Lymphoid-affiliated genes are associated with active histone modifications in human hematopoietic stem cells

doi:10.1182/blood-2008-02-140806Prepublished online July 14, 2008;

GoodhardtAndre-Schmutz, Marina Cavazzana-Calvo, Dominique Charron, Claire Francastel and Michele

Jerome Maes, Marta Maleszewska, Claire Guillemin, Francoise Pflumio, Emmanuelle Six, Isabelle in human hematopoietic stem cellsLymphoid-affiliated genes are associated with active histone modifications

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Lymphoid-affiliated genes are associated with active

histone modifications in human hematopoietic stem cells

Short title: Open chromatin at lymphoid loci in human HSC

Authors: Jerome Maës1,2, Marta Maleszewska1,2, Claire Guillemin3,4, Francoise Pflumio3,4,7, Emmanuelle Six5,6, Isabelle André-Schmutz5,6, Marina Cavazzana-Calvo5,6, Dominique Charron1,2, Claire Francastel3,4, Michele Goodhardt1,2

Affiliation: 1. Institut Universitaire d'Hématologie, Université Paris 7 Denis Diderot, 75010 Paris, France. 2. Institut National de la Santé et de la Recherche Médicale (INSERM) U662, 75010 Paris, France. 3. Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 75014 Paris, France. 4. INSERM, U567, 75014 Paris, France. 5. INSERM, U768, 75015 Paris, France. 6. Université Paris-Descartes, Faculté de Médecine René Descartes, IFR94, 75015 Paris, France. 7. Present address: CEA, 92260 Fontenay-aux-Roses, France. Corresponding author: Michele Goodhardt, INSERM U662, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, 1, av. Claude Vellefaux, 75010 Paris, France. tel: +33 1 42494889 ; fax: +33 1 42494641 ; email: [email protected] J.M. and M.M. contributed equally to this work. Scientific category: Hematopoiesis and Stem Cells

Blood First Edition Paper, prepublished online July 14, 2008; DOI 10.1182/blood-2008-02-140806

Copyright © 2008 American Society of Hematology




ABSTRACT

To address the role of chromatin structure in the establishment of hematopoietic stem cell

(HSC) multilineage potential and commitment to the lymphoid lineage, we have analyzed

histone modifications at a panel of lymphoid- and myeloid- affiliated genes in multipotent and

lineage-committed hematopoietic cells isolated from human cord blood. Our results show that

many B- and T-lymphoid genes, although silent in HSC, are associated with acetylated

histones H3 and H4. We also detected histone H3 lysine 4 methylation, but not repressive

lysine 9 or 27 methylation marks at these loci, indicative of an open chromatin structure.

Interestingly, the relative level of H3 lysine 4 dimethylation to trimethylation at B-specific

loci was high in multipotent CD34+CD38lo progenitors and decreased as they become actively

transcribed in B-lineage cells. In vitro differentiation of CD34+ cells towards the erythroid,

granulocyte and T cell lineages resulted in a loss of histone acetylation at non-lineage

associated genes. This study provides evidence that histone modifications involved in

chromatin decondensation are already in place at lymphoid-specific genes in primary human

HSC, supporting the idea that these genes are “primed” for expression prior to lineage

commitment. This permissive chromatin structure is progressively lost as the stem cell

differentiates.




INTRODUCTION

All blood cells are derived from multipotent hematopoietic stem cells (HSC) via a series of

differentiation steps during which the multilineage potential of the stem cell is gradually lost

as HSC differentiate into lymphoid or myeloid committed cells 1. These differentiation steps

require the coordinated activation of characteristic sets of genes and the silencing of others.

Increasing evidence indicates that epigenetic modifications play an important role in cell-fate

decision-making processes by regulating the chromatin state of genes and hence their

potential to be expressed 2,3. Remodeling of chromatin can be achieved in several distinct but

interconnected ways including covalent modifications of histones, such as acetylation,

phosphorylation, methylation and ubiquitinylation, often at N-terminal residues 4,5. These

modifications play an important role in the formation and maintenance of active and

repressive chromatin states 6. Acetylation of N-terminal lysine residues of histones H3 and H4

and methylation of lysine 4 of histone H3 have been associated with active chromatin

conformations, while methylation of histone H3 lysines 9 and 27 are hallmarks of repressive

chromatin states 7-13.

Recent studies of histone modifications in embryonic stem (ES) cells have revealed novel

epigenetic features, which are thought to contribute to the maintenance of pluripotency and

cell lineage determination in these stem cells 14-18. To date much less is known about the

chromatin structure of multipotent adult stem cells, such as HSC. Despite the fact that HSC

are probably the best characterized stem cells, the role of histone modifications in regulating

their multilineage potential is poorly understood. Many studies have shown that HSC and

early hematopoietic progenitors express multiple lineage-associated genetic programs at low

levels 19-22. This phenomenon, known as lineage priming is thought to reflect alterations in the

chromatin conformation of genes expressed in specific hematopoietic lineages, rendering

them accessible to transcription factors in multipotent cells prior to lineage commitment 23,24.




Interestingly, promiscuous expression of myeloid- but not lymphoid-affiliated genes was

observed in HSC 19,21, suggesting that opening of B- and T- lymphoid loci may occur later

during hematopoiesis.

