REVIEW ARTICLE

Complementary regulation of early B-lymphoid differentiationby genetic and epigenetic mechanisms

Takafumi Yokota • Takao Sudo • Tomohiko Ishibashi •

Yukiko Doi • Michiko Ichii • Kenji Orirani •

Yuzuru Kanakura

Received: 4 June 2013 / Revised: 21 August 2013 / Accepted: 23 August 2013 / Published online: 3 September 2013

� The Japanese Society of Hematology 2013

Abstract Although B lymphopoiesis is one of the best-

defined paradigms in cell differentiation, our knowledge

of the regulatory mechanisms underlying its earliest pro-

cesses, in which hematopoietic stem cells (HSCs) enter

the B lineage, is limited. However, recent methodological

advances in sorting progenitor cells and monitoring their

epigenetic features have increased our understanding of

HSC activities. It is now known that even the highly

enriched HSC fraction is heterogeneous in terms of lym-

phopoietic potential. While surface markers and reporter

proteins provide information on the sequential differenti-

ation of B-lineage progenitors, complex interactions

between transcription factors have also been shown to

play a major role in this process. Epigenetic regulation of

histones, nucleosomes, and chromatin appears to play a

crucial background role in this elaborate transcription

network. In this review, we summarize recent findings on

the physiological processes of early B-lineage differenti-

ation, which provides a new paradigm for understanding

the harmonious action of genetic and epigenetic

mechanisms.

Keywords Hematopoietic stem cells � Early B

lymphopoiesis � Developmental pathway �Transcription factors � Epigenetic regulation

Introduction

B lymphocytes are continuously replenished throughout

life, although their output ratios are influenced by infection

or age. Many studies have reported that their early differ-

entiation stages from hematopoietic stem cells (HSCs) are

largely influenced by the abovementioned specific cir-

cumstances [1, 2]. However, molecular mechanisms

underlying the age-related declines or the infection-related

changes in B lymphopoiesis remain elusive. If key mole-

cules responsible for the changes in early B-lymphoid

differentiation could be identified, manipulation of their

expression may facilitate in rejuvenating or activating the

immune system of elderly people and decrease the inci-

dence and/or impact of infections. The aim of this review is

to summarize recent findings in this field and to improve

our understanding of molecular mechanisms that regulate

the earliest steps involving B-lineage differentiation.

Developmental pathway of early B-lineage progenitors

Models on the relationships of B- and other lineage pro-

genitors have influenced the interpretations of experimental

results on changes in hematopoietic differentiation.

Extensive information has been gathered from studies on

surface molecules during early B-lineage differentiation. In

combination with genetically modified mice that show

changes in the expression of early B-lineage-related genes,

the developmental process of B-lineage cells has been

extensively studied.

An early study conducted by Hardy’s group fractionated

B-lineage cells from mouse bone marrow (BM) into

Fractions A to F based on the surface markers, CD45RA/

B220, CD43, heat-stable antigen, and BP1 [3]. Addition of

T. Yokota (&) � T. Sudo � T. Ishibashi � Y. Doi � M. Ichii �K. Orirani � Y. Kanakura

Department of Hematology and Oncology,

Osaka University Graduate School of Medicine,

2-2 Yamada-oka, Suita, Osaka 565-0871, Japan

e-mail: yokotat@bldon.med.osaka-u.ac.jp

123

Int J Hematol (2013) 98:382–389

DOI 10.1007/s12185-013-1424-7

AA4.1 and CD4 further sub-fractionated the early steps of

B-lineage differentiation in Fraction A [4], although later

studies showed that the earliest fraction, A, contained

progenitors for other lineages [5, 6]. Another study con-

ducted at the Basel Institute proposed a differentiation

scheme of the BM B-lineage cells based on immunoglob-

ulin gene rearrangements [7]. While those methods were

extremely useful in analyzing the molecular mechanisms

that precede B-lineage differentiation of committed pro-

genitors, investigations on earlier B-lineage differentiation

processes required an innovative method to discriminate

lineage-biased progenitors prior to cellular commitment.

Application of IL-7 receptor alpha (IL-7Ra) expression as

an early lymphoid-related marker brought about substantial

development. It was originally used by Kondo et al. [8] to

differentiate common lymphoid progenitors (CLPs) in mouse

BM. The definition of CLPs was based on the concept of a

binary fate decision in which B, T, and NK lymphoid cells

diverge from all other lineages, including myeloid, erythroid,

and megakaryocyte lineages. The IL-7Ra? cells in Lin-

c-KitLo Sca1? fraction met the criterion in terms of their

differentiation potential in culture and in the thymus [8].

