haematopoietic cell-fate decisions, chromatin regulation and ikaros

© 2002 Macmillan Magazines Ltd162 | MARCH 2002 | VOLUME 2 www.nature.com/reviews/immunol

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HAEMATOPOIETIC CELL-FATEDECISIONS, CHROMATINREGULATION AND IKAROSKatia Georgopoulos

The regulated production of several terminally differentiated cell types of the blood and immunesystems (haematopoiesis) has been the focus of many studies on cell-fate determination.Chromatin and the control of its structure have been implicated in the regulation of cell-fatedecisions and in the maintenance of the determined states. Here, I review advances in the field,emphasizing the potential role of chromatin in lineage commitment and differentiation. In thiscontext, I discuss Ikaros, an essential regulator of lymphocyte development and an integralcomponent of a functionally diverse chromatin remodelling network that operates from the earlystages of haematopoiesis to the mature lymphocytes.

CBRC, MGH East,13th St, Charlestown,Massachusetts 02129, USA.e-mail: [email protected]: 10.1038/nri747

states, they acquire and lose the expression of lineage-specific genes. These transitions correlate with adecreased ability to reverse the lineage choice and soallow terminal differentiation to a specific cell type7–9.

The fate of a cell is ultimately determined by the waythat its genetic material and its protein scaffold — col-lectively referred to as chromatin — are modified (FIG. 1).Modification of chromatin components that causeheritable changes in gene expression form the basis ofEPIGENETIC regulation. This has received particular atten-tion because of its association with development andcancer. Regulation of chromatin at the global and gene-specific levels contributes to changes in geneactivity10,11. Some of these events seem to be intimatelycoupled to specific developmental decisions by key reg-ulators of chromatin structure12. Regulation of chro-matin structure also underlies haematopoietic lineagedecisions and the balanced production of multiple dif-ferentiated cell types that are responsible for the properfunctioning of the blood and immune systems. Manyof the terminally differentiated states in this system arebuilt on previously determined cell identities, and so acomplex epigenetic regulation might be involved intheir genesis and maintenance. Some of the newinsights into chromatin regulation are reviewed here,with special emphasis on their potential role in lineage

At first glance, the most primitive of haematopoieticcells, the haematopoietic stem cell (HSC), seems to havea daunting task. It must generate, in a controlled fash-ion, all of the blood and immune cell types and alsomaintain its original pool by self-renewal. Prevailingmodels indicate that an HSC achieves this through aseries of binary decisions during which progressivelyrestricted precursors commit to alternative cell fates. So,cell-fate decisions and their regulation are not only tasksof the HSC and its immediate progeny but also a con-tinuous requirement imposed on several downstreamprecursors at distinct steps of haematopoiesis. A featurethat distinguishes the haematopoietic system frommany other developmental systems is that many of itspartially restricted intermediates have been extensivelycharacterized, which makes it an excellent choice forcell-fate determination studies.

In the haematopoietic system, development beginswith a quiescent HSC that is described as negative forknown lineage-specific markers1. The choice betweenone of the two main haematopoietic lineages, myelo-erythroid or lymphoid, is made by the immediate prog-eny of the HSC, but a fair amount of plasticity is stillapparent in the differentiation potential of earlymyeloid and lymphoid precursors2–6. As these precur-sors pass through a series of quiescent and proliferative

EPIGENETICS

The study of heritable changesin gene expression that occurwithout a change in DNAsequence.

© 2002 Macmillan Magazines LtdNATURE REVIEWS | IMMUNOLOGY VOLUME 2 | MARCH 2002 | 163

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CHROMATIN REMODELLING

A reversible alteration ofchromatin structure mediatedby ATP-dependent helicases,such as Mi-2 (NURD) andBrg1/Brm (SWI/SNF) that havean impact on several molecularprocesses, such asrecombination, repair andtranscription.

HETEROCHROMATIN

Highly compacted chromatinthat is found constitutively inregions such as the centromereand telomere, and is mostlyassociated with transcriptionallysilent genes. It is rich in A+Tsequences and can therefore bevisualized microscopically bystaining of nuclei with DNA-intercalating dyes.

CHROMATIN CODE

A covalent modification onhistones and DNA. They occurin different regions of thegenome by enzymes such asacetylases, methylases andkinases, and result in alterationsof gene transactions in a cell.These could include, forexample, changes intranscription, recombination,timing of DNA replication andthe degree of compaction ofDNA.

CHROMODOMAIN

The chromatin organizationmodifier domain is an ~50amino-acid motif originallyidentified as a region ofhomology shared between theDrosophila Hp1 and Polycombproteins, and that is now knownto exist in plants and animals.Chromodomain-containingproteins are usually componentsof chromatin remodellingcomplexes and are alsopresumed to have a role ingenome organization.

BROMODOMAIN

A 120-bp motif found in avariety of proteins, includinghistone acetyltranferases,kinases, basal transcriptionfactors (TAFII250) andchromatin remodelling factors.

acetylation is controlled by two antagonistic groups ofenzymes — HATs and histone deacetylases (HDACs) —each of which exists in several distinct complexes thatmight have different substrate specificities13,17,18.Structural studies of nucleosomes have indicated that theacetylation of histone tails might be responsible for amore ‘open’ chromatin structure that supports geneexpression19,20. Conversely, HDACs have been reported tofunction as co-repressors by deacetylating histones andpossibly providing a more rigid and less accessible nucle-osome structure21. Several studies have reported a func-tional link between this type of histone modificationand transcription or recombination events at variousdevelopmentally regulated and inducible gene loci22–25.

Attempts to delineate the modification status of his-tones have provided compelling support for the exis-tence of ‘histone codes’ that correlate with gene activ-ity15,26–30. Histone codes that are frequently found ininactive and silent genetic loci — and that are enrichedin HETEROCHROMATIN — include histone deacetylation,acetylation on Lys12 of H4 and methylation on Lys9 ofH3 (FIG. 2). Histone codes that correlate with gene activ-ity include H3 acetylation on Lys9 and Lys14, H3methylation on Lys4, H4 acetylation on Lys5 and H4methylation on arginine (Arg)3. The current definitionof the CHROMATIN CODE includes, but is not limited to, his-tone modifications. Other components of chromatin,protein and DNA that are chemically or structurallymodified can also provide codes to be deciphered bynuclear factors.

