The transcription factors E2A and HEB act in concert to
induce the expression of FOXO1 in the common
Eva Welindera,b, Robert Manssona, Elinore M. Mercera, David Bryderb, Mikael Sigvardssonc, and Cornelis Murrea,1
aDepartment of Molecular Biology, University of California at San Diego, La Jolla, CA 92093;bDepartment for Experimental Medical Science, Lund University,
22242 Lund, Sweden; andcDepartment for Clinical and Experimental Sciences, Linköping University, 58183 Linköping, Sweden
Edited* by Michael Levine, University of California, Berkeley, CA, and approved September 6, 2011 (received for review July 19, 2011)
Recent studies have identified a number of transcriptional regu-
lators, including E proteins, EBF1, FOXO1, and PAX5, that act to-
gether to orchestrate the B-cell fate. However, it still remains
unclear as to how they are linked at the earliest stages of B-cell
development. Here, we show that lymphocyte development in
HEB-ablated mice exhibits a partial developmental arrest, whereas
B-cell development in E2A+/−HEB−/−mice is completely blocked at
the LY6D−common lymphoid progenitor stage. We show that the
transcription signatures of E2A- and HEB-ablated common lym-
phoid progenitors significantly overlap. Notably, we found that
Foxo1 expression was substantially reduced in the LY6D−HEB-
and E2A-deficient cells. Finally, we show that E2A binds to en-
hancer elements across the FOXO1 locus to activate Foxo1 expres-
sion, linking E2A and FOXO1 directly in a common pathway. In
summary, the data indicate that the earliest event in B-cell speci-
fication involves the induction of FOXO1 expression and requires
the combined activities of E2A and HEB.
surrounding microenvironment. Hematopoiesis is initiated in he-
matopoietic stem cells (HSCs). HSCs have the ability to generate
the entire spectrum of hematopoietic cells as well as self-renew
without losing their multilineage potential (1). HSCs initially dif-
ferentiate into multipotent progenitors (MPPs). MPPs have lost
their ability to self-renew and can only transiently support hema-
topoiesis on transplantation (2). The MPP compartment itself is
heterogeneous and contains a subfraction of cells expressing high
surface levels of fms-like tyrosine kinase 3 (FLT3), often referred
to as lymphoid-primed multipotent progenitors (LMPPs) (3).
These cells have largely lost their megakarycyte/erythocyte po-
tential (3). Thus, LMPPs are considered to represent progenitor
cells en route to a lymphoid cell fate. LMPPs are the likely pre-
cursor of the common lymphoid progenitors (CLPs), with the
ability to develop into the entire spectrum of lymphoid cells (4, 5).
However, recent studies have shown that the CLP compartment is
also heterogeneous, containing immature cells along with a small
subset of cells committed to the B-cell lineage (6). The CLPs can
be segregated using lymphocyte antigen 6 complex, locus D
(LY6D) as a marker (7, 8). Specifically, the LY6D−CLPs repre-
sent the more immature compartment, whereas the B lineage-
committed cells can be found within the LY6D+fraction.
The development of lymphoid progenitors requires the activ-
ities of an ensemble of transcriptional regulators (9–13). Prom-
inent among these regulators are the E proteins E2A, E2-2, and
HeLa E-box binding protein (HEB) (14). The E2A proteins
maintain the HSC pool and promote the developmental pro-
gression of myelolymphoid and myeloerythorid progenitors
during early hematopoiesis (15–17). At the CLP cell stage, they
act upstream and in concert with Early B cell factor 1 (EBF1),
forkhead box O1 (FOXO1) and Paired box gene 5 (PAX5) to
promote specification and commitment to the B-cell lineage (10,
13, 18, 19). The E2A locus encodes for two isoforms, E12 and
E47, that arise through differential splicing (14). Whereas E47
plays a critical role in early B-cell development, the activity of
E12 is not essential (20). Although some T cells develop in E2A-
teady state hematopoiesis relies on fine tuned transcriptional
networks that are modulated by external signals from the
deficient mice, the E2A proteins have also been shown to play
critical roles in early thymocyte development (21). They induce
the expression of genes involved in Notch and pre-T cell receptor
signaling and in turn, act in concert with Notch signaling to
promote T-lineage development (21). A unique role for the E2-2
proteins has also recently been established. Specifically, it was
shown that plasmacytoid dendritic cell development is blocked in
E2-2–deficient mice (22).
