Dendritic cell fate is determined by BCL11A
Gregory C. Ippolitoa,1, Joseph D. Dekkera,1, Yui-Hsi Wangb, Bum-Kyu Leea, Arthur L. Shaffer IIIc, Jian Lina,
Jason K. Walla, Baeck-Seung Leea, Louis M. Staudtc, Yong-Jun Liud, Vishwanath R. Iyera, and Haley O. Tuckera,2
aDepartment of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712;bDivision of Allergy and
Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 44229;cMetabolism Branch, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892; anddBaylor Institute for Immunology Research, Dallas, TX 75204
Edited* by Richard A. Flavell, Yale School of Medicine and Howard Hughes Medical Institute, New Haven, CT, and approved February 5, 2014 (received for
review October 11, 2013)
The plasmacytoid dendritic cell (pDC) is vital to the coordinated
action of innate and adaptive immunity. pDC development has not
been unequivocally traced, nor has its transcriptional regulatory
network been fully clarified. Here we confirm an essential re-
quirement for the BCL11A transcription factor in fetal pDC de-
velopment, and demonstrate this lineage-specific requirement in
the adult organism. Furthermore, we identify BCL11A gene targets
and provide a molecular mechanism for its action in pDC commit-
ment. Embryonic germ-line deletion of Bcl11a revealed an abso-
lute cellular, molecular, and functional absence of pDCs in fetal
mice. In adults, deletion of Bcl11a in hematopoietic stem cells
resulted in perturbed yet continued generation of progenitors,
loss of downstream pDC and B-cell lineages, and persisting mye-
loid, conventional dendritic, and T-cell lineages. Challenge with
virus resulted in a marked reduction of antiviral response in con-
ditionally deleted adults. Genome-wide analyses of BCL11A DNA
binding and expression revealed that BCL11A regulates transcrip-
tion of E2-2 and other pDC differentiation modulators, including
ID2 and MTG16. Our results identify BCL11A as an essential, line-
age-specific factor that regulates pDC development, supporting
a model wherein differentiation into pDCs represents a primed
“default” pathway for common dendritic cell progenitors.
ered as a translocated locus in a lethal pediatric B-cell chronic
lymphocytic leukemia (1), and subsequently was identified as
a protooncogene implicated in numerous types and subtypes of
B-cell malignancies (2, 3). The N-terminal region common to all
BCL11A isoforms is evolutionarily conserved and can be used to
define a superfamily of transcription factors crucial to the de-
velopment, differentiation, and malignancy of several hemato-
poietic lineages (4). In vivo experimentation has confirmed an
essential requirement for Bcl11a in B-cell lymphopoiesis (5, 6)
and has implicated Bcl11a more broadly in hematopoietic stem
cell function and in development of lymphoid lineages (6, 7).
Initially thought to be exclusive to B cells, subsequent obser-
vations have demonstrated a wider range of function for BCL11A,
and surprisingly high levels of BCL11A transcripts in mouse and
human plasmacytoid dendritic cells (pDCs) suggested that BCL11A
might also play a key role in the biology of this dendritic cell type
(4, 8). Recently, BCL11A’s necessity has been specifically con-
firmed in fetal hematopoietic progenitors, yet its function in the
adult organism using conditional knockout models and functional
assays has yet to be clarified (9). Ranging from the production of
type I IFN (IFN-α) in response to infection by viruses, to the in-
duction of regulatory T cells, or the differentiation of germinal-
center B cells into plasma cells—the pDC encompasses a broad
range of immune functions and is pivotal to the coordination of
innate and adaptive immunity (10–12).
An understanding of the molecular control of pDC lineage de-
termination remains an enigma. Unlike its conventional dendritic
cell (cDC) counterpart, the pDC shares perplexing similarities with
lymphocytes (particularly B cells), including the transcription of
regulatory genes normally invoked during primary lymphocyte
development (BCL11A, mb-1/CD79A, B220/CD45RA, SPIB,
he B-cell chronic lymphocytic leukemia/lymphoma 11A
(BCL11A) zinc-finger transcription factor was first discov-
FOXP1, E2-2/TCF4). In this aspect of its molecular physiology,
pDCs resemble B cells more so than cDCs; moreover, the expres-
sion of key B-cell genes such as BCL11A can be used to distinguish
the pDC and cDC dendritic lineages in mice and humans (13).