Generation, function and propagation of codes. Giventhat histone codes are differentially distributed through-out the genome, how are they generated? A local orgene-specific mechanism can be provided by sequence-specific DNA-binding proteins that target histone-mod-ifying enzymes to their cognate sites (and their immedi-ate vicinity)31–34 (FIG. 3a). There are also more global waysof generating histone codes — for example, throughchromatin compartmentalization in nuclear subdo-mains that are enriched with specific modifiers (FIG. 3b).

Once histone codes are established, they can have arange of functions, including changing the accessibilityof the associated DNA to trans-acting factors orenzymes (FIG. 4). Histone codes can also act as markersfor specific interactions with protein modules and theirassociated regulatory factors; for example, betweenmethylated histones and CHROMODOMAIN modules35,36, aswell as acetylated histones and BROMODOMAIN mod-ules15,37–39. Such protein modules are found in a range ofnuclear regulatory factors including heterochromatinprotein 1 (HP1), TAFII250 (a component of the basaltranscription machinery, a variety of HAT) andBrg1/Brm (the ATPase components of SWI–SNF com-plexes). In the model shown in FIG. 3b, interactionsbetween CHROMATIN CODES and proteins that are residentin specific nuclear domains can provide chromatincompartmentalization. The interplay between chro-matin codes and protein modules can be constitutive orregulated (FIG. 4). It is therefore possible that a chromatincode generated early in development can function at a

restrictions in the haematopoietic system. In this con-text, I discuss Ikaros, which is the founding member of aunique family of zinc-finger DNA-binding proteins. It isrequired for lymphocyte development, is stably associ-ated with CHROMATIN REMODELLING and modifying activi-ties, and is therefore probably involved in chromatinregulation of gene expression.

Ways to regulate chromatinHistones and DNA, the components of the basic chro-matin unit (the nucleosome), are targeted by distinctenzymes for modifications that alter the structure of thenucleosome and its participation in a more or less com-pact chromatin assembly. The order of chromatinassembly can influence events during transcription,replication, recombination and DNA repair, and so itcan determine the developmental outcome.

Histone modifications. The known covalent modifica-tions of histones include phosphorylation, acetylation,methylation, ADP ribosylation and ubiquitylation13,14.Histone modifications can be synergistic or antagonis-tic to each other; for example, phosphorylation of ser-ine (Ser)10 on histone H3 is synergistically coupled tothe acetylation of lysine (Lys)9 and/or Lys14 but antag-onizes the methylation of Lys9 on the same histone15.The first insight into the role of histone acetylation ingene expression was provided by the finding that theTetrahymena homologue of the yeast co-activatorGcn5 was a histone acetyl transferase (HAT)16. Histone

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Figure 1 | Structure of a nucleosome. The assembly of DNAinto a compact structure termed chromatin is essential forpackaging the genome into the confines of the cell nucleus.The nucleosome, the basic unit of chromatin, consists of~200 bp of DNA coiled roughly twice around an octamer,which is composed of dimers of the core histones H2A, H2B,H3 and H4. The DNA that connects adjoining nucleosomes iscalled linker DNA and is associated with another histone,namely histone H1. The repeating structure of nucleosomes iswhat results in the visualization of DNA as ‘beads on a string’by electron microscopy. In a cell, the nucleosome is furthercompacted into a higher-order structure. Such compactioncan result in the regulatory elements of genes being largelyinaccessible to regulatory proteins, which interferes with geneexpression. Å, angstroms.


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a chromatin code on a small number of key lineage-spe-cific genes might take place in an HSC during its transi-tion into a multipotent progenitor (FIG. 5). Subsequentactivation of nuclear factors that can decipher the chro-matin code will allow transcription or recombinationevents that collectively support lineage restriction inthese cells.

For many of the haematopoietic lineages, the choiceof cell fate is available not only at the onset, but alsothroughout their pathways. Cell-fate events that occurlater in differentiation might rely on pre-existing lin-eage-specific chromatin markers in combination withnewly activated nuclear factors to fine tune the differen-tiated state. In some instances, the generation of a firstlayer of histone codes at specific gene loci can trigger acascade of events that lead to additional chromatincodes that collectively determine the final differentiatedstate. When most of these events have been executed,erasing the histone code might not reverse the fate ofthe terminally differentiated cell. However, there mightbe instances in which the histone code is required tomaintain identity throughout differentiation by allow-ing a backbone of gene activities that support the differ-entiated state. In this case, defacement of the early codecan reverse differentiation.

Chromatin fluidity and the chromatin codeChanges in chromatin structure are also provided byATP-driven enzymatic machines in a dynamic processdescribed as chromatin remodelling. Chromatinremodellers function by providing a dynamic equilib-rium between ‘open’ and ‘closed’ chromatin states thatis referred to as fluidity42. The functional differencesbetween the known remodellers lie in part withintheir substrate specificity, which ranges from histonesto nucleosomes to the more complex types of chro-matin, and their mode of action42–46. Remodellerschange the structure of chromatin in global and localways. The repertoire of activities in which globallyacting remodellers participate includes replication ofchromatin structures during the cell cycle (for exam-ple, by facilitating deacetylation of newly depositedhistones in heterochromatin and possibly elsewhere),replication of centromeric structures by exchange of

later time by associating with a nuclear factor that isinduced later in development.

For a chromatin code (and its associated activities)to exert its effects during development, it must be main-tained through proliferative and quiescent steps. Thiscan again be achieved by sequence-specific targeting ofhistone modifiers (FIG. 3a) to the daughter strands.However, the sequence-specific factor that is responsiblefor targeting the histone modifications might no longerexist at later stages of development. In this instance,code propagation can be achieved by delivering chro-matin to a nuclear compartment that can propagatethe parental code. Studies of silent genes moving intoPERICENTROMERIC HETEROCHROMATIN (PC-HC) as cells pre-pare for mitosis support this hypothesis40,41. Theaddressing of chromatin regions to specific nuclearcompartments might be provided by the code itselfand/or by its ability to interact with resident factorswithin a compartment (FIG. 3b). Interactions between themethylated Lys9 of H3 and HP1 in heterochromatinmight provide such an example. In this model, themaintenance of chromatin code through cell divisions isself-propagating. Once chromatin is delivered to anuclear compartment, the range of further modifica-tions that it can undergo and the events that it partici-pates in will be determined by the activities present atthis locale. It therefore follows that a change in the abil-ity of the cell to replicate its chromatin codes can pro-vide an opportunity for altering its identity. This canoccur by changing the activity of sequence-specific tar-geting factors or by changing the composition ofnuclear compartments.