While subsequent studies have shown that the divergence of

erythroid-megakaryocyte lineage occurred prior to that of

myeloid from lymphoid lineage [9] and that the separation

between myeloid and lymphoid potentials is rather gradual

[10], the expression of IL-7Ra has been consistently used to

study early events in B-lineage differentiation.

Recent improvements in immunodetection assays using

more sensitive antibodies and laser/fluorochrome/filter

combinations have enabled monitoring of IL-7Ra expres-

sion in c-KitHi cells [11]. In the same report, expression of

Ly6D was used to differentiate ‘‘all lymphoid progenitors

(ALPs)’’ and B-lineage restricted progenitors (named as

BLPs) among the IL-7Ra? progenitors, including the ori-

ginal CLPs. The observation supports our original claim

based on the data with Rag1-GFP knock-in mice, in which

the initiation of lymphoid-lineage differentiation occurs in

the Lin- c-KitHi Sca1? CD27? fraction, i.e., occurring in

more proximity to HSCs [12, 13]. This notion also corre-

sponds to a report demonstrating that an increased Flt3

expression is a characteristic of lymphoid-primed multi-

potent progenitors (LMPPs) present in the Lin- c-KitHi

Sca1? fraction [9]. Rag1-GFP? early lymphoid progenitors

(ELPs) are found in the Lin- c-KitHi Sca1? Flt3Hi fraction

[6, 14], suggesting the close and sequential relationship

between LMPPs and ELPs. Of note, those progenitors are

exclusively sensitive to steroid hormones among Lin-

c-KitHi Sca1? cells [14–16], whereas these generate B

lymphocytes in culture with slower kinetics than Lin-

c-KitLo Sca1Lo IL-7Ra? cells [17]. These evidences thus

suggest that an early differentiation pathway in B lineage is

likely to proceed sequentially from HSCs to BLPs (Fig. 1).

Network of transcription factors and cytokine signaling

Along with the recent progress in dissecting the differen-

tiation pathways of early B-lymphoid differentiation,

information on the role of transcription factors in regulat-

ing early stages has also increased. Five transcription fac-

tors namely, PU.1, Ikaros, E2A/E47, Ebf1, and Pax5 have

been traditionally recognized as essential regulators of

early B-lineage differentiation [18, 19]. Transcription fac-

tors PU.1, E2A, and Ikaros are expressed in primitive

progenitors and are involved in multiple lineage fate

determination, whereas Ebf1 and Pax5 regulate B-lineage-

specified progenitors.

Recent studies have identified the FOXO family as

crucial component in early B-lineage differentiation [20,

21]. The FOXO family enhances the expression of the IL7-

receptor, which transmits intracellular signals that result in

the inactivation of FOXO1. The pathway suggests the

involvement of an auto-regulatory loop for B-lineage

determination. Since FOXO1 is a direct target of E2A and

Ebf1, the family might act downstream of the two indis-

pensable factors of B-lineage differentiation [22, 23]. Early

B lymphopoiesis also depends on the function of c-Myb,

which acts upstream of Ebf1 and regulates the survival and

differentiation of pro-B cells [24, 25]. While c-Myb regu-

lates the expression of the IL7-receptor, the molecule also

affects pro-B cell survival independent of the IL7-receptor

signaling pathway.

Runx1 is a well-known critical factor in the develop-

ment of authentic HSCs. In addition to its role in hema-

topoiesis, a recent study by Taniuchi’s group has

demonstrated that loss of Runx1 in the mb1-cre conditional

knockout system causes a significant developmental block

in early B-lineage differentiation [26]. Expression of E2A,

Ebf1, and Pax5 was reduced in the Runx1-deficient pro-

genitors, indicating that the factor probably plays a role in

the early stages of B lymphopoiesis.

Based on the evidences on upstream or downstream

components of the signaling pathway, it is clear that the

sequential action of critical transcription factors plays an

important role in the early path of B-lineage differentiation.

