Genomic and Biochemical Insights into the Specificity of ...

BI80CH20-Graves ARI 20 May 2011 13:19

Genomic and BiochemicalInsights into the Specificityof ETS Transcription FactorsPeter C. Hollenhorst,1 Lawrence P. McIntosh,2

and Barbara J. Graves3,4

1Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405;email: [email protected] of Biochemistry and Molecular Biology, Department of Chemistry, and TheMichael Smith Laboratories, University of British Columbia, Vancouver, British Columbia,Canada V6T 1Z3; email: [email protected] of Oncological Sciences, Huntsman Cancer Institute, University of UtahHealth Sciences, Salt Lake City, Utah 84112; email: [email protected] Hughes Medical Institute, Chevy Chase, Maryland 20815

Annu. Rev. Biochem. 2011. 80:437–71

First published online as a Review in Advance onMay 3, 2011

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev.biochem.79.081507.103945

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4154/11/0707-0437$20.00

Keywords

autoinhibition, ChIP-Seq, DNA binding, ETS domain, PNT domain,protein dynamics

Abstract

ETS proteins are a group of evolutionarily related, DNA-binding tran-scriptional factors. These proteins direct gene expression in diverse nor-mal and disease states by binding to specific promoters and enhancersand facilitating assembly of other components of the transcriptional ma-chinery. The highly conserved DNA-binding ETS domain defines thefamily and is responsible for specific recognition of a common sequencemotif, 5′-GGA(A/T)-3′. Attaining specificity for biological regulationin such a family is thus a conundrum. We present the current knowledgeof routes to functional diversity and DNA binding specificity, includ-ing divergent properties of the conserved ETS and PNT domains, theinvolvement of flanking structured and unstructured regions appendedto these dynamic domains, posttranslational modifications, and proteinpartnerships with other DNA-binding proteins and coregulators. Thereview emphasizes recent advances from biochemical and biophysicalapproaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells.

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Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 438Evolution of the ETS Family . . . . . 439Biochemical and Biological

Diversity . . . . . . . . . . . . . . . . . . . . . . 439Extensive Coexpression . . . . . . . . . . . 440

ETS DOMAIN: BIOCHEMICALOVERVIEW OF DNA BINDNGAND AUTOINHIBITION . . . . . . 441Structure and DNA-Binding

Interface . . . . . . . . . . . . . . . . . . . . . . 441DNA-Sequence Preferences . . . . . . 442Structural Elements Appended

to the ETS Domain . . . . . . . . . . . 443Autoinhibition of ETS1 DNA

Binding: A DynamicMechanism . . . . . . . . . . . . . . . . . . . 444

Autoinhibition in Other ETSProteins . . . . . . . . . . . . . . . . . . . . . . 446

PNT DOMAIN: DIVERSEFUNCTION ANDREGULATION . . . . . . . . . . . . . . . . . 448PNT-Domain Structure and

Appended Elements . . . . . . . . . . . 448Ras Signal Transduction and

MAP-Kinase Docking . . . . . . . . . 448Role of the PNT Domain in

Phosphorylation-EnhancedInteractions of ETS1and CBP . . . . . . . . . . . . . . . . . . . . . . 449

PNT-Domain Self-Association. . . . 450GENOMIC APPROACHES TO

THE SPECIFICITYQUESTION: REDUNDANTAND SPECIFIC BINDING . . . . . 450Redundant Binding of ETS

Proteins in Living Cells . . . . . . . . 451Specific Binding of ETS Proteins

in Living Cells . . . . . . . . . . . . . . . . 453COOPERATIVE PARTNERSHIPS

PROVIDE ETS FAMILYSPECIFICITY: GENOMICAND BIOCHEMICALPERSPECTIVES . . . . . . . . . . . . . . . . 454Ternary Complex Factors/Serum

Response Factor . . . . . . . . . . . . . . . 454SPI1/IRF4 . . . . . . . . . . . . . . . . . . . . . . . 455ETS1/RUNX1 and

Autoinhibition . . . . . . . . . . . . . . . . 455ETS1/PAX5 . . . . . . . . . . . . . . . . . . . . . 456ETS/FOX . . . . . . . . . . . . . . . . . . . . . . . 456ETV6 Self-Association and

Autoinhibition . . . . . . . . . . . . . . . . 456Other Dimeric Configurations . . . . 457

GENOMIC INSIGHTS INTOONCOGENIC ETSFACTORS . . . . . . . . . . . . . . . . . . . . . . 458ERG and Androgen Receptor in

Prostate Cancer . . . . . . . . . . . . . . . 458EWS-FLI1 in Ewing’s Sarcoma . . . 458

ETS domain:conserved structuraldomain in all ETSproteins that isnecessary andsufficient for DNAbinding

PNT domain:structural domainconserved inapproximatelyone-third of ETSproteins, whichfunctions in proteininteractions andresembles the SAMdomain

INTRODUCTIONAn overarching conundrum for eukaryotictranscription factors is how a set of relatedproteins displaying highly conserved DNA-binding domains execute diverse biologicalroles. This review addresses the current state ofbiochemical and genomic investigation of thisspecificity question in the ETS family. Initially,we focus on the common and distinct DNA-binding properties of the ETS domain. Next,we discuss the PNT domain to illustrate thepotential of semiconserved domains to providea route to diverse function by different family

members. Then, we consider new studies thatprovide the first views of what is happening incells at the level of genome-wide ETS-factorDNA occupancy. We include genomic analysisof oncogenic ETS genes as these studieschallenge our thinking about specificity andredundancy. Both biochemical and genomicapproaches demonstrate the role of proteinpartnerships as a specificity mechanism. Weemphasize systems for which biochemical andbiophysical details are available because theseprovide a robust framework both for definingthe specificity problem and informing the many

438 Hollenhorst · McIntosh · Graves

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SRF: serum responsefactor

Intrinsicallydisordered proteinsequences: regions ofproteins that arebiologically active, yetlack a well-definedthree-dimensionalstructure underphysiologicalconditions

experimental investigations aimed at under-standing the role of ETS factors in biologicalregulation. To complement our structural andgenomics focus, we refer the reader to previousreviews that cover biological properties of theETS factors in more detail (1–6).

Evolution of the ETS Family

The ETS proteins are encoded by a genefamily that is defined by the conserved ETSdomain (Figure 1a,b) and present throughoutthe metazoan phyla (7). Family phylogenysuggests the duplication of an ancestral geneearly in metazoan evolution. The continuedgrowth of the family from invertebrates,represented by the 8 and 10 paralogs inDrosophila melanogaster and Caenorhabditiselegans, respectively (Supplemental Table 1:For all Supplemental Material, follow thelink from the Annual Reviews home page athttp://www.annualreviews.org), to verte-brates, represented by 28 human genes, waslikely facilitated by large-scale duplicationsof vertebrate genomic regions (8). Hallmarksof these evolutionary steps are seen in thedendogram of the family (Figure 2) as well asin the relative positions of structural domains(Figure 1a).

An understanding of the overall relatednessof ETS family members is critical for fram-ing the question of functional specificity. Forexample, there are subfamilies with higher de-grees of sequence similarity in both their ETSdomains and PNT domains (9) (Figures 1a

and 2). Closely related members of a subfam-ily may display redundant functions, whereasthose in different subfamilies may have uniquebiochemical properties that could be utilized indistinct biological pathways.

Biochemical and Biological Diversity

Structural or functional domains and posttrans-lational modifications that are found outside ofthe highly conserved ETS domain illustrate thepotential for unique properties of subfamiliesor individual ETS proteins. The PNT domain

(Figure 1a,c) has variable functions, discussedbelow, depending on the subfamily in whichit resides. The OST domain appears unique toGABPA (Figure 1a,d ) and serves in cofactorrecruitment (10). The B-box, found in thethree members of the ternary complex factor(TCF) subfamily, forms an induced structuralelement that interacts with serum responsefactor (SRF) on DNA (11). Although examinedin only a few cases by biophysical methods(12–15), the remaining regions of the ETSfactors, including those functioning as transac-tivation domains, likely display minimal stablesecondary or tertiary structure. These intrin-sically disordered regions can have numerouspossible roles, including serving as flexiblelinkers, linear peptide motifs for binding topartner proteins, and sites of posttranslationalmodifications (16). Figure 1 is annotated toshow only those examples of phosphorylationthat are covered in this review owing to theirinterplay with structured domains. Othermodifications found on ETS factors, includingubiquitinylation, sumoylation, glycosylation,and acetylation, have been reviewed elsewhere(17, 18). In conclusion, the overall diversifi-cation of the known biochemical propertiesof the family members suggests that differentfamily members will play distinct biologicalroles.

Diverse biological roles of individualETS family members are also suggested bythe broad range of phenotypes displayedin mouse genetic disruptions (Figure 2).Genetic studies in C. elegans (19) and Drosophila(20) demonstrate this same trend, with avariety of phenotypes suggesting specializedfunctions ranging from organ cell fate andneuronal development to oogenesis and aging(Supplemental Table 1) (21–26). Althoughclassic genetics approaches can be limitedas a result of potential redundancy withinthe family, this diversity strongly indicatesthat different ETS family members carry outdistinct biological roles. Indeed, recent reportsthat include analyses of transcriptional targetsimplicate specific ETS genes in development ofmammalian hematopoietic stem cells (27–29),

www.annualreviews.org • ETS Family of Transcription Factors 439

Supplemental Material

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endothelial cells (30–32), and T cells (33).Although many putative transcriptional targetshave been identified over the past 20 years forETS family members through experimentalanalyses of transcriptional control elements byin vitro and cell-based assays (3), we focus onlyon genome-wide occupancy approaches andevaluate their contribution to the evaluation ofthe authentic targets of ETS factors.

Extensive CoexpressionIn spite of the biological and biochemical evi-dence that different ETS proteins can executediverse roles, an extensive overlap in the ex-pression of various family members is found inmany human tissues. Coexpression of the ETSgenes has been systematically surveyed at themRNA level in more than 20 tissues and trans-formed cell lines (34–35). Half of the family

ETV1 (ER81)

ETV4 (PEA3, E1AF)

ETV5 (ERM)

FEV

ERF

ETV3 (PE1, PEP1, METS)

ETV3L

ETV2 (ER71)

ELF1

ELF2 (NERF)

ELF4 (MEF)

SPIB

SPIC

ETV6 (TEL)

ETV7 (TEL2)

FLI1

ERG

ELF3 (ESE1, ESX)

ELF5 (ESE2)

EHF (ESE3)

SPDEF (PDEF)

ETV6/RUNX1 (TEL/AML1)

EWS/FLI1

GABPA (GABPa, E4TF, ELG)

Human ETS family

ETS fusion oncoproteins

ELK1 P

ELK3 (NET, SAP2, ERP) P

ELK4 (SAP1) P

a

G

G

A A

NC

b

N

C

c

N

C

d

ETS1 P P

ETS2 P P

SPI1 (PU.1, Sfpi1) P

ETS domain PNT domain OST domain B-box C-terminal RUNX1 N-terminal EWS

ETS1 ETS domain

ERGPNT domain

GABPAOST domain

Carbon Oxygen Nitrogen


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members are ubiquitously expressed in all celltypes tested. In any particular cell type, 14to 25 family members are coexpressed. Thischallenges the simplest notion that each familymember is restricted to a tissue or cell type fora site of action. In some cases, however, high-level expression is confined to a single cell type(e.g., SPDEF in the prostate, SPI1 in myeloidcells). Additional levels of regulation beyondmRNA expression, such as translational con-trol, alternative splicing, posttranslational mod-ifications, cellular localization, and protein sta-bility, will add to the complexity of this picture.Therefore, systematic proteomic studies thatdetermine active protein levels will be neces-sary to describe the relative prevalence of ETSfactors in diverse tissues.