These features have made it difficult to define pDC lineage
affiliation (8, 14–17). One recent breakthrough, however, was
the finding that the basic helix–loop–helix (HLH) E-protein,
E2-2/TCF4, is an essential and specific transcriptional regulator of
pDC development (18). One current model proposes that E2-2
activity maintains the cell fate of mature pDCs through opposition
of a “default” pathway that would otherwise lead to cDC fate (19).
However, a more recent model has alternatively proposed that
pDCs represent the default pathway for common DC precursors
(CDPs) (20). Because BCL11A was found to be a binding target of
E2-2 in the CAL-1 human pDC cell line, the first model has
specifically postulated that E2-2 promotes murine pDC commit-
ment in part through Bcl11a-mediated repression of cDC differ-
entiation. However, another member of the E2-2 family of
E-proteins, E2A/TCF3, is a critical regulator of B-lymphoid de-
velopment and differentiation (21), and Bcl11a has been identified
as a direct target of E2a in murine B cells (22).
Inhibitor of DNA binding (ID) proteins are natural dominant-
negative HLH proteins, which, by protein–protein heterodimeriza-
tion with E-proteins, antagonize their ability to fulfill lineage-specific
functions by blocking their DNA activity. ID2 and ID3 are two
such HLH factors known to influence the differentiation of pDCs,
cDCs, and B- and T-lymphoid lineages (23–30). ID interactions
This work demonstrates a key role of the B lymphocyte tran-
scription factor BCL11A in dendritic cell (DC) development. Two
major DC subsets—the plasmacytoid DC (pDC) and the conven-
tional DC (cDC)—are believed to arise from a shared precursor
called the common DC progenitor (CDP). Potential precursor
differences between cDC and pDC generation might neverthe-
less remain to be elucidated. Here, we show that mutant mice
can generate CDPs and cDCs in the absence of BCL11A, whereas
pDCs (and also B cells) are abolished. This study also identifies
and validates BCL11A target genes using a variety of techniques,
and provides a molecular model for BCL11A activity in the B
lymphocyte and pDC lineages.
Author contributions: G.C.I., J.D.D., Y.-H.W., A.L.S., J.K.W., and H.O.T. designed research;
G.C.I., J.D.D., Y.-H.W., B.-K.L., A.L.S., J.L., J.K.W., and B.-S.L. performed research; G.C.I.,
J.D.D., A.L.S., L.M.S., Y.-J.L., V.R.I., and H.O.T. contributed new reagents/analytic tools;
G.C.I., J.D.D., Y.-H.W., B.-K.L., A.L.S., J.K.W., and H.O.T. analyzed data; G.C.I., J.D.D., and
H.O.T. wrote the paper; and G.C.I. and J.K.W. initiated the project.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The data reported in this paper have been deposited in the Gene Ex-
pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (Chip-seq accession no.
GSE55043 and microarray no. GSE55237).
1G.C.I. and J.D.D. contributed equally to this work.
2To 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.
| Published online March 3, 2014www.pnas.org/cgi/doi/10.1073/pnas.1319228111
specifically restrain the transcriptional activity of E-proteins (24,
26, 27, 31) as well as other factors crucial to pDC and B-lym-
phoid biology, including SPIB and PAX5 (21, 32).
The successive stages of commitment to the DC lineage in
bone marrow remain poorly understood and are just beginning
to be characterized (20). In this study we demonstrate a re-
quirement in vitro and in vivo for Bcl11a during fetal and adult
pDC development in the mouse. Complementary experiments
using human cell lines expand these observations made in mice
and imply determination of pDC cell fate, in part, by a molecular
network comprising BCL11A, ID2, ID3, MTG16, and E2-2/TCF4.
