Pioneer factors: directingtranscriptional regulators within thechromatin environmentLuca Magnani1, Jerome Eeckhoute2* and Mathieu Lupien1*

1 Norris Cotton Cancer Center, Institute for Quantitative Biomedical Sciences, Department of Genetics, Dartmouth Medical School,

Lebanon, NH 03756, USA2 Universite de Lille Nord de France, INSERM UMR1011, UDSL, F-59000 and Institut Pasteur de Lille, F-59019, Lille, France

Review

Glossary

Bookmarking: deposition of specific epigenetic modifications that label poised

or functional enhancers. This can take place during development to allow

delayed activation of enhancers.

Chromatin landscape: defined as the combination of parameters that influence

its function and that include the distribution of nucleosomes and epigenetic

modifications across the genome as well as higher-order folding and related

three-dimensional organization (Box 1).

Enhancer: a transcriptional regulatory element that activates its target gene

over a distance. Functional enhancers are epigenetically marked, sparse of

nucleosome, recruit transcription factors and can contact target gene

promoters through chromatin looping.

Insulator: a transcriptional regulatory element that delineates chromatin

domains acting as a barrier to the spread of epigenetic modifications or as

an anchor for chromatin looping. Insulators are typically bound by the CTCF

transcription factor.

Nucleosome: consists of approximately 147 base pairs of DNA wrapped

around histone octamers (made of H2A, H2B, H3 and H4).

Pioneer factors: defined as a specific class of transcription factors that is

required and sufficient to trigger competency of transcriptional regulatory sites

(Box 2).

Promoter: a transcriptional regulatory element found in the immediate

proximity of the transcriptional start site of genes. Promoters recruit the basal

transcriptional machinery and initiate transcription.

Regulatory element: sequence of DNA bound by transcription factors that

Chromatin is a well-known obstacle to transcription as itcontrols DNA accessibility, which directly impacts therecruitment of the transcriptional machinery. The recentburst of functional genomic studies provides new cluesas to how transcriptional competency is regulated in thiscontext. In this review, we discuss how these studieshave shed light on a specialized subset of transcriptionfactors, defined as pioneer factors, which direct recruit-ment of downstream transcription factors to establishlineage-specific transcriptional programs. In particular,we present evidence of an interplay between pioneerfactors and the epigenome that could be central to thisprocess. Finally, we discuss how pioneer factors, whoseexpression and function are altered in tumors, are alsobeing considered for their prognostic value and shouldtherefore be regarded as potential therapeutic targets.Thus, pioneer factors emerge as key players that connectthe epigenome and transcription in health and disease.

A role for pioneer factors in shaping the chromatinlandscape to activate functional regulatory elementsThe human body consists of hundreds of distinct cell typesthat are specified by their unique transcriptional pro-grams. These programs are dependent on the interplaybetween regulatory elements, including enhancers, promo-ters and insulators (see Glossary) [1–4]. Specific DNAsequences in these regulatory elements are recognizedby transcription factors that generate transcriptional reg-ulatory output signals [5–7]. These signals directly modu-late the transcription of mRNA from DNA by RNApolymerase II. Therefore, transcription factors are respon-sible for the spatial and temporal fine-tuning of transcrip-tional programs. Although they recognize and bind tospecific DNA sequences, the recruitment of transcriptionfactors is also dependent on the chromatin landscape (Box1). Indeed, the distribution of nucleosomes and epigeneticmodifications along the genome, as well as the three-dimensional chromatin organization, can regulate tran-scription factor recruitment and activities. For instance,transcription factor binding to chromatin is favored innucleosome-free regions [3]. Consequently, gene regulationcan be dynamically regulated by nucleosome positioning

Corresponding author: Lupien, M. (mathieu.lupien@dartmouth.edu)* These authors contributed equally to this work.

0168-9525/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2011.0

[8,9]. In this instance, enhancers differ significantly fromproximal regulatory elements; namely promoters. For ex-ample, promoters generally lie in open and nucleosome-free chromatin regardless of the cell type, whereas acces-sibility to the enhancer is highly variable and cell-typespecific [4]. Furthermore, different epigenetic modifica-tions characterize promoters and enhancers [1,5]. Thechromatin landscape is therefore a critical barrier thatguides the transcriptional potential in a cell type-specificmanner by modulating enhancer activity. Understandinghow the chromatin landscape is shaped at enhancers istherefore central to understanding cell type-specific com-mitment.

