In 1999, Stephen Nutt, Barry Heavey, Antonius Rolink, and Meinrad Busslinger unveiled how Pax5 masterminds B cell development (1). In doing so, they significantly deepened understanding about how competition between transcription factors can dictate cell fate.

Their discovery emerged from a decade of effort galvanized by the startling discovery of Davis, Weintraub, and Lassar that MyoD could entrain fibroblasts to become muscle cells (2). The concept of “master genes” controlling cell fate by activating a suite of lineage-specific genes was compelling, and molecular detectives had eagerly enlisted to track down the dictators.

The well-studied hematopoietic system provided a rich hunting ground, due to the excellent clonal culture systems, diversity of cell markers, and array of pure cytokines available. In this system, rare hematopoietic stem cells found in adult bone marrow give rise to multiple specialized blood cell lines, including the erythromegakaryocytic lineage, which produces oxygen-carrying RBCs and platelets to prevent bleeding; the various myeloid lineages, which provide early defense against invading pathogens; and the B and T lymphoid lineages, which generate specific long-lasting immunity (see Fig. 1 and Ref. 3).

FIGURE 1.

Developmental trajectories in hematopoiesis (adapted from Fig. 1 in Ref. 18), indicating transcription factors acting at distinct early steps. CLP, common lymphoid progenitor compartment; ETP, early thymocyte progenitor; GMP, granulocyte-macrophage progenitor compartment; Gran, granulocytes; HSC, hematopoietic stem cell; ILCs, innate lymphoid cells; LMPP, lymphoid primed multipotent progenitor; Mac, macrophage compartment; MEP, premegakaryocytic/erythroid progenitor compartment; MPP, multipotent progenitor. The trans- and dedifferentiation potential of Pax5-deficient B lymphoid cells is indicated by a dashed line.

FIGURE 1.

Developmental trajectories in hematopoiesis (adapted from Fig. 1 in Ref. 18), indicating transcription factors acting at distinct early steps. CLP, common lymphoid progenitor compartment; ETP, early thymocyte progenitor; GMP, granulocyte-macrophage progenitor compartment; Gran, granulocytes; HSC, hematopoietic stem cell; ILCs, innate lymphoid cells; LMPP, lymphoid primed multipotent progenitor; Mac, macrophage compartment; MEP, premegakaryocytic/erythroid progenitor compartment; MPP, multipotent progenitor. The trans- and dedifferentiation potential of Pax5-deficient B lymphoid cells is indicated by a dashed line.

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The approach initially taken was to clone a lineage-specific transcription factor and test whether its ectopic expression was capable of compelling another cell type to switch lineage identity. The subsequent advent of gene knockout technology greatly empowered the enterprise.

The first hematopoietic “master genes” identified were the transcription factors GATA-1 and PU.1. Ectopic expression of the erythroid factor GATA-1 induced a switch from monocytic to erythroid/megakaryocytic differentiation (46) and PU.1/Spi1 expression provoked the reverse (7, 8).

Initiation of B lymphoid development was known to depend on E2A and early B cell factor, as absence of either of these proteins stalled B lymphopoiesis at a very early stage (911). A third critical factor, B cell–specific activator protein, was shown to be encoded by the Pax5 gene (12). Inactivation of Pax5 blocked B lymphoid ontogeny at the pro–B cell (also termed pre-B1) stage, during which time DNA recombination between DH and JH elements initiates the process that generates functional Ab genes (13).

Striking and unexpected insight came when Nutt and his colleagues tried differentiating Pax5-deficient pro–B cells on stromal cells in vitro (1). As expected, Ab gene rearrangement and subsequent B cell maturation could not be completed unless Pax5 expression was restored. Surprisingly, however, when the Pax5-deficient cells were inadvertently cultured for several weeks on ST2 stromal cells in the absence of the cytokine IL-7, they changed in appearance, looking suspiciously like myeloid cells. Intrigued, Nutt et al. supplemented the IL-7–starved cultures with different cytokines and revealed an amazingly diverse differentiation capacity. Pax5-deficient pro–B cells could metamorphose into terminally differentiated macrophages in the presence of M-CSF, whereas GM-CSF produced dendritic cells, G-CSF produced granulocytes, TRANCE/RANKL produced osteoclasts, and IL-2 generated NK cells.

