isEEEEP Hslow.PFKGRRV-----IRRFOS_MmFOS_HsERPNAKPM SAAA A G AK YC SR RN RRRAG------ELTDTLQAETDVVQLKETDMETKGGSARL-QATQESIAGNRRLLGKEVKEEQKLLSEPFEEEEILAAKHRRRPIARRCKEIRPNDDKMLAAAGFPKEECR-N cJUN_MmFOSL2_MmFOSL1_Hs L RRRRTEGQSVETALSQTGLETKSQL------KQVAMENTHEEVNLSEEEGCQKLSMGLTQQQKELIQATEFLQKEKEKLEFMLVAHGPcJUN_HsVFOSL2_MmCKISPSEEP ------RR S PP LT RSEGQLVQASQLLRKGQTKGVSMAN------HVNSGCQLMLTQQLQTF VGPVVVKQEPPEEDSPS FOS_MmFOSL2_Hs IPPGT------VTAISTSPDLQVWILKVTQIPGTTTLV----SSVAVPGSRRRQTR-AR------DEQLSPEEEEPHPYKGRRL-----IRRFOSL2_HsFOS_MmERPNTKQL SAAA A G AK YC AR RN RRRAG------ELTEKLQAETEEMVLKEEETVSKGGSGRL-QAKQESIAGERRLQGKEVKEEQKLLSEPFEEEEMLVAKHRRGPIVRRCKEIRSNPKEEMAAARRSKPPCRAN JUND_HsFOSL1_HsFOSL1_Mm L RRRSEGQKSVETALSQTGLDAKFSQL------KQVALESTHDVKNLSEGDCEQKLLSGPLQHRQEVIPEEAYLQKQKERLELVLEAHRPcJUN_MmPLEASEIFOSL1_HsCKIPESGP A------K NOTE: E -- GLD RTEG QSShowingVTASQGLTKSQ------KV McolorsNHVNSG ConQL MlargeLTQQ LalignmentsQTF is slow. SP------FOSL2_Mm PG------VIKTIGTT----VGRRR-RDEQLSPEEEEKRRIRRFOSL2_MmFOSL2_HsERNKLAAA K C R N RRRPG------ELTEKLQAETEEVILKEEETIGKTTSG----LQKEVIGARRRELQ-KREDKEEQKLLSEPFEEEEMLVAKHRRGPIVRRCKEIRSNPKEELAAARRSKPPCRTN JUND_MmFOSL1_Mm L RRR E.QK VE AL QT LD KF QL KQ VA LE ST HD VK NL SE GD CE QK LLS G PL Q HR QE VI PEE A YL Q K Q K E R L E L V L E A H R P JUND_HsI FOSL1_MmC K I P E G D KK D P GGL RSEGQSVTASQGLAKSQ------KVLSHVNSGCQLLPQHQVPAY SP------FOSL1_Hs PG------VIRALGPP----PGVRRRPCEQISPEEEERRRVRRFOSL1_HsFOSL2_MmERNKLAAA K C R N RRPGK------ELTDFLQAETDVKILKETDIEGKTTSG----LQREVIGEERRRLQ-KRQDKEEQRLLSEPLEEEEVLEAKHRRRPIIRRCKEIRPNEKGLAAAAKE--KCGRDN ************:.**..****: **:***: *****::**:**: *: :** :::* *:**:**::* ** *JUND_Mm *** L R E.Q V A Q L K Q K V L S H V N S G C Q LL P Q H Q V P A Y FOSL1_Mm PG------VIRALGPP----PGVRRRPCEQISPEEEERRRVRRFOSL1_MmFOSL1_HsERNKLAAA K C R N RRPGK------ELTDFLQAETDVKILREADLEGKPPSG----LQREPIGEEVRRRLQKPQCKEEQRILSEPLEEEEVLEARRRHRPVIRRCKEIRPNEKGLDAAAKKDKPCGGRN FOSB_Hs T------SQDAPP-NLTASLFT------HSEVQ V L G D P F P V ---- V************:.**..****:N : :: * ** * ::*****:**:******:******* FOSL1_Mm **:***:PG------*****::**:**:VIRALGPP ----*: **PG V:*RRR *:**:**::*PCEQISPEEEE **RRR *V RR***ER N K L AAA K C R N FOSB_HsFOSB_Mm LSA------E-VRDLPGSARPD-----APP-NALKTEADSGLFSTW------LLPPPPPPP------HSEVQ VFOSB_Hs L G D P F P V---- L P FV QS T------SQDAPP-NLTASLFT------HSEVQVLGDPFPV----VN * ** * ::*****:**:******:******* FOS_Hs ASSRPSGSETARSVPDMDLSGSFYAADWEPLHSGSLGMGPMATELEPLCTP-VVTCTPSC FOSB_MmFOSB_Hs LRRRAE-EVLRTDLRPLGQSATEST-----DQLEEEAKEADEGLFEGSWELLIAPPPPPPPELQKEKE------RLEFVLVAHKPFOSB_MmGFOSB_HsCKIPYEE G PL GP F GQ PSL------AE-VRDLPGSRADPA-----PP-NLATKAESDLGFTS------WLLPPPPPPP------HSEVQVLGDPFPV----LPVFSQ FOS_HsFOS_Mm