Pioneer factors are an emerging class of chromatin-bound proteins grouped according to their biological func-tion, as opposed to homology of sequences and conserveddomains. They contribute to the implementation of celltype-specific transcriptional programs upstream of tran-scription factors binding to enhancers [10–17]. In general,pioneer factors can therefore be defined as factors that arefirst able to bind to enhancers and that subsequently

regulate gene transcriptional levels. Regulatory elements comprise promoters,

enhancers and insulators.

7.002 Trends in Genetics, November 2011, Vol. 27, No. 11 465

Box 1. Chromatin landscape

Chromatin is a macromolecule whose primary unit is the nucleosome.

Nucleosomes consist of approximately 147 base pairs of DNA wrapped

around histone octamers (made of H2A, H2B, H3 and H4) that form a

‘beads-on-a-string’ structure. However, nucleosomes are not regularly

spread along the DNA, as eukaryotic cells can dynamically reposition

nucleosomes to increase and/or decrease occupancy of specific

regions. For instance, transcriptional regulatory regions typically

consist of nucleosome-free DNA, making transcription factor recogni-

tion motifs more easily accessible. Moreover, chromatin adopts higher-

order compaction structures that result from modulation of internu-

cleosomal (linker) DNA bending levels through the linker histone H1.

Additionally, long-range interactions are also involved in shaping

chromatin three-dimensional conformation. Such contacts between

distant chromatin sites appear to be crucial to allow enhancers (long-

distance regulatory regions) to transmit signals to the promoters of

their target genes. Long-range interactions implicating insulatory

regions bound by the transcription factor CTCF are also important in

this context to allow and/or impede specific enhancer–promoter

contacts. Together, these different layers of chromatin complexity

and compaction levels therefore impact on the ability of transcription

factors to bind to chromatin and regulate gene expression.

An additional layer of information borne by chromatin consists of

epigenetic modifications of DNA and histones. This involves methyla-

tion and/or hydroxymethylation of cytosines within the DNA and

post-translational modifications (acetylation, methylation, etc.) of

histones. Various combinations of potential DNA and/or histone

modifications are found within the chromatin, depending on the

function of the regions under consideration. In addition, specific

combinations of epigenetic modifications are found at open versus

condensed chromatin regions and transcriptional regulatory events

involve changes in epigenetic modifications. Overall, combinations of

epigenetic modifications define a limited number of functionally

distinct chromatin domains across the genome. Importantly, epige-

netic modifications are involved in a dialog with transcription factors

where they modulate each other’s presence onto the chromatin.

Hence, work in recent years has pointed to a crucial role for epigenetic

modifications in control of transcription factor activities.

Thus, the chromatin landscape is defined as the combination of

parameters that influence its function and that include the distribution

of nucleosomes and epigenetic modifications across the genome, as

well as higher-order folding and related three-dimensional organiza-

tion [105].

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trigger their transcriptional competency (Box 2). In thisreview, we focus on recent advances in functional genomicsthat demonstrate how pioneer factors can impact thechromatin landscape to promote cell type-specific tran-scriptional programs. We also discuss evidence for theiremerging role in disease development and progression thatmakes them attractive prognostic markers and therapeu-tic targets in pathologies such as cancer.

Bookmarking enhancers: pioneer factors set the stageAs stated above, cell type-specific transcriptionalresponses are dependent on lineage-specific functionalenhancers [18–20]. Indeed, whereas the genetic informa-tion within an organism is conserved across cell types, agiven enhancer will only be functional in a subset of them.Furthermore, enhancers exhibit species-specific activities[21,22]. Deposition of specific epigenetic modificationscould play an important role in this process because theydistinguish functional from non-functional enhancers. For

Box 2. Pioneer factors

Pioneer factors are defined as a specific class of transcription factors

that are required and sufficient to trigger enhancer competency. In

line with this definition, pioneer factors are found bound to the

chromatin before activation of enhancers and gene expression

modulation. For example, FOXA factors bind the Alb1 enhancer in

primitive endoderm where it is not yet active and Alb1 is not yet

expressed [53]. Importantly, liver specification and Alb1 gene

expression are completely lost in mice when FOXA factors are

inactivated, providing genetic proof for their role as pioneer factors

during liver development [106]. More generally, pioneer factors have

crucial roles in development and differentiation. This implies that

pioneer factors have to recognize their DNA binding motifs in a

context that impedes binding of other transcription factors. Accord-

ingly, FOXA factors are able to bind to nucleosomal DNA [12].