Expression profiles in transformed cell linesshow strong similarity to those in matching nor-mal tissues, although exceptions are worth not-ing. There is high expression of SPDEF andESE subfamily members in mammary tumors(35), and a striking prevalence of ETV4 expres-sion in cell lines versus normal matching adulttissues (34). The high expression levels of ERGand PEA3 subfamily members in a subset ofprostate tumors led to the discovery of com-mon chromosomal rearrangements associatedwith this human cancer (36). Thus, relative dif-ferences in expression of ETS genes may havea significant role in both normal biology andcancer.

ETS DOMAIN: BIOCHEMICALOVERVIEW OF DNA BINDNGAND AUTOINHIBITION

Structure and DNA-Binding Interface

The overall structure and DNA-bindingmechanism of the ETS domain is based onhigh-resolution molecular models of ten familymembers derived from both X-ray crystal-lography and nuclear magnetic resonance(NMR) spectroscopy (Figure 1b, Supple-mental Table 2) (37–51). The conserved ETSdomain consists of three α-helices on a smallfour-stranded, antiparallel β-sheet scaffold.This variant of the prototypic helix-turn-helixDNA-binding motif, termed the wingedhelix architecture, is also found in eukaryoticFOX proteins, heat shock factors, and linkerhistones as well as the prokaryotic CAP protein(52). The ETS domain binds DNA over aregion spanning 12 to 15 base pairs (bp), butit displays sequence preference for only ∼9 bp(numbered 1–9 in this review) with a central,invariant 5′-GGA(A/T)-3′ core. The interfaceis characterized both by direct readout, whichinvolves base-pair contact by ETS-domainsidechains, and indirect readout, which utilizesthe sequence-dependent positioning of thephosphodiester backbone. The most importantdirect interactions in the major groove involvehydrogen bonding between the two invariantarginines in the recognition helix H3 and the

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1Structural and functional domains of the ETS family of transcription factors. (a) Nomenclature and domain organization of the 28paralogous human ETS proteins (grouped according to the dendogram of Figure 2). We use HUGO nomenclature (191) for all ETSgenes and proteins, with alternative names provided. Oncogenic ETS proteins, which are created by human chromosomerearrangements, are illustrated by two examples. Many ETS genes produce multiple protein products via alternative splicing/start sites,and in these cases, a single polypeptide was chosen arbitrarily. Boxes identify the DNA-binding ETS domain (red ), PNT domain( green), OST domain (blue), and B-box (magenta) of the ETS factors as well as the C-terminal portion of RUNX1 (light brown) and theN-terminal portion of EWS (dark brown). The circled P symbolizes a phosphorylated region discussed in the text. Not identified areadditional regions involved in transcriptional activation/repression, posttranslational modifications (including kinase docking andsumoylation), nuclear import/export, turnover, and so forth. (b) Ribbon diagram of the ETS1 ETS domain bound to a DNA duplex(1k79.pdb with the inhibitory helices deleted for clarity). The side chains of R391, R394, and Y395, which provide base-specificcontacts to core GGAA, are also shown in stick format. Ribbon diagrams of (c) the ERG PNT domain (1sxe.pdb) and (d ) the GABPAOST domain (1juo.pdb). See Supplemental Tables 2 and 3 for a referenced summary of all published ETS and PNT domainstructures. Molecular diagrams were rendered with PyMOL (http://pymol.org) with coordinate files from the Protein Data Bank(http://pdb.org).



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http://pdb.org



PEA3

TCF

ELF

ESE

SPI

TEL

ETS

ERG

ERF

Defective in mouse deletionETS geneETS subfamily

SPI1 Lethal, myeloid, B cells (197-203)

SPIB B cells (204)

SPIC Red pulp macrophages (205)

ETV6 Yolk sac, hematopoietic stem cells (206,207)

ETV7 N/A, no mouse ortholog ELF3 Intestine (208)

EHF N/AELF5 Lethal, embryo patterning, mammary gland (209,210)

ELF4 NK cells, NK-T cells (211)

ELF2 N/AELF1 No defect (212)

SPDEF Intestine (213,214)

ETV1 Neuronal connections (215)

ETV5 Male fertility (216)

ETV4 Male fertility, motor neurons, mammary gland (217,218)

ELK3 Vasculature (219)

ELK4 Thymocytes, peripheral T cells (220)

ELK1 No defect (221)

ETS1 T cells, NK cells, NK-T cells (159,222,223)

ETS2 Placenta, hair follicles (224)

ETV2 Lethal, blood, vasculature (225,226)

ETV3 N/AN/AETV3L

ERF Placenta (227) ERG Definitive hematopoiesis* (194)

FLI1 Vasculature, megakaryocytes, B cells (228-231)

FEV 5-HT neurons (232,233)

GABPA Lethal, T cells (234)

Class I Class II Class III Class IV

Figure 2Diversity in function of ETS family. (left) A dendogram shows the degree of relatedness of 28 human ETS domain sequences, built byClustalW (192) using the neighbor-joining method (193). The length of each horizontal line indicates estimated evolutionary distance.Branches that separate an individual subfamily are labeled in red. Classes defined by differences in the in vitro derived binding site (62)are indicated by background colors. (right) A list of selected tissues or functions that are defective in each mouse gene deletion, ascataloged at http://www.knockoutmouse.org. See also citations 159 and 197–234, which are linked to defects in mouse deletion infigure. For the sake of brevity, some phenotypes are not listed. N/A indicates the gene deletion has not been reported. The asteriskindicates that this phenotype results from a point mutation of ERG that affects transactivation ability rather than a gene deletion (194).Mice with deletions or mutations of two members of a subfamily have phenotypes that indicate overlapping roles in endothelial cells(ETS1 and ETS2) (30), hematopoietic stem cells (ERG and FLI1) (28), thymocyte development (ELK1 and ELK4) (33), B-cellreceptor signal transduction (SPI1 and SPIB) (195), and limb-bud development (ETV1 and ETV5) (196).

two guanines of the GGA(A/T) core. Addi-tional direct and water-mediated hydrogenbonding, hydrophobic, and electrostatic inter-actions involved in the interface are providedprimarily by residues in the “turn” between he-lices H2 and H3, the “wing” between strands S3and S4, and the N terminus of helix H1. Strik-ingly, there are no direct contacts to any basepair outside of the central GGA(A/T), thus im-plicating sequence-dependent backbone con-figurations for specificity in the flanking regions(53). Indeed, thermodynamic studies revealedan ∼400-fold variation of the affinity of SPI1depending on sequences flanking the GGA

core (54). The structural basis for this indirectreadout is currently not well defined, althoughDNA duplexes bound by ETS domains are typ-ically distorted from an ideal B-DNA geometry.

DNA-Sequence Preferences

The selectivity of multiple ETS proteins hasbeen determined by in vitro methods thatdemonstrate a binding preference for a partic-ular DNA sequence. Ten proteins were assayedin independent experiments in a variety oflaboratories that selected preferred sequencesfrom pools of DNA duplexes, using either the


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SELEX (systematic evolution of ligands byexponential enrichment) or length-encodedmultiplex approach (53, 55–61). More recently,26 mouse and 27 human ETS proteins weresubjected to either microwell binding assaysor protein-binding microarrays to determinesequence preferences (62). The results of suchanalyses performed by different laboratoriesand across the entire ETS family are strik-ing in their similarity. The most commonhigh-affinity, consensus sequence identified,ACCGGAAGT, consists of an invariant GGAcore surrounded by positions with lowerpreference (see also Genomic Approaches tothe Specificity Question, section below).

Sequence preferences observed in system-atic analysis of the whole family suggestfour classes of ETS proteins that track withsubfamilies (Figure 2) (62). Class I, con-taining more than half of the ETS family(PEA3, TCF, ETS, ERF, and ERG subfam-ilies), displays a consensus sequence identicalto the most common in vitro–derived sequence(ACCGGAAGT). Class II contains eight mem-bers (TEL, ESE, and ELF subfamilies) and hasa consensus that differs primarily in the firstnucleotide (CCCGGAAGT). Members of thissecond group tend also to be more sensitiveto the adenine-to-thymine substitution at po-sition 7, as has been previously reported forELF1 (63–65). However, variation in the affin-ity for the alternative GGAT core can also oc-cur between Class I members, such as ELK1and ELK4 (58). Class III is composed of onlythe SPI subfamily, whose members prefer bind-ing sites with an adenine-rich sequence 5′ tothe GGA (57). Class IV includes only SPDEF,which has a unique preference for a GGATcore sequence instead of GGAA (66). Quan-titative DNA-binding studies indicate that theaffinity of different ETS proteins for the sameconsensus sequence many vary by only ∼10-fold, whereas the affinity of the same proteinfor variant consensus sites differs by ∼5- to 80-fold (53, 56, 65).

A rationale for some sequence preferencescomes from detailed biochemical investi-gations. For example, two TCF subfamily

members, ELK1 and ELK4, have identicalDNA-recognition helices, but detailed crys-tallographic and mutagenic studies revealedthat residues at the DNA-binding interface,including a key tyrosine in helix H3, differconformationally owing to distinct tertiarycontacts with adjacent, nonconserved residues(41, 42). Collectively, these effects restrict theability of ELK1, but not ELK4, to bind vari-ants of the ETS consensus sequence includingGGAT.

In conclusion, the differences in DNA-sequence preference of most ETS proteinsidentified from in vitro studies are subtle. Fur-thermore, these sequences, which were selectedowing to their relative affinity in vitro, could oc-cur rarely in transcriptional-control elementsas a result of this lack of selectivity. Speci-ficity in cells could derive predominantly frominteractions with cofactors, including partnertranscription factors, as discussed below. Loweraffinity sites that vary from the high-affinityselected sequences could provide more speci-ficity by poising ETS factors to be dependenton these cofactors.