MASSSVARSPLSDGLSTEGGTSLRPSEVVPADTVPDELSEEGSAFFYTAALPDLLWENPDLPHESPNKSPLSGVMEGPVMKVSTIESSLEMPFOS_HsELLCKTTPE-PVV F DDT C FT LP FG PC ASSRPSGSETARSVPDMDLSGSFYAADWEPLHSGSLGMGPMATELEPLCTP-VVTCTPSC FOSB_Mm RRRELTDRLQAETDQLEEEKAELESEIAELQKEKERLEFVLVAHKPGFOSB_MmFOSB_HsCKIPYEE G P G P G P LRRRAE-EVLRTDLRPLGQSATEST-----DQLEEEAKEADEGLFEGSWELLIAPPPPPPPELQKEKE------RLEFVLVAHKPGCKIPYEEGPLGPFGQP FOS_MmFOSL2_Hs MSSSSVAS--LDGL------TGGLPEASTPESEEALFDTKLAPQLLRSNVDIPKEPPIKSPISAL-EGGPVFKYS-IGSEENVPFOS_MmELLHKTAPEIPVV F DDT S FT LP FA PV ASSRPSGSETSRSVPDVDLSGSFYAADWEPLHSNSLGMGPMVTELEPLCTP-VVTCTPGC FOS_Hs RRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPAFOS_HsFOSB_MmCKIPDD L G F P EE - MRRRSVAESLLTDLRTLGGQALEPTEDVQALTEEEPESKEEAEALFETSLEPILLAENLDQPKEPKKEPRSLVEEFPVVLKVSAIHSSKPMGECLKKITPEYPEEFDDGPFGLPFGP PLEASEFOSL2_HsFOSL2_Mm NOTE: PSG- LSShowingQAP--MRGS------GGG Scolors------on largeMDKT QalignmentsRSVIKPISIAGGG is Fslow.Y-VGGEEAVVVPFOSL2_HsLHKTQPEIPVVLEET S DT SP PA SI SSSA--G------LDKAQRSVIKPISIA-GGFY-GEEPLHTPIVVTSTPAV FOS_Mm RRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPAFOS_MmFOS_HsCKIPDD L G F P EE - MRRRSVAESLLTDLTTLGGQALEPTEDAQSLTEPDESKEESAALFQTLEPILLANNLLDPKEPKKEPKSLLEEFPIVLKAASIHSRNPVAECLKKIAPEDDPFLDDGFFPLEEFP- FOSL2_MmFOSL1_Hs S------GLQSLRGTGSA------PAPCRPVPCISLSPGPVL-VEGPPEPLEASEVVVAFOSL2_MmLHKTQPETPPLMEE- NOTE:TT D SP PS SL S- SShowingA--G------colors on largeMDKT QalignmentsRSVIKPISIAGGG is Fslow.Y-GEEPLHTPIVVTSTPAI FOSL2_Hs RRRELTEKLQAETEELEEEKSGLQKEIAELQKEKEKLEFMLVAHGPVFOSL2_HsFOS_MmCKISPEE RR S PP A PRRRGLQEPLMTRDSTGGGLQASE------TDQLEDEKSALQTEIANLLKEKEKLEFILAAVHGRAPVVVACKKIQPEDDPLEEGFDPSEEPS- FOSL1_HsFOSL1_Mm T------GSTSGTS------PAPGRPVPCISLSPGPVL-EPEAFOSL1_HsLHTPSTPL------M- TT P S L ------PAPCRPVPCISLSPGPVL-EPEALHTPTLM-TTPSL FOSL2_Mm RRRELTEKLQAETEELEEEKSGLQKEIAELQKEKEKLEFMLVAHGPVFOSL2_MmFOSL2_HsCKISPEERR S PP T SRRRGLQESLLTREGKTLGQSAAE------TEELEEEKSGLQKEIAELQKEKEKLEFMLVAVHGPVVVVCKKIQSEPPPEEEERRDSSPPPSA FOSL1_Mm S G S T S G A S ------: FOSL1_Mm* *S P ------: ------PAPGRPVPCISLSPGPVL-EPEALHTPTLM-TTPSL FOSL1_Hs RRKELTDFLQAETDKLEDEKSGLQREIEELQKQKERLELVLEAHRPIFOSL1_HsFOSL2_MmCKIPEGAK E -- G D TRRRGSTESLGTTESK------LQAETEELEEEKSGLQKEIAELQKEKEKLEFMLVAHGPVCKISSPPEE------RRSPPT . : * * : FOSL1_Mm RRKELTDFLQAETDKLEDEKSGLQREIEELQKQKERLELVLEAHRPIFOSL1_MmFOSL1_HsCKIPEGDKK D P GG SRRGSKTESLGTADSF------LQAETDKLEDEKSGLQREIEELQKQKERLELVLEAHRPICKIPSEPG------AKE--GD FOSB_Hs PSYTSSFVLTCPEVSA------FAGAQ---RTSGSDQPSDPLNSPSLLAL **:***: *****::**:**: *: ** :* *:**:**::* ** * FOSL1_Mm*** RR. K E L T D F L Q A E T D K L E D E K S G L Q R E I EE L Q K Q K E R L E L V L E A H R P I C K I P E G D KK D P GG FOSB_HsFOSB_Mm