In a companion paper, Rolink et al. (14) injected Pax5-deficient pro–B clones into immunodeficient RAG-2 null mice and showed that they could reconstitute T cell development, with the resulting T cells bearing clonal DH–JH DNA rearrangements, indicative of their B lymphoid origin, as well as fully rearranged TCR genes. Not all hematopoietic differentiation options remained open to the Pax5-deficient cells, however, because they could not rescue lethally irradiated mice.

The illuminating conclusion was that Pax5 not only activated vital B cell–specific genes, it also suppressed genes normally expressed in multiple other hematopoietic cell lineages (see Ref. 15). Ten years later, the theme of a transcriptional repressor enabling a progenitor cell to shed alternative cell fates was to be reiterated for Bcl11b and T cell commitment (16).

Pax5 does not act alone in specifying B cell fate but instead is a core component of a tightly controlled gene regulatory network with E2A and early B cell factor (17, 18). Its expression is sustained up to the B cell stage (15) and, despite their advanced differentiation state, Pax5-deficent mature B cells are still capable of rescuing T lymphopoiesis in T cell–deficient mice (19). The mechanism came as a big surprise: the B cells dedifferentiated back to early uncommitted progenitors, which then migrated from the bone marrow to the thymus to initiate T lymphopoiesis.

Mature B cells also retain their capacity to differentiate into macrophages, as inferred earlier from oncogene-transformed cells (20, 21). Xie et al. (22) showed that enforced expression of the myeloid transcription factors C/EBPα and C/EBPβ in mature B cells induced rapid reprogramming by inhibiting endogenous Pax5, thereby suppressing expression of B lineage target genes and synergizing with endogenous PU.1 to upregulate myeloid-specific genes such as Mac-1. Thus, Pax5 actively maintains cellular identity throughout the life of a B cell.

These hematopoietic lineage-switching experiments provided unequivocal evidence for differentiation plasticity or transdifferentiation, which had been long suspected but difficult to prove in primary cells (23). Evidence for transcription factor–mediated lineage conversions outside the hematopoietic system has also emerged (reviewed in Refs. 23, 24).

Such experiments prepared the way for the induced pluripotent stem cell revolution triggered by Takahashi and Yamanaka (25) in 2006 when they showed that fibroblasts can be converted into multipotent stem cells simply by providing them with four transcription factors: Oct4, Sox2, Klf4, and Myc. In a tour de force, Hanna et al. (26) subsequently showed that these factors could also reprogram terminally differentiated B cells into multipotential cells when, additionally, Pax5 levels were reduced, either directly or by overexpression of C/EBPα. Furthermore, these mouse B cell–derived induced pluripotent stem cells gave rise to late-term embryos when injected into tetraploid blastocysts. Clearly, much has been learned since John Gurdon first produced fertile frogs by transplanting somatic cell nuclei into enucleated oocytes (27).

Today, considerable effort is being devoted to deciphering how to deprogram mature cells and then reprogram them into therapeutically useful cell types. However, much remains to be understood before we can efficiently and safely deploy reprogrammed cells for regenerative medicine. Many “master genes” are also proto-oncogenes or tumor suppressors (28). Thus, in our zeal to restore health, we must guard against inadvertently producing cancer.

Here, again, Pax5 studies are illuminating. Pax5 is the most mutated transcription factor in B-progenitor acute lymphoblastic leukemia (B-ALL), with diverse mutations lowering but not ablating its expression (29, 30). Furthermore, hypomorphic mutations of Pax5 have recently been associated with familial B-ALL (31). The inference is that reduced levels of Pax5 impose a differentiation block, thereby putting the cycling progenitor cell pool at increased risk of acquiring frankly oncogenic mutations (3234). In strong support of this hypothesis, mice lacking Pax5 in mature B cells develop aggressive progenitor cell lymphomas (19).

Excitingly, Liu et al. (32) have recently used a mouse model to show that, even in established B-ALL, the differentiation program can be re-engaged by elevating Pax5, despite the presence of other mutations. In light of the remarkable success of differentiation therapy for acute promyelocytic leukemia (35), this result encourages a search for comparable agents for treating B-ALL. Clearly, the progress spawned by the work of Nutt et al. is still unfolding, even 16 years down the road.