Structural Elements Appendedto the ETS Domain

Structural elements appended to the ETS do-main regulate its function and demonstrate di-versification in the family (Figures 3a and 4).In several ETS proteins, a short α- or 310-helix, or in the case of ELF3 an ordered coilsequence, lies immediately C terminal to the fi-nal β-strand of the ETS domain. This may bean even more common feature than currentlyrecognized because the analogous C-terminalregions have not been characterized structurallyfor all family members. These flanking residuescontribute to the tertiary structure and stabil-ity of several ETS domains (51, 67). GABPA,ETS1, ETS2, and ETV6 have two C-terminalhelices, termed H4 and H5. In the case ofGABPA, these serve as a platform for bind-ing GABPB1 (GABPβ), which indirectly en-hances DNA affinity, slowing the half-time fordissociation by at least 100-fold (Figure 4a)


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H1 S1 S2 H2 H3 S3 S4 H4 H5HI-2HI-1

H0 H1 H2 H3 H4 H5H2'

ETS domain

**

ETS1, ETS2

GABPA, ETV6

ELK4

FLI1, SPI1, SPDEF, ELF3, ELF5, ELK1

ERG, FLI1, ETV6, Yan

GABPA, SPDEF

ETS1, ETS2, Pnt-P2

PNT domain

SAM domain

H1

S1

S2

H2

H3S3

S4

H4

H5

HI-2

HI-1

H0

H1

H2H3

H4

H5

H2'

**

a

b

SRR

N

PNT

C

N

ETS

Figure 3Appended structures to the conserved ETS and PNT domains expand regulatory potential. (a) The core ETS domain (red ) containsthree α-helices (rectangles) on a four-stranded, antiparallel β-sheet (arrows). Appended N- and C-terminal helices (cyan), as well as anordered C-terminal coil region in ELF3 (not shown) and dynamic sequences such as the serine-rich region (SRR) of ETS1, provideadditional functional diversity. Shown (right) is a ribbon diagram of residues 301–441 of ETS1 (1r36.pdb), with the appended helices(cyan) packed against helix H1 of the core ETS domain. Helices HI-1, HI-2, H4, and H5 inhibit DNA binding of ETS1. Helix HI-1,which is distal from the recognition helix H3, unfolds upon DNA binding as part of the autoinhibition mechanism (Figures 4c,d and5a). (b) The core PNT domain ( green) consists of four α-helices and a small α- or 310-helix (H2′) with a fold similar to that of the SAMdomain. Although not shown for clarity, residues preceding H2 form a conserved helical-like turn. Again, appended N-terminal (H0,H1) helices (orange) provide additional routes to functional diversity. Shown (right) is the ribbon diagram of residues 29–138 of ETS1(2jv3.pdb), including the PNT domain, which constitute a docking site for both MAP kinase and the TAZ1 domain of an ETS1cofactor, CBP (Figure 5b). Two conserved phosphoacceptors (asterisk) preceding the dynamic helix H0 of ETS1 are indicated. SeeSupplemental Tables 2 and 3 for a referenced summary of all published ETS and PNT domain structures. The codes shown are fromthe Protein Data Bank (http://pdb.org).

(40). The two appended C-terminal helices inETV6 participate in DNA-binding autoinhi-bition, likely via a steric mechanism (50). InETS1 and, by sequence similarity, ETS2 andtheir Drosophila ortholog Pnt-P2, two more he-lices, HI-1 and HI-2, reside N terminal to theETS domain and pack with helices H4 and H5(Figure 3a, right) (45, 48, 68). The resultinghelical bundle is central to the allosteric au-toinhibition of DNA binding by these familymembers (discussed below).

Autoinhibition of ETS1 DNA Binding:A Dynamic MechanismSeveral ETS proteins exhibit autoinhibition ofDNA binding. Diagnostic of this phenomenonis a reduction in the overall affinity for DNA ofthe full-length protein relative to smaller frag-ments that retain the ETS domain. Autoinhi-bition offers many routes for the regulation ofproteins, including alternative splicing or pro-teolysis that would remove inhibitory elements,and posttranslational modifications or protein



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ETS domain

Appended helices

B-box

Partner

Monomer units of ETV6 polymer

GABPA / GABPB1

ETS1 / PAX5

ETV6 / ETV6 / ETV6

ELK4 / SRF

ETS1 / ETS1

a

c

b

e

d

f

SPI1 / IRF4

Figure 4Structurally characterized ETS protein partnerships. Shown are molecular models of the ETS partnerships involving the ETS domainsof (a) GABPA with GABPB1 (1awc.pdb), (b) ELK4 with serum response factor (SRF) (Ihbx.pdb), (c) ETS1 with PAX5 (1 mdm.pdb),(d ) the ETS1 homodimer (2nny.pdb), and (e) SPI1 with IRF4 (46). In each case, the ribbon diagrams are colored as ETS domain (red ),appended helices (cyan), B-box (magenta), and partner ( yellow). ( f ) Three monomer units of an ETV6 PNT domain polymer associatedhead-to-tail ( greens; 1ji7.pdb). See Supplemental Tables 2 and 3 for a referenced summary of all published ETS and PNT domainstructures. The codes listed are from the Protein Data Bank (http://pdb.org).


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partnerships that can either reinforce or antag-onize inhibition (69).

The best-characterized example of au-toinhibition among the ETS proteins is thatof ETS1, in which all the above regulatorymechanisms are at play. Autoinhibition ofETS1 DNA binding was discovered initiallyby deletion analyses (56, 70, 71). However,two naturally occurring variants confirm thebiological relevance of inhibitory sequences.The oncogenic version of ETS1 found in thep135 oncoprotein of the E26 retrovirus has theC-terminal inhibitory sequences deleted. Itshigher DNA-binding activity may contributeto oncogenesis (72). Likewise, an alternativelyspliced ETS1 isoform, which lacks the N-terminal inhibitory sequences encoded by exonVII, binds DNA with higher affinity and slightlybroader specificity (73). As discussed below,ETS1 autoinhibition is reinforced by phospho-rylation (74, 75) and counteracted by proteinpartnerships, such as those with RUNX1 (alsotermed AML1/CBFα2) and PAX5.

Detailed analyses implicate the three struc-tural components of ETS1 in the mech-anism of autoinhibition, namely the ETSdomain, the appended inhibitory module(Figure 3a), and an adjacent intrinsically dis-ordered serine-rich region (SRR) (Figure 5a).The inhibitory module comprises a helical bun-dle (HI-1, HI-2, H4, and H5) and interfaceswith H1 of the ETS domain. Collectively,these two components, the inhibitory moduleand ETS domain, are described as the regu-latable unit of ETS1. The basic framework ofETS1 autoinhibition was established by earlystudies demonstrating that HI-1 unfolds uponDNA binding, thus suggesting a thermody-namic penalty to reduce net DNA affinity (76).Subsequent structural analyses showed that theinhibitory module is distal to the DNA-bindinginterface of ETS1, indicative of an allostericmechanism linking binding and helix unfolding(45, 48).

More recent NMR-based studies have re-vealed that dynamic properties across a widetimescale range are central to ETS1 autoinhibi-tion (Figure 5a). Helix HI-1 is only marginally

stable and thus poised to unfold. The recogni-tion helix H3 also shows significant local fluc-tuations (48) supporting the hypothesis thatDNA-binding domains, in general, requireflexibility for high-affinity DNA recognition(77, 78). Furthermore, a network of hydropho-bic residues spanning from helix HI-1 to H3 un-dergoes millisecond to microsecond timescalemotions (79). Most importantly, the ETS do-main appended with all four inhibitory helicesbinds DNA with half the affinity of the isolatedETS domain, whereas the presence of an adja-cent SRR reduces binding 10- to 20-fold. Moststrikingly, the SRR is intrinsically disorderedand interacts only transiently with the ETS do-main and inhibitory module (80).

Collectively, these data lead to the currentmodel of ETS1 autoinhibition in which theregulatable unit exists in a conformationalequilibrium between a flexible state competentfor DNA binding and a more rigid inactivestate (Figure 5a). Transient interactions withthe SRR allosterically favor the latter state.Increasing levels of multisite phosphorylationof the SRR by CaM kinase II leads to aprogressive reduction in DNA affinity bypromoting these transient interactions (79).Consistent with in vivo studies that show a rolefor phosphorylation (81), this rheostatic mech-anism provides a graded control of ETS1 DNAaffinity (82). Although the mechanism linkingHI-1 unfolding with DNA binding is notreadily apparent in static molecular structures(45), molecular dynamics simulations point tocorrelated motions involving HI-1, H4, andH1 that change upon hydrogen bonding of H1to the phosphodiester backbone of DNA (83).This theoretical evidence for a conduit fromthe DNA interface through the regulatableunit is consistent with the dynamics of thisregion observed in the absence of DNA byNMR.

Autoinhibition in Other ETS Proteins

Other ETS factors also exhibit DNA-bindingautoinhibition. A C-terminal inhibitory se-quence, bearing helix H5, strongly attenuates


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P

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PP

PP

PPPP

P

HI-1

HI-2

HI-1

HI-2

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helicalbundle

Appendedinhibitory module

Appendedhelices

Figure 5Dynamic properties of the ETS and PNT domains regulate ETS1 function. (a) ETS1 autoinhibitioninvolves a conformational equilibrium between a rigid inactive state and a flexible active state. Upon DNAbinding, helix HI-1 unfolds. Transient phosphorylation-dependent interactions of the unstructuredserine-rich region (SRR) with the regulatable unit, formed by the core ETS domain (red ) and the appendedinhibitory module (cyan), stabilize the inactive state (80). This stabilization increases progressively withincreasing levels of CaM kinase II multisite phosphorylation of the SRR, leading to rheostatic control ofETS1 DNA binding (79). (b) The PNT domain of ETS1 interacts with the TAZ1 domain of CBP.Phosphorylation of ETS1 causes a conformational change and increases the affinity of the interaction (97).The ETS1 PNT domain consists of a core helical bundle ( green) with appended helices H0 and H1 (orange).MAP kinase phosphorylation of Thr38 and Ser41 shifts a conformational equilibrium of the dynamic helixH0 toward a more open state. This conformational switch, along with increased complementary electrostaticinteractions, favors binding of the TAZ1 domain ( yellow). Note that the structure of the PNT-TAZ1complex has not been determined, and though not shown in panel a or b, both the unmodified andphosphorylated forms of ETS1 can bind CBP and DNA, respectively.

ETV6 DNA binding by steric blockage ofits ETS domain DNA-binding interface(Figure 3a) (50). Members of the PEA3subfamily, including ETV4 (12, 84) andETV5 (85), contain autoinhibitory sequencesappended to both sides of their ETS domains.However, based on proline-scanning muta-genesis, these sequences do not appear to behelical. A cooperative partnership with USF-1enhances the affinity of ETV4 for DNA, at

least in part by interacting with these inhibitorysequences (84).

