Chromatin Modifications in Hematopoietic Multi Potent And

7/31/2019 Chromatin Modifications in Hematopoietic Multi Potent And

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Chromatin Modifications in Hematopoietic Multipotent andCommitted Progenitors Are Independent of Gene Subnuclear

Positioning Relative to Repressive CompartmentsCLAIRE GUILLEMIN,a,b MARTA MALESZEWSKA,c,d ADELINE GUAIS,a,b JEROME MAES,c,d

MARIE-CHRISTINE ROUYEZ,a,b AZZEDINE YACIA,a,b SERGE FICHELSON,a,b MICHELE GOODHARDT,c,d

CLAIRE FRANCASTELa,b

aInstitut Cochin, Universite Paris Descartes, Centre National de la Recherche Scientifique (Unite Mixte de

Recherche [UMR] 8104), Paris, France; bInstitut National de la Sante et de la Recherche Medicale, U567, Paris,

France; cInstitut Universitaire dHematologie, Hopital Saint-Louis, Paris, France; dInstitut National de la Sante et de

la Recherche Medicale, U662, Paris, France

Key Words. Epigenetic processes Regulation of gene expression Cell differentiation Hematopoietic stem cells Hematopoietic progenitor cells

ABSTRACT

To further clarify the contribution of nuclear architecture inthe regulation of gene expression patterns during differentia-tion of human multipotent cells, we analyzed expression status,histone modifications, and subnuclear positioning relative torepressive compartments, of hematopoietic loci in multipotentand lineage-committed primary human hematopoietic progen-itors. We report here that positioning of lineage-affiliated locirelative to pericentromeric heterochromatin compartments(PCH) is identical in multipotent cells from various origins andis unchanged between multipotent and lineage-committed he-matopoietic progenitors. However, during differentiation of

multipotent hematopoietic progenitors, changes in gene expres-sion and histone modifications at these loci occur in committedprogenitors, prior to changes in gene positioning relative topericentromeric heterochromatin compartments, detected atlater stages in precursor and mature cells. Therefore, duringnormal human hematopoietic differentiation, changes in genesubnuclear location relative to pericentromeric heterochroma-tin appear to be dictated by whether the gene will be perma-nently silenced or activated, rather than being predictive ofcommitment toward a given lineage. STEM CELLS 2009;27:108115

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

The multilineage potential of human hematopoietic stem cells(HSC) is gradually lost as these multipotential progenitors makecommitment choices to differentiate to non-self-renewing andthen lineage-committed progenitors along the different bloodlineages (reviewed in [1, 2]). When a stem cell is committed todifferentiating toward a given lineage, global genome repro-gramming involves both repression of nonaffiliated genes andselective activation of genes involved in the establishment ofthis lineage. Accumulating evidence indicates that lineage-spe-

cific gene expression is determined not only by the availabilityof specific transcription factors but also by epigenetic modifi-cations, including both local modifications of DNA and chro-matin and global topological changes in chromosome and genepositioning in the nucleus (reviewed in [36]). Combined, thesedifferent levels of gene regulation allow for fine control thatintegrates environmental and intracellular signals to establish

appropriate gene expression programs, and hence ultimatelydetermine the identity of the cell.

Specific histone amino-terminal modifications can generatesynergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitionsbetween transcriptionally active and transcriptionally silentchromatin states [7]. For example, acetylation of certain N-terminal lysine residues in histones H3 and H4 (H3ac and H4ac)is generally associated with a transcriptionally active chromatinconfiguration, whereas methylation of lysine 9 of histone H3(H3K9me3) appears to be a hallmark of condensed chromatin atsilent loci. These modifications play an important role in prim-

ing of gene activity and in maintenance of gene expression overseveral cell divisions (reviewed in [8, 9]). Chromatin accessi-bility might therefore be the key to regulating the activity oftranscription factors and hence ultimately determine cell fate.

Accumulating evidence indicates that the subnuclear loca-tion of a gene also influences its activity. This has been shownfor a number of genes expressed in hematopoietic cells (recently

Author contributions: C.G.: performance of research, data analysis and interpretation; M.M., A.G., and J.M.: performance of research;M.-C.R.: contributions to real-time PCR; A.Y.: contributions to expansion of erythroid progenitors; S.F.: provision of study material; M.G.:data analysis and interpretation, manuscript drafting; C.F.: conception and design, financial support, data analysis and interpretation,manuscript writing, final approval of manuscript. C.G. and M.M. contributed equally to this work.

Correspondence: Claire Francastel, Ph.D., Institut Cochin, Departement dHematologie, INSERM U567CNRS UMR 8104, MaternitePort-Royal, 5eme etage, 123, Boulevard Port-Royal, 75014 Paris, France. Telephone: 33-1-53-10-43-84; Fax: 33-1-43-25-11-67; e-mail:

[email protected] Received August 5, 2008; accepted for publication October 16, 2008; first published online in STEM CELLSEXPRESS October 30, 2008. AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2008-0755

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reviewed in [1014]). Furthermore, active and inactive regionsof the genome, as well as protein factors involved in activationor repression of gene expression, are compartmentalized withinthe nucleus [15, 16]. Several studies indicate that the active stateof a gene is associated with permissive nuclear compartments(i.e., enriched in proteins of the transcriptional machinery [17

19] or splicing factors [20]). For some loci, the transcriptionallyactive state may also be associated with chromatin deconden-sation and looping out of chromosome territories [2124]. Incontrast, silent loci are often found in close proximity to repres-sive compartments (i.e., the nuclear periphery [25, 26] or peri-centromeric heterochromatin [2730], a compartment wheretranscriptional repressors concentrate [31]).