Lineage choice and the chromatin code. The evolvingfield of chromatin codes provides us with new ways tostudy mechanisms that initiate and support lineage deci-sions. It is reasonable to propose that events that generatea first layer of chromatin code (in the form of histonemodifications) at key lineage-specific genes provide newinformation on their regulation and support a lineagechoice (FIGS 4,5). Subsequent protein interactions thatinvolve these codes can further modulate the activity ofthe associated genes and restrict differentiation into thechosen pathway. Such a hypothetical model of generating

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Figure 2 | Histone modifications as chromatin codes. a | Schematic representation of histone modifications encountered oninactive and active gene loci. Acetylation (Ac) of lysine (K) 12 on histone H4 and methylation (Me) of K9 on H3 are found at inactivegene loci and are also enriched in heterochromatin (orange circles and squares). b | Acetylation on K9 and K14 of H3, methylationon K4 of H3, acetylation on K5 of H4, and methylation on arginine 3 of H4 are found at gene loci that are either active or have thepotential to be (green circles and squares).


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Figure 3 | Models for the generation and propagation of histone codes. a | Recruitment of chromatin modifiers including histone acetyl transferases (HATs),histone deacetylases (HDACs) and histone methyl transferases (HMTs) (coloured boxes) through sequence-specific DNA-binding factors (DBFs; coloured ovals)generate chromatin codes in the vicinity of their cognate sites (similarly coloured rectangles on the DNA ribbon). Histone modifiers that generate codes consistent withgene inactivity are shown in orange, whereas those that correlate with gene activity are shown in green. b | Nuclear compartmentalization of chromatin regions canalso generate codes that are consistent with the activity of resident factors. For example, histone methylation in pericentromeric heterochromatin (PC-HC) bySUV39H1 (REFS 96,97), histone deacetylation by HDACs enriched in PC-HC72 and histone acetylation by HATs and other hypothetical HMTs that reside ineuchromatin (EC). Nuclear compartmentalization can be achieved through the interactions of chromatin codes with protein modules (chromo- and bromo-domains) ofchromatin-regulating factors that are resident in nuclear subdomains. For example, interactions between methylated lysine (K) 9 of histone H3 (orange square) with thechromodomain of heterochromatin protein 1 (HP1) might promote PC-HC localization36, whereas interactions between acetylated K9 or K14 of H3 (green circles) andbromodomains present on HATs and components of the SWI–SNF complex might promote euchromatic localization. Chromatin codes and interacting factors inheterochromatin (orange) and euchromatin (green) are colour coded to distinguish between the two environments. Chromatin codes can be initiated and propagatedeither by the recruitment of chromatin modifiers (a) or by nuclear compartmentalization of chromatin regions (b). Which mechanism prevails at a given time indevelopment might depend on the presence of sequence-specific targeting factors, as well as on the composition of nuclear compartments. Ac, acetylation; CENPA,centromere protein A; Me, methylation.


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inherent sensitivity of some transcription factors tochromatin, chromatin configuration might determinewhere they act. Chromatin accessibility might there-fore be the key to regulating the activity of transcrip-tion factors that are crucial for distinct lineagechoices, some of which might co-exist in early prog-enitors. Which one of the lineage-regulating factors‘wins’ in determining the lineage choice can thereforebe decided not only by their relative concentrationbut also by the chromatin accessibility at their sites ofaction. Some of these factors might be responsible forgenerating chromatin code and providing lineagechoice by targeting HATs, HDACs and methylases tospecific gene loci (FIG. 6).

H3 with centromere protein A (CENPA) and chromo-some condensation and decondensation before andafter mitosis47,48. Globally acting remodellers also facil-itate the action of enzymatic complexes, such as DNAand RNA polymerases, and DNA repair and recombi-nation enzymes in chromatin environments.

Remodellers also act in gene-specific ways toenable both positive and negative regulatory factorsto access their sites42 (FIG. 6). In vitro studies haveshown that transcriptional regulators differ in theextent to which their activity is affected by chromatinconfiguration49–51. This difference in activity might befurther augmented in vivo, where chromatin is of ahigher order than that assembled in vitro. Given the

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Figure 4 | Deciphering the histone code. a | Histone codes can control DNA accessibility and the ability of DNA-binding factors(DBFs; purple oval) to target their cognate sites on nucleosomes (coloured rectangles on DNA ribbon). b | Histone codes can alsoprovide interaction interfaces for protein modules (chromo- and bromodomains) that are present in functionally diverse chromatin-regulating factors and environments. Chromatin codes and interacting factors in heterochromatin and euchromatin are colour coded(orange and green) to distinguish between the two environments. Constitutive interactions between chromatin code and nuclearfactors have been shown to exist (FIG. 3b). c | There might be instances in which these interactions are regulated by cell-cycle ordevelopmental stage (highlighted interaction modules). This hypothetical model enables a chromatin code to act at a point indevelopment that is distinct to that of its generation. Ac, acetylation; Me, methylation.


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restrict the developmental potential of the cell (FIGS 5,6).The action of a remodeller can therefore be crucial forthe series of events that lead to lineage determinationthrough the generation of chromatin codes in a lineage-gene-specific way.

Remodellers can also act later in differentiation,after lineages and their chromatin codes have beenestablished. In these cases, their sites of action might bedetermined by the combination of pre-existing histonecodes, the protein modules present in the remodellingcomplex and their nuclear location. Interactionsbetween bromodomains in the SWI–SNF complex andacetylated histones can provide examples of this model(FIG. 4). Late-acting chromatin remodellers might beinvolved in enabling further diversification of the dif-ferentiation state within a lineage (for example,remodelling of interleukin 4 and interferon-γ loci dur-ing the differentiation of CD4+ T cells into helper-1 orhelper-2 types57,58).