TP------SYTSSFVLTSCQPDEAVPPSA------NLTASLFT------FAGAQ---RTSGSEQHPSEDVPQLVNFOSB_HsLSGPDSPLLFPAVL ---- V N PSYTSSFVLTCPEVSA------FAGAQ---RTSGSDQPSDPLNSPSLLAL **:***: *****::**:**: *: ** :* *:**:**::* ** * *** FOS_Hs TAYTSSFVFTYPEADS------FPSCAAAHRK-GSSSNEPSSDSLSSPTLLAL FOSB_MmFOSB_Hs SL------AE-VRDLPGSRADPA-----PP-NLATKAESDLGFTS------WLLPPPPPPP------HSEVQVFOSB_MmFOSB_HsLGDPFPV ---- L PV FS QPTS------YTSSFVLTCSPQEDVASPPA------NLTASLFT------FAGAQ---RTSGSEQPHSDEPVLQNVSLPGSDLLPFAPLV----VN FOS_HsFOS_Mm ATTSSYRTPSSSGFSVEFTAYRPSEVAPDDSM------DLSGSFYFAAPSDCWAAAEPLHHRSKG-SGLSSSGMGNPEMPASSTEDLSELPSSFOS_HsLCPTPLL-VVA L T C T P S C TAYTSSFVFTYPEADS------FPSCAAAHRK-GSSSNEPSSDSLSSPTLLAL FOSB_Mm LAE-VRDLPGSTS-----AKEDGFGWLLPPPPPPP------FOSB_MmFOSB_Hs L P F Q SL------AE-VRDLPGSRADPA-----PP-NLATKAESDLGFTS------WLLPPPPPPP------HSEVQVLGDPFPV----LPVFSQ FOS_MmFOSL2_Hs ATSSPGRTPSSNGLSVEFTSYRPSVPLDEVQDELSSPGASFPYSAAESDCWSEKPALHHRRSN-SSSSSLGMGPDMQVSSTEDLSELPNFOS_MmLSCPTPLL-VVA L T C T P G C TTYTSSFVFTYPEADS------FPSCAAAHRK-GSSSNEPSSDSLSSPTLLAL FOS_Hs MSVASLDLTGGLPEVATPESEEAFTLPLLNDPEPKPSVEPVKSISSMFOS_HsFOSB_MmELKTEP F DD F L F P ALSSAER-PVSRGDSLEPTGASRTSV-----PDMDLASKGESDFGYFAAGWDLLWEPPPPPPPPLHSGSL------GMGPMATELEPLCTP-VVTCTLPSFCQ FOSL2_HsFOSL2_Mm SSSTPGAT--SNGL------VFTYPNVLEQESPSSLPDSKEASQCRSSKVAIHKRRPI-SSSSSIA-GGGDFQYSS-GDEESLPNFOSL2_HsLSHPTPLLIVVALT S T P A V TPGTSNLVFTYPSVLEQESPASPSESCSKAHRR-SSSSGDQSSDSLNSPTLLAL FOS_Mm MSVASLDLTGGLPEASTPESEEAFTLPLLNDPEPKPSLEPVKSISNVFOS_MmFOS_HsELKAEP F DD F L F P AMSSSVRAPSSLGDSLETTGGSRLSPVEPVDAVTDPLESGEESFAYFAATLDPWLLEPNLDHPSENPSKLPGSMVGEPMVVKTSEILSSEPMLECLTKPT-EVVPFTDDCTFPLGFCP FOSL2_MmFOSL1_Hs ST-PSFAT--PSGL------VFTYPSTP------MDKETPQCRASVAIHKRPKISSSSSSIAGGGGDFPYSS-GDEEPLPGFOSL2_MmLSHPTPLLIVVALT S T P A I TPGTSNLVFTYPNVLEQESPSSPSESCSKAHRR-SSSSGDQSSDSLNSPTLLAL FOSL2_Hs PGLQPMRSGGGS------VGAVVVFOSL2_HsFOS_MmKQEP L EE D S P S SSSMSVA--SLGD------LTGGLPEASTPESEELADFKTALQPRLLSVNIDKPPEIPSKIPAS-LGGEPFVYK-SGIEESNPVLEHLTKPAIEVVPFTDDSTFPLAFVP FOSL1_HsFOSL1_Mm ------TPFTPSLVFTYPSTP------PEAPPCCSSRPAVHPRCKISSSSSSLSPGPDVPLSS-EDPELAGFOSL1_HsLSHPTPLLTLAML- TT P S L TPFTPSLVFTYPSTP------EPCASAHRKSSSSSGDPSSDPLGSPTLLAL FOSL2_Mm SGLQSLRGTGSA------VGPVVVFOSL2_MmFOSL2_HsKQEPPEE D S P S SP-GSLAQ--PMGR------SGGGS------MDKTQRSVIKPISIAGGGFY-GVEEGAPVVVLHTKPQIEVVPLTEESTDPSAPIS FOSL1_Mm ------* .:*:* *.. P A P G:R P*:VP C I S L:*..:SPGPV L -**EP E*.**:****AFOSL1_MmLHTPTLM- TT P S L TPFTPSLVFTYPSTP------EPCSSAHRKSSSSSGDPSSDPLGSPTLLAL FOSL1_Hs TGSTSGTS------FOSL1_HsFOSL2_MmSP------SGLQSLRGTGSA------PAPCRPVPCISLSPGPVL-EVPGEPAVVVLHTKPQTELPPM-EETTDPSSPLS : * * : * .:*:* *.. : *: :*..: ** *.**:**** FOSL1_Mm SGSTSGAS------FOSL1_MmFOSL1_HsSP------TGSTSGTS------PAPGRPVPCISLSPGPVL-EPEALHTPTSLPM------TTPSL