However, recent studies have also revealed that each

transcription factor affects a broader range of B-lineage

cells than previously expected. Furthermore, the regulatory

mechanisms of transcription factors are not influenced by a

simple hierarchy but by a cross-regulatory network (Fig. 2)

[27–29]. Ikaros plays an important role in establishing the

prime lymphoid-lineage fate of HSCs and this factor

associates with other B-lineage-related transcription factors

and upregulates various genes involved in the commitment

stage, in which CLPs differentiate into proB cells [30].

E2A induces T-lineage-related genes to act in concert with

Notch1 signaling [31], as well as plays critical roles in both

Early B-lymphocyte lineage differentiation 383

123

early and late stages of B lymphopoiesis, including ger-

minal center B cells [32–35]. Ebf1 and Pax5 were also

found to affect a wider range of stages during B-lineage

differentiation [36, 37]. While the two factors indepen-

dently induce B-lineage specification and commitment [38,

39], these mutually regulate their transcription in a feed-

forward manner. FOXO1 also participates in this positive

feedback loop [40]. Studies involving chromatin immu-

noprecipitation (ChIP) sequencing have revealed the

association of these transcription factors and their ensem-

ble with the promotion of B-lineage differentiation [22].

Epigenetic regulation of early B-lineage differentiation

in the bone marrow

In an effort to elucidate the elaborate network of key

transcription factors, studies on the epigenetic regulatory

mechanisms for gene expression have significantly

increased. A recent study by Murre’s group has shown that

PU.1, E2A, PAX5 and the histone acetyltransferases p300

are involved in folding genomes, and thus, the transcription

of the associated genes is regulated through its repression

or activation [41]. Interestingly, this process is develop-

mentally regulated by a spectrum of genes that switch their

location within the nucleus during early B-lineage differ-

entiation. Particularly, the Ebf1 locus, which are generally

associated with the nuclear lamina and prevent its pre-

mature activation in multipotent progenitors, move away

from the lamina during HSC differentiation into pro-B

cells. These positional changes of genes within the nucleus

affect their transcription, as shown in a previous report on

the immunoglobulin heavy chain (Igh) locus [42–46]. The

importance of spatial shift is probably a universal occur-

rence for biological regulation of gene expression [47, 48],

and has been extended to early B-lineage differentiation.

However, the underlying mechanisms that govern the

location of critical genes in the nucleus remain elusive.

It is also evident that histone modifications are intimately

involved in the regulation of the early process of B-cell

differentiation. Rag2, an indispensable molecule for the

rearrangement of Igh or T cell receptor (Tcr), was found to

target the H3K4me3 marks on D and J segments of Igh and

Tcrb genes [49–51]. Unsuccessful rearrangement or

HSC:Lin- c-kitHi Sca1+ Flt3-

Balanced

Lymphoid biased

Myeloid biased

CMPMEP

GMP LMPP:Lin- c-kitHi Sca1+ Flt3+

Flt3Lo

Flt3Hi

c-kitLo

c-kitHi

IL7-R Lo

IL7-R Hi

CLP:Lin- c-kitLo Sca1-/Lo

Flt3Hi IL7-R +

ALP:Ly6D-

BLP:Ly6D+

ELP:Rag1LoRag1Lo

Rag1Hi

HSC

LMPP

CLP

Fraction A Pre-pro-B:B220+ CD43+ AA4.1+ CD19-

Fig. 1 Schematic diagram of

the early B-lineage

differentiation pathway. The

differentiation pathway of

murine B lymphopoiesis from

HSCs to Fraction A/pre-proB

cells that are committed to the B

lineage is shown. Terminology

and important hallmarks to

distinguish each differentiation

stage are included: HSC

hematopoietic stem cells, LMPP

lymphoid-primed multipotent

progenitors, ELP early

lymphoid progenitors, CLP

common lymphoid progenitors,

ALP all lymphoid progenitors,

BLP B-lineage restricted

progenitors, CMP common

myeloid progenitors, MEP

megakaryocyte erythroid

progenitors, and GMP

granulocyte monocyte

progenitors

384 T. Yokota et al.

123

defective class switch of the Igh locus caused by dysfunc-

tion of the polycomb protein Ezh2, the H3K36me3-specific

histone methyltransferase MM-SET or the CTCF looping/

insulator factor further supports the notion that epigenetic

regulatory mechanisms are essential for normal B-lineage

differentiation [52–54]. Since the role of epigenetic changes

in immunosenescence remains largely unknown, it is

noteworthy that the manipulation of rhoGTPase Cdc42

activity reverts the levels and patterns of histone H4 acet-

ylation of aged HSCs to those of young HSCs and rejuve-

nates their lymphopoietic potential [55]. Thus, we might

think that the epigenetic mechanism is the key to understand

the regulatory steps involved in HSC differentiation into

B-lineage progenitors.