In this study, we have investigated the chromatin structure at a panel of lymphoid- and

myeloid-affiliated genes in HSC and lineage-committed cells isolated from human umbilical-

cord blood by analyzing their pattern of histone modifications. We show that lymphoid-

affiliated genes are associated with a combination of active histone modifications, including

histone acetylation and histone H3 lysine 4 dimethylation, but very low levels of negative

marks in HSC. This accessible chromatin conformation precedes gene expression and lineage

commitment during HSC differentiation. Further active histone modifications, notably H3

lysine 4 trimethylation are added at these loci in lymphoid-committed cells expressing high

levels of the genes, while the histone marks present in HSC are replaced by repressive H3

lysine 9 or 27 methylation upon commitment to other lineages. These results show that active

epigenetic marks are already present at lymphoid loci in HSC and highlight the role of

chromatin structure in the maintenance of multilineage potential in these stem cells.




MATERIALS AND METHODS

Cell isolation

Umbilical-cord blood samples were collected from normal full-term deliveries, after informed

consent of the mothers, according to the approved institutional guidelines of GHU Nord

(APHP, Paris). After Ficoll density gradient separation (Lymphocytes Separation Medium,

Eurobio, Les Ullis, France), CD34+ cells were purified by immunomagnetic selection (Stem

Cell Technologies, Vancouver, BC, Canada). CD34+ cells (purity ≥ 95%) were used

immediately or after storage in liquid nitrogen. CD34+CD38lo cells, which make up to 10% of

the CD34+ cell population, were routinely isolated from pools of 5-10 cord blood samples by

FACS using a Vantage SE - DiVa (Becton Dickinson, USA). Isolation of cord blood B cells

was performed on CD34- cell fraction based on CD19 expression (Miltenyi Biotec, Paris,

France).

Human muscle satellite cells (a generous gift from Dr. V. Mouly, Institut de Myologie, Paris,

France) were isolated and cultured as described 25.

Chromatin Immunoprecipitation Assays

Purified hematopoietic cells were cross-linked with 0.5% formaldehyde in RPMI medium

containing 10% FCS for 10 minutes at 37°C, then washed in 10 volumes of RPMI medium.

Cells (4 x 105 – 106) were then lysed in 400µl 1%SDS, 10mM EDTA, 50mM Tris-HCl

(pH8.0) lysis buffer and sonicated with a Branson Sonifier in 1.5ml Eppendorf tubes.

Chromatin immunoprecipitation (ChIP) experiments were performed on solubilized

chromatin diluted 10-fold in ChIP dilution buffer (Upstate #20-153) using 2 x 105 cells per

histone modification. All immunoprecipitation and washing steps were performed in

Eppendorf tubes at 4°C. Chromatin was pre-cleared by incubating with 80µl salmon sperm

DNA/protein A agarose beads (Upstate #16-157) for 3 hours then incubated overnight with




antibodies against anti-acetyl histone H4 (Upstate; #06-866), anti-acetyl histone H3 (Upstate;

#06-599), anti- dimethyl histone H3K4 (Upstate; #07-030), anti-trimethyl histone H3K4

(Upstate; #07-473), anti-trimethyl histone H3K9 (Upstate; #07-523) or anti-trimethyl histone

H3K27 (Upstate; #07-449). Immune complexes were collected by incubating with 60µl

salmon sperm DNA/protein A agarose beads, washed as described (Upstate #17-295) using

900µl of each buffer and modified histone/DNA complexes eluted with 500µl 1%SDS,

0.1MNaHCO3. Following reversal of cross-link, DNA was purified by phenol extraction. An

aliquot of the sonicated chromatin was treated identically for use as “input”. ChIP

experiments for the 6 histone modifications were performed on the same chromatin sample.

Chromatin from 1.2x106 CD34+CD38lo cells isolated from 20-30 cord blood samples was

pooled for each complete experiment.

Quantitative PCR was performed to determine the relative enrichment of gene segments in

ChIP compared to input DNA. Reactions were performed in triplicate using SYBR Green and

the ABI 7000 Sequence Detection System (Applied Biosystems). Percent

immunoprecipitation was calculated by dividing the average value of the IP by the average

value of the corresponding input normalized by the dilution factor. To compare histone

modifications in different cell types, results were routinely normalized with respect to the

promoter of the ubiquitously expressed β2-microglobulin gene for histone H3 and H4

acetylation and histone H3 lysine 4 methylation or the non-hematopoietic THP gene for

histone H3 lysine 9 and 27 methylation.

PCR primers were designed within the promoter region or known regulatory sequences using

the Primer Express software (Applied Biosystems) (Supplemental Table I). For some loci,

due to particular base sequence whether highly or poorly rich in G/C or containing repeats,

the amplified fragment was located within coding regions, essentially in the first exon.




In vitro differentiation

Freshly isolated CD34+CD38lo cells were differentiated towards the T lineage by in vitro

culture on OP9 stromal cells expressing human Notch ligand Delta-1 (OP9-hDelta1) as

described 26,27. Differentiation was monitored by FACS analysis based on phenotypic changes

in CD34, CD7, CD4 and CD8 cell surface expression. In vitro differentiation of CD34+

progenitors towards the erythroid and granulocyte lineages were performed as described 28.




RESULTS

Lymphoid genes are associated with acetylated histone H3 and H4 in cord blood

CD34+CD38lo cells.

We focused our study on a panel of more than 20 lymphoid- and myeloid-specific genes

(Supplemental Table I) that are not or very poorly expressed in HSC, but are expressed at

later stages of HSC lineage commitment and differentiation 29-32. As controls, we also

analyzed non-hematopoietic genes such as the neuronal Pax6 and kidney-specific THP genes,

as well as genes that are highly expressed in HSC, such as TAL1 33 and the ubiquitous β2-

microglobulin and GAPDH genes.