Gene-specific targeting of chromatin remodellers hasbeen the subject of intense study. Studies on mating-typeswitching in yeast have shown that remodellers can betargeted to the mating-type loci by DNA-binding factors,thereby affecting cell-fate choices52,53. In these and possi-bly other instances54,55, targeting of chromatin remodel-ling to a specific gene through a sequence-specific DNA-binding factor is the first in a cascade of chromatinregulation events that leads to cell-fate determination(FIG. 6). In this model, chromatin fluidity at disparate lin-eage-determining gene loci might underlie the plasticityof early progenitors. In support of this idea, several genesexpressed in distinct lineages seem to be transientlyaccessible in early progenitors5,6,56. However, chromatinfluidity is lost because it enables the establishment of his-tone codes that favour either an open or a closed chro-matin state. This is brought about by the concertedaction of trans-acting factors and covalent modifiers togenerate codes that stabilize a chromatin state and

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Figure 5 | A model of how histone codes can influence lineage decisions. A hypothetical model of how the acquisition of restrictive (orange) and permissive(green) histone codes by two groups of lineage-determining genes (M1, M2, L1, L2) promotes two distinct lineage choices (myeloid or lymphoid) at the level of amultipotent progenitor (MP). In this model, histone codes are not present on lineage-determining genes in the haematopoietic stem cell (HSC) but they are generated(through instructive or stochastic mechanisms) during its transition into a multipotent progenitor. The codes acquired on lineage-determining genes in a multipotentprogenitor will influence its ability to progress further into a differentiation pathway by modulating the activity of the underlying gene (light orange or green DNA ribbon).Interactions between constitutive or developmentally regulated nuclear factors with chromatin codes will further modulate the activity of the gene (dark orange or darkgreen DNA ribbon) and cause lineage restriction. DBF, DNA-binding factor.


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PRIMITIVE HAEMATOPOIESIS

The transient production oflarge, nucleated erythrocytesthat express embryonichaemoglobin, which takes placein the yolk sac. Primitivemegakaryocytopoiesis is alsoreported, but no other types ofblood cell have ever beenreported to be generated inprimitive haematopoiesis.

DEFINITIVE HAEMATOPOIESIS

Haematopoiesis frompluripotent haematopoieticstem cells (HSCs), in which alltypes of blood cell are produced.HSCs emerge first in theaorta–gonad–mesonephrosregion, whereas the main sites ofdefinitive haematopoiesis infetuses and adults are the liverand bone marrow, respectively.

SPLANCHNOPLEURA

The layer formed by the union ofsplanchnic mesoderm andendoderm that gives rise tomuscle, digestive tract and yolk sac.

Expression. Ikaros expression is evident in the jawlessvertebrates (the sea lamprey) and is highly conserved upto humans60,61. Within the mouse embryo, IkarosmRNA is first detected at sites of PRIMITIVE and DEFINITIVE

HAEMATOPOIESIS. At 8.5 days gestation, it is seen in theSPLANCHNOPLEURA in a limited number of mesodermalprecursors, probably those that give rise to haematopoi-etic precursors. Ikaros is expressed in primitive anddefinitive haematopoietic precursors that reside withinearly haematopoietic sites — the yolk sac and fetalliver62. It is also present in a rare bone marrow popula-tion that is enriched for HSCs (Lin−ckit+Sca1+)63–65.Ikaros expression is downregulated on differentiationalong the monocyte/macrophage and erythroid path-ways but is maintained throughout granulocyte matu-ration65. By contrast, Ikaros is upregulated during lym-phocyte differentiation, with highest expression indouble-positive (CD4+CD8+) thymocytes63,64.

Ikaros proteins are normally found in the nucleusof resting lymphocytes in a diffuse, dot-like pattern.Interestingly, as quiescent lymphocytes become acti-vated, the dot-like Ikaros pattern that is characteristicof the resting state gives way to distinctive ring-shaped (toroid) structures that surround the periph-ery of densely staining PC-HC. These structures were

IkarosOur understanding of chromatin regulation hasadvanced in parallel with our knowledge of the nuclearfactors that control development. Targeted mutations inthe mouse germ line have identified an expanding list ofubiquitous and cell-type-specific nuclear factors, theactivities of which are required at distinct steps ofhaematopoiesis to control differentiation, proliferationand survival. The remainder of this article concerns onefactor in particular — Ikaros — highlighting its effectsand its possible involvement in chromatin regulation inseveral steps of this pathway. I discuss evidence indicat-ing that Ikaros complexes might promote chromatinfluidity and can thereby enable the generation of chro-matin code in the haematopoietic system. Given theability of Ikaros to bind nucleosomal DNA in asequence-specific manner (B. Heller, unpublishedobservations), its effects on chromatin are expected tobe gene specific. However, enrichment of Ikaros withone of its remodelling associates in PC-HC in cyclingcells59 indicates that it might be involved in a more gen-eral mechanism, possibly the maintenance of hete-rochromatic code through cell divisions. This might bea key aspect of the role of Ikaros as a tumour suppressorin lymphocytes.

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Figure 6 | Chromatin remodellers in the generation of histone code during development. In this model, a remodeller in association with an early-lineage-determining DNA-binding factor (e.g. Ikaros) is depicted as providing chromatin fluidity in the vicinity of its cognate sites. Chromatin fluidity is the equilibriumbetween open and closed chromatin states. During early stages of development (haematopoietic stem cell, multipotent progenitors and early-lineage-boundprecursors), genes of disparate lineages are often present in both open (green) and closed (orange) chromatin configurations. This equilibrium between disparatechromatin states enables a second wave of DNA-binding factors to have access to their sites and to recruit histone modifiers. This event locks chromatin into the‘open permissive’ or ‘closed restrictive’ configuration by generating the appropriate histone code. The Ikaros complex can by itself provide a restrictive chromatincode because of its associated histone deacetylase (HDAC) activity. Ac, acetylation; DBF, DNA-binding factor; HAT, histone acetyl transferase; HMT, histonemethyl transferase; Me, methylation.


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stem-cell and progenitor populations sorted fromIkaros-null mice revealed the deregulation of two tyro-sine kinase receptors that control the size of theHSC/progenitor pool75. Expression of the tyrosinekinase receptor fetal liver kinase 2 (Flk-2) was absentand c-kit levels were reduced 5–10-fold. Inactivation ofIkaros, however, has a more profound effect on the pro-duction and differentiation of lymphocyte precursors.All components of the B-cell pathway are absent, fromthe earliest described precursors in fetal liver and bonemarrow to the mature populations in peripheral lym-phatic centres and in the peritoneum76. Cells at all stagesof fetal T-cell differentiation are also absent, indicatingthat fetal multipotent haematopoietic progenitors areunable to provide any lymphoid precursors.