Global modulators of chromatin structure are involved

in the early lymphoid differentiation

We searched key molecules directing HSCs towards

B-lineage fate by comparing the gene expression signature

between HSCs and ELPs [56]. We found that numerous

lymphoid-related genes, including Igh and Tcrb, were

remarkably upregulated even in the very early lymphoid

progenitors, whereas HSC-related or myeloid-related ones

were downregulated. Therefore, we speculated that a

master regulator is present that orchestrates the expression

of various genes during the first step of B-lineage differ-

entiation. Since the molecule needs to regulate several

genes that may be proximal and distal simultaneously, we

assumed that it should possess the ability to modulate

nuclear architecture. Among the list of upregulated genes

in ELPs compared with HSCs, special AT-rich sequence

binding protein 1 (Satb1) strongly attracted our attention

because it was reported to coordinate spatial and temporal

expression of various genes. Using both loss-of-function

and gain-of-function strategies, we have demonstrated that

Satb1 expression warrants lymphopoietic potential in HSCs

and indeed induces early lymphoid differentiation even

from aged HSCs or embryonic stem cells [56]. In addition,

ectopic Satb1 induction in HSCs induced the expression of

various lymphoid-related genes while it suppressed mye-

loid-related ones.

Satb1 is a DNA-binding protein, which specifically

target genomic DNA in a specialized DNA context with

AT-rich high base-unpairing potential [57]. Although

Satb1 was originally identified as Igh gene-associated

protein, it is predominantly expressed in the thymus, and

subsequent studies have revealed its critical roles in the

T-lineage cells [58–60]. This molecule exists in the nuclear

matrix and regulates global gene expression by establishing

chromatin-loop architecture in collaboration with other

nuclear matrix components [61, 62].

Protein families that can interact with AT-rich sequen-

ces of genomic DNA appear to play important roles in

lymphocyte development. Satb2, a protein closely related

with Satb1, has been shown to regulate Igh gene expression

under appropriate conjugation of the small ubiquitin-rela-

ted modifier [63]. Bright/ARID3a/Dril1, the founder

member of the AT-rich interaction domain family, was

originally identified as a protein that was dominantly

expressed in B-lineage cells and important for the Igh

PU.1

Ikaros

E2A

Ebf1

Pax5

Runx1

FOXO1

Flt3 Notch1IL7R

Rag1c-Myb

Fig. 2 A network of

transcription factors and cell

surface receptors governing

early B-lineage differentiation.

Key transcription factors for

early B-lineage differentiation

of murine lymphopoiesis are

shown with green circles. Three

cell surface receptors, Flt3,

IL7R, and Notch1, are drawn on

an orange bar, which represents

the cell membrane. Arrows

represent positive regulation,

whereas blunt ends indicate

repression

Early B-lymphocyte lineage differentiation 385

123

expression [64]. Subsequent studies have revealed that the

protein is expressed in very early B-lineage progenitors,

even HSCs, and is indispensable for normal cellular

development [65–67]. A recent paper has shown that

appropriate Satb1 expression in HSCs is critical for the

maintenance of cellular integrity [68]. Determining whe-

ther AT-rich sequence binding proteins interact in primi-

tive progenitors or independently play roles in lineage- and

stage-specific manners is, therefore, a very interesting

future research theme.

Complementary relationship between genetic

and epigenetic regulation

From the accumulated evidences mentioned, it is highly

likely that genetic and epigenetic regulations are comple-

mentarily involved in the early process of B-lineage dif-

ferentiation. As illustrated in Fig. 3, nuclear matrix

proteins such as Satb1 and histone modification seem to

construct a background frame for the expression of tran-

scription factors, which then work together on the tran-

scription of various lymphoid lineage-related genes.

Previous studies have employed the term ‘‘promiscu-

ous’’ in describing the expression of lineage-related genes

in hematopoietic progenitors and have collectively coined

the entire phenomenon as ‘‘lineage-priming’’ [69, 70].