In cases of the ESE and TCF subfamilies,DNA binding is repressed by sequences dis-tant from the ETS domain. The C-terminalETS domain of ELF3 is inhibited by its cen-tral transactivation domain (86), whereas re-lated ELF5 is inhibited by sequences at its Nterminus (87). The TCF subfamily members,ELK1, ELK3, and ELK4, exhibit even more


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MAP kinase:mitogen-activatedprotein kinase

complex inhibitory behaviors involving an in-terplay of the B-box, the C-terminal transac-tivation domain, and a NID (NET inhibitorydomain), as well as interactions with the helix-loop-helix Id proteins (88–90). Furthermore,phosphorylation of the transactivation domainresults in enhanced DNA binding (91).

An interesting question is whether theautoinhibition mechanisms in these otherETS proteins have features in common withETS1. A critical feature of autoinhibition inETS1 is the dynamic character of the ETSdomain. The dynamic character of DNA-binding domains, in general, is proposed to benecessary for nonspecific scanning of DNA insearch of specific, high-affinity sites (77, 78),with motions quenched upon specific complexformation. Thus, all ETS domains could havethese dynamic properties. Different appendedinhibitory elements of diverse ETS factorscould impinge on these dynamic propertiessimilarly to how the inhibitory helices of ETS1regulate its motions. Importantly, autoinhi-bition sets up the potential for regulation ofDNA binding through protein partnerships, asdescribed below.

PNT DOMAIN: DIVERSEFUNCTION AND REGULATION

PNT-Domain Structure andAppended Elements

Approximately one-third of ETS familymembers encode a second structured do-main, termed the PNT domain (Figure 1a).This ∼80-residue conserved domain playsunique roles in different ETS proteins.High-resolution molecular models of the PNTdomain are derived from both X-ray crystallog-raphy and NMR spectroscopy performed onone Drosophila and multiple vertebrate proteins(Supplemental Table 3) (92–97). The coreof this domain is formed by four α-helices(H2–H5) along with a short 310- or α-helix(H2′) (Figures 1c and 3b), which is highlysimilar to that of the more widespread SAMdomain. SAM domains exhibit remarkablydiverse association states and function in a

variety of protein-protein and -RNA inter-actions (98). Similar to the ETS domain,additional helical elements (H0 and H1)appended to the conserved-core of the PNTdomain provide unique regulatory paths fordistinct family members (Figure 3b).

Ras Signal Transduction andMAP-Kinase Docking

The transcriptional activation by vertebrateETS1 and ETS2 and the Drosophila orthologPnt-P2 of Ras-responsive genes is increased bymitogen-activated protein (MAP) kinase phos-phorylation of threonine and serine residuesimmediately preceding their highly similarPNT domains (68, 99). Detailed in vitro ki-netic studies revealed that the PNT domainsfacilitate signaling by enhancing substrate bind-ing (ETS1 or ETS2 to the MAP kinaseERK2 and Drosophila Pnt-P2 to the ortholo-gous Rolled kinase) rather than altering thekinetic properties of the enzymes (100–102).Thus, the PNT domains are docking mod-ules, interacting with complementary dock-ing sites on the kinases (103, 104). This pre-sumably increases the effective local concen-tration of the adjacent phosphoacceptors atthe catalytic sites of the enzymes, thereby en-hancing both the specificity and rate of theirmodification (105).

Although a detailed structural model ofa PNT-domain/MAP-kinase complex hasnot been solved, insights into the interfaceshave been obtained from mutagenesis studies,chemical footprinting, and competition exper-iments with peptides that bind known dockingsites on kinases. Key to PNT-domain bindingare residues from helices H2, H4, and H5 sur-rounding an exposed phenylalanine side chain(100, 102). Thus, unlike the well-characterizedlinear peptide motifs that bind docking sites onkinases (106, 107), the PNT domains interactvia a three-dimensional surface. Conversely,this region of the ETS1 PNT domain has beenmodeled to associate with the ERK2 substrate-binding groove centered near loop 12 and theαG helix (108). Additional contacts involving



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the unstructured N-terminal tail of ETS1 withthe common D-recruitment site on the kinasealso have been proposed (109, 110). To bothaccommodate this docking mode and positionthe phosphoacceptors in the active site of thekinase, a structural change in the PNT domain,such as the unfolding of helix H0, would berequired. Indeed, as discussed below, this helixis dynamic and forms part of a phospho-switchmechanism for CBP recruitment.

In contrast, members of the TCF subfam-ily (ELK1, ELK3, and ELK4) lack a PNT do-main and dock on the D- and F-recruitmentsites of MAP kinases via linear peptide se-quences (88, 89). These sequences, termedthe D- and FxFP-motifs, respectively, flankthe phosphoacceptor-containing transactiva-tion domains of the TCF factors. In additionto increasing phosphorylation efficiency and di-recting acceptor specificity, these motifs differ-entially target TCF subfamily members to theMAP-kinase isoforms ERK2, JNK, or p38. Theresulting phosphorylation regulates the TCFproteins at the levels of DNA binding, coac-tivator or -repressor recruitment, and cellularlocalization (2, 88, 91, 111).

Role of the PNT Domain inPhosphorylation-EnhancedInteractions of ETS1 and CBP

The mechanism by which Ras/MAP-kinasesignaling enhances ETS1-regulated geneexpression via phosphorylation-enhancedrecruitment of the general transcriptionalcoactivator CBP has been characterized re-cently using cell-based assays and biophysicalmeasurements (65, 97, 112). ERK2 phosphory-lates Thr38, as well as a nonconsensus acceptorSer41, within the unstructured N-terminalregion of ETS1, immediately adjacent to itsPNT domain. NMR spectroscopic studiesrevealed that, in addition to the four α-helicesthat match the SAM domain (H2-H5), thePNT domain of ETS1 is appended with twoadditional helices (H0-H1) (Figure 3b). Im-portantly, helix H0 is only marginally stable and

undergoes significant local structural fluctua-tions (97).

Dual phosphorylation perturbs a “closed-open” conformational equilibrium of the PNTdomain, displacing the dynamic helix H0 fromthe core bundle (Figure 5b) (97). These mod-ifications increase the affinity of ETS1 for theTAZ1 (or CH1) domain of CBP by ∼30-fold(97). NMR-monitored titration experimentsmapped TAZ1 binding to both the phospho-acceptors and helices H0, H2, and H5 of theETS1 PNT domain. Reciprocal experimentsmapped the interaction surface of the TAZ1domain bound by ETS1. This same CBP re-gion is also implicated in several peptide modelsof transactivation domains bound to the TAZ1domain (113). The charge complementarity ofthese surfaces indicates that electrostatic forcesact in concert with the conformational equi-librium to mediate phosphorylation effects. Asdiscussed above, the same interface of the PNTdomain also serves as a docking site for theERK2 kinase. Thus, the PNT domain of ETS1is key to relaying signal transduction, both“accepting” and “transmitting” the effects ofphosphorylation.

As is the case for many transcription factors,other ETS proteins function in associationwith CBP or its closely related coactivator,p300. GABPA contains an OST domain,which displays a ubiquitin-like fold andbinds the TAZ1 and TAZ2 domains of CBP(Figure 1a,d ) (10). ELK1 binds p300, andRas/MAP-kinase-dependent phosphorylationwithin a transactivation domain enhances theinteraction (89, 114–116). In addition, directinteractions between CBP and p300 havebeen reported for SPI1 (117–118) and ETV1(119, 120). Note, however, ELK1, ETV1, andSPI1 have no PNT domain (Figure 1a), andthe structural bases for CBP or p300 bindingremain to be established. The Mediator hasalso been identified as a coactivator for theTCF subfamily (121–124). The interplay ofmultiple coactivators for transcription factors,including the ETS proteins, remains an areaof current investigation.


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Promoter: a genomicregion in relativelyclose proximity to thetranscription start sitethat regulatestranscription of thenearby gene

Enhancer: a genomicregion that regulatestranscription atvariable distances fromthe transcription startsite, often greater than1 kb

ChIP:chromatinimmunoprecipitation

ChIP-Seq: agenomics techniquethat combineschromatinimmunoprecipitationwith massively parallelsequencing to identifyDNA sequencesbound by proteins inliving cells

PNT-Domain Self-Association

The PNT domain of ETV6 and its Drosophilaortholog Yan form helical homopolymers ina head-to-tail configuration as deduced fromcrystallographic studies (Figure 4f ) (93, 94,102). The polymer results from the associa-tion of two complementary surfaces of neigh-boring PNT domains, termed mid-loop (ML)and end-helix (EH). Mutant proteins bearing asubstituted hydrophobic to charged residue oneither the ML or EH surface are monomeric.However, a mixture of two mutants, onewith an ML-mutated surface and one with anEH-mutated surface, yields dimers by stableassociation of their remaining wild-type inter-faces. This dimer has been used as a model poly-mer and subjected to functional analysis of therole of self-association by ETV6 and Yan (26,50, 96, 102). As discussed below, the dimericform of ETV6 facilitates DNA binding, butother roles for a longer polymer are possible,such as recruitment of cofactors. With the ex-ception of Yan, ETV6, and likely ETV7 (125),all other PNT domains characterized to datehave one or both of these surfaces blocked byamino acid substitutions or appended helicesand, thus, are monomeric (95, 126).

The EGF-receptor signaling pathwayin Drosophila eye development provides awell-studied example of the interplay of PNTdomains from ETS family members that relieson both kinase docking and self-association(reviewed in Reference 127). Yan functionsas a repressor and self-associates via its PNTdomain (26). Pnt-P2 acts as an activator ofcommon developmental genes. These twoETS factors are targets of the DrosophilaMAP kinase Rolled. Upon signaling, Rolledphosphorylates Yan, leading to its export fromthe nucleus and subsequent degradation in thecytoplasm. Mae, which bears a PNT domainbut no ETS domain (128, 129), facilitates Yanphosphorylation by serving as a docking site forRolled. In addition, the affinity of Mae for Yanis 1000-fold higher than Yan for itself; thus, thepresence of Mae favors Yan depolymerization(96). In parallel, phosphorylation of Pnt-P2

adjacent to its PNT domain stimulates acti-vation of its target genes presumably owing toenhanced recruitment of CBP. The Mae PNTdomain binds to the PNT domain of Pnt-P2,blocking its Rolled docking site and, thereby,preventing its phosphorylation-dependentactivation (102). Thus, PNT-domain homo-and hetero-typic interactions provide a robustresponse to signaling.

The PNT domain is present in certain sub-families (ETS, TEL, ERG) plus GABPA andSPDEF, but absent in others (SPI, ERF, TCF,ELF) (Figures 1a and 2). Multiple evolution-ary events likely led to the loss of this domain asthe family duplicated and diversified. The PNTdomains in the ERG and ELF subfamilies, aswell as in SPDEF, have no reported activities.We anticipate that potential novel functions areyet to be discovered.