After birth, however, a few T-cell precursors aredetected in the thymus, providing us with an importantdistinction between the fetal and postnatal haemato-poietic systems. The Ikaros-deficient postnatal T-cell precursors undergo aberrant differentiation to the dou-ble- and single-positive stage (see below). In sharp con-trast to the severe impairment in the production ofB- and T-cell precursors, common myeloid precursorsand the more restricted granulocyte precursors are pre-sent at near normal numbers75. When the 30–40-folddecrease that is detectable in HSCs in these mice is takeninto account, this indicates a possibly increased propen-sity to form or maintain myeloid precursor populations.Furthermore, their mature progeny of monocytes,macrophages and granulocytes are abundantly presentin the bone marrow and spleen of the Ikaros-null mice.

These studies indicate that Ikaros activity is evidentfrom the very first steps of haematopoietic development.Ikaros regulates HSC activity and enables the differentia-tion of its multipotent progeny along the lymphoid path-ways, whereas it might prohibit the myeloid fate. Furthersupport for this is provided by precursor analysis in micethat are heterozygous for the Ikaros-null mutation.Despite having normal numbers of peripheralhaematopoietic populations, heterozygous Ikaros-nullmice show an increase in myeloid and a decrease in lym-phoid precursors (M. Cortes and M. Trevisan, unpub-lished observations). Within the myeloid lineage, Ikarosis expressed in differentiating and mature granulocytesbut lack of expression is not detrimental to their matura-tion. Nonetheless, in the absence of Ikaros, the granulo-cyte-specific marker Ly6G is downregulated, indicating apossible role for Ikaros in regulating the expression ofsome non-essential granulocyte-specific genes76.

Ikaros also controls progression through subsequentsteps of the T-cell differentiation pathway. Thymocytesthat lack expression of recombinase-activating gene(RAG) 1 or 2 are normally arrested at the late double-negative stage of their differentiation because of lack ofpre-T-cell receptor (pre-TCR) signalling. However, thy-mocytes that are deficient in Ikaros and RAG1 seem toprogress to the double-positive and single-positivestages of their differentiation, indicating a breakdownin pre-TCR- and TCR-signalling requirements77. Lackof Ikaros also causes an increase in the number ofCD4 single-positive thymocytes at the expense of

originally reported to form predominantly in B cells, inmid-to-late G1 phase, and to become diffuse throughthe S phase of the cell cycle66,67. However, independentstudies carried out with primary B and T cells show theformation of the Ikaros toroids from mid-G1 to late Sphase, when they become coincident with late DNAreplication foci68,69. Ikaros toroids are not observed inthe G1 and S phases of many transformedhaematopoietic and lymphoid cell lines. Instead, Ikarosis found in a diffuse nuclear pattern (J. Koipally,unpublished observations). It is therefore important todistinguish between experiments that study the func-tion of Ikaros in cell lines and those using primary tis-sue culture models.

Structural features of Ikaros proteins. Ikaros exerts itseffects in development as a set of differently spliced iso-forms that contain two functionally distinct Kruppel-type zinc-finger domains70,71 (FIG. 7). Of the Ikaros iso-forms described so far, Ik-1 and Ik-2 are the mostabundantly expressed throughout development63,70. So,in normal haematopoietic cells and mature lympho-cytes, most of the Ikaros protein produced can bindDNA. The DNA-binding specificity of Ikaros is causedby the DNA-binding isoforms being incorporated into ahigher-order complex (B. Heller, unpublished observa-tions), which contains 10–12 Ikaros units as well asother proteins (see below)72. Non-DNA-binding Ikarosisoforms are also generated by splicing that excludes theDNA-binding, zinc-finger-containing exons. These iso-forms are also incorporated within the same complexbut are normally present at a much lower amount. Thenon-DNA-binding Ikaros isoforms are often referred toas dominant negative because of the pronounced nega-tive effect they have on the DNA-binding properties ofan Ikaros complex when present in excess73,74.

Ikaros in haematopoietic development. The role ofIkaros in the haematopoietic system has been studied indetail (TABLE 1). A mutation that generates an Ikaros -nullallele causes a 30–40-fold reduction in HSC activity inthe fetus and in the adult75. Gene-expression studies in

Protein interaction

F1 F2 F3 F4 F5 F6Ikaros1

Nuclear localization,DNA binding

Repression domain I Repression domain II

Zinc-finger domain Zinc-fingerdomain

Figure 7 | The most full-length Ikaros isoform (Ik-1) and its functional domains. The firstzinc-finger domain spans the amino-terminal half of the protein, varies in composition betweenisoforms and is involved in sequence-specific interactions with DNA containing theC/TGGGAAT/C core motif and in nuclear localization. The second zinc-finger cluster, located atthe carboxyl terminus, is shared by all Ikaros isoforms and is involved in interactions with self andwith other Ikaros family members74. Interestingly, this self-interaction motif is shared with theDrosophila gap protein Hunchback and with the mammalian proteins Pegasus and the tricho-rhino-phalangeal syndrome 1 (TRPS1)98–100. So, the carboxy-terminal Ikaros zinc fingers areexamples of an evolutionarily conserved interaction module. Domains involved in repression andnuclear localization are indicated. F, zinc-finger motif.


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POSITION EFFECT VARIEGATION

Stochastic silencing of a genethat is adjacent toheterochromatin in a proportionof cells.

The similarity of the phenotypes manifested by theIkaros DNA-binding homozygous mutants and the nullmice proves the centrality of the DNA-binding domain ofIkaros to its regulatory function during haemolym-phopoiesis. The greater severity of the DNA-bindingmutants led to the hypothesis that there were other struc-turally related factors that physically interact with Ikarosand participate in the same molecular mechanisms thatcontrol differentiation in the haematopoietic system. Thisled to the identification of three structurally related family members (Aiolos, Helios and Dedalos/Eos) withpartially overlapping expression profiles63,64,67,78.