. FOSL1_Mm S G S T S G A S ------: * * S P:------FOSB_Hs PSYTSSFVLTCPEVSA------FAGAQ---RTSGSDQPSDPLNSPSLLAL . Fig.FOSB_MmFOSB_Hs S9 Prediction PTS------YTSSFVLTCSPQEDVA SofPPA------NJUNLTASLFT ------andFAGAQ--- FOSRTSGSE QregionsPHSDEPVLQNVFOSB_HsSLPGSDLLPF APthatLV ---- V N contactPSYTSSFVLTC PMED25.EVSA------SequenceFAGAQ---RTSGSD QalignmentPSDPLNSPSLLAL of FOS_Hs TAYTSSFVFTYPEADS------FPSCAAAHRK-GSSSNEPSSDSLSSPTLLAL FOSB_Mm S------SRDAPP-NLTASLFT------HSEVQVFOSB_MmFOSB_HsLGDPFPV ---- V S PTS------YTSSFVLTCSPQEDVASPPA------NLTASLFT------FAGAQ---RTSGSEQPHSDEPVLQNVSLPGSDLLPFAPLV----VN FOS_MmPLEASE NOTE: TT YShowingTSSFVFTY PcolorsEADS------on largeFPS CalignmentsAAAHRK-GSSS isNE Pslow.SSDSLSSPTLLAL JUNFOS_Hs ( a ) and ASSRP FOSSGSETARS V(PbDM)D LhumanSGSFYAADWE P(Hs)LHSGSLG MaGPndMAT EmouseLEPFOS_HsFOSB_MmLCTP-VV T C(Mm) T P S C TSA------YT proteins.SSFVFTYSPREDADPPS------NTheLTASLFP TSDNA------CAAAHRK--bindingGSSSNEPSSHSDESV LQdomainSSVLPGTDLLPFAPLV---- VS FOSL2_Hs TPGTSNLVFTYPSVLEQESPASPSESCSKAHRR-SSSSGDQSSDSLNPLEASESPTLLAL NOTE: Showing colors on large alignments is slow. FOS_Mm ASSRPSGSETSRSVPDVDLSGSFYAADWEPLHSNSLGMGPMVTELEPFOS_MmFOS_HsLCTP-VV T C T P G C TTASSYTRSSPSFGVSFETYAPRESAVDPSD------MDLSGSFFYPAASCDAAAWEPHLRHKS-GGSSSSLGMNGEPPMSSATDESLLESSPLPCTLLP-AVVLTCTPSC FOSL2_Mm TPGTSNLVFTYPNVLEQESPSSPSESCSKAHRR-SSSSGDQSSDSLNSPTLLAL ofFOSL2_Hs each protein SSSA--G------is boxed.L DPredictedKAQRSVIKPISIA-GG regionsFY-GEEPFOSL2_HsFOS_MmLHTP IforVV T S T bindingP A V TAPSSGTRSPNSLGVS FEtoTYSPR SMED25VLPEDQVEDSLPSAGSPFSY EAAareSCDSWKEAP HLboxedRRHS-NSSSSSLGMGDPQ MSSandVTDESLLENP SLshadedPCTLLP-AVVLTCTPG C FOSL1_Hs TPFTPSLVFTYPSTP------EPCASAHRKSSSSSGDPSSDPLGSPTLLAL FOSL2_Mm S-SA--G------MDKTQRSVIKPISIAGGGFY-GEEPFOSL2_MmFOSL2_HsLHTPIVVT S T P A I TSSSPGTAS--NLGV------FTYPNVLEQESPSSPLSDEKSACQSRKSAVHIRRKP-ISSSSSIA-GGGDQFSSY-DGSEELNPSLPHTLLPIAVVLTSTPAV FOSL1_Mm TPFTPSLVFTYPSTP------EPCSSAHRKSSSSSGDPSSDPLGSPTLLAL redFOSL1_Hs (JUN) ------or blue (FOS). PTheseAPCRPVPCI SregionsLSPGPVL-EPE AFOSL1_HsFOSL2_MmwereLHTPTLM- TT predicted P S L TSP-FSTAP--SLGV------FT YbasedPSTP------onMDEK PTfittingCQARSAVHIRKKPSSSSSI StheIAGGGGD PFfollowingSSY-DGPEELGPSLPHTLLPIAVVL TSTPAI * .:*:* *.. : *: :*..: ** *.