Epigenetic changes in chromatin structure and subnuclearlocation, therefore, most likely precede gene activation and playa critical role in the choices of a stem cell to continue toself-renew or to differentiate. On the basis of our previousstudies on the -globin locus, we proposed a sequential modelof gene activation, first involving relocation away from pericen-tromeric heterochromatin to establish or maintain an open chro-matin configuration and locus-wide acetylation, followed by

local histone H3 hyperacetylation and promoter activation [3,29]. This model suggests that commitment of multipotent pro-genitors to the red-cell lineage may involve a preactivation stepin which the globin locus is open, acetylated, and localized awayfrom pericentromeric heterochromatin in the nucleus of a red-cell progenitor.

In this study, we tested this model using primary humanmultipotent and lineage committed hematopoietic progenitorsisolated from umbilical cord blood. We examined the sub-nuclear location and histone modifications associated with lym-phoid- or myeloid-specific genes following differentiation ofprimary human multipotent hematopoietic cells toward differenthematopoietic lineages. In essence, our results show that acet-ylation of histones at lineage-affiliated genes occurs in commit-

ted progenitors and further increases as cells progress throughdifferentiation, whereas subnuclear positioning of these locirelative to repressive compartments does not significantlychange upon commitment of multipotent hematopoietic progen-itors to a given lineage. In addition, hematopoietic loci adopt thesame positioning in multipotent cells from different origins,whereas they show distinct histone marks. Therefore, the chro-matin state in multipotent cells and committed progenitors isestablished independently of gene positioning relative to repres-sive pericentromeric heterochromatin compartments (PCH) ornuclear periphery. Changes in subnuclear gene positioning dur-ing human hematopoietic differentiation occur at later stages, inprecursor and mature cells, suggesting that they are implicatedin maintenance of a tissue-specific chromatin state rather than insetting this particular chromatin state.

MATERIALS AND METHODS

Cell Separation, Antibodies, and Culture Conditions

Human umbilical cord blood (UCB) samples from normal full-termdeliveries were collected, with informed consent of the mothers,according to approved institutional guidelines. CD34 cells wereenriched from mononuclear cells to greater than 85% by immuno-magnetic selection (StemCell Technologies, Grenoble, France,http://www.stemcell.com) using the MidiMACS system (MiltenyiBiotec, Paris, http://www.miltenyibiotec.com).

Multipotent hematopoietic cells (CD34CD38lo) and B-lym-phoid progenitors (pro-B, CD34CD10CD19) were enrichedfrom the CD34 population by fluorescence-activated cell sorting

(Epics ALTRA; Beckman Coulter, Villepinte, France, http://www.beckmancoulter.com) based on the expression of surface markers.

Multipotent CD34CD38lo progenitors contain the majority oflong-term engrafting cells and represent 5%10% of total UCBCD34 cell fraction. Precursors and mature B-lymphoid cells(CD34CD10CD19) were directly sorted from mononuclearcells (supporting information Fig. 1). To study histone modifica-tions by chromatin immunoprecipitation (ChIP), we also sortedhuman CD45 pro-B and pre-B cells from the marrow of NOD/

SCID mice engrafted with UCB CD34

cells, as described [32]. Forerythroid cells we used the in vitro cell culture procedure describedby Freyssinier et al. [33] to produce large amounts of human erythroidprogenitor and precursor cells (supporting information Fig. 1).

The purity of the sorted cells was verified by flow cytometryreanalysis. For flow cytometry analysis and cell sorting, we usedphycoerythrin- or allophycocyanin-conjugated mouse anti-humanmonoclonal antibodies CD38 (clone LS198 4-3), CD34 (clone581), CD36 (clone FA6.152), and CD19 (clone J4.119) (all fromBeckman Coulter). Thymic CD34 progenitor cells were purified(purity, 94%) from a thymus sample collected after heart surgery ona 3-month-old baby with informed consent of the parents. Humanmuscle satellite cells (a generous gift from Dr. V. Mouly, CentreNational de La Recherche Scientifique (CNRS) UMR 787, Institutde Myologie, Institut National de la Sante et de la RechercheMedicale [INSERM] and Universite Pierre et Marie Curie, Paris,

France) were isolated from healthy donors upon biopsy in agree-ment with the French legislation on ethical rules and cultured asdescribed [34]. Mesenchymal stem cells were obtained from healthydonors bone marrow (generous gift from Dr. P. Charbord,INSERM, Equipe ESPRI/EA-3855, University Francois Rabelais, Fac-ulty of Medicine, Tours, France) [35].

Probes for In Situ Hybridization

The -globin probe is a 185-kilobase (kb) genomic fragment (gen-erous gift from Dr. P.A. Ioannou, The Murdoch Institute for Re-search into Birth Defects, Royal Childrens Hospital, Melbourne,VIC, Australia) covering the whole -globin locus and additionalsequences in 3 of the genes and in 5 of the locus control region(LCR) [36]. The -globin probe was made from a 150-kb BACclone (generous gift from Dr. D.R. Higgs, MRC Molecular Hema-tology Unit, Weatherall Institute of Molecular Medicine, John Rad-

cliffe Hospital, Oxford, U.K.), and it contained the whole -globinlocus and its regulatory elements [37]. We used BAC and PACclones from the Sanger Institute (Wellcome Trust Sanger Institute,Cambridge, U.K, http://www.sanger.ac.uk) as probes to detect thehuman Ig locus (BAC RP11-316G9), human IgH locus (BACaccession no. RP11-151B17), and human RhesusAG gene (PACRP3-442L6).