GENOMIC APPROACHES TOTHE SPECIFICITY QUESTION:REDUNDANT ANDSPECIFIC BINDING

Recent genome-wide analyses of ETS-factoroccupancy provide a new approach to thespecificity question by identifying genomicregions, both promoters and enhancers, boundby individual ETS proteins in living cells.Genomics technology combines chromatinimmunoprecipitation (ChIP) with eithermicroarrays (ChIP-chip) or massively parallelsequencing (ChIP-Seq) for identification ofthe factor-bound DNA regions (130, 131).Nine ETS proteins have been assayed forgenome-wide occupancy by either promotermicroarrays (ETS1, GABPA, ELF1, ELK1,EWS-FLI1, and SPI1) (132–135) or high-throughput sequencing (GABPA, ETS1, ERG,FLI1, EWS-ERG, EWS-FLI1, SPI1, SPDEF,ETV1, and ELF1) (29, 62, 65, 136–140). (SeeSupplemental Table 4 for public databaseaccess.) Although there are caveats with thecurrent state of this technology (see Caveatsof Genome-Wide Occupancy Techniques,sidebar), recent progress with these approacheshas generated interesting occupancy patterns



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Housekeepinggenes: a looselydefined set of genesthat are expressed in allcell types and functionin normal cell growthand maintenance

CpG islands:genomic regions withmany occurrences ofthe DNA sequence5′-CG-3′, which ismodified by DNAmethyltransferase tobear a methyl-cytosine

for ETS proteins. Furthermore, the availabilityof multiple data sets from several familymembers is providing insights into specificityand genomic views of cooperative partnerships.

Redundant Binding of ETS Proteinsin Living Cells

Genome-wide occupancy data for several ETSproteins have been compared and found to havea high degree of similarity (Figure 6). The re-ported overlap for ETS1 and GABPA showsmost GABPA-bound regions are also occupiedby ETS1 in Jurkat cells (65, 136). Many of thesesame regions are occupied by ELK1 in HeLacells (134, 141). In Figure 6b, we comparedata for ELF1 (62) and GABPA from Jurkat Tcells (136) and SPI1 from mouse macrophages(138). From even these limited data sets, whichuse different techniques, cell lines, and species,overlapping occupancy is observable with sta-tistically significant frequency. We speculatethat the redundancy may encompass the en-tire family because the diverse ETS proteins,which have already been implicated, includingGABPA, ETS1, ELK1, ELF1, and SPI1, rep-resent five subfamilies.

Overlapping binding within the resolutionof the ChIP technology could be the result ofclusters of binding sites specific for each familymember. Alternatively, interchangeable occu-pancy of multiple ETS factors at one site overtime could be sampled in the pool of multiplecells (Figure 7). Favoring the latter hypoth-esis, high-resolution comparisons of bindingdata for ETS1 and GABPA reveal sharp colo-calization of binding events (65). Furthermore,bioinformatics analyses of regions with broadredundancy identified a striking enrichment ofthe sequence CCGGAAGT (132, 134). Thissequence motif matches exactly the in vitro-derived DNA-binding consensus of all but fourETS proteins (Classes I and II) (Figure 6a).Thus, the sequence motifs in redundantly oc-cupied regions are likely able to bind most, ifnot all, ETS proteins.

DNA regions occupied redundantly bymany ETS proteins are most frequently found

CAVEATS OF GENOME-WIDE OCCUPANCYTECHNIQUES

Microarrays and massively parallel sequencing can identify the se-quences of DNA derived from ChIP experiments. Mapping thesesequences to the genome results in peaks of reads above back-ground that indicate the genomic region bound by the immuno-precipitated transcription factor. Lists of bound regions identi-fied by peak-calling algorithms are not comprehensive, but theygenerally reflect cut-off values that compromise between false-negative and false-positive results. Resolution ranges from tensto hundreds of base pairs depending on the quality of the peak(136, 190). Bioinformatics approaches can predict binding se-quences by searching for overrepresented patterns in large num-bers of bound regions or by directly searching for a particularmotif. Neither method proves the use of a particular motif. It isalso important to consider that bound regions may differ by celltype or growth condition. Another caveat of the current technol-ogy is the limited information regarding the function of a boundtranscription factor, including information about which gene(s)may be targets of regulation. Currently, bound regions are usu-ally assigned to the closest genes to make functional correlations.Compilation of correlative genomics and bioinformatics data isessential at this stage with anticipation that technology can drivehigh-throughput tests of functionality in the future.

a short distance (∼20 to 40 bp) upstream oftranscription start sites and are correlatedwith characteristics of active promoters, asevidenced by DNase I sensitivity and presenceof histone H3K4 tri-methylation (65, 142,143). There is also an indication from geneontology studies that “housekeeping-type”genes are more likely to be characterized by thisredundant ETS-factor binding. Searching foroverrepresented motifs in the human genomeidentified an ETS-binding sequence located∼25 bp upstream of the transcription start siteof CpG island-containing housekeeping genepromoters (144–146). It has been proposedthat housekeeping genes, which are activelyexpressed in most cell types, could be regulatedby different ETS factors in different cell types.The frequency of this sequence (in ∼25% ofhuman promoters) (65), the association withcommonly expressed housekeeping genes, and


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GABPA

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Figure 6Genomic analyses via ChIP-chip and ChIP-Seq detect specific and redundant ETS binding. (a) Redundantlyoccupied regions in cells have a DNA sequence similar to preferred ETS-binding sequences derived in vitro.Sequence frequencies are compiled (top) from all 27 in vitro–derived ETS-binding sites (62) or (bottom) fromthe most overrepresented sequence in regions co-occupied in cells by GABPA, ETS1, and ELF1 (62, 65,136). Frequencies are shown in logo form, which depicts base pair (A:T, T:A, G:C, C:G) frequency asrelative heights of A, T, G, and C, respectively. (b) Redundant occupancy is observed for even the mostdivergent family members and between species. The relative distribution of specific and overlapping boundregions are shown for GABPA (136) and ELF1 (62) in human Jurkat T cells and SPI1 in mouse macrophages(138) by numbers of bound regions within a Venn diagram display. Analysis of SPI1 mouse data was limitedto promoter regions, which could be compared with orthologous human regions using the LiftOver tool athttp://genome.UCSC.edu (235). Data were reprocessed for this comparison, as previously reported (65).(c,d ) Serum response factor (SRF) and RUNX preferentially co-occupy regions bound specifically by ELK1and ETS1, respectively. Diagrams (c,d ) illustrate data from Boros et al. (134) and Hollenhorst et al. (65),respectively. See Supplemental Table 4 for database sources of all genomics data.

its occurrence near transcription start sitessuggest a common role for ETS factors in pro-moter proximal regions, such as transcriptioninitiation, promoter clearance, or maintenanceof active chromatin. Functional studies will benecessary to resolve whether individual ETS

proteins have distinct activities at these sitesor whether a redundant function could beexecuted by alternative family members.

Redundancy within subfamilies has not beenextensively investigated by genome-wide tech-niques. However, such redundancy may be


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http://genome.UCSC.edu


expected and has been investigated in a detailedanalysis of the EGR1 promoter. This promoteris regulated by the TCF subfamily, in part byrecruitment of Mediator, as mentioned above.Direct ChIP experiments for ELK1, ELK3, andELK4 demonstrated alternate occupancy thatappeared to correspond to relative abundanceof the factors in different cell types (121). Inthis case, findings suggest the TCF proteinsdiffered in Mediator binding, thus providingdistinct EGR1 expression and responsiveness tosignaling in different cell types.

Specific Binding of ETS Proteins inLiving Cells

Specific binding by an ETS factor is defined asthe binding of only one or possibly only highlyrelated subfamily members. Because compar-isons of ETS transcription factor genomicoccupancy data reveal a significant amount ofredundant binding, it can be difficult to differ-entiate specific binding sites from false-negativeor false-positive results. Furthermore, untilall ETS family members expressed in a singlecell are assayed by ChIP, specificity cannot bedefinitively concluded. Despite these limita-tions, current genome-wide studies have identi-fied specific genomic occupancy that correlateswith predicted ETS protein roles. For example,in hematopoietic stem cells, the closely relatedETS proteins ERG and FLI1 bind to a largelyredundant set of targets, whereas targets of themore divergent ETS protein SPI1 have muchless overlap (29). However, the mechanism ofspecificity is yet to be determined.

In some cases the subtle differences inDNA-sequence preference observed in vitroare supported by genomic studies. Regionsbound specifically by ETS1 are found in Jurkatcells and have a higher proportion of sites witha thymine at position 7 compared with regionsco-occupied by ETS1 and GABPA (65).This observation may be due to the relativetolerance of this change by ETS1 comparedwith other ETS proteins (147). Genomicoccupancy data for SPI1 in myeloid cells andSPDEF in prostate cells enabled bioinformatics

AR AR

FLI1

EWS

FLI1

EWS

ETS1

ELK1

GABPA

ELF1

Housekeeping genes

T-cellactivation

genesRUNX

Serumresponsive

genesSRF SRF

Prostatedifferentiation

genesERG

Ewing’s sarcoma

genes

CCGGAAGT

AGGATGTGG

GGAAGGAAGGAAGGAA

CC(A/T)6GG

ELK1

CCGGAAGT

GNACANNNTGTNCC AGGAAG

ETS1

Figure 7Partnerships provide a route to specificity in normal and disease states. Modelsdepict ETS protein interactions with other DNA-binding proteins at thepromoters or enhancers of distinct target classes. ETS proteins exhibitredundant occupancy of a consensus ETS-binding site in housekeepingpromoters (132, 134). In contrast, ETS proteins bind specifically near genesthat agree with predicted biological functions in either normal or cancer cells(65, 133–134, 139). Specific interactions are associated with activation ( greenarrows) or repression (red bar) of target genes. Specific targets are highlightedby co-occupancy of multiple transcription factors [RUNX, serum responsefactor (SRF), androgen receptor (AR)] implicating partnerships as drivers ofspecificity. EWS-FLI1 tandem occupancy is inferred from functional andbiochemical analyses of binding to GGAA microsatellite repeats (133, 137,187). See Supplemental Table 4 for database sources of all genomics data.

analyses for these divergent family members(62). Position-weight matrices derived fromunbiased searches of top-scoring regions showsimilarities to the subtly different sequencesderived from the high-throughput proteomicsapproaches that are discussed above. However,analysis of ELK1, SPI1, GABPA, ERG, FLI1,and ETV1 by other groups report no distinctETS motifs (29, 134, 136, 138–140). Moreparsing of bound regions into different classes,such as redundant proximal promoter versusspecific distal enhancers, may be necessary toascertain signatures of specificity determinants.