it is requiredto promote efficient maturation beyond thepre-TCR
checkpoint (21) and induce the invariant natural killer T cell fate
(23). During fetal life, HEB is required to promote developmental
progression of early thymocyte progenitors and pro-B cells (24,
25). However, the roles of HEB in adult hematopoesis and lym-
phocyte development and how HEB acts in concert with E2A to
promote B-cell development have not yet been carefully exam-
ined. Here, we report that HEB is broadly expressed at substantial
levels within the entire spectrum of adult hematopoietic progeni-
tors. Whereas erythroid and myeloid development seemed nor-
that was similar to the block described for E2A-ablated CLPs (7,
B-cell development; HEB−/−and E2A+/−mice exhibit a partial
block at the LY6D−CLP cell stage, whereas E2A+/−HEB−/−mice
exhibit an arrest. Furthermore, HEB- and E2A-deficient LY6D−
transcription factor FOXO1. Finally, we identify enhancer ele-
ments, characterized by the presence of H3K4me1 islands, across
the FOXO1 locus that are responsive to E2A activity. These
observations directly link E2A, HEB, and FOXO1 in B-cell pro-
genitors into a common framework that specifies the B-cell fate.
Expression of E2A, HEB, Id2, and Id3 in Progenitor Cells. To charac-
terize the patterns of E2A, HEB, Id2, and Id3 expression in bone
marrow progenitor cells, mRNA was isolated from FACS-sorted
progenitor populations and analyzed by real-time PCR. Both E2A
and HEB were expressed in the Lineage−/lowSCA1+KIT+(LSK)
compartment known to harbor the majority of the HSCs’ potential
(1) as well as substantial levels within the entire spectrum of he-
matopoietic progenitors (Fig. 1A). Id2 was expressed at low levels
in the CLP compartments, whereas Id3 expression was only de-
tectable in the LY6D+CLP compartment (Fig. 1A). Whereas the
roles of E2A in early adult hematopoiesis have been well-docu-
Author contributions: E.W., R.M., E.M.M., D.B., M.S., and C.M. designed research; E.W.,
R.M., E.M.M., D.B., and M.S. performed research; E.W., R.M., E.M.M., D.B., M.S., and C.M.
analyzed data; and E.W. and C.M. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The microarray data reported in this paper have been deposited in the
Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 18, 2011
| vol. 108
| no. 42www.pnas.org/cgi/doi/10.1073/pnas.1111766108
mented, the function of HEB in adult hematopoietic development
has remained to be determined. As a first approach to this ques-
tion, mice in which the HEB DNA binding regions were flanked by
loxP sites (27) were bred to transgenic mice carrying a Tie2-Cre
transgenic construct to generate mice where HEB is deleted from
the entire hematopoietic system (Fig. S1A). HEBf/fTie2Cre mice
were viable and fertile, and they lacked obvious developmental
defects. However, on examination of bone marrow cellularity,
total number of nucleated cells (P = 0.025) (Fig. 1B).
HEB Activity Is Not Essential to Promote HSCs and Myelo-Erythroid
Development. BecauseHEBisexpressedinboththe primitive LSK
compartment as well as hematopoietic progenitors derived from
HSCs, we analyzed the hematopoietic compartment of HEB-
The majority of the long-term HSCs can be found within a sub-
populationofthe LSKsexpressingCD150onthesurface (28).The
CD150+LSK and the CD150−LSK compartments were not af-
fected in the bone marrow of HEBf/fTie2Cre mice (Fig. 1C). In
addition, no significant changes were found in the numbers of the
Lineagelow/−SCA−KIT+(LK) population known to harbor
myelo-erythroid progenitors (29) (Fig. 1C). Furthermore, because
HEB has been reported to be associated with T cell acute lym-
phocytic leukemia 1 (TAL1), E2A, and ETO2 in erythroid cells
(30), we examined the heterogenous LK compartment (31). No
statistically significant differences were observed among these
subsets, and all subpopulations were present in normalnumbers in
the bone marrow of HEBf/fTie2Cre compared with WT mice (Fig.