Probes were either biotin-labeled by random priming (BioprimeDNA Labeling System; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) or directly labeled by nick translation (Nick Transla-tion Mix; Roche Diagnostics, Meylan, France, http://www.roche-applied-science.com) using Alexa Fluor 488 incorporation (AlexaFluor 488-dUTP; Invitrogen), according to the manufacturers instruc-tions. Human centromeres were detected using a mixture of 89-mer and83-mer covering the entire -satellite consensus sequence, labeled intheir 5 ends with either a digoxigenin (DIG) or a biotin molecule.

DNA Fluorescence In Situ Hybridization

DNA fluorescence in situ hybridization in conditions that preservethree-dimensional (3D) nuclear architecture was performed as pre-viously described, with slight modifications [38]. Cells (2 104

cells) were fixed for 10 minutes at room temperature (RT) with 4%paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA,http://www.emsdiasum.com)/1 phosphate-buffered saline (PBS),permeabilized for 10 minutes in 1 PBS/0.1% Triton X-100,treated with 100 g/ml RNaseA (Fermentas Life Sciences, St-Remyles Chevreuse, France, http://www.fermentas.com) in 2 standardsaline citrate (SSC) for 30 minutes at 37C, and further permeabil-ized at 37C for precisely 5 minutes with 0.002%0.01% pepsin(Sigma-Aldrich, Saint-Quentin, France, http://www.sigmaaldrich.com) depending on the cell type, in 10 mM HCl. Cells were thenpostfixed with 1% paraformaldehyde/1 PBS for 10 minutes at RT.

Denaturation was performed in 70% formamide/2 SSC at 75Cfor precisely 3 minutes.

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We approximately used 200 ng of DNA probe combined with 8g of human Cot-1 DNA (Cot-1; Invitrogen) and 2 g of salmonsperm DNA (Sigma-Aldrich) per slide in 50% formamide/10%dextran sulfate/2 SSC/1% Tween-20. The mixture of probe withcompetitive DNA was heat denatured at 95C for 5 minutes, pre-annealed at 37C for 1 hour, then hybridized overnight on the slides,at 37C in a humid chamber. After washes in 50% formamide/2

SSC at 42C and then 2 SSC (or 0.1 SSC, depending on theprobe) at 42C, oligonucleotides (40 ng/slide) against human -sat-ellite were hybridized in 50% formamide/10% dextran sulfate/2SSC/1% Tween-20 for 2 hours at 37C in a humid chamber and thenwashed in 2 SSC at 42C. Probes were revealed using Alexa Fluor594-conjugated streptavidin (1:1,000; Molecular Probes, Eugene,OR, http://probes.invitrogen.com) and anti-DIG-fluorescein Fabfragments (1:200; Roche Diagnostics) in 4 SSC/3% bovine serumalbumin for 45 minutes at RT and mounted in Vectashield (VectorLaboratories, Paris, http://www.vectorlabs.com) containing 1 g/ml4,6-diamidino-2-phenylindole (DAPI).

Microscopy and Measurements

All images were taken through a Plan-Neofluar 63 oil objective(numerical aperture, 1.25; Carl Zeiss, Le Pecq, France, http://www.zeiss.com) using MetaView 7.04 software (Roper Scientific, Evry,France, http://www.roperscientific.com). Optical z-sections werecollected at 0.2-m steps through each nucleus on an Axioplan 2Imaging fluorescence microscope (Carl Zeiss) with a cooled Cool-snap camera (Roper Scientific) and processed in Metamorph 7.04software (Roper Scientific). Examples of images obtained from thevarious cell types used are shown in supporting information Figure 2.

The proximity to the closest cluster of pericentromeric hetero-chromatin revealed by an -satellite probe was analyzed in Meta-morph. For each population studied, at least three independentseries of slides for at least two independent cell sorting wereassessed, and 70220 nuclei for each cell type or maturation stagewere scored.

Gene Expression Analysis

RNA was isolated from 105 cells using 1 ml of TRIzol reagent

(Invitrogen). Total RNA was reverse transcribed using randomhexamers and the Superscript reverse transcriptase kit (Invitrogen)according to the manufacturers instructions. cDNA reactions wereused as templates in real-time polymerase chain reactions (PCRs)using the light cycler-DNA MasterPLUS SYBR Green I mix (RocheDiagnostics) supplemented with 0.5 M specific primer pairs (sup-porting information Table 1). Real-time quantification PCRs wererun on a light cycler rapid thermal system (Roche Diagnostics) with5 seconds of annealing at 60C and 10 seconds of extension at 72Cfor all primers. Quantitative results for each sample were obtainedby using a standard curve. Each sample was tested at least twotimes.