One sequence change that is often seenin specific binding sites is the presence of anadenine instead of a cytosine at position 3


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(ACAGGAAGT). This change may influencespecificity in some cases. However, it mayalso be a result of selection against a CpG,which would be vulnerable to DNA methy-lation and, thus, inhibition of ETS-proteinbinding (148, 149). This selection would benecessary at enhancers, where ERG (62) andETS1 specificity (65) is observed, but not atredundant promoter-binding sites where theETS-binding sequence can be protected fromDNA methylation as a result of proximity to,or inclusion in, hypomethylated CpG islands.

In conclusion, genomic-binding studieshave revealed both redundant and specific occu-pancy by ETS factors. The power of genomicsto detect redundancy contrasts with the limits ofgenetic analyses in which such redundancy mayeasily be masked. However, the relevance of allgenomic occupancy awaits more detailed func-tional tests, especially ones that can distinguishroles of distinct ETS factors. In addition, manyETS sites predicted on the basis of biochemicalunderstanding, and bioinformatics tools are notoccupied in vivo, and conversely, many actualsites of genomic occupancy are not predicted.These discrepancies are consistent with stud-ies in other transcription factor families (130,150) and likely reflect in vivo complexities suchas competition with chromatin packaging andother DNA-binding proteins. Thus, identifica-tion of other chromatin-associated factors andspecificity partnerships, as described below, willprovide a necessary backdrop to understandETS-protein binding detected in living cells bygenomics approaches.

COOPERATIVE PARTNERSHIPSPROVIDE ETS FAMILYSPECIFICITY: GENOMIC ANDBIOCHEMICAL PERSPECTIVES

A well-established theme in the regulationof eukaryotic gene expression is combinato-rial assembly of multiple factors within thesame control elements (e.g., a promoter orenhancer). This phenomenon can provideadded specificity and thus synergistic controlif there is positive or negative cooperative

DNA binding between juxtaposed factors orjoint recruitment of additional components ofthe transcriptional machinery. ETS proteinsdisplay a variety of such cooperative part-nerships, as previously reviewed (1, 2, 151,152). To provide a mechanistic frameworkfor understanding this route to specificity, wefocus on the high-resolution structural modelsof ETS partnerships that delineate underlyingprotein-protein interactions (Figure 4; Sup-plemental Table 2) and on cases supported byunbiased identification of composite elementsthrough genome-wide analysis (Figure 7).

Ternary Complex Factors/SerumResponse Factor

Serum response elements that mediate acti-vation of immediate-early genes such as c-FOS include both ETS- and SRF-binding sites.The cooperative binding interactions betweenSRF and the TCF subfamily of ETS tran-scription factors, including ELK1, ELK3, andELK4, have been well characterized (11; re-viewed in Reference 89). Crystallographic stud-ies demonstrate that DNA binding is enhancedby a direct protein contact between ELK4 andthe SRF MADS domain (44). The partnershipalso depends on the TCF B-box, a 21-residueregion that adopts a helix-sheet-helix motifwhen bound to the MADS domain of SRF. Thisconserved box is limited to the TCF subfamilyand, thus, illustrates a route to both redundancywithin a subfamily and specificity within the en-tire ETS family (Figures 1a and 4b).

Chromatin occupancy studies that relied onChIP-chip reveal co-occupancy of ELK1 andSRF at more than 200 promoters (Figure 6c)(134, 153). These co-occupied regions are morelikely than other sites to be bound specificallyby ELK1 and not by another ETS protein.This specific binding mode occurs at only 22%of ELK1-bound regions, whereas the redun-dant binding mode, characterized by a strongconsensus ETS-binding sequence and overlap-ping occupancy with other ETS transcriptionfactors, is observed more frequently (50%).Thus, similar to ETS1, the TCF subfamily can



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undergo both specific and redundant binding(Figure 7).

SPI1/IRF4

The ETS factor SPI1 partners with theinterferon regulatory family transcriptionfactor IRF4 at immunoglobulin light-chaingene enhancers. The low affinity of the wingedhelix-turn-helix DNA-binding domain of IRF4for the λB enhancer is increased 20–40-fold bythe presence of SPI1 at this site (154, 155). Thecrystallographically determined structure ofthe two DNA-binding domains on a compositeenhancer sequence reveals that this coopera-tivity arises from both direct protein-proteincontacts across the central minor grooveand induced DNA conformational changes(Figure 4e) (46). More dramatically, however,the phosphorylated PEST sequence of SPI1interacts with an autoinhibitory region of IRF4to enhance DNA binding by almost threeorders of magnitude (154). This enhancedinteraction awaits structural characterization.

ETS1/RUNX1 and Autoinhibition

ETS1 and RUNX1 form a partnership that iswell characterized by biochemical and genomicapproaches (156–158). The level of cooperativ-ity of ETS1 and RUNX1 on ETS sites sim-ilar to that in the Moloney murine leukemiavirus enhancer approximates the 10-fold re-pression of ETS1 DNA binding by autoinhibi-tion. Also, ETS1 variants with a deleted or dis-rupted inhibitory module no longer show suchenhancement. These observations suggest thatcooperativity results from a relief of autoinhi-bition. Intriguingly, the effect also requires au-toinhibitory sequences in RUNX1 (157). At thetime of writing this review, molecular coordi-nates for several ternary complexes of ETS1 andRUNX1 are on hold in the Protein Data Bank.Thus, we can anticipate informative structuraldata on this partnership in the near future.

Both redundant and specific occupancywere characterized for ETS1 in Jurkat Tcells with more than 14,000 bound regions

detected (65). Specific ETS1 occupancy oc-curs primarily in regions relatively distal totranscription start sites that have enhancercharacteristics, such as DNase I sensitivityand histone H3K4 mono-methylation (142,143). More than 1000 of these specificallybound regions detected by ChIP-Seq displaythe sequence AGGA(A/T)GTGG. This motifhas been characterized in the T-cell receptorenhancers as an ETS1/RUNX1 cooperativebinding element with the RUNX1 site com-prising the (A/T)GTGG (Figure 7). Indeed,co-occupancy by RUNX factors at many ofthe ETS1-specific regions was verified byChIP-Seq (Figure 6d ). More than 30-foldcooperative binding is detected with compositesequences bearing this close spacing (156);thus, there may be additional mechanisms atplay beyond simply counteracting the 10-foldeffect of autoinhibition. Ontology analysis ofgenes with the composite motif included com-ponents of the T-cell activation machinery,including ZAP70, LCK, CXCR3, CXCR4, andCD38. Thus, the ETS/RUNX sequence motifis predictive of the function of nearby genes(65). These sites also recruit the transcrip-tional coactivator CBP, which adds furtherevidence for their functional relevance. Thesegenomic studies are consistent with geneticsexperiments in which mice with a deletionof ETS1 have defects in T-cell activation(159).

Despite the evidence that ETS1 regulatesT-cell genes via ETS/RUNX-binding sites, it isnot clear that these sites are completely specificfor ETS1. Currently, only GABPA and ELF1ChIP-Seq data are available in Jurkat T cells tocompare with ETS1 (62, 136). However, otherETS proteins such as ELF2, ELF4, and FLI1have been reported to interact with RUNXproteins (160, 161). Furthermore, ChIP-Seqin hematopoietic stem cells identifies extensiveco-occupancy of RUNX1 with FLI1 and ERG(29). Thus, it is possible that ETS/RUNX bind-ing sites have only partial specificity for sub-sets of ETS proteins and that combinations ofETS and RUNX factors may vary with cell type.The forthcoming structural analysis of ETS and


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RUNX combinations will be important for de-ciphering rules of their partnering.

ETS1/PAX5

ETS1 and PAX5 bind cooperatively to a com-posite sequence located in the promoter ofmb-1, a gene expressed in early B cell differenti-ation (Figure 4c) (162). This ETS-binding sitedisplays a variant GGAG core. Crystallo-graphic studies of this cooperative partnershipdemonstrate a protein contact that leads to analtered conformation of a tyrosine conserved inmost ETS domains (Tyr395 in ETS1). Gln22,found in the β-hairpin of the PAX5 paired do-main, hydrogen bonds to Tyr395 in the recog-nition helix H3 of ETS1 to stabilize this alteredconformation. This enables new DNA contactsfor ETS1, while avoiding unfavorable interac-tions with the variant GC bp (43). Interest-ingly, this same conserved tyrosine is involvedin ELK4’s ability to bind the variant GGATcore (41).

This partnership also provides insights intothe linkage of protein interactions and ETS1autoinhibition. The PAX5 paired domain en-hances the binding of both the autoinhibitedand uninhibited variants of ETS1 to the sameaffinity (163). Thus, the PAX5 partnership ap-pears to counteract the effects of autoinhibi-tion, possibly through the ETS domain ratherthan direct interactions with inhibitory ele-ments. Gln22 of the paired domain also con-tacts Gln336 near the N terminus of helix H1 ofthe ETS domain (43), providing a conduit intothe dynamic core of the ETS domain. There isspecificity of this PAX5 partnership for ETS1rather than other family members, thus illus-trating the use of protein interactions to selectindividual ETS proteins (162). However, thebiochemical basis for this selectivity is not clear,as the key residues Tyr395 and Gln336, whichcontact PAX5, are present in other family mem-bers. Furthermore, there are no genomic datain the B-cell system to ascertain the frequencyand specificity of this partnership in cells or thecommon occurrence of a PAX5-ETS compos-ite element.

ETS/FOX

The major historical route to discovery of part-nerships in the ETS family, including thosementioned above, has been mapping of tissue-specific enhancer regions that leads to the iden-tification of a functional ETS-binding site andneighboring synergistic elements. A number ofadditional ETS partnerships have been impli-cated at this level, but they are not the focus ofthis review. However, one is mentioned to il-lustrate emerging topics and critical directionsfor investigation of partnerships. The ETS andFOX families converge, as illustrated by the roleof ETV2 and FOXC2 in the endothelial-cellgene-expression program (31, 32). The part-nership is characterized by a composite motif,detected in genome-wide bioinformatics anal-yses, which can be co-occupied by ETV2 andFOXC2 in DNA-binding assays. Endothelial-cell-specific genes, including FLK1, TAL1,NOTCH4, and VE-CADHERIN, display themotif. Importantly, a genome-wide search withthis motif identified endothelial-specific genes.A combination of cell-based transcription as-says, mouse transgenics, and zebrafish reversegenetics demonstrates the function of this part-nership. The basis of transcriptional synergy isnot yet established, and thus it will be importantto perform biochemical and structural studieson an ETS-FOX ternary complex.