Taken together, these genetic studies have estab-lished that Ikaros has a crucial role at several lineagedecision points during haemolymphopoiesis. These in vivo studies are, however, not well suited to determin-ing whether Ikaros influences specific lineage decisionsor acts subsequently to influence the survival, prolifer-ative expansion or differentiation of cells. However,unpublished clonal assays for lymphoid and myeloidprecursors in Ikaros heterozygous null mice supportthe model that Ikaros influences the outcome of lin-eage decisions by promoting lymphoid differentiationat the expense of myeloid differentiation. Within thelymphoid lineage, Ikaros also has a crucial role in reg-ulating the proliferative expansions that differentiatingand mature lymphocytes undergo in response toengagement of their antigen receptors.

Ikaros in chromatin remodelling. Early biochemical stud-ies with Ikaros protein showed that it can interact withitself 74. This property underlies the pronounced accu-mulation of wild-type Ikaros protein in heterochro-matin-associated structures and the inability of mutantsin this interaction domain to do so79. In lymphocytes,most Ikaros is present in a 2-MDa complex that contains10–12 Ikaros molecules as well as several other proteins72.Characterization of the main Ikaros-containing complexin T cells revealed the presence of Mi-2β, an ATP-depen-dent chromatin remodeller (also known as Chd4), aswell as the HDACs HDAC1 and HDAC2. These threeproteins are components of the nucleosome remodellingand deacetylation (NURD) complex that is active inboth chromatin remodelling and histone deacetylation.

double-positive thymocytes76. One explanation for thisphenotype is the requirement for Ikaros to give stableexpression of CD8 in double-positive thymocytes.Support is provided by two independent lines of investi-gation, one showing that Ikaros is required to counter-act POSITION EFFECT VARIEGATION on CD8 regulatory ele-ments in transgenic mice (D. Kioussis, unpublishedobservations) and a second showing that these regula-tory elements are associated with chromatin-boundIkaros (T. Naito, unpublished observations).

Proper lymphocyte differentiation is affected notonly by the presence or absence of Ikaros, but also byits relative expression levels. Haploinsufficiency ofIkaros causes a halving of lymphocyte precursors and asmall increase in myeloid precursors (M. Cortes andM. Trevisan, unpublished observations). However,homeostatic mechanisms that operate at later stages ofthe haemolymphoid pathway provide mature lympho-cyte and myeloid populations that seem to be normal innumber and cell-surface phenotype. Nonetheless, theseapparently ‘normal’ mature T cells enter the cell cycleunder minimal in vitro engagement of their TCR andproliferate robustly compared with their wild-typecounterparts69,77. Consistent with this hyperproliferativephenotype, mice that are haploinsufficient for Ikarosdevelop T-cell leukaemias and lymphomas.

The importance of the Ikaros DNA-bindingdomain in its ability to control haemolymphoiddevelopment was also established in a series of geneticstudies (TABLE 1). Analysis of mice that are homozy-gous for a germ-line deletion in the Ikaros DNA-bind-ing domain revealed a trend of phenotypes similar tothe homozygous Ikaros-null mutants, but moresevere73. For example, HSC activity was reduced bymore than 100-fold and postnatal T-cell developmentwas not detected in these mice73,75. Furthermore, micethat were heterozygous for this mutation showed amore profound hyperpoliferative phenotype and devel-oped T-cell leukaemias and lymphomas at a higherrate54,66. Ectopic overexpression of Ikaros non-DNA-binding isoforms during T-cell differentiation causessimilar phenotypes, the severity of which depends on the level of transgene expression (S. Winandy,unpublished observations).

Table 1 | Ikaros effects in haematopoiesis and lymphocyte development

Mutation Phenotype

Ikaros null−/− Reduction in HSC activity 30–40-foldLack of all B-cell and fetal T-cell precursors and downstream progeny; lack of NK cellsRelative increase in myeloid precursors and their progenySkewing of postnatal T-cell differentiation to the CD4+ populationClonal T-cell expansions

Ikaros DN−/− (∆ DNA BD) Reduction in HSC activity >100-foldLack of all B- and T-cell precursors and downstream progenyLack of NK cells and DCs

Ikaros null+/− Reduction in B-cell precursors by 50%B- and T-cell populations are normal in number and cell-surface phenotypeAugmented antigen-receptor-mediated proliferationDevelopment of T-cell malignancies

Ikaros DN+/− Same as Ikaros null+/– but more severe

DC, dendritic cell; ∆DNA BD, DNA-binding-domain (BD)-deleted mutant; DN, dominant negative; HSC, haematopoietic stem cell; NK,natural killer.


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in its complex. The increased expression of the granulo-cyte–macrophage colony-stimulating factor (GM-CSF)receptor detected in the Ikaros-null progenitor com-partment provides support for this hypothesis75. Indeed,Ikaros proteins that are fused to GAL4 behave as potentrepressors, with an activity that depends on HDACs83.Mutations in Ikaros that affect its ability to localize toPC-HC do not interfere with its ability to repress in anHDAC-dependent fashion, indicating that Ikaros neednot be located in PC-HC for this function59.

However, gene-expression studies on Ikaros-nullstem cells and lymphocytes have revealed that a signifi-cant number of genes that support lymphocyte differen-tiation are downregulated75 (S. Ng, unpublished obser-vations). It is therefore possible that the Ikaros–NURDcomplex, which can provide fluidity to chromatin,might potentiate gene expression in the vicinity of targetsites that also accommodate transcriptional activators.In this model, Ikaros acts as a potentiator of geneexpression through the activity of its associated chro-matin remodellers by enabling a second layer of tran-scription factors (and, for example, associated HATs) toprovide an open chromatin code and over-ride theactivity of the associated HDAC (FIG. 8a).

Given the ability of Ikaros to move into PC-HC-associated structures in cycling B and T cells66,68,69 andits stable association with the NURD complex72,86, tar-geting of NURD into PC-HC by Ikaros became astrong possibility. This was verified by nuclear localiza-tion studies on Mi-2β, one of the central componentsof the NURD complex. Most Mi-2β is localized to thePC-HC in wild-type activated T cells, whereas itremains diffusely distributed throughout the nucleus ofthe Ikaros-null T cells72. A similar study was carried outin NIH-3T3 fibroblasts, cells which do not normallyexpress Ikaros59. In these cells, Mi-2β is diffusely distrib-uted in the nucleus throughout the cell cycle. However,on ectopic expression of Ikaros in these cells, most Mi-2β relocates into PC-HC-associated toroids togetherwith Ikaros. So, the Mi-2β (NURD) complex is targetedinto PC-HC-associated areas only in cycling cells thatexpress Ikaros, presumably through its association withIkaros protein.