We first investigated the pattern of histone H3 and H4 acetylation by chromatin

immunoprecipitation (ChIP) experiments in lineage committed and in CD34+CD38lo cord

blood cells enriched in HSC and multipotent progenitors. To compare histone modifications

in different cell types, results were normalized with respect to the promoter of the

ubiquitously expressed β2-microglobulin or GAPDH gene. Similar modification patterns

were observed with the two control genes (Supplemental Figure 1). Results are presented

relative to the β2-microglobulin control. In CD19+ B-lineage cells, we found that histone H3

and H4 modifications were lineage specific with only the set of B-specific genes marked by

acetylated H3 and H4 at levels similar to that of β2-microglobulin (Figure 1, middle panels).

Similarly, histone acetylation was only observed at erythroid loci in glycophorin-positive

(GpA+) erythroid precursors and at T-specific genes in CD4+ T cells isolated from cord blood

(Figure 1, bottom panels and data not shown).

A distinct acetylation profile was observed in multipotent CD34+CD38lo cord blood cells,

with many B- and T-lymphoid genes associated with acetylated histones H3 and H4 (Figure

1, top panels). The level of H4 acetylation was similar or even greater than that of the

expressed β2-microglobulin or TAL1 genes for most of the loci analyzed. Although fairly




low levels of acetylated histone H4 were found at the β-globin locus, the EPOR and c-fms

genes were clearly acetylated, indicating that erythroid and myeloid genes are also acetylated

in CD34+CD38lo cord blood progenitors, consistent with results from previous studies 24,34.

Acetylated histone H3 was detected at a more restricted number of genes in CD34+CD38lo

cells. Nevertheless, similar levels of histone H3 acetylation were observed at most B-specific

genes in CD34+CD38lo and in CD19+ B cells, indicating that this modification is already fully

present at certain lymphoid loci in HSC. Interestingly, histone H4 acetylation levels at these

genes were somewhat higher in CD34+CD38lo than in B-committed cells, suggesting that H4

acetylation is the prominent mark at transcriptionally silent B lymphoid genes in HSC.

For the B-specific CD79B gene, we assayed histone H3 and H4 acetylation at DNaseI

hypersensitive sites (HS) throughout the locus (Supplemental Figure 2). High levels of both

acetylated H3 and H4 were found at the promoter and 0.7kb HS sites in CD34+CD38lo cells.

Acetylation of histone H3 decreased at 3’ HS sites, whereas levels of H4 acetylation

remained high throughout the locus.

Altogether, our results show that multipotent CD34+CD38lo cells have a much broader histone

acetylation profile than lineage committed cells with many lymphoid- and myeloid-affiliated

genes associated with acetylated histones. The genes analyzed are either not transcribed or

expressed at very low levels in CD34+CD38lo cells. This indicates that histone acetylation at

these loci occurs prior to transcription and lineage commitment in HSC and may be associated

with lineage potential rather than gene expression per se.

Lymphoid loci are not associated with repressive histone methylation marks in cord

blood CD34+CD38lo cells.

Methylation of lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3) have been

described as negative marks mediating gene repression and chromatin condensation via the

recruitment of heterochromatin protein 1 (HP1) and polycomb complexes, respectively 7,8,10.

We therefore examined the presence of these repressive modifications at the panel of




lymphoid and myeloid genes in CD34+CD38lo cells. All loci analyzed had low levels of

H3K9me3 in CD34+CD38lo cells (Figure 2). In cord blood CD19+ cells, however, H3K9me3

was observed at the non-hematopoietic gene PAX6, as well as the Ig kappa locus, consistent

with previous reports of H3K9me3 and HP1 binding at Ig kappa genes in murine B cells,

implicated in heterochromatin recruitment and allelic exclusion 35,36. Similarly, we did not

detect appreciable levels of H3K27me3 in CD34+CD38lo cells at the lymphoid and myeloid

loci analyzed, although H3K27me3 was observed at the non-hematopoietic genes PAX6 and

THP (Figure 2). A notable exception was for the B cell transcription factor PAX5, although

the level of H3K27me3 detected at the Pax5 gene in CD34+CD38lo cells was 10-fold lower

than in GpA+ erythroid cells. In these erythroid cells, high H3K27me3 levels were also

observed at genes coding for neuronal (PAX6) and other lymphoid (EBF and GATA-3)

transcription factors. Together these results show that most lymphoid and myeloid genes are

associated with very low levels of repressive histone marks in CD34+CD38lo cells, supporting

the idea that these loci have an open chromatin conformation in HSC.

B-specific genes are associated with higher levels of H3K4 di- to trimethylation in

CD34+CD38lo than in CD19+ cells.