ChIP Assays

ChIP experiments were carried out essentially according to themanufacturers instructions (ChIP Assay Kit; Upstate, Charlottes-ville, VA, http://www.upstate.com) with slight modifications [39].Cells (12 106) were fixed with 0.5% formaldehyde for 10minutes at 37C. We typically used chromatin prepared from 2 105 cells per histone modification to be analyzed. To perform ChIPon rare CD34CD38lo cells, we pooled up to 20 cord blood sam-ples. Solubilized chromatin was diluted 10-fold in ChIP dilutionbuffer and, after removal of an aliquot (input), immunoprecipitatedovernight at 4C with antibody against anti-acetyl histone H4 (06-866; Upstate), anti-acetyl histone H3 (06-599; Upstate), anti-di-methyl H3-K4 (07 030; Upstate), anti-trimethyl histone H3-K9(07-523; Upstate), or anti-trimethyl histone H3-K27 (07-449; Up-state). Analysis of positive and negative modifications was alwaysperformed on the same chromatin samples. Quantitative PCR wasperformed to determine the relative enrichment of gene segments inChIP compared with input DNA. Reactions were performed intriplicate using SYBR Green and the ABI 7000 Sequence Detection

System (Applied Biosystems, Courtaboeuf, France, http://www.appliedbiosystems.com). We designed primers for real-time quan-

titative PCR within the promoter region or known regulatory se-quences using the Primer Express software (Applied Biosystems)(supporting information Table 1). To compare histone modificationsin different cells types, results were normalized with respect to theubiquitously expressed 2-microglobulin gene.

RESULTS

Changes in Subnuclear Positioning of Erythroid-Affiliated Genes During Erythroid DifferentiationOccur at the Precursor Stage

We first determined subnuclear positioning of erythroid-specific-globin, -globin and RhesusAG markers relative to repressivecompartments. In mammalian cells, two types of repressivenuclear compartments have been described, the nuclear periph-ery and PCH. Positioning of erythroid-affiliated loci, detectedby specific genomic probes, was therefore measured relative tothe nuclear periphery, determined by a sharp drop in DAPIstaining, or to the closest cluster of pericentromeric foci, de-tected by the human pancentromeric probe. We measured posi-tioning of erythroid-specific loci in 3D reconstructed nuclei asdescribed in Materials and Methods.

Distances were first measured relative to the center of massof the nucleus as described [40] and corrected for nuclear radiusto account for variations of nuclear volume that occur duringdifferentiation. Specific fluorescent signals were then sorted asbeing localized within one of the four concentric nuclear shellsof equal volume. In multipotent progenitor cells, the -globinand RhesusAG loci appeared to be nearly randomly distributedwithin these four nuclear shells, whereas most of the -globinloci were preferentially positioned in the inner shells of thenucleus (supporting information Fig. 3A). In addition, differen-tiation of multipotent cells toward the erythroid lineage did notlead to significant changes in their subnuclear distribution (sup-

porting information Fig. 4).We then scored positioning of the above-mentioned loci

relative to PCH; signal was scored as associated with PCH whenthere was no visible separation between the signal and thepericentromeric domain boundary (an example shows a cellcontaining both -globin alleles associated with PCH on Fig.1A). In multipotent progenitors, positioning of -globin lociclose to PCH appeared to be a rather rare event since only 28%of the -globin loci were spatially associated with PCH in thesecells (supporting information Fig. 3B), whereas 42% of the-globin loci and up to 55% of the RhesusAG loci were asso-ciated with this repressive compartment (Fig. 1B, red lines).During erythroid differentiation, whereas 70% of the -globinloci remained dissociated from PCH (not shown), -globin and

RhesusAG loci underwent repositioning away from PCH (Fig.1B, red lines). In glycophorin A positive (GpA) precursorcells, the number of loci associated with PCH decreased to 30%for the -globin loci and 40% for the RhesusAG loci. Todetermine whether the dissociation of-globin loci from PCHwas specifically observed during erythroid differentiation, weassessed positioning of these loci in pro- and pre-B cells. Not onlydid -globin loci not undergo dissociation from PCH during B-lymphoid differentiation, but we observed a significant pericentro-meric repositioning, with the percentage of alleles being associatedwith PCH increasing from 42% to 60% (Fig. 1C, red line).

It appears then, that regulation of the human -globin lociduring normal erythroid differentiation is not associated withchanges in subnuclear location relative to PCH. In contrast,-globin and RhesusAG loci underwent dissociation from

PCH, only in the lineage in which they are transcriptionallyactive. However, differences in nuclear positioning were

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detected only at the stage of precursor or mature cells (ery-

throid CD34

GpA

) and not in committed progenitors (ery-throid CD34GpA cells). Indeed, the distribution of theseloci relative to PCH did not vary between the multipotentprogenitor and committed erythroid progenitor stages. Thesedata suggest that in contrast to the proposed model [3],changes in nuclear location of erythroid-specific genes arenot associated with commitment of human multipotent he-matopoietic cells to the erythroid lineage.

Gradual Increase in Histone Acetylation at the-Globin Locus During Erythroid Differentiation

To gain insight as to the causal relationship between subnuclearpositioning of a gene and its chromatin structure and/or tran-scriptional activity, we assessed histone modifications at various

positions at the human -globin locus, as well as its transcrip-tional status. Changes in expression levels of various lineage-

specific markers were followed by conventional quantitativereverse transcriptase-PCR techniques. After normalization to thehousekeeping gene transcript 2-microglobulin (B2m), whichshowed relatively stable expression during differentiation ofUCB cells compared with other housekeeping genes (notshown), we found that expression of -globin loci was not

detectable in the most immature multipotent progenitors(CD34CD38lo) and in nonerythroid cells (CD19 B cells)(supporting information Fig. 5). -globin expression was firstdetectable in erythroid progenitors (CD34GpA), and it pro-gressively increased in precursor and mature GpA erythroidcells.