Although the Ikaros-mediated relocation of Mi-2βinto PC-HC in cycling cells is clearly established, its roleand significance are not. Several correlations can, how-ever, be made that provide us with testable hypotheses.The hyperproliferative phenotype of Ikaros-deficient Tcells correlates with the inability of Mi-2β in these cellsto be targeted effectively into the PC-HC-associatedstructures owing to a loss of Ikaros. Three mechanismshave been suggested to account for this. One possibilityis that the targeting of Mi-2β/NURD into PC-HC byIkaros ensures regulated proliferation by removing thecomplex from its sites of action on genes that reside out-side this compartment until a later stage in the cellcycle59. At the same time, it might directly affect expres-sion of its gene targets that reside within PC-HC (FIG. 8b).Targeting of Mi-2β/NURD into PC-HC by Ikaros mightbe crucial for regulated changes in gene expression inactivated lymphocytes.

The previously characterized NURD complex inepithelial cells also contains the MTA2 (metastasis-associated protein 2; also known as MTA1L1), MBD3(methyl-CpG-binding domain protein 3) and Rbp48/46(RNA-binding proteins 48/46) proteins80–82. With theexception of MBD3, most of these factors are also presentin the Ikaros complex isolated from lymphocytes. Like itscounterpart in epithelial cells, the Ikaros–NURD com-plex has potent chromatin remodelling activity in vitroand can deacetylate histones72. Although most Ikaros inmature lymphocytes exists in a NURD complex, a smallamount is associated with the SWI–SNF remodellingcomplex and with two other co-repressors (the Sin3 andCtBP proteins)72,83,84. The recent finding that theSWI–SNF complex also contains the co-repressors Sin3,HDAC1 and HDAC2 (REF. 85) provides a possible linkbetween the minority of Ikaros protein that associateswith the SWI–SNF and Sin3–HDAC components. It isnoteworthy that most Ikaros purifies in these complexes(as determined by gel-filtration studies on both unfrac-tionated nuclear extracts and the immunopurified pro-tein), indicating that most Ikaros protein in these cells isstably integrated in these chromatin remodellers.

In differentiating thymocytes and mature T cells,most Ikaros proteins are present in a NURD complex (A.Jackson, unpublished observations). However, it is possi-ble that, in other cell types (for example, in haematopoi-etic progenitors or precursors), Ikaros has distinct associ-ations. A similar association between Ikaros and theNURD complex was reported in an erythro-leukaemiacell line86. Furthermore, forced expression of Ikaros inhaematopoietic and non-haematopoietic cells have alsoshown important interactions between Ikaros andNURD components59 (S. Ng, unpublished observa-tions). The Ikaros–NURD association therefore seems todominate in a range of cell types and does not rely onany lymphoid-specific factors.

Taken together, these biochemical studies show thatIkaros is stably associated with enzymatic machines,remodellers and HDACs, which could function duringhaematopoiesis by providing chromatin fluidity andestablishing chromatin codes (FIG. 6).

Role of Ikaros in the NURD complex. Given that mostIkaros proteins associate with the NURD complex, onecan assume that the Ikaros–NURD complex is responsi-ble for many of the effects of Ikaros on haematopoiesis.But how does a NURD complex that contains Ikarosbring about specific events during haematopoiesis? One obvious possibility is that Ikaros affects the target-ing of the complex, with its DNA-binding capacity andits strong presence (10–12 units) in a haematopoieticversion of NURD.

The Ikaros–NURD complex can be targeted toIkaros-binding sites that are present in haematopoietic-specific genes. Targeting of the Ikaros–NURD complexto lineage-specific genes in early progenitors mightunderlie lineage decisions effected by Ikaros (FIG. 8a). Inan early haematopoietic progenitor, Ikaros mightrepress the expression of genes that promote myeloiddifferentiation through the action of an HDAC present


R E V I E W S

from Ikaros-deficient T cells provide some support forthis second hypothesis69. In Ikaros-deficient T cells, dele-tions of regions in proximity to the centromere, as wellas chromatide breakpoints and extrachromosomal frag-ments, are observed. These aberrant events in chromo-some propagation might be caused by inappropriatereplication of chromatin structures. Mutations in the de novo methyltransferase DNMT3b, which is involvedin the methylation and stable propagation of cen-tromeric structures, also causes the development ofleukaemias and lymphomas87,88.

A third possibility is that the Ikaros–NURD com-plex attaches to specific gene targets in resting lympho-cytes and recruits them into PC-HC. This allows prop-agation of the closed, repressed chromatin code by thelocal environment (that is, methylation of DNA or his-tones). The presence of several Ikaros units in theIkaros–NURD complex72 would allow a simultaneousinteraction with gene-specific targets and heterochro-matin-associated DNA sequences79. Support for such amodel is provided by studies that have shown that sev-eral potential Ikaros gene targets are associated withIkaros in PC-HC66. However, these studies have alsoindicated that a neuron-specific gene and Hox genesthat are probably not regulated by Ikaros are also pre-sent in the vicinity of PC-HC with Ikaros40. In theabsence of further evidence for a direct associationbetween a gene and Ikaros, their PC-HC co-localizationmight, in some cases, reflect sharing of this nuclearcompartment or indicate a possible global role forIkaros (or its NURD associate) in gene silencing in thiscompartment. The NURD complex might thereforerely on Ikaros for some of its activities in thehaemolymphoid system. Deregulation of NURDactivities might underlie many of the developmentaland proliferation phenotypes manifested in thehaematopoietic system in the absence of Ikaros.

Future directionsAlthough much has been gleaned from past studies onchromatin regulation and Ikaros, we are still in the earlystages of understanding how they together contribute todevelopment and proliferation. One obvious question ishow much of Ikaros’ effort is directed towards globalrather than gene-specific effects on chromatin and howthese tie into the development and homeostasis of thehaematopoietic system. Initial analysis of Ikaros-defi-cient hyperproliferative T cells has provided evidencefor defects in chromosome propagation. However, itremains to be settled whether these are due to global orgene-specific events mediated by Ikaros.