Subsequently, pioneer factors have to trigger recruitment of

additional transcription factors directly responsible for the transcrip-

tional regulatory output signals. Direct protein interactions could help

tether additional transcription factors to regulatory sites. However,

even if this opportunity exists, in many instances pioneer factors

promote transcription factor binding to their own DNA recognition

motifs [19]. This implies that pioneer factors have to remodel the

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example, mono- and dimethylation of lysine 4 on histone 3(H3K4me1/me2) are commonly associated with functionalenhancers, whereas H3K9me2 is found at non-functionalsites [18,19,23–28]. Therefore, how these enhancers arebookmarked by these epigenetic modifications is central tounderstanding the process guiding the commitment of cellsto a specific fate.

Importantly, pioneer factors can actively contribute tothis process. Indeed, the pioneer factor PU.1 was recentlyshown to promote H3K4me1 deposition at enhancers inthe course of macrophage and B-cell differentiation [20,29](Figure 1). PU.1 binding to the chromatin can be observedwithin an hour following its expression and is accompa-nied by nucleosome repositioning and chromatin opening[20,29]. Subsequently, H3K4me1, which is initially lack-ing at PU.1 binding sites, is gained [20]. H3K4me1 depo-sition coincides with the recruitment of transcriptionfactors guiding the cell type-specific transcriptional pro-gram promoting B cell or macrophage differentiation [20].

chromatin landscape locally (Box 1). First, pioneer factors could

reposition and/or evict nucleosome(s) to promote binding to nucleo-

some-free DNA [107]. However, a recent study suggested that FOXA

factors are not crucial for nucleosome distribution across the genome

of hepatocytes [61]. Second, pioneer factors could modulate higher-

order chromatin structures. For instance, FOXA factors could replace

the linker histone H1 and modulate linker DNA bending [12]. However,

the importance of higher-order chromatin structure in the regulation

of transcription factor accessibility to DNA still remains to be firmly

established [105]. Another attractive explanation for the role of

pioneer factors is that they could be required to trigger epigenetic

changes at enhancers. These changes can affect the chromatin-

binding affinity of proteins (with DNA or histone binding domains),

potentially resulting in direct or indirect regulation of transcription

factor binding [105].

Most potential pioneer factors known to date have been shown to

precede enhancer activation and/or to be required for recruitment of

prototypic transcription factors, which can not directly bind to

compacted chromatin. However, how pioneer factors fulfill this role in

vivo is still elusive and may involve different mechanisms, depending

on the chromatin landscape and the pioneer factor considered.

Pre B-cellMacrophageHepatocyteBreast cell

FOXD3

PU.1

FOXA1

AP-2γ

PBX1 GATA-3

and/orPF

PF

PF

PF

PFTF

etc.Prostate cel

FOXA1 FOXA1

FOXA2

CTCF

FOXD3

CTCF

TLE1

Differen

tiation

TRENDS in Genetics

Figure 1. Pioneer factors and activation of enhancers during cellular differentiation. During differentiation, cells progress through a diverse set of stages associated with

distinct transcriptional programs. These programs are implemented during development in a cell type-specific fashion by marking enhancers with histone modifications

(enhancer bookmarking). Pioneer factors (PF) play an active role in this process. In pluripotent cells (top DNA strand), the chromatin at cell type-specific enhancers is

selectively bookmarked. This is accomplished by arrays of epigenetic modifications, such as mono- or dimethylation of lysine 4 on histone H3 (H3K4me1/me2; green

circles), H3K9me2 (red squares), DNA methylation (red stars) or by transcription factors, such as the pioneer factor FOXD3 or the insulator CTCF. During the transition from

pluripotent to progenitor cells (center strand), pioneer factors (indicated by the colored ovals) are expressed in a cell type-specific manner. They can bind the chromatin at

bookmarked regions engaging in the translation of the histone modification into functional enhancers. This can involve sequential binding of pioneer factors, co-occupancy

or cross-regulation. In addition to simple binding at epigenetically marked enhancers, pioneer factors can also specify changes to the chromatin landscape directing the

deposition of a new set of epigenetic modifications. These include loss of DNA methylation and gain of H3K4me1/me2 (center strand). Furthermore, nucleosome

displacement also contributes to enhancers becoming active for transcription factor (TF) recruitment in committed cells (bottom strand). Hence, as cells commit to a specific

fate, the transcription factors they express will be able to bind the chromatin and potentiate target gene expression.

Review Trends in Genetics November 2011, Vol. 27, No. 11

Indeed, these events allow subsequent binding and activi-ty of the nuclear receptor liver X receptor b (LXR b) inmacrophages [20]. Therefore, PU.1 binding functions as amarker that defines the set of potential cis-regulatoryelements that will subsequently be bound by specifictranscription factors. The concept that PU.1 can repro-gram the chromatin landscape through epigenetic modifi-cation is consistent with the observation that its ectopic

expression can transform fibroblasts into macrophage-like cells [30].