To determine whether changes in chromatin structure at the-globin loci precede or follow changes in subnuclear locationduring erythroid differentiation, we performed ChIP experi-ments to analyze histone modifications. We first followed acet-ylation of histones H3 and H4 at fetal and adult -globinpromoters, which are both active in UCB erythroid precursorcells, LCR elements hypersensitive sites 2 to 4, at the erythro-poietin receptor (EPO-R) promoter, as well as at nonerythroidcontrol genes, such as the B-specific Ig locus and the kidney-

specific Tamm-Horsfall protein (THP) gene. In multipotentcells, acetylation of histone H3, and to a lesser extent H4, at allthe -globin regulatory regions tested, was lower than at B2mand EPO-R promoters (Fig. 2). Differentiation toward the ery-throid lineage was associated with an increase in acetylation ofboth histone H3 and H4 at -globin regulatory regions. Thisincrease was gradual since we observed a three- to sixfoldincrease in H3 acetylation and a three- to ninefold increase inH4 acetylation in CD34/GpA progenitors, followed by afurther two- to fourfold increase for both histones in CD34/GpA precursors and mature cells after 5 days in erythropoietin.No increase in H3 or H4 acetylation was observed at thenonerythroid Ig and THP genes during erythroid differentia-tion (Fig. 2).

Our data show that the increase in histone acetylation ob-served from committed progenitors onward (Fig. 2A, 2B) par-alleled that of gene expression (supporting information Fig. 5),suggesting that gene priming occurred right before this stage.We therefore analyzed histone H3 lysine 4 dimethylation(H3K4me2), a mark recently shown to be associated with tran-scriptionally poised hematopoietic genes in multipotent hema-topoietic cells [39, 41]. We found that in multipotent cells,H3K4me2 levels at erythroid (Fig. 2C) and lymphoid (Fig. 2C,J; [39]) loci were similar to that of the control gene B2m. Themaximum enrichment at erythroid loci was observed in earlycommitted erythroid CD34 CD36GpA progenitors (Fig.2C).

Collectively, these data show that during erythroid differenti-ation, an increase in both transcription and activating histone mod-

ifications at -globin loci is first detected between the immatureCD34CD38lo stage and the progenitor CD34GpA stage. Thisis in contrast with the observed changes in subnuclear positioningthat occur later, at the CD34 GpA precursor stage.

Histone Acetylation at the IgLoci DuringB-Lymphoid Differentiation Also Precedes Changesin Subnuclear Positioning

Our analysis of histone marks and subnuclear positioning of thehuman -globin loci during erythroid differentiation of primaryUCB progenitors suggests that, in contrast to the model weoriginally proposed [29], locus relocation away from repressiveheterochromatin compartments follows locus-wide acetylation.We therefore tested whether this was also the case for other

hematopoietic loci. We analyzed subnuclear positioning andhistone marks at the B-lymphoid-specific IgH and Ig loci

Figure 1. Lineage-specific gene positioning relative to pericentromericheterochromatin during erythroid and B-lymphoid differentiation ofmultipotent progenitors. (A): Example of a nucleus showing positioningof both -globin loci close to PCH in a B-lymphoid precursor(CD34CD10CD19) nucleus. Samples were hybridized with a bio-tinylated genomic probe specific for the -globin loci revealed usingAlexa Fluor 594-conjugated streptavidin (red) and digoxigenin (DIG)-labeled oligonucleotides specific for -satellite DNA revealed by anti-DIG antibodies (green) as a marker of PCH. 4,6-diamidino-2-phenylin-

dole is shown in blue. The insets show the corresponding fluorescencein situ hybridization (FISH) signals and their nuclear environmentsenlarged. Scale bar 5 m. (B, C): The frequency of association ofspecific DNA-FISH signals with PCH was scored for individual eryth-roid-specific alleles (-globin: solid red line; RhesusAG: dotted red line)and B-lymphoid-specific alleles (Ig: green line) at various erythroid(B) and B-lymphoid (C) stages of differentiation. ErythroidCD34GpA progenitors and CD34GpA precursors were obtainedas described in Materials and Methods. Data represent the mean valuefrom two to four independent experiments for each samples on at leasttwo independent preparations of cells. Error bars represent the SD.Abbreviations: D3, 3 days in culture with erythropoietin; D5, 5 days inculture with erythropoietin; PCH, pericentromeric heterochromatin com-partments; RhAG, RhesusAG.


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during human B-lymphoid differentiation, since they have beenshown to be regulated at the level of subnuclear compartmen-talization in murine cells [25]. In contrast to the reported posi-tioning ofIgHloci in murine B-lymphocytes [25], we found thatthe human IgHloci are mostly localized toward the center of thenucleus and dissociated from PCH (supporting information Fig.