We also analyzed the methylation status of histone H3 lysine 4 (H3K4me), which has been

reported to be an activating modification, facilitating transcription by the recruitment of

remodeling complexes or by preventing transcriptional repressors from binding to chromatin

37. Both di- and trimethylation forms are associated with active loci, although H3K4me3 is

found at promoters of actively transcribed genes, while H3K4me2 can be present on poised,

inactive genes 11,38,39. Methylated H3K4, especially H3K4me2, was detected at lymphoid loci

in CD34+CD38lo cells but not at the nonhematopoietic THP gene or in GpA+ cells (Figure 3

and data not shown), again suggesting that lymphoid loci have an accessible chromatin

conformation in multipotent hematopoietic progenitors. As expected, B- but not T-lymphoid

genes were associated with methylated H3K4 in CD19+ B-committed cells. A direct




comparison of absolute levels of di- to trimethylation cannot be made, as the efficiency of

the two antibodies used in the ChIP experiments may not be the same. However, the relative

level of H3K4me2 to H3K4me3 was higher at B-specific loci in CD34+CD38lo progenitors,

where they are not yet transcribed, than in CD19+ B cells, expressing high levels of these

genes (Figure 3). The presence of high levels of H3K4me2 in CD34+CD38lo cells suggests

that it may be a marker of transcriptional or developmental “competence” in human HSC, as

previously reported for yeast and avian cells 11,39, while H3K4me3 appears to be more directly

related to transcriptional activity.

Muscle satellite cells and HSC have different histone modification profiles

To investigate whether the chromatin structure observed in CD34+CD38lo HSC at lymphoid

loci is related to the differentiation potential of the stem cell, we compared the histone

modification profile in HSC and in human muscle satellite cells, which are responsible for

regeneration of postnatal skeletal muscle 25. Unlike CD34+CD38lo cells, hematopoietic genes

were not marked by active histone modifications in primary muscle satellite cells (Figure 4).

We found very low levels of histone H3 acetylation and H3K4me2 at most B, T and erythroid

genes in the muscle satellite cells, with the exception of hematopoietic transcription factors

that retained significant H3K4me2 marks (Figure 4). Furthermore, high levels of repressive

H3K9me3 and H3K27me3 marks were detected at lymphoid and erythroid loci in the muscle

satellite cells. Thus, we found high levels of H3K9me3 at the IgH locus, while transcription

factors expressed in hematopoietic and neuronal cells were associated with high levels of

H3K27me3. In contrast, the muscle determining myogenic factor-5 (MYF5) was marked with

acetylated histone H3 and H3K4me2 but not with repressive H3K9me3 or H3K27me3

modifications in muscle satellite cells; while in CD34+CD38lo cells, MYF5 is associated with

high levels of H3K27me3 marks but not H3K4me2 or histone H3 acetylation (Figure 4). The

histone modification profile observed in HSC and muscle satellite cells therefore seems to

reflect the differentiation potential of the stem cells.




Comparison of histone modification profiles in CD34+ and CD34+CD38lo cord blood cells

We compared histone modification profiles in CD34+CD38lo cells and in total CD34+ cord

blood population, containing multipotent and lineage committed progenitors. To our surprise,

the pattern of histone H4 and H3 acetylation and H3K4me2 in CD34+ cells was similar to that

observed in CD34+CD38lo cells (Figure 5). These results suggest that active histone

modifications are maintained in CD34+ progenitor cells at lineage-inappropriate loci. To test

this, we isolated CD34+CD38+CD19-CD10- cells to remove multipotent and B-lineage

committed CD34+ progenitors and analyzed histone modifications at B-specific loci. The

pattern of histone acetylation and H3K4me2 marks in these cells was similar to that observed

in total CD34+ and CD34+CD38lo cells (Figure 5). This shows that B-specific genes are

associated with active histone modifications in CD34+ progenitors committed to lineages

other than the B cell lineage, suggesting that Lin+CD34+ progenitors retain a certain degree of

lineage plasticity.

Differentiation of CD34+ cells is associated with decreased histone acetylation of non-

lineage associated genes.

We next looked at histone modifications at lineage-affiliated genes during in vitro

differentiation of HSC. Given the low numbers of CD34+CD38lo cells and the similar

chromatin structure to CD34+ cells, histone H3 and H4 acetylation was analyzed following in

vitro differentiation of CD34+ cells toward the erythroid, granulocyte and T cell lineages

(Figure 6). For erythroid differentiation, CD34+ cells were first cultured in vitro in the

presence of SCF, IL-3, IL-6 for 7 days, after which CD36+ erythroid progenitors were purified

and cultured for a further 5 days in the presence of Epo, which promotes the differentiation of

CD34-CD36+GpA+ erythroid cells 28. We observed a decrease in histone acetylation at all B

and T-affiliated genes following erythroid differentiation, while acetylation levels at erythroid

genes was increased or remained the same (Figure 6A). Interestingly, loss of histone H3

acetylation at lymphoid loci occurs in CD36+ progenitors before addition of Epo, whereas the




decrease in H4 acetylation is more gradual and is only marked after 5 days of Epo treatment.

Differentiation of CD34+ cells towards the granulocyte lineage was associated with a marked

decrease in the level of histone acetylation at both lymphoid and erythroid genes (Figure 6B).

In vitro culture of HSC on OP9 stromal cells expressing human notch ligand Delta-1 (OP9-

hDelta1) leads to cellular expansion and differentiation towards the T cell lineage 27. Analysis

of histone acetylation after 21 days of culture showed a decrease in histone H3 acetylation at

all non-T specific genes (Figure 6C). Of note, acetylated histone H4 was still observed at B-

lineage loci but not at erythroid or myeloid genes, perhaps reflecting the proximity of B and T

lymphoid lineages.

Taken together, these results show that histone modifications involved in chromatin

decondensation are already in place at lymphoid-affiliated genes in HSC and that commitment

to one of the myeloid or lymphoid lineages is associated with loss of active histone marks at

lineage inappropriate loci.