1D). Taken together, these observationsindicate that HEB, unlike
E2A, is dispensable for the steady state maintenance of HSCs and
the generation of myelo-erythroid progenitors.
HEB Promotes the B-Cell Fate. An early step to the development of
lymphoid-restricted cells is the development of LMPPs that are
defined by the up-regulation of FLT3 in a subset of the LSK
compartment. Interestingly, the LMPP compartment was signifi-
cant reduced in HEBf/fTie2Cre relative to WT mice (P = 0.0026)
(Fig. 2 A–C).The CLP compartment was alsosignificantlyaffected
in HEBf/fTie2Cre mice. Specifically, both the LY6D−CLP (P =
0.0024) and LY6D+CLP compartments (P < 0.0001) were sub-
stantially reduced (Fig. 2 B and C) and strongly resembled the
E2A+/−mice(Fig. S1B).Cellularityofthe CD19+compartment in
HEBf/fTie2Cre bone marrow was significantly altered compared
with WT bone marrow (Fig. 2 D and E). The total reduction in
CD19+bone marrow B-cell progenitors corresponded well to the
overall reduction seen in bone marrow cellularity, suggesting that
in vivo HEBf/fTie2Cre progenitors did not adopt alternate cell
fates at the expense of B-cell differentiation.
Previous studies have shown that E2A-ablated HSCs enforce a
cell cycle checkpoint in HSCs (15, 16). To explore the possibility
that HEB performs a similar role in progenitor cells, the fraction
of cycling progenitors was analyzed in HEBf/fTie2Cre bone
marrow. Lack of HEB in the CLP compartment did not alter the
cell cycle distribution, and in contrast to E2A-deficient mice, no
differences in cell cycle progression were found in the LSK and
LK compartments of HEBf/fTie2Cre mice (Fig. 2F and Fig. S2 A
and B). Furthermore, Annexin V staining revealed that HEB
seemed to not be required for the viability of the cells in the
CLP, LSK, and LK compartments (Fig. 2F and Fig. S2 A and B).
These data indicate that the development of early hematopoietic
progenitors in the absence of HEB resembles the development
observed in E2A-deficient mice.
HEB Deficiency Interferes with the LY6D−CLP to LY6D+CLP Transition.
B-cell development can be separated into phenotypically defined
stages based on surface markers and Ig rearrangement status
(Fig. 3A). To identify the compartments where developmental
progression was affected by a lack of HEB, the ratio of cell
numbers of consecutive developmental steps were plotted (Fig.
3B). This analysis indicated that a critical function of HEB is
exerted during transition from the LY6D−to LY6D+cell stage.
No significant changes were found in the pro-, pre-, and imma-
ture B-cell transitions, indicating that HEB is dispensable for
developmental progression beyond the LY6D+stage. Rather, we
suggest that the reduced numbers of CD19+bone marrow cells
present in the bone marrow of HEBf/fTie2Cre originate from the
developmental defects in the CLP compartment.
A recent study using Rag1-GFP reporter mice identified
functionally distinct low and high reporter-expressing cells within
the LY6D+CLP population (8). These observations raise the
question as to whether the reduced frequency of LY6D+CLPs is
caused by a partial block in the LY6D−to LY6D+transition
and/or a transition within the LY6D+fraction. To explore this
question, single LY6D+cells were examined for the presence of
progenitor populations were analyzed by real-time PCR for the abundance of
phosphoribosyl transferase (HPRT) expression and are shown as mean ± SEM
using purified mRNA from two independent sorts. (B) Absolute numbers of
bone marrowcells (fromfemurs,tibias,andcrestailiac)of sex-and age-matched
WT and HEBf/fTie2Cre mice. Each dot represents the number from a single
mouse. The horizontal lines indicate the mean in each group. The asterisk
indicates a P value < 0.05. C Left represents FACS plots of the gating strategies
used to identify LK, MPP, and HSC populations. Lineage (LIN) includes CD11B,
GR1, CD3ε, and NK1.1. C Right displays the absolute cell numbers of each pop-
ulation in WT and HEBf/fTie2Cre mice. Each dot represents the number from
are pooled from two independent experiments. D Upper shows representative
FACS plots of the gating strategy to identify erythromyeloid progenitor pop-
ulations within the LK population. D Lower shows the absolute cell number of
each population in WT and HEBf/fTie2Cre bone marrow. Each dot represents the
number from a single mouse, and the horizontal lines are the mean in each
group. Data shown are pooled from two independent experiments. MkP,
megakaryocyte progenitor; GMP, granulocyte macrophage progenitor; preGM,
pregranulocyte macrophage; preMegE, premegakarycocyte erythrocyte; pre-
CFUE, pre-CFU erythrocyte; CFUE, CFU erythrocyte; proEry, proerythrocyte.