3A, 3B). In addition, positioning of the IgH loci did not changesignificantly during differentiation (not shown). We thereforefocused on the Ig loci and found that although they are nearlyrandomly distributed within the four nuclear shells of the nu-cleus (supporting information Fig. 3A), more than 60% of theIg alleles associated with PCH in multipotent hematopoieticcells (Fig. 1C, green line). During B-lymphoid differentiationthis percentage dropped to 35% (Fig. 1C, green line). Again, thischange in subnuclear positioning was not observed betweenmultipotent and committed progenitor stages but betweenCD34CD10CD19 pro-B and CD34CD10CD19 pre-B/B cells, the stage at which Ig gene recombination andexpression occurs. In contrast to the dissociation from PCHobserved during B-lymphoid differentiation, the percentage of

Ig alleles associated with PCH increased to 75% during ery-throid differentiation (Fig. 1B, green line).

The levels of B-cell progenitors in UCB are too low to

perform ChIP experiments on sorted progenitor populations.However, human UCB multipotent progenitors efficiently re-constitute the B-lymphoid compartment of immunodeficientNOD/SCID mice, giving rise to CD45 human B-lymphoidcells [32]. To study histone modifications at the Ig locus duringB-cell differentiation, NOD/SCID mice were therefore en-grafted with UCB CD34 cells. Histone modifications wereanalyzed in CD45CD34CD19 pro-B and CD45CD34

CD19 pre-B cells isolated from the bone marrow of mice3-month postengraftment, as well as in CD34CD38lo multipo-tent progenitors isolated from human UCB. We assessed histoneacetylation at Ig J segments, the C constant region, and theintronic E enhancer. We found that levels of histone H3acetylation at the Ig locus remained low throughout B-lym-phoid differentiation. However, as for the -globin locus during

erythroid differentiation, we found a two- to sixfold increase inthe levels of H4 acetylation between the multipotent progenitorand pro-B cell stage, whereas histone acetylation levels re-mained low at the kidney-specific THP gene (Fig. 3). Therefore,as observed at the -globin locus during erythroid differentia-tion, our data show that changes in histone H4 acetylation at theIg locus precede changes in nuclear positioning relative toPCH during B-lymphoid differentiation.

Subnuclear Positioning of Hematopoietic Loci DoesNot Change in Immature Progenitors

Since the distribution of the -globin and Ig loci relative to PCHappeared to be similar in immature progenitors committed to eitherthe erythroid or B-lymphoid lineages, we wanted to determine

whether that was the case in other immature hematopoietic pro-genitors. We therefore also determined the subnuclear repartition of

Figure 2. Activating histone marks during in vitro differentiation ofmultipotent progenitors toward the erythroid lineage. Chromatin iso-lated from CD34CD38lo multipotent progenitors (black bars),CD34GpA erythroid progenitors (pink bars), and erythroid precur-sors CD34GpA (red bars) was analyzed by chromatin immunopre-cipitation (ChIP) using antibodies to H3ac (A), H4ac (B), or H3K4me2(C) followed by real-time polymerase chain reaction using primersamplifying gene segments at B-lymphoid (J region of the Ig locus),erythroid (-globin locus control region: hypersensitive sites 2 to 4; fetal

and adult -globin proms; EPO-R prom), N, and control B2m andTHP genes. Histograms show enrichment values (bound/input) relativeto the B2m control (set at 1). Results are means and SDs of two to fourindependent ChIP experiments analyzed in triplicate. Abbreviations:B2m, 2-microglobulin; EPO-R, erythropoietin receptor; H3ac, acety-lated histone H3; H4ac, acetylated histone H4; H3K4me2, histone H3lysine 4 dimethylation; J, junction segment of the Ig locus; N,nonhematopoietic; ND, data not determined in multipotent progenitors;prom, promoter; THP, Tamm-Horsfall protein.

Figure 3. Histone acetylation during human B-lymphoid differen-tiation. Chromatin from CD34CD38lo multipotent progenitors(black bars), CD34CD10CD19 pro-B (light green bars) andCD34CD10CD19 pre-B cells (darker green bars) were analyzed bychromatin immunoprecipitation (ChIP) using antibodies to acetylatedhistone H3 (A) and H4 (B) followed by real-time polymerase chainreaction using primers amplifying segments at B-lymphoid (J, E, andC), N, and control B2m or THP genes. Results were calculated as inFigure 2 and are means and SDs of two independent ChIP experimentsanalyzed in triplicate. Abbreviations: B2m, 2-microglobulin; C, con-stant region of the Ig locus; E, enhancer region of the Ig locus; J,J region of the Ig locus; N, nonhematopoietic; THP, Tamm-Horsfallprotein.



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the -globin and Ig loci in CD34 progenitors isolated fromhuman thymus. Surprisingly, we found that the positioning of boththe -globin and Ig loci relative to PCH was similar in all thehematopoietic progenitors tested: multipotent CD34CD38lo,committed erythroid CD34GpA, committed B-lymphoidCD34CD10CD19 (Fig. 1B, 1C), and T lymphoid CD34

progenitors (Fig. 4). We found that, as observed in CD34CD38lo

multipotent hematopoietic progenitors, progenitors committed tovarious lineages contain approximately 40% of the -globin allelesand 60% of theIg alleles associated with PCH, suggesting that thesubnuclear distribution of these loci is similar in all the CD34

cells, regardless the lineage toward which these progenitors arecommitted.

We then assessed the distribution of these hematopoieticgenes in nonhematopoietic multipotent cells (i.e., in muscularsatellite cells and in primary mesenchymal stem cells). Again,we found that the positioning of-globin and Ig genes relativeto PCH was similar to that described in multipotent hematopoi-etic progenitors (Fig. 4). Collectively, our data suggest thatsubnuclear distribution of these loci is independent of the originof the multipotent cells tested and that repression of -globin

and Ig loci in CD34

progenitor cells from various lineages ofhematopoietic differentiation or from various tissues does notcorrelate with association with the repressive PCH.