DISCUSSION

In this study, we have investigated the chromatin structure of primary HSC and lineage

committed cells isolated from human cord blood. By pooling cord blood samples and

improving the sensitivity of conventional ChIP assays, we have analyzed 6 histone

modifications specific of active or repressive chromatin at a panel of more than 20 lymphoid-

and myeloid-affiliated genes. Although the results presented here are not based on a genome-

wide analysis, clear trends are observable. Unlike lineage-committed cells, which have a

tissue-specific histone acetylation profile, multipotent cord blood CD34+CD38lo cells have a

much broader profile with many lymphoid- and myeloid-affiliated genes associated with

acetylated H4 and to a lesser extent H3 acetylation. We found that these genes are also

marked by histone H3K4me2, another modification associated with chromatin

decondensation and transcriptional activation, but not with repressive H3K9me3 or

H3K27me3 marks. This indicates that many lymphoid- and myeloid-specific genes have an

open/accessible chromatin conformation in human HSC, consistent with the notion that

lymphoid, like myeloid, genes are “primed” for expression prior to lineage commitment in

multipotent hematopoietic progenitors 19-21.

The histone modification profile in HSC appears to reflect the differentiation potential of the

stem cell and is different from that of other adult stem cells. Thus in human muscle satellite

cells, active histone modifications were found at the muscle specifying MYF5 gene, whereas

the set of lymphoid and myeloid genes analyzed were marked by high levels of repressive

histone modifications. H3K9me3 was present at the IgH locus, suggesting that silencing of Ig

genes in muscle satellite cells is regulated by interaction with HP1 and recruitment to

heterochromatin, as previously reported in terminally differentiated mouse B cells 35,36,40.

Neuronal and lymphoid transcription factors were associated with H3K27me3, indicating that




lineage inappropriate transcription factors are repressed via a polycomb protein–mediated

pathway in these cells 6.

We found that differentiation of multipotent hematopoietic progenitors towards one of the

myeloid or lymphoid lineages leads to loss of histone H3 and H4 acetylation and addition of

repressive H3K9 or H3K27 methylation marks at lineage-inappropriate genes. These results

show that progressive lineage restriction and cell fate specification of HSC is accompanied by

epigenetic silencing of lineage-inappropriate genes, as originally proposed by transcription

profiling studies 20,21,41. Interestingly, lineage-committed CD34+ progenitors still have a broad

histone acetylation profile, suggesting that early CD34+ precursors retain a certain degree of

lineage plasticity. Differentiation of HSC was also associated with a change in the relative

levels of H3K4 di- to tri-methylation. We found that B lymphoid genes are marked by

H3K4me2, but relatively low levels of H3K4me3 in HSC. As the genes become expressed in

B committed cells, H3K4me3 levels increased, while the levels of H3K4me2 were

maintained. Similarly, Attema and coworkers found lower levels of H3K4me3 in murine HSC

than in more mature progenitors at GATA 1, GATA 3, c-fms and Ptcrα promoters 42. Histone

H3K4me2 and H3K4me3 are found at promoters of transcriptionally active and “poised”

inactive genes, however, H3K4me3 correlates closely with transcription rate and polymerase

II occupancy 11,38,43. Consistent with this, our results indicate that high H3K4me2/H3K4me3

ratios mark potentially active lymphoid-specific genes in HSC and may therefore define a

“permissive” chromatin conformation, whereas transcription is associated with increased

H3K4me3 levels that may allow a more fully active chromatin state.

Recent studies mapping histone modifications in ES cells have considerably advanced our

understanding of the relationship between chromatin state and cell fate decisions. These

studies revealed that a set of genes, including key developmental and lineage-specifying genes

are marked by opposing H3K4me3 and H3K27me3 modifications in ES cells 14-18. Bivalent




chromatin marks occur at promoters with high CpG island content and is proposed to keep

genes transcriptionally silent in ES cells but “poised” for lineage-specific activation or

repression later during development. We find that this bivalent chromatin is largely resolved

in cord blood HSC. Non-hematopoietic transcripion factors, such as Pax6 and Myf5, lose

H3K4me3, but retain the H3K27me3 mark, indicating that they become stably repressed in a

polycomb-dependent manner in hematopoietic cells. In contrast, we found low levels of both

H3K4me3 and H3K27me3 marks at lymphoid-specific transcription factors in HSC. The lack

of H3K27me3 suggests that these genes are kept silent in HSC by a polycomb protein-

independent pathway. This chromatin structure resembles that reported for a second category

of transcriptionally silent genes in ES cells, which are marked by neither H3K4me3 nor

H3K27me3 marks. This set of genes have a low CpG island content and contain tissue-

specific genes involved in responses to external stimuli, including immune responses. Our

results suggest that in HSC, transcriptionally silent hematopoietic genes with both high and

low CpG content are devoid of both H3K4me3 and H3K27me3. The presence of high H4

acetylation and H3K4 dimethylation in the absence of active H3K4me3 or repressive

H3K27me3 or H3K9me3 marks appears to describe a transcriptional competent “ground

state” for these genes in human HSC keeping them silent but poised for expression at later

stages of differentiation. A key question that remains to be resolved is how these epigenetic

states are established during hematopoiesis. Notably, activating histone modifications are

already in place at B-affiliated genes in CD34+CD38lo HSC prior to expression of the major B

cell transcription factors E2A, EBF and PAX5. The hematopoietic factor PU.1 that is

expressed in multipotent progenitors may play a role in initiating these early epigenetic

marks, since there is evidence to suggest that PU.1 interacts with the promoter of the myeloid

c-fms gene in murine HSC and is implicated in chromatin reorganization of the c-fms and the

B-specific λ5-VpreB locus 34,44,45. Further, characterization of the epigenetic states of




hematopoietic cells at different stages of differentiation and studies aimed at identifying non-

histone proteins associated with lymphoid loci in HSC should help elucidate this question.