The Forkhead family member, Forkhead box protein A1(FOXA1) also acts as a pioneer factor, bookmarkingenhancers by reprogramming the chromatin landscape(Figure 1) [31]. Indeed, FOXA1 induction and binding tochromatin upon neuronal differentiation of pluripotentcells is followed by accumulation of the H3K4me2

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epigenetic modification at bound enhancers [31]. Interest-ingly, this is also accompanied by the loss of DNA methyl-ation at those occupied enhancers [31,32].

Pioneer factors can also bookmark enhancers by pre-venting deposition of repressive epigenetic modifications.This has been observed for the Forkhead factor FOXD3(Figure 1). In embryonic stem cells, the pluripotencytranscription factor FOXD3 occupies an enhancer associ-ated with the albumin (Alb1) gene, maintaining it in apoised state (Figure 1) [14,33]. As embryonic stem cellsdifferentiate into endoderm, FOXD3 is replaced byFOXA1, which is required for subsequent activity ofthe enhancer and expression of the Alb1 gene [14,34].By contrast, in cell-lineages where FOXA1 expression isnot induced upon FOXD3 loss, the enhancer gains DNAmethylation. This blocks transcription factor recruitmentand prevents Alb1 expression [14]. Ectopic expression ofFOXD3 in these cells leads to loss of DNA methylation ofthe enhancer, whereas FOXA1 ectopic expression has noeffects [14]. This suggests that these factors are notinterchangeable and provides a rationale for FOXD3binding before FOXA1.

Taken together, these results demonstrate that book-marking by pioneer factors is dependent on facilitating thedeposition of active epigenetic signatures and preventingthat of repressive epigenetic modifications at regulatoryelements (Figure 1). Moreover, these data suggest thatenhancer bookmarking occurs at different stages of cellulardifferentiation.

Translating bookmarks: positive feedback betweenpioneer factors and epigenetic signaturesWhereas pioneer factor binding to the chromatin associ-ates with epigenomic reprogramming in differentiatedcells, epigenetic modifications also provide signals forpioneer factors. Indeed, pioneer factors can act as ‘readers’of epigenetic modifications. In agreement with FOXA1bookmarking functions, in vitro assays have previouslydemonstrated that FOXA1 can bind recombinant histonesdeprived of any post-translational modifications [35].However, nucleosome-harboring H3K4me1/me2 favorsbinding of FOXA1 in vivo (Figure 1) [19,36–38]. Indeed,removal of this epigenetic modification through the ectopicexpression of the histone demethylase KDM1 (lysine-spe-cific demethylase 1; LSD1) significantly abrogates FOXA1binding to chromatin [19,38]. FOXA2, another relatedForkhead family member, recognizes compacted chroma-tin at enhancers marked with H3K4me1 nucleosomes inislet and liver cells [39]. At most enhancers, FOXA2 isfound between two nucleosomes bearing methylation ofH3K4 [39], reminiscent of what was reported for thepioneer factor FOXA1 [40]. By contrast, recent work as-cribed the presence of H3K9me2 epigenetic modification toFOXA1 sites not involved in transcriptional regulation[37]. Comparing FOXA1 binding sites in breast, prostateor colon cancer cells reveals that cell type-specific tran-scriptional regulation is associated with FOXA1 sites thatlack H3K9me2 [37], in agreement with the repressivenature of H3K9 methylation [41]. Overall, this demon-strates that the binding of pioneer factors to the chromatinbenefits from their interplay with epigenetic modifications

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to define functional enhancers driving transcriptionalregulation.

Other non-histone proteins also affect the activity ofpioneer factors. Indeed, the insulator protein CCCTC-bind-ing factor (CTCF) can influence the binding of pioneerfactors to the chromatin. Through genome-wide studies,it was recently demonstrated that a small fraction ofFOXA1 chromatin-bound sites are co-occupied by CTCF[42]. CTCF depletion reduces FOXA1 binding to thosesites, but favors its recruitment to sites bound by CTCFbut not by FOXA1 in control cells [42,43]. Therefore, CTCFbinding to chromatin appears to be able to both positivelyand negatively impact FOXA1 recruitment. CTCF wasrecently shown to modulate the position of nucleosomessurrounding its binding site [43]. How CTCF modulatespioneer factor recruitment to chromatin and what theimpact is on the chromatin structure remain to be investi-gated.