Welinder et al.PNAS
| October 18, 2011
| vol. 108
| no. 42
DHJHrearrangements (Fig. 3C). We found that the frequency of
DHJHrearrangements was not altered in HEBf/fTie2Cre mice,
indicating that the distribution of subpopulations within the
LY6D+CLP compartment remained unperturbed (Fig. 3C).
These data indicate that, although the lack of HEB caused
a partial block in the transition from LY6D−to LY6D+CLPs,
development within the LY6D+compartment was not signifi-
cantly altered in the absence of HEB.
The data described above indicate that HEB acts to promote
specification to the B-cell fate. To directly evaluate how HEB
actsin progenitors, weevaluated the in vitro B-celldevelopmental
potential of HEBf/fTie2Cre LY6D−progenitors using a limiting
dilution assay approach; 1, 3, 5, or 10 uncommitted LY6D−CLPs
were plated on OP9 stroma cells in the presence of the cytokines
FLT3-Ligand,IL-7, and stem cell factor.On culturing the cells for
14 d, the number of cells plated per well was plotted against the
percentages of wells negative for B-cell growth, which were
measured by CD19 expression. The frequency of LY6D−CLPs
containing in vitro B-cell potential was determined as the con-
centration of cells in which 37% of the wells lacked B cells (32).
One of five WT cells developed into B cells (Fig. 3D). In contrast,
only one of nine cells of the HEB-deficient cells progressed to the
committed B-cell stage (Fig. 3D). These data indicate a 50% re-
duction in the ability of HEB-ablated progenitors to differentiate
into committed B cells. These data correspond well with the de-
crease observed in the HEB-deficient bone marrow. In contrast,
HEBf/fTie2Cre LY6D−CLPs efficiently differentiated into early
T-lineage cells on culture in the presence of OP9-DL1 cells (Fig.
LMPPs from WT and HEBf/fTie2Cre bone marrow. Lineage (LIN) includes CD11B,
of LY6D−CLPs and LY6D+CLPs from WT and HEBf/fTie2Cre bone marrow. (C)
Total cellularity of LMPP, LY6D−CLPs, and LY6D+CLPs in WT and HEBf/fTie2Cre
bone marrow. Each dot represents the number from a single mouse, and the
horizontal lines indicate the mean in each group. Data shown are pooled from
plots of CD19+B220+cells from WT and HEBf/fTie2Cre bone marrow. LIN includes
CD11B, GR1, and TER119. (E) Total cell numbers of B-cell progenitor populations
within the CD19+B220+bone marrow B-cell populations. All populations were
first gated as CD19+B220+. Pro-B, KIT+IgM−IgD−; Pre-B, KIT−IgM−IgD−; Im. B,
KIT−IgM+IgD−; Mat. B, KIT−IgM+/lowIgD+. Each dot represents the number from
are pooled from two independent experiments. *P < 0.05; ***P < 0.001. F Left
WT and HEB-deficient CLPs (n = 3 in each group). F Right shows a representative
plot of Annexin V staining comparing WT and HEB-deficient CLPs (total n = 3 in
each group). For F Left and F Right, CLPs are defined as depicted in B.