Abbreviation used in this article:

B-ALL

B-progenitor acute lymphoblastic leukemia.

1
Nutt
S. L.
,
Heavey
B.
,
Rolink
A. G.
,
Busslinger
M.
.
1999
.
Commitment to the B-lymphoid lineage depends on the transcription factor Pax5
.
Nature
401
:
556
562
.
2
Davis
R. L.
,
Weintraub
H.
,
Lassar
A. B.
.
1987
.
Expression of a single transfected cDNA converts fibroblasts to myoblasts
.
Cell
51
:
987
1000
.
3
Rothenberg
E. V.
2014
.
Transcriptional control of early T and B cell developmental choices
.
Annu. Rev. Immunol.
32
:
283
321
.
4
Visvader
J. E.
,
Elefanty
A. G.
,
Strasser
A.
,
Adams
J. M.
.
1992
.
GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line
.
EMBO J.
11
:
4557
4564
.
5
Kulessa
H.
,
Frampton
J.
,
Graf
T.
.
1995
.
GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts
.
Genes Dev.
9
:
1250
1262
.
6
Heyworth
C.
,
Pearson
S.
,
May
G.
,
Enver
T.
.
2002
.
Transcription factor-mediated lineage switching reveals plasticity in primary committed progenitor cells
.
EMBO J.
21
:
3770
3781
.
7
Nerlov
C.
,
Graf
T.
.
1998
.
PU.1 induces myeloid lineage commitment in multipotent hematopoietic progenitors
.
Genes Dev.
12
:
2403
2412
.
8
DeKoter
R. P.
,
Singh
H.
.
2000
.
Regulation of B lymphocyte and macrophage development by graded expression of PU.1
.
Science
288
:
1439
1441
.
9
Zhuang
Y.
,
Soriano
P.
,
Weintraub
H.
.
1994
.
The helix-loop-helix gene E2A is required for B cell formation
.
Cell
79
:
875
884
.
10
Bain
G.
,
Maandag
E. C.
,
Izon
D. J.
,
Amsen
D.
,
Kruisbeek
A. M.
,
Weintraub
B. C.
,
Krop
I.
,
Schlissel
M. S.
,
Feeney
A. J.
,
van Roon
M.
, et al
.
1994
.
E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements
.
Cell
79
:
885
892
.
11
Lin
H.
,
Grosschedl
R.
.
1995
.
Failure of B-cell differentiation in mice lacking the transcription factor EBF
.
Nature
376
:
263
267
.
12
Adams
B.
,
Dörfler
P.
,
Aguzzi
A.
,
Kozmik
Z.
,
Urbánek
P.
,
Maurer-Fogy
I.
,
Busslinger
M.
.
1992
.
Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis
.
Genes Dev.
6
:
1589
1607
.
13
Nutt
S. L.
,
Urbánek
P.
,
Rolink
A.
,
Busslinger
M.
.
1997
.
Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus
.
Genes Dev.
11
:
476
491
.
14
Rolink
A. G.
,
Nutt
S. L.
,
Melchers
F.
,
Busslinger
M.
.
1999
.
Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors
.
Nature
401
:
603
606
.
15
Cobaleda
C.
,
Schebesta
A.
,
Delogu
A.
,
Busslinger
M.
.
2007
.
Pax5: the guardian of B cell identity and function
.
Nat. Immunol.
8
:
463
470
.
16
Li
L.
,
Leid
M.
,
Rothenberg
E. V.
.
2010
.
An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b
.
Science
329
:
89
93
.
17
Boller
S.
,
Grosschedl
R.
.
2014
.
The regulatory network of B-cell differentiation: a focused view of early B-cell factor 1 function
.
Immunol. Rev.
261
:
102
115
.
18
Mercer
E. M.
,
Lin
Y. C.
,
Murre
C.
.
2011
.
Factors and networks that underpin early hematopoiesis
.
Semin. Immunol.
23
:
317
325
.
19
Cobaleda
C.
,
Jochum
W.
,
Busslinger
M.
.
2007
.
Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors
.
Nature
449
:
473
477
.
20
Klinken
S. P.