Enhancer activation through pioneer factor activitiesChromatin compaction varies greatly across the genomeand between cell types. Recent assessment of chromatinaccessibility through genome-wide DNAse I assays coupledwith massively parallel sequencing revealed distinctionsbetween functional and nonfunctional enhancers. Indeed,functional enhancers tend to lie in a open chromatinconformation, whereas nonfunctional distant regulatoryelements are found in compacted chromatin [44,45]. Iden-tical results were obtained using formaldehyde-assistedisolation of regulatory elements (FAIRE)-seq, a techniquemeasuring chromatin accessibility based on phenol-chlo-roform extraction of formaldehyde cross-linked chromatin[46]. Hence, in most instances, compacted chromatin is notable to direct binding of prototypic transcription factors[47].

One of the strategies adopted by transcription factors toovercome the chromatin barrier is to recruit proteins di-rectly that can remodel chromatin upon their recruitment[48–51]. Pioneer factors could also possess intrinsic chro-matin remodeling activities, as suggested by the ability ofFOXA1 to relax condensed chromatin structures in vitro inan ATP-independent manner [34]. Even if the mechanismsinvolved are not yet precisely defined, several studiesindicate that chromatin opening is central to pioneer factorfunctions. For instance, whereas PU.1–/– derived hemato-poietic stem cells fail to express the c-fms gene, re-expres-sion of PU.1 directly translates into chromatin remodelingboth at the promoter and enhancer associated with the c-fms gene, as assessed by DNase I in vivo foot printing [52].More importantly, PU.1 binding at the enhancer is re-quired to facilitate recruitment of early growth response2 (EGR-2), another transcription factor essential for mac-rophage development [52]. Similar conclusions were drawnbased on a genome-wide analysis in pre-B cells, wherePU.1 inducible expression resulted in nucleosome reposi-tioning around the linker region (the DNA sequence foundbetween two nucleosomes), as assessed by MNase diges-tion coupled with massively parallel sequencing [20].

FOXA1 was also reported to reposition nucleosomes invitro [53]. Interestingly, FOXA1 contains a winged helixmotif (FKH/WH) that is highly homologous to that found in

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histone H1 [12]. This homology allows FOXA1 to displacelinker histone H1 in vitro on chromatin templates and invivo in embryonic stem (ES) cells after retinoic acid stimu-lation [12,32,34,54]. Given that histone H1 normally favorschromatin compaction by bending the linker DNA andlinking distant nucleosomes to one another, this wouldresult in chromatin opening. Relaxation of chromatin byFOXA1 was evidenced using DNase I digestion [34].FOXA1 genome-wide remodeling actions have also beendemonstrated through FAIRE coupled with massivelyparallel sequencing [42,55]. In line with the existence ofinactive FOXA1-bound sites, it has been previously dem-onstrated that FOXA1 alone is not sufficient to promotecomplete relaxation of the chromatin [37]. However,FOXA1-bound regions deprived of nucleosomes were high-ly associated with cell type-specific transcription [41]. Suchchromatin remodeling ability associated with DNase Ihypersensitivity is also common to several other Forkheadfamily members, including FOXE1, FOXI1 and FOXO1[54–57]. Similarly, binding of the pioneer factor pre-B cellleukemia homeobox 1 (PBX1), previously reported to occu-py chromatin prior to transcription factor recruitment inthe course of muscle differentiation [58], and of the trans-ducin-like enhancer protein 1 (TLE1) in breast cancer cellsassociates with nucleosome depletion [59]. Indeed, FAIREassays in breast cancer cells depleted of TLE1 revealincreased nucleosome occupancy at their binding sites[59]. Chromatin remodeling abilities also distinguish an-other class of protein with pioneer factor characteristics;namely the GATA family. Ectopic expression of GATA-3 innaive primary CD4+ cells directly increases chromatinopenness at the IL-10 enhancer [60]. However, a recentstudy indicates that maintenance of nucleosome distribu-tion in mouse liver might not rely on pioneer factors [61].Indeed, FOXA1 and FOXA2 gene inactivation did not leadto nucleosome repositioning in liver cells [61]. Further-more, at some genomic regions, FOXA1 and FOXA2 bind tonucleosome-occupied sites [38,61]. This suggests that pio-neer factors have different roles at distinct classes ofenhancer [36]. The relative importance of the impact ofpioneer factors on nucleosome repositioning, displacementof the linker histone H1 or deposition of epigenetic marksat different subsets of enhancers, remain to be established.