ACKNOWLEDGMENTS

We thank Drs Vincent Mouly and Serge Fichelson for the human satellite muscle cells and help with in vitro erythrocyte differentiation experiments and are grateful to Dr Christine Dosquet for critical reading of the manuscript. This work was supported by research funding from INSERM and grants from Association pour la Recherche sur le Cancer “ARECA” network (M.G., C.F.) and INSERM Adult Stem Cell AIP A03187DS (M.G., M.C.-C., P.F.) and AIP A03203DS (M.G., C.F.).

AUTHORSHIP Contributions: J.M. performed research, analyzed and interpreted data, drafted the manuscript. M.M. performed research, analyzed and interpreted data, drafted the manuscript. C.G. performed research. F.P. contributed vital new analytical tools. E.S. performed research. I.A.-S. analyzed and interpreted data. M.C.-C. contributed vital new analytical tools. D.C. head of department, contributed to research strategy. C.F. analyzed and interpreted data, drafted the manuscript. M.G. designed research, analyzed and interpreted data, drafted the manuscript. J.M. and M.M. contributed equally to this work. Conflict-of-interest disclosure: The authors declare no competing financial interests.

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FIGURE LEGENDS Figure 1. Pattern of histone H3 and H4 acetylation at lymphoid- and myeloid-affiliated genes in cord blood cells. Chromatin from multipotent CD34+CD38lo, B-committed CD19+ and erythroid GpA+ cells were analysed by ChIP using antibodies to acetylated histone H4 (A) and H3 (B) followed by real time PCR with primers specific for B lymphoid (B), T lymphoid (T), erythroid (E), myeloid (M), non-hematopoietic (N) and β2-microglobulin (Β2m) genes. Histogram shows enrichment values (bound/input) normalized to B2m control (set at 1). Results are means and standard deviations of 3 to 5 independent ChIP experiments analyzed in triplicate. Figure 2. Histone H3K9me3 and H3K27me3 modifications at lymphoid genes in CD34+CD38lo cells. ChIP analyses of multipotent CD34+CD38lo (white bars), erythroid GpA+ (grey bars) and B-committed CD19+ (black bars) cells with antibodies to H3K9me3 and H3K27me3. Histone modifications at B lymphoid (B), T lymphoid (T), erythroid (E) and non-hematopoietic (N) genes were normalized to the non-hematopoietic ΤΗP gene (set at 1). Results are means and standard deviations of 2 to 5 independent ChIP experiments analyzed in triplicate. ND, not determined. Figure 3. Histone H3K4 di- and tri-methylation at B-specific genes in CD34+CD38lo and CD19+ cells. Multipotent CD34+CD38lo and B-committed CD19+ cells were subjected to ChIP analyses with antibodies to H3K4me2 (white bars) and H3K4me3 (black bars). Results were calculated as in Figure 1 and are means and standard deviations of at least 2 independent ChIP experiments analyzed in triplicate. Figure 4. Comparison of histone modifications in HSC and muscle satellite cells. ChIP analyses of CD34+CD38lo HSC (white bars), and human muscle satellite cells (black bars) with antibodies to histone H3Ac, H3K4me2, H3K9me3 and H3K27me3 at representative B lymphoid (B), T lymphoid (T), erythroid (E), non-hematopoietic (N), β2-microglobulin (Β2m) and muscle specific (Mus) genes. Results are shown as enrichment values (bound/input) relative to the B2m gene (for H3Ac and H3K4me2) or to the ΤΗP gene (for H3K9me3 and H3K27me3) and are means and standard deviations of 2 to 5 independent ChIP experiments analyzed in triplicate. Figure 5. Comparison of histone modifications in CD34+CD38lo, CD34+ and CD34+38+10-

19- cord blood progenitors. ChIP analyses of multipotent CD34+CD38lo (white bars), total CD34+ (black bars) and B-lymphoid depleted CD34+38+10-19- (grey bars) progenitors with antobodies to histone H4Ac, H3Ac and H3K4me2 at B lymphoid (B), T lymphoid (T), erythroid (E), non-hematopoietic (N) and β2-microglobulin (Β2m) genes. Results are shown as enrichment values (bound/input) relative to the B2m control and are means and standard deviations of 2 to 5 independent ChIP experiments analyzed in triplicate. Figure 6. Changes in histone acetylation during in vitro differentiation of cord blood CD34+ progenitors. Top: Scheme of in vitro differentiation. Bottom: (A) Differentiation towards the erythroid lineage. Freshly isolated CD34+ cells (white bars) were cultured in the presence of IL-3, IL-6 and stem cell factor and after 7 days CD36+ cells (grey bars) were isolated and cultured for a further 5 days in the presence of Epo to give GpA+ cells (black bars). (B) Differentiation towards the granulocyte lineage. Freshly isolated CD34+ cells (white bars) were cultured in the presence of G-CSF, IL-3, stem cell factor and Flt3-L for 14 days to




give CD11b+ cells (black bars). (C) Differentiation towards the T-lymphoid lineage. In vitro culture of CD34+CD38lo cells (white bars) was performed on OP9-hDelta1 stromal cells in the presence of Flt3-L, stem cell factor and IL-7. After 21 days over 90% of cells were CD7+

committed T cell precursors (black bars). ChIP experiments were performed with antibodies to acetylated histones H3 and H4 at B lymphoid (B), T lymphoid (T), erythroid (E), myeloid (M), non-hematopoietic (N) and β2-microglobulin (Β2m) genes. Representative results of 2 to 4 independent experiments are shown.