,
Nicola
N. A.
,
Johnson
G. R.
.
1988
.
In vitro-derived leukemic erythroid cell lines induced by a raf- and myc-containing retrovirus differentiate in response to erythropoietin
.
Proc. Natl. Acad. Sci. USA
85
:
8506
8510
.
21
Borzillo
G. V.
,
Ashmun
R. A.
,
Sherr
C. J.
.
1990
.
Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes
.
Mol. Cell. Biol.
10
:
2703
2714
.
22
Xie
H.
,
Ye
M.
,
Feng
R.
,
Graf
T.
.
2004
.
Stepwise reprogramming of B cells into macrophages
.
Cell
117
:
663
676
.
23
Graf
T.
2011
.
Historical origins of transdifferentiation and reprogramming
.
Cell Stem Cell
9
:
504
516
.
24
Graf
T.
,
Enver
T.
.
2009
.
Forcing cells to change lineages
.
Nature
462
:
587
594
.
25
Takahashi
K.
,
Yamanaka
S.
.
2006
.
Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors
.
Cell
126
:
663
676
.
26
Hanna
J.
,
Markoulaki
S.
,
Schorderet
P.
,
Carey
B. W.
,
Beard
C.
,
Wernig
M.
,
Creyghton
M. P.
,
Steine
E. J.
,
Cassady
J. P.
,
Foreman
R.
, et al
.
2008
.
Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency
.
Cell
133
:
250
264
.
27
Gurdon
J. B.
,
Elsdale
T. R.
,
Fischberg
M.
.
1958
.
Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei
.
Nature
182
:
64
65
.
28
Mullighan
C. G.
2012
.
Molecular genetics of B-precursor acute lymphoblastic leukemia
.
J. Clin. Invest.
122
:
3407
3415
.
29
Mullighan
C. G.
,
Goorha
S.
,
Radtke
I.
,
Miller
C. B.
,
Coustan-Smith
E.
,
Dalton
J. D.
,
Girtman
K.
,
Mathew
S.
,
Ma
J.
,
Pounds
S. B.
, et al
.
2007
.
Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia
.
Nature
446
:
758
764
.
30
Kuiper
R. P.
,
Schoenmakers
E. F.
,
van Reijmersdal
S. V.
,
Hehir-Kwa
J. Y.
,
van Kessel
A. G.
,
van Leeuwen
F. N.
,
Hoogerbrugge
P. M.
.
2007
.
High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression
.
Leukemia
21
:
1258
1266
.
31
Shah
S.
,
Schrader
K. A.
,
Waanders
E.
,
Timms
A. E.
,
Vijai
J.
,
Miething
C.
,
Wechsler
J.
,
Yang
J.
,
Hayes
J.
,
Klein
R. J.
, et al
.
2013
.
A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia
.
Nat. Genet.
45
:
1226
1231
.
32
Liu
G. J.
,
Cimmino
L.
,
Jude
J. G.
,
Hu
Y.
,
Witkowski
M. T.
,
McKenzie
M. D.
,
Kartal-Kaess
M.
,
Best
S. A.
,
Tuohey
L.
,
Liao
Y.
, et al
.
2014
.
Pax5 loss imposes a reversible differentiation block in B-progenitor acute lymphoblastic leukemia
.
Genes Dev.
28
:
1337
1350
.
33
Heltemes-Harris
L. M.
,
Willette
M. J.
,
Ramsey
L. B.
,
Qiu
Y. H.
,
Neeley
E. S.
,
Zhang
N.
,
Thomas
D. A.
,
Koeuth
T.
,
Baechler
E. C.
,
Kornblau
S. M.
,
Farrar
M. A.
.
2011
.
Ebf1 or Pax5 haploinsufficiency synergizes with STAT5 activation to initiate acute lymphoblastic leukemia
.
J. Exp. Med.
208
:
1135
1149
.
34
Dang
J.
,
Wei
L.
,
de Ridder
J.
,
Su
X.
,
Rust
A. G.
,
Roberts
K. G.
,
Payne-Turner
D.
,
Cheng
J.
,
Ma
J.
,
Qu
C.
, et al
.
2015
.
PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia
.
Blood
125
:
3609
3617
.
35
Alimoghaddam, K. 2014. A review of arsenic trioxide and acute promyelocytic leukemia. Int. J. Hematol. Oncol. Stem Cell Res. 8: 44–54
.

The author has no financial conflicts of interest.