Importantly, the transcriptional competency of regula-tory elements could derive from collaborations betweendifferent pioneer factors (Figure 1). This is observed duringliver development, where GATA and Forkhead familymembers cooperate to regulate the expression of theAlb1 gene. In addition to the transition between FOXD3and FOXA1 occupancy of the Alb1 enhancer during celldifferentiation to endoderm, GATA-4 can also be recruitedto the FOXA1-occupied enhancer. In fact, both in vitro andin vivo assays confirm that FOXA1 and GATA-4 co-occupythe Alb1 enhancer [12,13,16,62]. In agreement with therequired presence of both pioneer factors at the Alb1enhancer, depletion of either prevents the expression ofthe Alb1 gene [16]. FOXA1 cooperation with other pioneerfactors also applies in other cell types. For instance, inbreast cells, FOXA1 is found at sites occupied by theactivating protein 2 (AP-2)g pioneer factor [63]. The inter-dependency between these pioneer factors is highlighted

by the fact that each is required for the recruitment of theother [63]. By contrast, whereas FOXA1 can also cooperatewith TLE1 in breast cancer cells, the depletion of eitherpioneer factor does not affect the binding to chromatin ofthe other [59]. Cooperation between pioneer factors hasalso been reported during B cell development. Indeed, SRY(sex determining region Y)-box 2 (SOX2) is required for therecruitment of FOXD3 to the chromatin in embryonic stemcells. This than allows for the deposition of the H3K4me2epigenetic modification [64]. Similarly, in pro-B cells,SOX4 is required for the recruitment of the forkhead factorFOXP1 [64]. However, the co-occupancy of enhancers bypioneer factors does not always translate into cooperation.Indeed, in erythroid differentiation, PU.1 binding atGATA-1-bound regulatory elements leads to the repressionof GATA-1 target genes through the implementation ofrepressive epigenetic modifications [65]. Conversely,GATA-1 binding at PU.1-bound regulatory elements dur-ing myelopoiesis impairs the expression of PU.1 targetgenes [65].

In summary, epigenetically marked functional enhan-cers are remodeled and maintained in an open chromatinconformation through the action of pioneer factors. Theinterplay between pioneer factors through collaboration,substitution or cross-regulation further contributes to theestablishment of specific transcriptional programs. Thesemechanisms are important for development and cellulardifferentiation, and may be altered in diseases such ascancer, contributing to establishment of aberrant tran-scriptomes.

Pioneer factors in cancerCancer commonly associates with altered transcriptionalprograms. For example, it is possible to discriminate sev-eral subtypes of breast tumor based on their transcription-al profiles [66]. Changes to the chromatin landscapethrough chromatin remodeling and epigenetic modifica-tions play an active role in the establishment of theseanomalous programs [67–70]. Through their chromatinremodeling activity and their capacity to modulate thedeposition of epigenetic modifications, pioneer factorsare prime candidate drivers of changes in transcriptionalprograms seen in cancer. Accordingly, reports have dem-onstrated that genes encoding pioneer factors and theirgenomic activities are altered in multiple types of cancer(Figure 2).

Alterations to pioneer factor genes

Mutations within pioneer factor genes can disrupt theiractivity and contribute to cancer. For instance, the PU.1gene is mutated in 7% of patients with acute myeloidleukemia (AML) [71]. Although the effect of these muta-tions on the function of PU.1 as a pioneer factor has notbeen investigated clearly, they decrease its capacity tointeract with transcription factors, such as runt-relatedtranscription factor 1 (RUNX1) and JUN, and directlyimpact target gene expression [71]. In addition, transloca-tion events can target genes encoding pioneer factors. Forexample, a translocation event frequently involves PBX1in leukemia, where a fusion protein harboring the trans-activation domain of the transcription factor E2A and the

469

SNP-induceddifferential PF binding

PF

T/G

Altered chromatin landscape

Other cancer-related genetic and/or epigenetic alteration

Other cancer-relatedevents on partners

Altered PF function

PF-FUSION or PF

PF

PF-FUSION

or

PF

or

Overexpression

PF gene PF-fusion gene

TranslocationMutation

PF gene

Increased PF occupancy

PF PFPFPF

Gene level

Proteinlevel

Target level

PF

PF

PF

TRENDS in Genetics

Figure 2. Role of pioneer factors in cancer development. Cancer is characterized by aberrant expression profiles resulting in uncontrolled cell proliferation. Activities of

pioneer factors (PF) are altered in several different ways in cancer. At the gene level, pioneer factors may be overexpressed, mutated or rearranged through translocation

events (cancer-associated alteration is presented in red). As a consequence, pioneer factors see their protein level increase, their DNA binding specificity altered and their

interaction with partners modified. These events can be enhanced by additional cancer-related events targeting their partners. In addition, the genomic activity of pioneer

factors can be targeted in the course of cancer development. For instance, mutations or genetic variations within their DNA binding motif can modulate their binding

capacity. Overexpression of pioneer factors can result in increased occupancy at certain genomic locations and binding to de novo sites. Mutations and translocation events

in pioneer factor coding sequences can also alter their function by driving their recruitment to de novo sites or altering their activity at endogenous binding sites. Finally,

cancer-dependent de novo epigenetic reprogramming may guide pioneer factors to new sites. For example, de novo deposition of the epigenetic modification H3K4me2

(red/green circles) provides enhancers with a signature favoring FOXA1 binding in prostate cancer. Hence, these events highlight the multitude of way through which

pioneer factors can be affected in cancer. Abbreviation: SNP, single nucleotide polymorphism.