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CD

79B

ex

EB

F

PA

X5

TDT

RA

G1

5'D

B1

JB1.

2

Ebe

ta

CD

3

GA

TA

3

HS

4

HS

2

Bpr

om

EP

O R

TA

L1

C-F

MS

PA

X6

THP

B2m

0

1

2

3

Mu

O

JH1

Em

u

Cm

u

3'E

mu Jk

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.

2

Eb

eta

CD

3

GA

TA

3

HS

4

HS

2

Bpr

om

EP

O R

TA

L1

C-F

MS

PA

X6

TH

P

B2m

0

1

2

MuO JH

1

Em

u

Cm

u

3'E

mu Jk

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1

.2

Ebe

ta

CD

3

GA

TA

3

HS

4

HS

2

Bp

rom

EP

O R

TA

L1

C-F

MS

PA

X6

TH

P

B2

m

BCD34+CD38lo

CD19+

GpA+

rela

tive

enric

hmen

tre

lativ

e en

richm

ent

rela

tive

enric

hmen

t

rela

tive

enric

hmen

tre

lativ

e en

richm

ent

rela

tive

enric

hmen

t

B T E M N

B2mB T E M N

B2m B T E M N

B2mB T E M N

B2m

BB BB

EE EE

Figure 1




0

5

10

15

20

25

30

35

40

MuO JH

1

Cm

u

3'E

mu Jk

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.2

CD

3

GATA

3

HS

4

Bpr

om

EPO

R

TAL1

PA

X6

TH

P

B2m

0

10

20

30

40

50

60

70

Mu

O

JH1

Cm

u

3'E

mu Jk

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L1

PA

X6

TH

P

B2m

Figure 2

H3K9me3

H3K27me3

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

B T E N B2m

B T E N B2m

ND

ND

0

5

10

15

20

25

30

35

40

MuO JH

1

Cm

u

3'E

mu Jk

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.2

CD

3

GATA

3

HS

4

Bpr

om

EPO

R

TAL1

PA

X6

TH

P

B2m

0

10

20

30

40

50

60

70

Mu

O

JH1

Cm

u

3'E

mu Jk

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L1

PA

X6

TH

P

B2m

Figure 2

H3K9me3

H3K27me3

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

B T E N B2mB T E N B2mB T EE N B2m

B T E N B2mB T E N B2mB T EE N B2m

ND

ND




Figure 3

0

2

4

6

8

10

MuO

Cm

u

3'E

mu

Ck

CD

79A

CD

79B

EB

F

PA

X5

CD

3

5'D

B1

JB1.2

TH

P

0

1

2

3

4

MuO

Cm

u

3'E

mu

Ck

CD

79A

CD

79B

EB

F

PA

X5

CD

3

5'D

B1

JB1.

2

TH

P

CD34+CD38-

CD19+

B

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent B

Figure 3

0

2

4

6

8

10

MuO

Cm

u

3'E

mu

Ck

CD

79A

CD

79B

EB

F

PA

X5

CD

3

5'D

B1

JB1.2

TH

P

0

1

2

3

4

MuO

Cm

u

3'E

mu

Ck

CD

79A

CD

79B

EB

F

PA

X5

CD

3

5'D

B1

JB1.

2

TH

P

CD34+CD38-

CD19+

B

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent B

0

2

4

6

8

10

MuO

Cm

u

3'E

mu

Ck

CD

79A

CD

79B

EB

F

PA

X5

CD

3

5'D

B1

JB1.2

TH

P

0

1

2

3

4

MuO

Cm

u

3'E

mu

Ck

CD

79A

CD

79B

EB

F

PA

X5

CD

3

5'D

B1

JB1.

2

TH

P

CD34+CD38-

CD19+

BBB

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent BB




Figure 4

0

5

10

15

20

MuO JH

1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

5

10

15

20

25

30

MuO JH

1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA3

EP

OR

TAL1

PA

X6

TH

P

B2m

MY

F5

0

2

4

6

MuO

JH1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

1

2

3

MuO JH

1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

H3K9me3

H3K4me2

H3Ac

H3K27me3

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

N

B2m

Mu

sB T E

Figure 4

0

5

10

15

20

MuO

JH1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

5

10

15

20

25

30

MuO

JH1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

2

4

6

MuO

JH1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

1

2

3

MuO JH

1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

H3K9me3

H3K4me2

H3Ac

H3K27me3

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

N

B2m

Mu

sB T E

0

5

10

15

20

MuO

JH1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

5

10

15

20

25

30

MuO

JH1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

2

4

6

MuO

JH1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

0

1

2

3

MuO JH

1

Cm

u

CD

79A

CD

79B

CD

79B

ex

EB

F

PA

X5

TD

T

5'D

B1

JB1.2

CD

3

GA

TA

3

EP

OR

TA

L1

PA

X6

TH

P

B2m

MY

F5

H3K9me3

H3K4me2

H3Ac

H3K27me3

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

N

B2m

Mu

sB T E N

B2m

Mu

sB TB T E




Figure 5

0

1

2

3

4

5

6

7

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

0

1

2

3

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

0

1

2

3

4

5

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

H4Ac

H3Ac

H3K4me2

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

B T E N B2m

Figure 5

0

1

2

3

4

5

6

7

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

0

1

2

3

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

0

1

2

3

4

5

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

H4Ac

H3Ac

H3K4me2

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

B T E N B2m

0

1

2

3

4

5

6

7

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

0

1

2

3

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

0

1

2

3

4

5

MuO JH

1

Cm

u Jk

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

5'D

B1

JB1.