ment. (A) Schematic overview of B-cell development in the bone marrow. (B)
Plot of cell number ratios from the consecutive developmental stages depicted
in A. The total cellularity of an early population for a mouse is divided by the
total cell number of the proceeding population. Each dot represents the ratio
from a single mouse, and the horizontal lines are the mean in each group. Data
shown are pooled from two independent experiments. *P < 0.05; ***P < 0.001.
n.s., not significant. C Left shows an example of DH–JHrearrangement PCR
readouts from LY6D+CLPs. C Right shows the distribution of cells within the
LY6D+CLP compartment with respect toDH–JHrearrangementstatus. A total of
mean ± SEM of cells producing at least one PCR product. (D) Result of limiting
dilution assay for in vitro B-cell potential. CLPs from WT and HEB-deficient mice
were sorted onto preplated OP9 stroma cells and cultured in conditions pro-
moting B-lymphoid development.The numberof cells plated perwell is plotted
vs. the percentages of wells negative for B-cell growth (CD19+cells). Full lines
represent thebest fitregression. Thefrequencyofcellswith B-cell potential was
determined as the cell concentration where 37% of the cells were not gener-
ating B cells. The frequency for WT and HEBf/fTie2Cre cells is indicated. Data
shown are froma representative experimentfrom a total of threeexperiments.
HEB promotes the transition from the LY6D−to LY6D+CLP compart-
| www.pnas.org/cgi/doi/10.1073/pnas.1111766108Welinder et al.
S3). Collectively, these data indicate that HEB acts in the CLP
compartment to specify the B-cell fate.
HEB and E2A Act in Concert to Specify the B-Cell Fate. The data
described above indicate a critical role for HEB in promoting B-
lineage specification in a manner similar to the role described for
E2A. To determine whether E2A and HEB act together to specify
the B-cell fate, E2A and HEB compound mice were generated and
Because Tie2Cre expression did not efficiently excise E2A alleles in
E2Af/fmice (27), we used Vav-iCre transgenic mice to achieve a
higher degree of excision. E2Af/fVaviCre and HEBf/fVav-iCre dis-
played the same hematopoietic phenotype as the E2A−/−and
HEBf/fTie2Cre mice (Fig. 4A and Fig. S4). Next, we compared
HEBf/fVav-iCre, E2Af/+Vav-iCre, and E2Af/+HEBf/fVav-iCre
mice for abnormalities in CLP development (Fig. 4A). As
expected, E2Af/+Vav-iCre and HEBf/fVav-iCre mice exhibited a
partial block at the CLP cell stage (Fig. 4A). However, the
LY6D+compartment was close to absent in E2Af/+HEBf/fVav-
iCre mice (Fig. 4A). These data indicate that E2A and HEB act in
concert to initiate B-cell development.
To determine how E2A and HEB act in the CLP compartment
to prime the B-cell fate, we performed microarray analysis using
mRNA from sorted LY6D−CLPs derived from HEBf/fTie2Cre
mice. Notably, only 18 genes were altered by a factor of more
than twofold in HEBf/fTie2Cre vs. WT LY6D−CLPs (Fig. 4B).
To verify that chemokine (C-C motif) receptor 9 (CCR9),
a previously identified E2A target, is also regulated by HEB,
HEB-ablated CLPs were analyzed for the expression of Ccr9 by
flow cytometry (17). As predicted, Ccr9 expression was sub-
stantially reduced in HEBf/fTie2Cre LY6D−CLPs (Fig. 4C). To
directly evaluate the extent to which E2A and HEB share
a common set of target genes, mRNA from E2A−/−LY6D−
CLPs was next examined by microarray analysis. The majority of
the genes altered in the absence of HEB exhibited a similar
change as depletion of E2A activity (Fig. 4B).