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DNA-binding domain of PBX1 is generated [72]. Thisresults in the expression of target genes through PBX1binding sites [72].

In addition to mutations and translocation events, pio-neer factors are generally overexpressed in cancer(Figure 2). This is seen in breast [73], ovarian [74] andprostate cancers [75] for PBX1. This also applies to AP-2g,which is commonly overexpressed in breast cancer [76–78],

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where it facilitates estrogen receptor a (ERa) signaling[63]. FOXA1 overexpression has also been reported invarious types of cancer [79–81]. Moreover, DNA copy num-ber amplification of FOXA1 is found in metastatic prostatetumors [81] and in breast tumors [82]. Overexpression oramplification of FOXA1 protein levels in breast and pros-tate cancer cells is directly linked to its ability to specifychromatin binding and function of ERa and androgen

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receptor (AR) [10,15,19,37,42,83]. FOXA1 is also amplifiedand overexpressed in thyroid carcinoma, where it promotescell cycle progression by controlling the expression ofp27kip1 [84]. Hence, changes in the protein levels of pio-neer factors through their overexpression or amplificationare common in cancer. However, the mechanisms drivingthese events and how they impact on pioneer factor normalfunctions are, for the most part, yet to be identified.

Disrupting pioneer factor genomic activity

The chromatin-binding activity of pioneer factors is as-cribed to their capacity to recognize specific DNA motifs inenhancers [19,20,61]. Recently, genome-wide associationstudies have linked genetic variants, such as single nucle-otide polymorphisms (SNPs), to cancer development. In-terestingly, some of the SNPs associated with prostatecancer map to FOXA1 binding sites [85,86]. Importantly,these SNPs were shown to disrupt the genomic activity ofFOXA1 by modulating its binding to chromatin (Figure 2)[85]. Disruption of pioneer factor binding by SNPs istherefore potentially involved in cancer development.The ongoing whole-genome resequencing efforts led bythe 1000 Genomes Project is constantly expanding thelist of genetic variants found within the human genome[87]. Many of these are likely to map to pioneer factorbinding sites. Therefore, it will be critical to investigatethe functional implication of these genetic variants onpioneer factor activity and their potential role in diseasedevelopment. Similarly, cancer genome resequencingefforts led by the International Cancer Genome Consor-tium (ICGC) are likely to identify mutations in tumorsthat map to pioneer factor binding sites [88]. The func-tional implication for these mutations will also need to bedefined.

As an alternative to genetic variations, pioneer factorbinding can also associate with changes to the chromatinlandscape imposed by epigenetic modifications (Figure 2).For instance, prostate cancer progression from castration-sensitive to castration-resistant status is related to de novoFOXA1 binding favored by H3K4 methylation at sitesunique to castration-resistant prostate cancer cells [38].These new FOXA1 binding sites promote the expression ofthe ubiquitin-conjugating enzyme E2C gene (UBE2C),which is essential for the proliferation of castration-resis-tant prostate cancer cells [2,38]. Removal of H3K4 meth-ylation in these cells significantly reduced FOXA1 bindingand UBE2C expression, thereby decreasing cancer cellproliferation [38].

Taken together, these studies demonstrate that pioneerfactors are direct targets in cancer through modification oftheir coding sequence, modulation of their expression oralteration of their genomic activities (Figure 2). This canhave consequences for the initiation, proliferation or pro-gression of tumors. These characteristics make pioneerfactors very attractive from a translational point of viewfor providing prognostic and/or therapeutic values.

Pioneer factors as therapeutic targets and biomarkersProgress in the field of personalized medicine for cancer isintimately linked to the discovery of novel biomarkers forprognostic purposes and to an expansion in drugs available

to clinicians to treat patients. The list of relevant mecha-nisms that can be targeted in cancer is rapidly expandingthrough the discovery of compounds that modulate epige-netic events [89,90]. Although these compounds arecurrently restricted to modulation of histone post-transla-tional modifications (e.g. histone acetylation [90,91]) andDNA methylation [92], the central role of pioneer factors incancer makes them an attractive entry point for therapeu-tic intervention.