**:**** FOSL1_Mm ------PAPGRPVPCISLSPGPVL-EPEAFOSL1_MmFOSL1_HsLHTPTLM- TT P S L T------PFTPSLVFTYPSTP------EPPACPSSCRAPHVRPKCSSSSSISLSPGDPPVSSL-DEPLEGASLPHTLLPTALLM-TTPSL motif: Φ Ω xxx Ω or Ω xxx ΩΦ , with Ω indicating : FOSL1_Mm* * : any ------aromatic * .:*:* *.. residue, P A P :GΦ R*:PV Pindicating C I S:*..:LSPGP V L**-E P*.**:****E AanyLHTPTL M-TTPSL : * * : hydrophobicFOSB_Hs PSYTSS residueFVLTCPEVSA------, and xF AindicatingGAQ---RTSGSDQPS DanyPLNSP Sresidue.LLAL Importantly, all of these predictions FOSB_Mm PSYTSSFVLTCPEVSA------FAGAQ---RTSGSEQPSDPLNFOSB_HsSPSLLAL PSYTSSFVLTCPEVSA------FAGAQ---RTSGSDQPSDPLNSPSLLAL occurPLEASEFOS_Hs withinNOTE: TA YShowingT SSregionsFVFTY PcolorsEADS ------that on large FhavePS CalignmentsAAAHR previouslyK-GSSS isNE Pslow.SSDSLSSFOSB_Mm PbeenTLLAL assigned PSYTSSFVLTC PasEVSA ------activationFAGAQ ---domainsRTSGSEQPSDP LforNSPS LLJUNAL FOS_Mm TTYTSSFVFTYPEADS------FPSCAAAHRK-GSSSNEPSSDSLSSFOS_HsPTLLA L TAYTSSFVFTYPEADS------FPSCAAAHRK-GSSSNEPSSDSLSSPTLLAL andFOSL2_Hs FOS TandPGTSN LpointVFTYPSVL EmutationsQESPASPSESCSKAH RRof-SSSS PheGDQSS andDSLNPLEASEFOS_MmS PTrpTLLA L residuesNOTE: TT ShowingYTSSFV FinTY Pcolors EtheADS------onfirst largeF PpredictedS CalignmentsAAAHRK-GSSS isN E siteslow.PSSDS LforSSPT LLFOSAL FOSL2_Mm TPGTSNLVFTYPNVLEQESPSSPSESCSKAHRR-SSSSGDQSSDSLNFOSL2_HsSPTLLAL TPGTSNLVFTYPSVLEQESPASPSESCSKAHRR-SSSSGDQSSDSLNSPTLLAL disruptFOSL1_Hs transcripti TPFTPSLVFTYPonalSTP------activationEPCASAHRKSSSSS [5,G D6]PSS.D PLGFOSL2_MmSPTLLAL TPGTSNLVFTYPNVLEQESPSSPSESCSK AHRR-SSSSGDQSSDSLNSPTLLAL FOSL1_Mm TPFTPSLVFTYPSTP------EPCSSAHRKSSSSSGDPSSDPLGFOSL1_HsSPTLLAL TPFTPSLVFTYPSTP------EPCASAHRKSSSSSGDPSSDPLGSPTLLAL * .:*:* *.. : *: :*..: ** *.**:****FOSL1_Mm TPFTPSLVFTYPSTP------EPCSSAHRKSSSSSGDPSSDPLGSPTLLAL * .:*:* *.. : *: :*..: ** *.**:****