ETV6 Self-Associationand Autoinhibition

In contrast to most ETS proteins, ETV6 isa transcriptional repressor. ETV6 monomersbind DNA extremely weakly owing to au-toinhibition of the ETS domain, as discussedabove (50). In contrast, an engineered ETV6dimer shows higher affinity, but only onmultiple ETS-binding motifs. It is proposedthat self-association via the PNT domain (seeFigure 4f ) mediates cooperative DNA bind-ing, which compensates for the low affinity ofthe monomeric ETS domain. Furthermore,this cooperativity is very tolerant of the orien-tation and spacing (within five helical turns) of


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the multiple ETS-binding sites, suggesting asubstantial degree of flexibility of the middle re-gion of ETV6 (∼200 residues), which links theETS and PNT domains (Figure 1a). Collec-tively, these results suggest that autoinhibitionand polymerization could define the specificityof ETV6 by targeting promoters bearingmultiple binding sites. Such sites need not havea well-ordered array of GGA core sequences.Furthermore, polymerization of ETV6 isspeculated to create an extended complex,which, along with recruitment of corepressors,may facilitate transcriptional repression (93).

Reports on Drosophila Yan, the apparent or-tholog of ETV6, strongly suggest that self-association is a key to the function of this classof ETS factors (26). Biological relevance isalso drawn from the chromosome rearrange-ments of the ETV6 locus that encode onco-genic fusion proteins associated with B cellleukemias. The fusions include the PNT do-main of ETV6 and the DNA-binding domainof either RUNX1 (Figure 1a) (164) or PAX5(165, 166). We speculate that the rules re-garding DNA binding learned from the nativeETV6 polymer, as modeled by the dimer, mayalso apply to these oncogenic fusion proteins.Genomic and additional biochemical data willbe helpful to understand fully how the biophys-ical properties of ETV6 impact target selectionof both native and oncogenic versions of ETV6.

Other Dimeric Configurations

In addition to ETV6, several ETS proteinshave been implicated in dimeric interactions.ETS1 binds cooperatively as a homodimer totandem ETS motifs, such as that found inthe MMP3 promoter. The cooperativity ofdimerization is the same 10-fold effect as au-toinhibition, suggesting that the dimer config-uration may counteract autoinhibition (167).The crystallography-based model of the ternarycomplex reveals that residues in the loop be-tween inhibitory helix HI-2 and helix H1 ofone monomer interact with those in the turnbetween helices H2 and H3 of the ETS do-main in the other monomer (Figure 4d ) (49).

An alternatively spliced isoform of ETS1, lack-ing inhibitory elements N terminal to theETS domain, fails to dimerize, thus implicat-ing the contact made by the inhibitory elementsin the cooperativity mechanism (168). How-ever, as with ETS1/PAX5 and ETS1/RUNX1partnerships, a complete mechanism for linkingpartnerships and autoinhibition is still lacking.

Other ETS proteins possibly use multi-ple ETS-binding motifs. On the basis of thecrystallographic structure of a single ELF3ETS domain bound to the B site of a tan-dem type II TGF-B receptor promoter, en-hanced binding of a second ELF3 to the ad-jacent A site was proposed to result fromprotein-induced DNA conformational changes(51). This configuration has not been investi-gated by either biochemical or genomics ap-proaches. In addition, GABPA has two alter-native non-DNA-binding partners GABPB1and GABPB2, which enhance its DNA-bindingaffinity and provide a homodimeric interfaceto form GABPA-(GABPB)2-GABPA heterote-tramers on DNA (40). The spacing require-ment for the tetrameric GABPA/B complex hasnot been determined, and genomic data forGABPA have not yet elucidated whether suchcomplexes distinguish specific sites in promot-ers. Note that most of the GABPA sites are alsoused by monomeric ETS1 or other ETS pro-teins (65), thus implicating no GABPA speci-ficity determinants.

The ability of ETS factors to use multi-ple ETS motifs either by ETV6 polymers orother dimeric configurations begs the questionof whether clusters of ETS consensus sites oc-cur in the genome. We found no evidence foruse of tandem ETS sites in Jurkat cells withthe dimeric spacing of the MMP3 promoter(P.C. Hollenhorst, unpublished data). How-ever, a high density of many transcription factorbinding motifs, including ETS sites, is notedin promoter and enhancer regions (134, 136,169). Testing of ETS-factor polymeric bindingin cells awaits discovery of additional authenticETS targets.

In conclusion, combinatorial control oftranscription employs multiple DNA-binding


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AR: androgenreceptor

transcription factors in close proximity. Al-though the DNA recognition sequences forETS proteins and their partners may be closelyjuxtaposed or even overlapping, the resultingprotein-protein interfaces are often small andthe effects on their DNA binding rather subtle.Nevertheless, the thermodynamic result of thedirect protein-protein interactions and/or theindirect changes in protein-DNA interactionsis net-positive cooperativity relative to theindependent binding of each transcription fac-tor. The added specificity of larger compositeDNA-binding sites, which is gained throughthe cooperative binding of two factors, mayconstitute the greatest biological impact ofsuch partnerships. Extrapolating from the de-tailed mechanism of ETS1 autoinhibition, thedynamic character of the ETS domain couldbe impacted within the context of a cooperativepartnership. It remains to be determined howmany of these ETS factors and their respectivepartnerships tap into the potential to regulatean ETS domain by autoinhibition and/or byaffecting protein dynamics.

GENOMIC INSIGHTS INTOONCOGENIC ETS FACTORS

ERG and Androgen Receptor inProstate Cancer

Rearrangements of ETS loci are a hallmark ofprostate cancer with ∼50% of tumors show-ing alterations at an ETS gene locus (36). Inthe most common scenario, a promoter regionfrom an androgen-responsive, prostate-specificgene, TMPRSS2, is attached to the ERG locusto drive aberrantly regulated ERG expressionin prostate cells. Chromosomal rearrangementsthat result in the overexpression of members ofthe PEA3 subfamily (ETV1, ETV4, or ETV5)are found less frequently (36, 170, 171). Thesegenetic changes are implicated in tumorigene-sis (170, 172–176). ChIP-Seq of ERG in bothcell lines and tumors that overexpress this tran-scription factor identifies a striking level of co-occupancy (44%) with the androgen receptor(AR) (62, 139). The involvement of the AR

in normal prostate development, coupled withthe prevalence of ETS-binding sequences atAR-bound regions (177), implies a coopera-tive interaction that could play a role in ETS-mediated prostate oncogenesis (Figure 7).Expression studies indicate that a potentialmechanism of oncogenic ETS proteins, suchas ERG and ETV5, at these sites is antagonismof normal AR function (139, 178, 179). Sup-porting this idea, tumor-suppressor ETS pro-teins, such as SPDEF, synergize with the AR(66). However, the genetic finding that ETSand AR genes cooperate in oncogenesis (175)indicates that this mechanism is more complexthan our current understanding and may differon a target-by-target basis (180).

The biochemical mechanism for interac-tions between ETS factors and the AR is alsonot known. There is some evidence that theAR can physically interact with ERG (139),ETV5 (179), SPDEF (66), ETV1 (181), andETS1 (177) through their ETS domains, evenin the absence of DNA. However, whetherthis interaction has any ETS family specificityor how it impacts ETS function remainsunknown. DNA-binding assays have nottested cooperative binding, and bioinformaticsanalyses have not identified an overrepre-sented spacing of ETS- and AR-binding sitesfunctionally analogous to that observed withthe ETS1/RUNX partnership. Therefore, theinteraction between ETS proteins and the ARmay lead to specificity of function, but notnecessarily cooperative DNA binding. Thus,ETS proteins may influence cancer via ARtarget genes, but the mechanism of this effectremains to be discovered.

EWS-FLI1 in Ewing’s Sarcoma

Ewing’s sarcoma, a common pediatric cancer,is invariably associated with ETS gene translo-cations (182) that result in a fusion oncogeneconsisting of the 5′ end of EWS and the 3′ endof FLI1, or less frequently ERG, ETV1, ETV4,or FEV (Figure 1a) (183, 184). The strongtransactivation activity in the N terminusof EWS contributes to the function of the


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Microsatellite: agenomic region with10 to 100 tandemrepeats of a 2–6-bpsequence

resulting oncogenic fusion protein (185). How-ever, insights from genomic studies suggestthat the fusion protein also directs regulationof new transcriptional target genes. ChIP-chipand ChIP-Seq have identified the genomicregions bound by the most common ETS fu-sion proteins in Ewing’s sarcoma, EWS-FLI1and EWS-ERG (62, 133, 137). Two overrep-resented DNA sequences are found in theseregions: a consensus ETS-binding site that issimilar to promoters discussed above, whichhave redundant occupancy of ETS factors, andmicrosatellites that bear tandem repeats of thesequence GGAA. Of these two classes of targetsequences, only genes near the microsatelliterepeats correlate with expression changes asso-ciated with transformation, including NROB1(133) and GSTM4 (186). Thus, GGAA repeattargets, but not redundant promoter targets,mediate the oncogenic role of these fusions.

GGAA microsatellites, which can havemore than 10 copies of the tetrameric repeat,constitute arrays of ETS-binding sites thatcould mediate cooperative binding betweenneighboring factors. In fact, four or morerepeats are necessary for EWS-FLI1 transcrip-tional activation in reporter assays, and twoEWS-FLI1 monomers bind to this four-motifarray in biochemical assays (Figure 7) (133,137, 187). However, whether cooperativity isat play is unclear. A recent study revealed thatthe EWS portion of the fusion oncoproteincontributes only to the transcriptional acti-vation, not the DNA-binding activity. Thisimplicates the ETS-domain component of thefusion protein in microsatellite binding (187).Indeed, multiple ETS proteins, includingexamples not involved in Ewing’s sarcoma, canbind to GGAA microsatellites. Furthermore, asdiscussed above, ETS proteins ETV6, ETV7,

GABPA, and ELF3 bind as homo-oligomersto neighboring ETS-binding sites. Whetherthe GGAA microsatellites that are involved inEwing’s sarcoma are the natural targets of anyof these ETS proteins remains to be tested.

In conclusion, the oncogenic potential ofETS factors is likely derived from theirtranscriptional properties. In addition to thechromosome rearrangements described above,there is mounting evidence for an oncogenicrole for dysregulation of ETS genes in prostate(188, 188a), melanoma (189), and gastroin-testinal stromal tumors (140). Alterations inany ETS protein can have multiple down-stream effects, amplifying the primary geneticdefect. Thus, a potentially effective interven-tion in ETS-driven cancers would be a dis-ruption in the function of the oncogenic ETSprotein. The range of macromolecular inter-actions, which include DNA-protein and var-ious homo- or hetero-typic protein-proteincomplexes, all serve as potential interfaces fortargeted therapeutics.

In considering the route to oncogenesis asa window into the specificity question of theETS family, it is noteworthy that multiple fam-ily members can be involved in sarcoma andprostate cancer with an interesting overlap be-tween the two diseases. There are no substantialdata to indicate that the various family memberscause different disease progression or outcome.Future questions include whether these familymembers are selected as a result of susceptibilityof the genetic locus to rearrangements or dis-tinct properties of these, but not other, ETSfamily members. Finally, continued genomicand biochemical analysis of the oncogenic ETSfactors can help elucidate oncogenesis pathwaysas well as shed light on specificity routes withinthe family.