Pioneer factors as therapeutic targets

The activity of the pioneer factor PU.1 can be modulated toimpact the growth of different subtypes of AMLs. Forexample, PU.1 expression is required for the expressionof the colony stimulating factor 1 receptor (CSF1R) gene inthe AML subtype harboring the (MOZ)-TIF2 translocation[93]. CSF1R expression allows for maintenance of theleukemia stem cell population that gives rise to (MOZ)-TIF2 AML. Depletion of PU.1 blocks CSF1R expression,leading to a decrease in the leukemia stem cell populationand, therefore, in leukemogenesis [93]. By contrast, in theAML subtype lacking the (MOZ)-TIF2 fusion protein, PU.1acts as a pioneer factor to promote the genomic activity oftranscription factors promoting differentiation [20]. Ectop-ic PU.1 expression can also promote the differentiation ofhighly proliferative myeloid cells into nondividing maturemacrophage-like cells prone to apoptosis [94]. Interesting-ly, vitamin K2, which promotes PU.1 upregulation inmyeloid progenitor cells, induces differentiation and apo-ptosis in AML cell lines [95].

FOXA1 has also been proposed as a therapeutic targetowing to its role in ERa signaling in breast cancer [96].Unfortunately, therapeutic approaches to antagonizeFOXA1 activity are still missing. However, PBX1 mightbe an actionable target in cancer. Its genomic activity hasbeen linked to its capacity to interact with HOX factors[97]. Recently, small peptides designed to antagonize theinteractions between PBX1 and HOX factors were shownto promote apoptosis efficiently in melanoma, ovarian andlung cancer cell lines [98–100]. They were also revealed toblock proliferation of leukemia and pancreatic cancer cells[101]. Although more studies are required to define thespecificity of these small peptides against PBX1, recentwork using small interfering (si)RNAs against PBX1 hasdemonstrated that its depletion abrogates proliferation ofovarian and breast cancer cells both in vitro and in vivo,supporting its role as a prime candidate for intervention incancer [74].

The development of novel compounds that antagonizepioneer factor activity is likely to be impaired by theinherent difficulties of targeting proteins that localizepredominantly to the nucleus and that do not possessligand-binding abilities. Given that pioneer factors arecommonly overexpressed in cancer, leads for the develop-ment of alternative therapeutic applications may originatefrom an understanding of the mechanisms that regulatetheir expression.

Pioneer factors as prognostic biomarkers

Recent studies have revealed the prognostic potential ofpioneer factors. For example, FOXA1 acts as a biomarker

471

Box 3. Outstanding questions

� What are the remaining pioneer factors and how can they be

screened for?

� How do pioneer factors collaborate with one another?

� How do pioneer factors modulate chromatin condensation

(ex: impact on higher-order chromatin folding and long-range

chromatin loops)?

� How do pioneer factors lead to de novo deposition of epigenetic

signatures?

� How different are pioneer factor activities in normal versus

cancerous cells?

� What are the mechanisms regulating pioneer factor expression?

Review Trends in Genetics November 2011, Vol. 27, No. 11

in prostate cancer, where its overexpression is associatedwith metastasis [81]. FOXA1 and GATA-3 expressioncharacterizes luminal breast tumors and is a predictor ofhigher survival [79,80,102]. This is in agreement with theircoexpression with ERa, a clinically validated marker ofgood outcome in patients with breast cancer [103]. Simi-larly, PBX1 is a prognostic marker for ovarian cancers [74].Finally, the prognostic value of the pioneer factor PU.1 hasrecently been proposed. Indeed, PU.1 expression levelspositively correlate with remission in chronic myeloidleukemia (CML) [104]. Hence, although the study of pio-neer factors is still in its infancy, it conceals the potential toderive novel translational applications.

Concluding remarksDefining the functional relationship between chromatinand transcription factors is at the heart of current under-standing of transcriptional regulation. Pioneer factors playan essential role in this process because they initiatefunctional competency of enhancers through chromatinremodeling. However, much work remains to be done toidentify all pioneer factors and their mode(s) of action (Box3). In particular, it is still not precisely understood howpioneer factors cooperate with one another to remodelchromatin and induce transcriptional competency, howthey modulate chromatin condensation or lead to de novodeposition of epigenetic signatures to define functionalregulatory elements. Finally, it will be critical to delineatethe function of pioneer factors in disease development andprogression, and to take into account how genetic variantsand mutations currently being identified through genomeresequencing efforts will impact their activity.

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