PLEASE NOTE: Showing colors on large alignments is slow. PLEASE NOTE: Showing colors on large alignments is slow. (a) + FOS - FOS 0.50 )

[FOS] 0.25 Response (nm

0.00 0 200 400 Time (s)

(b) + JUN/FOS - JUN/FOS 0.50 [JUN/FOS] )

0.25 Response (nm

0.00 0 200 400 Time (s)

Fig. S10 FOS and JUN/FOS have high-affinity interactions with MED25. Sensorgrams correspond to six different concentrations of FOS (a) and JUN/FOS (b) with a single concentration of MED25. Highest sensor is 50 nM of FOS (a) or 50 nM JUN/FOS (b); every sensor thereafter is diluted 1.5-fold from the previous sensor. The bottom sensor, colored gray, corresponds to no FOS or JUN/FOS. These are representative examples of this experiment; three replicates resulted in calculated KD values of 9 ± 3 nM for FOS- MED25 and 5 ± 3 nM for JUN/FOS-MED25 (Table S6). MED25

JUN FOS ETV4

Fig. S11. Model for cooperative recruitment of MED25 by ETV4 and JUN/FOS. The AD of FOS strongly binds to Site 1 of MED25. Sites 2 and 3 of MED25 are available to interact with the DBD of ETV4 and the AD of JUN. In this manner, MED25 could be robustly recruited to ETS-AP1 composite DNA binding sites even when ETV4 is lacking its N-terminal activation domain due to oncogenic chromosomal rearrangements [7, 8]. Supplementary References

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