Hematopoietic Genes Are Associated withRepressive Chromatin Marks in NonhematopoieticMultipotent Progenitors

The above results show that nuclear architecture of progenitorcells, studied through subnuclear positioning of lineage-specificgenes relative to PCH, did not show a tissue- or lineage-specificorganization. We therefore asked whether this was the case forhistone marks at the regulatory regions of these genes.

To determine whether silencing of hematopoietic genes innonhematopoietic multipotent progenitors was associated with

addition of repressive marks, we assayed for histone H3 lysine9 and lysine 27 trimethylation (H3K9me3 and H3K27me3) by

ChIP experiments. Multipotent CD34CD38lo hematopoieticprogenitors showed relatively weak marks for repressive chro-matin at lymphoid- and erythroid-affiliated genes (Fig. 5A, 5B,black bars), whereas the levels of H3K27me3 were 510 timeshigher at the nonhematopoietic THP and muscle-specific Myf5

genes than at the Ig and -globin loci in these cells (Fig. 5B).Interestingly, hematopoietic genes showed high levels of repres-sive histone marks in muscle satellite cells (Fig. 5A, 5B, whitebars; [39]), consistent with these genes being transcriptionallyrepressed in nonhematopoietic lineages.

DISCUSSION

The spatial distribution of genes within the nuclear space or relativeto nuclear landmarks has been implicated in the establishment andmaintenance of active, poised or repressed chromatin states (re-cently reviewed in [12, 42]). However, the causality relationshipbetween spatial positioning of genes within the nucleus, or relative

to nuclear landmarks, and their transcriptional activity appears todepend in large part on the gene studied and the cellular systemused. In addition, the contribution of nuclear architecture to mul-tipotency of primary human cells remains questionable. In thisstudy, analysis of subnuclear positioning, chromatin structure, andexpression status from the most immature stage of human hema-topoietic differentiation through committed progenitors and thenprecursor and mature stages has allowed us to assess epigeneticcharacteristics of hematopoietic multipotent cells and follow andorder epigenetic events that take place during lineage commitmentand differentiation.

In human multipotent hematopoietic cells, both -globin andIg loci were randomly distributed relative to the nuclear interior.Interestingly, positioning of-globin and Ig loci relative to PCH

is distinct, but this positioning was similar in all early CD34

committed progenitor and multipotent cells tested, regardless of the

Figure 4. No significant change in subnuclear location of lineage-specific loci in hematopoietic multipotent, committed, and nonhemato-

poietic progenitor cells. Histogram shows frequency of -globin

(redbars) and Ig (green bars) locus association with PCH in hematopoieticprogenitors (multipotent CD34CD38lo and T-lymphoid CD34 pro-genitors), and in nonhematopoietic multipotent cells (muscular satellitecells and mesenchymal stem cells). Error bars represent the SD. Meansand SDs are from at least two independent fluorescence in situ hybrid-ization experiments. Abbreviation: PCH, pericentromeric heterochroma-tin compartments.

Figure 5. Comparison of repressive histone modifications in hemato-poietic multipotent progenitor and muscle satellite cells. Chromatinimmunoprecipitation (ChIP) analyses of multipotent hematopoietic pro-

genitor cells (black bars) and muscle satellite cells (white bars) forrepressive histone modifications using antibodies to H3K9me3 (A) orH3K27me3 (B) followed by real-time polymerase chain reaction usingprimers amplifying the same segments analyzed in Figures 2 and 3 or amuscle-specific gene (Myf5). Results are means and SDs of two to fiveindependent ChIP experiments analyzed in triplicate. Abbreviations:B2m, 2-microglobulin; C, C region of the Ig locus; H3K27me3,histone H3 trimethylated on lysine 27; H3K9me3, histone H3 tri-methylated on lysine 9; J, J region of the Ig locus; Mus, muscle; N,nonhematopoietic; THP, Tamm-Horsfall protein.


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lineage to which they were committed or the origin of the multi-potent cell. In contrast, the histone modification profiles at hema-topoietic loci were clearly distinct between multipotent cells ofhematopoietic and muscle origins ([39] and this study) and betweencommitted progenitors from erythroid and lymphoid origins.Therefore, silencing of hematopoietic genes in multipotent progen-itors and the higher levels of repressive histone marks found atthese loci in muscle satellite cells are not favored by positioning inthe vicinity of repressive PCH or nuclear periphery compartments.In contrast, repression of nonaffiliated genes (i.e., erythroid genesin mature B [this study] or T cells [26]) or B-lymphoid genes inerythroid precursor cells and addition of repressive histone marks[39] are associated with increased proximity to PCH. Gene posi-tioning in multipotent cells may therefore represent a default loca-tion, intrinsic to the locus, rather than being involved in setting aparticular chromatin state. Similarly, spatial compartmentalizationof inactive genes was not observed in ES pluripotent cells either[43, 44]. A number of experiments have shown that progenitorcells, although committed to differentiate to a given lineage, wereable to differentiate to a different lineage, a phenomenon known astrans-differentiation [45]. As proposed earlier, CD34 hematopoi-

etic progenitor cells could represent a pool of cells that retain someplasticity and show epigenetic similarities [6], and it is tempting topropose, in the light of data presented here, that the observed activechromatin structure at lineage-affiliated genes (this study and [39,41, 46, 47]) combined with a default subnuclear location relative torepressive compartments, may participate in the plasticity of humanmultipotent cells and committed CD34 hematopoietic progenitors.