2

CD

3

GA

TA

3

HS

4

Bpr

om

EP

O R

TA

L

PA

X6

TH

P

B2m

H4Ac

H3Ac

H3K4me2

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

rela

tive

enric

hm

ent

B T E N B2mB T EE N B2m




0

1

2

3

4

MuO JH

1

Cm

u

Ck

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RAG

1

5'D

B1

JB1.

2

CD

3

GA

TA

3

Bpr

om

EP

O R

TA

L1

PA

X6

TH

P

B2m

H4Ac H3Ac

0

1

2

3

MuO JH

1

Cm

u

Ck

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RAG

1

5'D

B1

JB1.

2

CD

3

GA

TA

3

Bpr

om

EP

O R

TAL1

PA

X6

TH

P

B2m

0

1

2

3

4

JH1 Jk Ck

CD

79A

CD

79B

EB

F

PA

X5

RA

G1

5'D

B1

JB1.

2

CD

3

GA

TA

3

Bpr

om

EP

O R

TA

L1

PA

X6

B2m

0

1

2

3

MuO JH

1

Cm

u Jk

CD

79A

CD

79B

p

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.

2

Ebe

ta

JPga

mm

a

Ega

mm

a

CD

3

GA

TA

3

HS

4

EP

O R

TA

L1

C-F

MS

PA

X6

TH

P

B2m

0

1

2

3

JH1 Jk Ck

CD

79A

CD

79B

EB

F

PAX5

RAG

1

5'D

B1

JB1.

2

CD

3

GAT

A3

Bpr

om

EP

O R

TAL1

PAX6

B2m

0

1

2

3

MuO JH

1

Cm

u Jk

CD

79A

CD

79B

p

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.

2

Ebe

ta

JPga

mm

a

Ega

mm

a

CD

3

GA

TA

3

HS

4

EP

O R

TA

L1

C-F

MS

PA

X6

TH

P

B2m

rela

tive

enric

hmen

tre

lativ

e en

richm

ent

rela

tive

enric

hmen

t

E N

B2mB TE N

B2mB T

E N

B2mB T E N B2mB T

E N B2mB T M E N B2mB T M

HSC Granulocyte differentiation (B)

Erythroid differentiation (A)

T-lymphoid differentiation (C)

IL-3, IL-6, SCF + EPO

G-CSF, IL-3, SCF, Flt-3

Flt3-L, IL-7, SCF, OP9-hDelta1

A

B

C

0

1

2

3

4

MuO JH

1

Cm

u

Ck

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RAG

1

5'D

B1

JB1.

2

CD

3

GA

TA

3

Bpr

om

EP

O R

TA

L1

PA

X6

TH

P

B2m

H4Ac H3Ac

0

1

2

3

MuO JH

1

Cm

u

Ck

3'E

mu

CD

79A

CD

79B

EB

F

PA

X5

TD

T

RAG

1

5'D

B1

JB1.

2

CD

3

GA

TA

3

Bpr

om

EP

O R

TA

L1

PA

X6

TH

P

B2m

0

1

2

3

4

JH1 Jk Ck

CD

79A

CD

79B

EB

F

PA

X5

RA

G1

5'D

B1

JB1.

2

CD

3

GA

TA

3

Bpr

om

EP

O R

TA

L1

PA

X6

B2m

0

1

2

3

MuO JH

1

Cm

u Jk

CD

79A

CD

79B

p

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.

2

Ebe

ta

JPga

mm

a

Ega

mm

a

CD

3

GA

TA

3

HS

4

EP

O R

TA

L1

C-F

MS

PA

X6

TH

P

B2m

0

1

2

3

JH1 Jk Ck

CD

79A

CD

79B

EB

F

PAX5

RAG

1

5'D

B1

JB1.

2

CD

3

GAT

A3

Bpr

om

EP

O R

TAL1

PAX6

B2m

0

1

2

3

MuO JH

1

Cm

u Jk

CD

79A

CD

79B

p

EB

F

PA

X5

TD

T

RA

G1

5'D

B1

JB1.

2

Ebe

ta

JPga

mm

a

Ega

mm

a

CD

3

GA

TA

3

HS

4

EP

O R

TA

L1

C-F

MS

PA

X6

TH

P

B2m

rela

tive

enric

hmen

tre

lativ

e en

richm

ent

rela

tive

enric

hmen

t

E N

B2mB T E N

B2mB TB TE N

B2mB T E N

B2mB TB T

E N

B2mB T E N

B2mB TB T E N B2mB T E N B2mB TB T

E N B2mB T ME N B2mB TB T M E N B2mB T ME N B2mB TB T M

HSC Granulocyte differentiation (B)

Erythroid differentiation (A)

T-lymphoid differentiation (C)

IL-3, IL-6, SCF + EPO

G-CSF, IL-3, SCF, Flt-3

Flt3-L, IL-7, SCF, OP9-hDelta1

A

B

C

Figure 6




Lymphoid-affiliated genes are associated with active histone modifications in human hematopoietic stem cells

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