To further explore the possibility that HEB and E2A share
a common set of target genes in the LY6D−compartment, we
examined the entire spectrum of genes changed in the absence of
E2A; 197 transcripts were up- or down-regulated more than two-
fold in E2A-deficient LY6D−CLPS compared with the corre-
sponding WT cells (Fig. 4D Left and Table S1). We next examined
revealed a striking pattern of E-protein dose dependency (Fig. 4D
Right). Genes up- or down-regulated by E2A were, in general, also
affected by the absence of HEB, albeit to a lower degree. Among
the changed transcripts were Ccr9, Id2, Blnk, Dntt, Notch1, IL-7ra,
and most notably, Foxo1. These data indicate that E2A and HEB
(A) Representative FACS plots comparing bone marrow
stained for LY6D+CLPs in WT, HEBf/fVaviCre, E2Af/+VaviCre,
HEBf/fE2Af/+VaviCre, and E2Af/fVaviCre mice. (B) Microarrays
analysis displaying genes that are changed by a factor of at
least twofold between WT and HEBf/fTie2Cre LY6D−CLPs.
E2A−/−displays how this subset of genes is affected in E2A−/−
LY6D−CLPs. Displayed data are derived from the means
from two or more microarray replicas using material from
independent sorts. (C) Graph shows the number of CCR9+
LY6D−CLPs in WT and HEBf/fTie2Cre mice. Each dot repre-
sents the number from a single mouse, and the horizontal
lines are the mean in each group. Representative data from
one of a total of two experiments are shown. **P < 0.01. D
Left, right column shows the result of microarray analysis,
displaying genes that are changed by a factor of at least
twofold between WT and E2A−/−LY6D−CLPs. In addition,
the center column displays how these genes are affected in
HEBf/fTie2Cre LY6D−CLPs. Highlighted in the left column is
the location of genes of interest for lymphocyte de-
velopment (a full gene list is in Table S1). D Right displays
box plots of cluster analysis showing how genes patterns in
D Left are changed in WT and HEB- and E2A-deficient LY6D−
CLPs. ***P < 0.001. Displayed data are the means derived
from two or more microarray replicas using material from
HEB and E2A act in concert to specify the B-cell fate.
Welinder et al. PNAS
| October 18, 2011
| vol. 108
| no. 42
E proteins that promotes the development of LY6D+CLPs.
Regulation of Foxo1 Expression by the E Proteins. The microarray
data described above indicate that both E2A and HEB are re-
quired to activate normal FOXO1 expression in the CLP com-
partment. To quantitatively compare FOXO1 transcript levels in
WT and HEB- and E2A-deficient CLPs, mRNA derived from
each of the populations was examined by real-time PCR. Con-
sistent with the microarray data, FOXO1 transcript levels were
reduced in both HEBf/fTie2Cre and E2A−/−CLPs (Fig. 5A).
To explore the possibility that E proteins directly regulate
FOXO1 expression, we examined the regulatory regions of the
Foxo1 locus for E2A occupancy within putative enhancer regions
as characterized by H3K4 monomethylation (H3K4me1). We
previously reported the distribution of H3K4me1 and H3K4me3
as well as E2A occupancy in EBF1−/−hematopoietic progenitors
(10). On inspection of epigenetic marks across the FOXO1 locus,
we identified high-affinity E2A binding sites in putative enhancers
as well in the promoter region (Fig. 5B) (10). Subsequently, the
putative enhancerandpromoterregions,containingthe identified
WTaswellasmutated E-boxsites,wereinserted intoanenhancer
luciferase reporter construct, transfected into pro-B cells, and
assayed for transcriptional activity. Notably, deletion of the E-box
sites substantially interfered with reporter activity (Fig. 5C)
(Ppeak1= 0.0178, Ppeak2= 0.047). Thus, these data indicate that
E-protein activity directly regulates Foxo1 expression (Fig. 5D).
Previous observations have indicated that the E2A proteins play
critical rolesthroughouthematopoiesis.IntheHSC compartment,
the E2A proteins maintain the hematopoietic stem cell pool (15,
16), and in developing progenitors, the E2A proteins promote the
developmental progression of erythroid/megakaryocytic and my-
eloid progenitors (15). However, perhaps most prominent is the
block observed in the CLP compartment in E2A-deficient mice at
although the LY6D−CLP compartment in E2A-deficient mice is
present, albeit in reduced numbers, the LY6D+compartment is
complete absent (7). Here, we show that E2A and HEB mecha-
nistically act together to specify the B-cell fate.