SUMMARY POINTS

1. Conservation of the ETS domain leads to similar DNA interactions among all ETSfactors as detected by in vitro biochemical assays. However, deviation from these highest-affinity sites is encountered in biological settings.


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2. The PNT domain provides diversity to ETS proteins, both by its variable presence anddiffering functions, ranging from kinase and coactivator binding to self-association.

3. The DNA-binding affinity of many, if not most, ETS proteins is tempered by autoin-hibition. Specific DNA motifs used in cells can diverge from the optimal consensus,thus lowering affinity further. This combination of circumstances poises an ETS fac-tor to participate in partnerships with other factors that have additional preferencesfor DNA sequence and architecture, thus enhancing specificity for genomic locations.High-resolution molecular models demonstrate the relatively small protein-interactionsurfaces that can mediate such cooperativity.

4. Protein dynamics underlie phosphorylation-dependent regulatory processes, includingautoinhibition of the ETS domain and enhanced binding of CBP by the PNT domain.Flexible regions, which are likely sites of modification, function in concert with morestructured regions, which also exhibit dynamic conformational equilibria.

5. ETS binding sites displaying a strong match to the highest-affinity consensus motifs arecommon in CpG island-containing housekeeping promoters. These sequences serve asinterchangeable binding sites for many ETS proteins. Every cell type expresses multipleETS factors that could potentially participate in this redundant occupancy.

6. Genome-wide occupancy studies identify many (hundreds to thousands) binding sitesfor a single ETS protein in a particular cell type. Although the functional relevance ofmany of these sites awaits further analysis, bioinformatics approaches have identifiedETS consensus motifs and composite elements that are beginning to define genomicdeterminants for specificity and cooperative partnerships.

7. Oncogenic ETS proteins alter numerous transcriptional targets within cells that expressmany other ETS factors. Selection of oncogenic target genes is likely achieved by altereduse of the specificity mechanisms for normal cells described in this review.

FUTURE ISSUES

1. Dynamics are increasingly implicated as a powerful regulatory mechanism in proteinfunction. Studying how intra- and intermolecular interactions establish the amplitudeand timescale of these dynamics—and, thus potentially, the DNA-binding properties oftranscription factors—is a new avenue for understanding the regulation of gene expres-sion. Biophysical studies of increasing sophistication will help define the physicochemicalbases for ETS autoinhibition and dissect the role of dynamics in protein and target DNArecognition by the PNT and ETS domains, respectively.

2. A high level of redundant genomic occupancy in the ETS family was detected in promoterproximal regions. Further work will determine whether ETS proteins play a redundantmechanistic role in transcription regulation at these targets and whether these sites havefunctions distinct from more distal enhancer elements.

3. As genome-wide binding data become available for more ETS proteins, as well as othertranscription factors, comparisons will allow classification of redundant, specific, andpartially specific binding sites. This categorization will improve the robustness of thenew data sets to investigate mechanisms that enable specificity within the ETS family,


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including composite elements for cooperative binding partnerships and other routes tosynergistic transcriptional regulation. Redundancy within subfamilies versus the entirefamily needs to be clarified.

4. The ETS genes that are involved in chromosomal abnormalities in solid tumors arelimited largely to those in ERG and PEA3 subfamilies. It is not known to what degree theoncogenic functions of these genes overlap. Determining the specific roles of these twosubfamilies, compared with the rest of the ETS family, will be important to understandingthe mechanisms of ETS-mediated oncogenesis.

5. The development of therapeutic strategies, including small-molecule modulators of pro-tein interactions, to regulate ETS proteins could have high clinical impact. Currentlyavailable and new structural and biochemical information about ETS proteins will bevaluable in achieving this challenging research goal.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding or financial holding thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Tim Parnell and Krista Meyer for critical comments on the manuscript, David Nixand Katherine Chandler for help with data analysis, and Carlos Escalante and Aneel Aggarwalfor providing the coordinates of the SPI1/IRF4/DNA complex. This work was supported by theNational Institutes of Health through awards GM38663 (to B.J.G.) and CA42014 (to the Hunts-man Cancer Institute for support of shared resources) as well as the Canadian Cancer Society Re-search Institute (017308 to L.P.M.). Huntsman Cancer Institute/Huntsman Cancer Foundationfunding (to B.J.G.); Indiana University School of Medicine support (to P.C.H.); and instrumentsupport from the Canadian Institutes for Health Research, the Canadian Foundation for Inno-vation, the British Columbia Knowledge Development Fund, the Michael Smith Foundation forHealth Research, and the UBC Blusson Fund (to L.P.M.) are gratefully acknowledged.

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BI80-FrontMatter ARI 16 May 2011 14:13

Annual Review ofBiochemistry

Volume 80, 2011Contents

Preface

Past, Present, and Future Triumphs of BiochemistryJoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �v

Prefatory

From Serendipity to TherapyElizabeth F. Neufeld � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Journey of a Molecular BiologistMasayasu Nomura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �16

My Life with NatureJulius Adler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �42

Membrane Vesicle Theme

Protein Folding and Modification in the MammalianEndoplasmic ReticulumIneke Braakman and Neil J. Bulleid � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Mechanisms of Membrane Curvature SensingBruno Antonny � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 101

Biogenesis and Cargo Selectivity of AutophagosomesHilla Weidberg, Elena Shvets, and Zvulun Elazar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 125

Membrane Protein Folding and Insertion Theme

Introduction to Theme “Membrane Protein Folding and Insertion”Gunnar von Heijne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Assembly of Bacterial Inner Membrane ProteinsRoss E. Dalbey, Peng Wang, and Andreas Kuhn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

β-Barrel Membrane Protein Assembly by the Bam ComplexChristine L. Hagan, Thomas J. Silhavy, and Daniel Kahne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

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Transmembrane Communication: General Principles and Lessonsfrom the Structure and Function of the M2 Proton Channel, K+

Channels, and Integrin ReceptorsGevorg Grigoryan, David T. Moore, and William F. DeGrado � � � � � � � � � � � � � � � � � � � � � � � � 211

Biological Mass Spectrometry Theme

Mass Spectrometry in the Postgenomic EraBrian T. Chait � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 239

Advances in the Mass Spectrometry of Membrane Proteins:From Individual Proteins to Intact ComplexesNelson P. Barrera and Carol V. Robinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

Quantitative, High-Resolution Proteomics for Data-DrivenSystems BiologyJurgen Cox and Matthias Mann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 273

Applications of Mass Spectrometry to Lipids and MembranesRichard Harkewicz and Edward A. Dennis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Cellular Imaging Theme

Emerging In Vivo Analyses of Cell Function UsingFluorescence ImagingJennifer Lippincott-Schwartz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Biochemistry of Mobile Zinc and Nitric Oxide Revealedby Fluorescent SensorsMichael D. Pluth, Elisa Tomat, and Stephen J. Lippard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 333

Development of Probes for Cellular Functions Using FluorescentProteins and Fluorescence Resonance Energy TransferAtsushi Miyawaki � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 357

Reporting from the Field: Genetically Encoded Fluorescent ReportersUncover Signaling Dynamics in Living Biological SystemsSohum Mehta and Jin Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Recent Advances in Biochemistry

DNA Replicases from a Bacterial PerspectiveCharles S. McHenry � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

Genomic and Biochemical Insights into the Specificity of ETSTranscription FactorsPeter C. Hollenhorst, Lawrence P. McIntosh, and Barbara J. Graves � � � � � � � � � � � � � � � � � � � 437

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Signals and Combinatorial Functions of Histone ModificationsTamaki Suganuma and Jerry L. Workman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 473

Assembly of Bacterial RibosomesZahra Shajani, Michael T. Sykes, and James R. Williamson � � � � � � � � � � � � � � � � � � � � � � � � � � � � 501

The Mechanism of Peptidyl Transfer Catalysis by the RibosomeEdward Ki Yun Leung, Nikolai Suslov, Nicole Tuttle, Raghuvir Sengupta,

and Joseph Anthony Piccirilli � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 527

Amyloid Structure: Conformational Diversity and ConsequencesBrandon H. Toyama and Jonathan S. Weissman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

AAA+ Proteases: ATP-Fueled Machines of Protein DestructionRobert T. Sauer and Tania A. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 587

The Structure of the Nuclear Pore ComplexAndre Hoelz, Erik W. Debler, and Gunter Blobel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 613

Benchmark Reaction Rates, the Stability of Biological Moleculesin Water, and the Evolution of Catalytic Power in EnzymesRichard Wolfenden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 645

Biological Phosphoryl-Transfer Reactions: Understanding Mechanismand CatalysisJonathan K. Lassila, Jesse G. Zalatan, and Daniel Herschlag � � � � � � � � � � � � � � � � � � � � � � � � � � � 669

Enzymatic Transition States, Transition-State Analogs, Dynamics,Thermodynamics, and LifetimesVern L. Schramm � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 703

Class I Ribonucleotide Reductases: Metallocofactor Assemblyand Repair In Vitro and In VivoJoseph A. Cotruvo Jr. and JoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 733

The Evolution of Protein Kinase Inhibitors from Antagoniststo Agonists of Cellular SignalingArvin C. Dar and Kevan M. Shokat � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 769

Glycan Microarrays for Decoding the GlycomeCory D. Rillahan and James C. Paulson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 797

Cross Talk Between O-GlcNAcylation and Phosphorylation:Roles in Signaling, Transcription, and Chronic DiseaseGerald W. Hart, Chad Slawson, Genaro Ramirez-Correa, and Olof Lagerlof � � � � � � � � � 825

Regulation of Phospholipid Synthesis in the YeastSaccharomyces cerevisiaeGeorge M. Carman and Gil-Soo Han � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 859

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Sterol Regulation of Metabolism, Homeostasis, and DevelopmentJoshua Wollam and Adam Antebi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 885

Structural Biology of the Toll-Like Receptor FamilyJin Young Kang and Jie-Oh Lee � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 917

Structure-Function Relationships of the G Domain, a CanonicalSwitch MotifAlfred Wittinghofer and Ingrid R. Vetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 943

STIM Proteins and the Endoplasmic Reticulum-PlasmaMembrane JunctionsSilvia Carrasco and Tobias Meyer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 973

Amino Acid Signaling in TOR ActivationJoungmok Kim and Kun-Liang Guan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1001

Mitochondrial tRNA Import and Its Consequencesfor Mitochondrial TranslationAndre Schneider � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1033

Caspase Substrates and Cellular RemodelingEmily D. Crawford and James A. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1055

Regulation of HSF1 Function in the Heat Stress Response:Implications in Aging and DiseaseJulius Anckar and Lea Sistonen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1089

Indexes

Cumulative Index of Contributing Authors, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � �1117

Cumulative Index of Chapter Titles, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1121

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found athttp://biochem.annualreviews.org/errata.shtml

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Genomic and Biochemical Insights into the Specificity of ...

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