Several studies have suggested that positioning away fromrepressive pericentromeric heterochromatin compartments favorschromatin opening and full promoter activation and that differen-tiation-mediated activation of tissue-specific genes leads to theirdissociation from PCH [25, 27, 38, 4850]. We report that com-mitment of hematopoietic multipotent progenitors to differentiatetoward a given lineage is associated with a gradual increase inactivating histone marks at lineage-affiliated loci from the progen-itor stage onward (this study and [39]), whereas changes in sub-nuclear positioning of these genes occur at later stages, in CD34

precursor cells. Although high-level expression in precursor andmature cells was associated with an increased dissociation fromPCH, neither acquisition of histone marks indicative of gene prim-ing (H3K4me2) or active transcription (H3ac and H4ac) nor tran-scriptional activation per se initiated in committed progenitors wasassociated with changes in positioning relative to the nuclear pe-riphery nor to PCH. Likewise, activation of Ig during murine Bcell development takes place in a stepwise manner, with histoneacetylation following an intranuclear shift [49]. The percentage ofmurine -globin alleles associated with PCH was also very low inmurine primary cells [18], and activation of transcription thatoccurs between early and late committed progenitors correlated

with a further dissociation from PCH. Thus, our data show thatchanges in both chromatin structure and gene expression thataccompany differentiation were detected before changes in genepositioning relative to PCH. This is in contrast to the proposedmodel based on studies of the human -globin locus in murine cellhybrids, in which the locus was clearly dissociated from PCH whenin an open chromatin configuration, regardless of its transcriptionalactivity, suggesting that location away from PCH was a prerequi-site for chromatin opening [29]. The present analysis of endoge-nous human loci, in primary hematopoietic cells, suggests thatchanges in subnuclear positioning of human -globin and Ig locifollow changes in gene expression rather than being involved insetting a particular chromatin state. Studies of gene silencing duringdifferentiation of murine B cells led to the notion that heritable butnot transient gene silencing (i.e., transmitted through generations)

was associated with positioning close to PCH [51]. Similarly, weproposed that stable gene expression or chromatin opening required

positioning away from PCH [28, 29]. Our present data indicate thatin primary human hematopoietic cells, gene positioning relative toPCH may be important for maintaining chromatin states in precur-sor and mature cells, rather than for establishing these chromatinstates in committed progenitors. Silent chromatin states, which areprogressively established at lineage-inappropriate genes in commit-

ted progenitor cells by the addition of repressive chromatin marks[39], may be further maintained by positioning of the gene close toheterochromatin compartments in late precursor and mature cells.Similarly, positioning away from repressive compartments is asso-ciated with maintenance of the active state, allowing lineage-affiliated loci to stay active throughout differentiation. It remains tobe determined when during hematopoiesis the multilineage poten-tial of progenitor cells is established, and whether high levels ofhistone modifications (this study and [39, 41, 46, 47]) and tran-scriptional regulators [52] are sufficient to maintain developmen-tally important genes in particular nuclear subcompartments;namely, lineage-affiliated genes that need to stay active throughoutthe differentiation process in permissive compartments and genesthat need to be silenced in repressive compartments.

CONCLUSION

Deciphering how HSC pluripotency and lineage commitment isachieved at the chromatin level is likely to be very informativeto further understand stem cell plasticity and reprogrammingand for successfully applying stem cell-based therapies. Thechromatin and nuclear profiling that we initiated (this studyand [39]) will provide a valuable signature for stem cellidentity and will allow somatic postnatal stem cells (i.e., hema-topoietic, muscular satellite, mesenchymal, and so forth) to bereliably distinguished from their proliferative, committed prog-eny. We believe that these studies could be a way of predictingthe developmental potential/plasticity of stem cell populations

isolated from different sources. This may become increasinglyuseful for characterizing stem cells when the functional verifi-cation of differentiation potential in vivo is not possible or maybe detectable only after weeks.

ACKNOWLEDGMENTS

We thank Florent Hube and Guillaume Velasco for critical readingof the manuscript. We are grateful to Dr. F. Pflumio for the gift ofhuman B cells isolated from engrafted NOD-SCID mice, Dr. V.Mouly for the gift of human satellite muscle cells, Dr. P. Charbordfor the gift of human mesenchymal stem cells, Dr. D.R. Higgs forthe-globin genomic probe, and Dr. P.A. Ioannou for the -globingenomic probe. This work was supported by research funding fromINSERM and grants from Association pour la Recherche sur leCancer (ARECA) network (to M.G. and C.F.), INSERM AdultStem Cell Grant AIP A03203DS (to M.G. and C.F.), EU fp6program Eurythron MRTN-CT-2004-005499 (to C.F.), and theAssociation Francaise contre les Myopathies (AFM) (to C.F.). C.G.was supported by a doctoral fellowship from the French Ministry ofResearch, A.G. was supported by a postdoctoral fellowship fromthe Ligue Nationale Contre le Cancer and a contract from INSERM,and J.M. was supported by postdoctoral contracts from theINSERM/AFM and ARECA networks.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.



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