Whereas HSCs were not significantly perturbed in HEB-de-
ficient mice, we observed a clear reduction in B-cell progenitors
in the adult bone marrow. The defect in B-lymphoid differenti-
ation was initiated in the LMPP compartment but manifested
predominantly during the LY6D−to LYD6+CLP transition.
However, committed B-lineage cells derived from the LY6D+
CLP compartment developed in expected ratios. Thus, these
data indicate that the partial block in the LY6D−to LY6D+CLP
transition is the major cause for the observed reduction in B-cell
progenitor numbers in HEB-deficient bone marrow. Further-
more, HEBf/fVaviCre and E2Af/+VaviCre mice show a partial
block at the CLP cell stage, whereas HEBf/fE2Af/+VaviCre mice
display a more complete arrested block in the LY6D−com-
partment. Consistent with these observations, using microarray
analysis, we show that the transcription signatures of E2A- and
HEB-deficient CLPs substantially overlap.
at the LY6D−CLP cell stage to initiate a B lineage-specific pro-
gram of gene expression. Here, we show that Foxo1 expression is
reduced in the absence of E proteins. A genome-wide screen for
E2A binding sites revealed that the E2A proteins bind to regula-
tory elements present in the FOXO1 locus (10), and we show that
these E2A binding sites indeed play a role in modulating FOXO1
expression. In addition to the E proteins, an ensemble of tran-
scriptional regulators has been shown to play critical roles in early
B-cell development. Among these regulators are EBF1, PU.1,
FOXO1, and PAX5 (13, 33). Furthermore, IL-7R–mediated sig-
naling also has been proposed to modulate EBF1 expression (34–
36). How is this spectrum of transcriptional regulators connected
observations described here, we would like to propose that, in
LY6D−CLPs, E2A and HEB act in concert to induce the ex-
pression of FOXO1. FOXO1, in turn, together with E2A, HEB,
and PU.1 as well as IL-7R–mediated signaling, then activates the
expression of EBF1 in LY6D+CLPs. E2A, FOXO1, EBF1, and
IRF4 as well as IRF8 subsequently induce the expression of PAX5
(10, 13, 37, 38). These data bring into question how the E proteins
are activated in theCLPLY6D−compartment.E2Aprotein levels
do not change significantly during the developmental progression
that Id2 and Id3 mRNA levels do not change substantially during
the developmental progression from the LSK to the CLP cell
stage. The question then arises as to why a B lineage-specific
program gene expression is not induced at the HSC or LMPP cell
stage. We would like to suggest that transcriptional repression
rather than activation regulates the expression of FOXO1 during
early hematopoiesis, because the low levels of FOXO1 expression
are not affected in E2A-ablated LMPPs (17). Thus, before
reaching the CLP compartment, FOXO1 levels are kept low by
active repression. On reaching the CLP compartment, the activi-
ties of transcriptional repressors are blocked, permitting E2A and
HEB to induce FOXO1 transcription and specify the B-cell fate.
The critical problem now will be to determine how the activation
of a B lineage-specific program of gene expression by the E pro-
teins is suppressed during the earlier stages of hematopoiesis.
LY6D−CLPs from WT, HEBf/fTie2Cre, and E2A−/−mice were analyzed by real-
time PCR for the abundance of Foxo1 mRNA. Values were normalized to HPRT
expression and are shown as mean ± SEM using purified mRNA from two in-
dependent sorts. (B) E2A (red; top row) occupancy, H3K4me1 (blue; middle
row), and H3K4me3 (blue; bottom row) epigenetics mark across the Foxo1
locus in EBF1-deficient cells identified by ChIP followed by genome-wide deep
on the leftindicates a Foxo1 locus promoter region with E-box sites; the box on
the right indicates a region where E2A occupancy is associated with H3K4me1.
(C) Transcriptional activity of H3K4me1 islands (corresponding to the boxes in
B) with associated E2A occupancy and presence of E boxes. Δ, deletion within
the E-box sequences. Data shown are the mean ± SEM derived from two in-
dependent experiments. *P < 0.05. (D) Schematic diagram depicting B-cell
development and the activities of E2A and HEB in B-cell specification.