Examining the role of Ikaros in gene-specific eventshas revealed the need to establish criteria for what is acrucial target. Following the example of other develop-mental systems, we must consider: first, the presence ofIkaros-binding sites in the regulatory regions of apotential gene target; second, the in vivo associationbetween Ikaros and the regulatory sites in question; andthird, the deregulation of the gene in the absence ofIkaros. Together, these criteria provide a compellingargument for a gene target. Studies of the regulatory

A second possibility is that the Ikaros–NURD com-plex sequestered into PC-HC during the cell cycle catersto the needs of this nuclear compartment (FIG. 8b). Rapidlycycling lymphocytes might rely in part on theIkaros–NURD remodelling activity to replicate their PC-HC structures and to propagate its code. Giemsa stainingreveals degrees of chromosome condensation (and chro-matin compaction) in a banding pattern, with regions ofeuchromatin staining lightly, and regions of heterochro-matid staining darkly.Alterations in the banding patternof subcentromeric areas seen on mitotic chromosomes

a

b

Gene transcriptionrepressed

GenetranscriptionActivators

RNA Pol IIholoenzyme

PC-HC

Propagation of silentstate in PC-HC

Modulation of geneexpression

Ikaros

HDAC

Mi-2β (NURD)

Figure 8 | Gene-specific and global targeting of chromatin remodelling by Ikaros.a | Models of gene-specific targeting and its effects. The Ikaros–NURD complex that is recruitedto Ikaros-binding sites through chromatin remodelling and histone deacetylation provides arestrictive chromatin code and places genes in a repressed state (orange nucleosomes).However, on remodelling, transcriptional activators might gain access to their sites and recruithistone modifiers that lock into an open and transcriptionally permissive chromatin state (greennucleosomes) and over-ride the restrictive code mediated by the histone deacetylase (HDAC).b | As cells enter G1 phase, much of the Ikaros–NURD complex is found surroundingpericentromeric heterochromatin (PC-HC, depicted as a ring of balls). This correlates with theability of the cell to undergo regulated proliferation. Ikaros–NURD compartmentalization into PC-HC might be crucial for the replication of this chromatin-dense region and its associated genes. Itmight also modulate the expression of Ikaros-specific gene targets that reside in thiscompartment. Mi-2β, chromodomain helicase DNA-binding protein 4 (also known as Chd4);NURD, nucleosome remodelling and deacetylation.


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gene-expression systems. These studies might providethe first steps in the cascade of events that take place in vivo on Ikaros gene targets. Further studies that exam-ine their chromatin status in vivo in the vicinity of thetarget sites of Ikaros can provide more steps in this path-way. Significant correlations can then be made betweenchromatin codes and possible nuclear movements, andaid our understanding of the mechanisms that supportthe generation and propagation of chromatin states.

Ikaros provides us with a prototype of a novel classof cell-fate and chromatin regulators. It will be impor-tant to know whether this is a class of regulators andmechanisms used only by the haematopoietic systemor whether it is also used by other developmental sys-tems. The Ikaros family members Helios and Eos areexpressed in epithelial and neuronal tissues and mightserve similar regulatory pathways in non-haemato-poietic developmental systems. There are no knownIkaros homologues in model organisms such as fruitflyor worm, but DNA-binding factors with similar prop-erties to Ikaros have been reported. In the fly, the gapgene hunchback encodes a zinc-finger DNA-bindingprotein with functional domain similarities to Ikarosand that also interacts with Drosophila Mi-2 (REF. 95).Ikaros and Hunchback might therefore exist in parallelregulatory networks that control cell-fate decisions inthe mouse and fruitfly. In coming years, this combi-nation of genetic, biochemical and cell-biologicalapproaches to understanding this complex of regula-tory factors and their chromatin targets will provideunique insights into the epigenetic regulation oflineage decisions.

elements of genes expressed in lymphocytes, such as λ5,TdT and the long-terminal repeat of Mcf89–91 have iden-tified Ikaros-binding sites, which are important for theiractivity in lymphocytes. Although Ikaros proteins canbind to these sites in vitro, there is still no direct evidencefor in vivo association. Deregulation of Flk-2, c-kit, GM-CSF receptor, GR1, L-selectin and IL-2 receptor-β in theIkaros-null haematopoietic system75, or the expressionof a dominant negative Ikaros isoform92–94 also indicatethat these might be targets of Ikaros. The effect might,however, be indirect and caused by the deregulation of aregulator of these genes. So far, the only gene that meetsthe above criteria and the definition of a target is CD8.More must follow. It is through the knowledge of Ikarosgene-specific targets in the haematopoietic system thatwe will further delineate the molecular pathways thatcontrol its regulation.

It is well established that Ikaros proteins are integralcomponents of at least one major chromatin remodelling complex that is decorated by co-repressors. Therole of this complex and its associated activities inhaematopoiesis need to be further established by geneticstudies. These might address the ability of Ikaros mutantsthat cannot interact with, for example, NURD compo-nents to rescue the Ikaros-null phenotypes, and theeffects of targeting mutations in other components of theNURD complex in a haemolymphoid-specific manner.

A first-level insight into the mechanisms by which theIkaros-remodelling complexes exert their function canbe obtained by examining their effect on chromatinstructure and transcriptional activity on their targetingto specific regulatory regions in in vitro chromatin-based


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AcknowledgementsI am grateful to members of my laboratory and to D. Kioussis forallowing me to cite unpublished results. I am indebted to J.Koipally, E. Wong, B. Heller, T. Yoshida and B. Morgan for con-structive discussions and other support that helped to shapethis manuscript.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink CD4 | CD8 | DNMT3b | FLK2 | GM-CSF | GR1 | HDAC1 | HDAC2 |HP1 | Hunchback | Ikaros | IL-2 receptor-β | interferon-γ | interleukin4 | MBD3 | Mi-2β | MTA2 | Pegasus | Sin3 | SUV39H1 | TAFII250OMIM: http://www.ncbi.nlm.nih.gov/Omimtricho-rhino-phalangeal syndrome 1Access to this interactive links box is free online.

haematopoietic cell-fate decisions, chromatin regulation and ikaros

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