Multipotent progenitors are generally capable of differen-

tiating into any of the possible lineage options and are

induced to follow a specific differentiation pathway based

on the organism’s physiological demand. Thus, the priming

process appears to exist inherently in those cells. However,

a recent investigation has otherwise observed that the

priming expression of lineage-related genes follows a

systemic approach, thus reflecting a reversible fluctuation

of transcriptome activities [71]. Novershtern et al. [72]

have subjected human HSCs to extensive gene arrays and

bioinformatics analyses. They have confirmed the antici-

patory loading of promoters of lineage-related genes even

with primitive HSCs, corresponding to the notion of

‘‘lineage-priming.’’ Furthermore, they have revealed that

such anticipatory loading of promoters becomes simple

when it simultaneously occurs with lineage specification.

Since both chromatin architecture and transcription factors

are important for multiple gene expression, epigenetic and

genetic regulations should be complementarily involved in

such lineage and stage-specific loading of promoters.

Epigenetic abnormality involved in early B-lineage

neoplasms

Recent revolutionary development in the DNA sequencing

technology has enabled us to reach comprehensive under-

standing of genome-wide abnormality in cancers [73, 74]. A

variety of novel gene mutations have been revealed in

hematopoietic malignancies. In particular, mutations of epi-

genetic regulators and RNA splicing factors have been

involved in the pathogenesis of myeloid-lineage neoplasms

including acute myeloid leukemia and myelodysplastic syn-

drome [75–77]. Compared with those advances in myeloid-

lineage neoplasms, the information of epigenetic abnormality

in lymphoid leukemia seems to be limited. However, there are

some important findings to be introduced.

Mullighan et al. [78] performed genome-wide analyses

for 242 pediatric acute lymphoid leukemia (ALL) patients.

They have identified high mutation frequencies of genes

encoding regulators for early B-cell lineage development in

acute lymphoblastic leukemia. Indeed, 40 % of pediatric

B-cell lineage ALL cases harbored mutations in the prin-

cipal regulators including PAX5, E2A/E47, EBF and I-

KAROS. Among them, mutations of IKZF1, a gene

Nucleus

Heterochromatin Euchromatin Nuclear matrix proteins

Chromatin loopTranscription factor

Transcription factor

Fig. 3 A proposed model of

Satb1 function on the chromatin

loop structure. Left a cartoon

showing the simplified nuclear

architecture consisting of

chromatin and the nuclear

matrix. Satb1 protein is present

in the nuclear matrix and binds

to the AT-rich DNA sequences

attached to nuclear matrix.

Right a schematic diagram of

the putative mechanism of

nuclear matrix proteins such as

Satb1 in regulating multiple

gene expressions through the

construction of chromatin loop

structures and transcriptional

complexes

386 T. Yokota et al.

123

encoding IKAROS protein, have been drawing attention

because they are found to relate with a high relapse rate

and a poor prognosis [79]. Two recent studies have sug-

gested that the IKZF1 mutations are more involved in adult

rather than in pediatric B-ALL [80, 81]. Furthermore, the

IKZF1 gene abnormality is very frequent and severe in

BCR-ABL positive cases [82–84]. It is important to stress

here that, besides the essential role as a transcription factor

in B-lymphocyte differentiation, an IKAROS protein

serves as an integral component of chromatin-remodeling

networks and prevents leukemogenesis [85, 86]. Thus, the

loss of IKAROS function is assumed to cause aberrant

association between genetic and epigenetic networks,

thereby mediate high susceptibility to aggressive leukemia.

Future direction and concluding remarks

To date, it has become evident that the roles of transcrip-

tion factors are essential for physiological B lymphopoie-

sis. Substantial progress has been recently made in

understanding how epigenetic regulation has influenced

this process. More precise information of the comple-

mentary relation between the genetic and epigenetic

mechanisms may potentially boost lymphocyte production

after chemotherapy and stem cell transplantation in the

future. Environmental cues regulating the two mechanisms

during the early stage of B-lineage differentiation remain

elusive. Understanding the physiological process of early

differentiation is essential to appreciate when and how

aberrant incidences occur in cancer. Future research studies

can shed light on epigenetic problems associated with

malignant transformation.

Acknowledgments We would like to thank Dr. Noriko Saitoh

(Kumamoto University) for providing a figure of the cell nucleus and

Dr. Kiyoe Ura (Osaka University) for discussions and for critical

reading of this manuscript. We also thank Dr. Yusuke Satoh (Kobe

Shoin Women’s University) for the effort in Satb1-related

experiments.

Conflict of interest The authors declare that they have no conflict

of interest.

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