Direct regulation of Foxo1 expression in CLPs by the E proteins. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1111766108Welinder et al.
Materials and Methods Download full-text
Mice. HEBf/f and E2Af/f mice were a gift from Yuan Zhuang (Duke University,
Durham, NC). Tie2Cre and VaviCre mice were purchased from The Jackson
Laboratory. E2A-deficient mice have been previously described (9). Mice were
Animal Care and Use Committee of the University of California at San Diego.
FACS Staining and Purification of Bone Marrow Cells. Bone marrow DNA was
prepared aspreviouslydescribed (26)cellswere Fc-blocked(CD16/CD32;2.4G2)
and stained with combinations of the antibodies FLT3(A2F10), CD11B(M1/70),
GR1(RB6-8C5), TER119 (Ter119), CD3e(145-2c11), CD11C(N418), LY6C (AL-21),
(11-26c), IgM(11/41), CD41(MWReg30), CD16/32(2.4G2), CD150(TC15-12F12.2
(BioLegend), and CD105(MJ7/18) followed by streptavidin-conjugated
Qdot655 (Invitrogen) to visualize biotinylated antibodies and propidium
iodine to exclude dead cells. For CLP detection, cells where stained with LY6D
(49-H4) (BD Bioscience) followed by Qdot605 goat anti-rat IgG (Invitrogen)
before Fc blocking. Antibodies were purchased from eBioscience unless
otherwise noted. For progenitor isolation, cells were subjected to magnetic-
activated cell sorting enrichment of CD27+or KIT+immunomagnetic beads
(Miltenyi Biotec) before antibody staining. Analysis was performed using an
LSRII, and cell sorting was performed on FACSAria II (BD Biosciences).
RNA Analysis. Quantitative RT-PCR analysis of sorted cells was performed as
previously described (6). Assays on Demand (Applied Biosystems) probes used
Id2 (Mm00711781_m1), and Id3 (Mm00492575_m1). All experiments were per-
formed in duplicate at least two times using cells from independent sorts.
IgH D–J Rearrangements in Single Cells. Nested single-cell PCR has been pre-
viously described (26) and was performed with slight modifications. Briefly,
single-cell LY6D+CLPs were sorted directly into 96-well plates containing PCR
buffer containing 20 mM Tris·HCl 50 mM KCl (pH 8.4), snap-frozen, and
stored in −80 °C. In the first round, PCR amplification was performed with,
0.2 mM dNTP, 1 μL homemade Taq, 0.5 mg/mL BSA, and 0.4 μM of each
primer in a total of 50 μL/well; 1 μL first-round PCR was used as a template in
the second round of PCRs. Second-round primers were used at 2 μM con-
centrations. Only samples producing either a germline product and/or
a specific rearrangement product are included in the analysis.
Annexin V Staining. After staining with appropriate surface markers, cells
were stained using Annexin V Apoptosis detection kit (eBioscience) according
to manufacturer’s instructions.
Cell Cycle Analysis. Cells were fixed with the BD Cytofix/Cytoperm solution kit
(BD Bioscience) followed by incubation with anti-human Ki67 (BD Bioscience)
and 7AAD according to manufacturer’s instructions.
In Vitro Evaluation of B- and T-Cell Potential by OP9 Coculture. For evaluation
of in vitro B-cell potential, 1, 3, 5, or 10 cells/well were deposited (using
experiment, 10 or more wells were analyzed per cell concentration. For
FACS evaluations have been previously described (6). Frequencies from
limiting dilution assays were calculated as previously described (32).
Affymetrix Gene Expression and Data Analysis. Gene expression assays have
been previously described (8).
ACKNOWLEDGMENTS. We thank Yuan Zhuang for providing the HEBf/fand
E2Af/fmice, Dr. Chris Brenner and Annamaria Kauzlaric for help with statis-
tical analyses, Dr. Yin Lin for help with accessing chip-sequencing data, and
members of the laboratories of D.B., M.S., and C.M. for helpful discussions.
We are grateful to Gerd Sten and Liselotte Lenner for technical help. C.M. is
supported by the National Institutes of Health.
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| October 18, 2011
| vol. 108
| no. 42