Genome-wide ChIP-sequencing (ChIP-seq) revealed BCL11A
binding to the promoter and other gene-proximal regions of
several other key factors implicated in pDC and lymphoid
development, including SPI1, SPIB, IKZF1, and E2A/TCF3. We
advance a model that is consistent with existing data demon-
strating reliance on additional pDC transcription factors, which
postulates that the default pathway of CDP differentiation is to
Confirmation That Bcl11a-Deficient Fetal Liver and Spleen Is Devoid
of pDC. In agreement with previous analyses of conventional
Bcl11a−/−knockout (KO) mice by Liu et al. (5), we observed an
absence of B220+cells in liver and spleen isolated from fetal KO
mice (Fig. S1A). The loss of B220+cells appeared dose-dependent
because Bcl11a+/−heterozygotes contained B220+popula-
tions intermediate between Bcl11a+/+wild-type and Bcl11a−/−
cDC pDCcDC pDC
Total cells x 105
Total cells x 105
-/- +/- +/+
0.7% 4.7% 5.5%
2% 20 % 24%
Bcl11a+/+mice were cultured in vitro with Flt3-L to induce the growth of cDC and pDC from progenitor cells. (A) Day 3 of culture (Upper) resulted in efficient
production of cDCs (Cd11b+Cd11c+) among Bcl11a+/+and Bcl11a+/−genotypes, but a near absence of pDCs (Cd11b–Cd11c+) in the Bcl11a−/−homozygous
knockout. Day 10 cultures (Lower) continued to exhibit this marked reduction in pDC generation (>10-fold compared with wild type). Results depicted are
representative of n = 3 independent experiments. (B) Additional markers (B220, Ly6c) were used to track the outgrowth of cells in additional experiments (n = 3),
confirming as above that only a minute portion of pDC-like cells (Cd11b–Cd11c+) could be detected in Bcl11a−/−cultures. (C) The pDC transcript Siglec-H was
undetectable by RT-PCR in day 3 Flt3-L culture of Bcl11a−/−fetal liver progenitors. (D) Bcl11a−/−fetal liver culture (Flt3-L 7 d) failed to mount a measurable IFN-α
response to type A CpG oligonucleotide (ODN D19) after 24 h of stimulation. (E–J) Adult bone marrow cells from pI:pC-treated Bcl11aF/FMx1-creYFP+conditional
knockout (cKO) mice or littermates (8 wk old) were cultured in vitro with Flt3-L to induce the growth of cDC and pDC from progenitor cells. (E) Flow cytometry of
day 10 Flt3-L culture exhibited no pDC production from YFP+(Bcl11a-deleted) bone marrow cells, whereas as shown in F the production of cDC was unimpeded
and equivalent to control (n = 3 independent experiments). (G and H) Cultures of GM-CSF–supplemented cells (day 7) produced statistically similar frequencies of
cDCs in both YFP+(Bcl11a-deleted) and wild-type bone marrow. (I) Total cell numbers in Flt3-L cultures were drastically reduced (day 10; P = 0.008; Student t test),
whereas total cells in GM-CSF stimulations were reduced only modestly (day 7; P = 0.04). (J) cDC-to-pDC ratios in Flt3-L–supplemented cultures were significantly
different in YFP+Bcl11a-deleted cells vs. total bone marrow cells (207.6 ± 87.7 vs. 3.05 ± 0.63, respectively, P = 0.018).
In vitro lack of pDC development in cultures of Bcl11a-deficient fetal liver or adult bone marrow. (A–D) Fetal liver cells from Bcl11a−/−, Bcl11a+/−, and
Ippolito et al. PNAS
| Published online March 3, 2014
homozygous-deficient siblings. In addition to the previously
documented loss of B220+B lymphocytes in these mice, we con-
jectured that B220+pDCs might also be absent from these tissues.
To test the role of Bcl11a in murine pDC development, we
isolated hematopoietic cells from the fetal liver and spleen of
Bcl11a+/+, Bcl11a+/−, and Bcl11a−/−E18 embryos. Examination
of fetal organs was mandatory because constitutive homozygous
deficiency of Bcl11a is neonatal lethal. In mouse, pDCs are
B220+Cd11c+Cd11b–, and express the specific marker Bst2/
PDCA1/CD317. In n = 3 independent experiments, pDCs were
missing from Bcl11a−/−KO mice. Collectively, the missing cells
were Cd11c+CD11b–Cd19–RB6-8C5+B220+PDCA1+. In one
experiment, pDC were reduced 25-fold in the Bcl11a KO(−/−)
compared with the wild-type littermate control (+/+) (Fig. S1B).
We could not detect Siglec-H transcripts in Bcl11a−/−fetal
liver precursors in vivo (Fig. S1C) or ex vivo (Fig. 1C), whereas
Siglec-H expression was detectable in heterozygous and wild-type
controls. Though immature pDCs (PDCA1–) have been reported
to be negative for Siglec-H transcripts, mature pDCs (PDCA1+)
are routinely positive for expression of this gene (18). In light of
this observation, we could not exclude the possibility that pDC
progenitors were blockaded at an intermediate stage of differenti-
ation; this could result from cell-extrinsic Bcl11a-dependent effects
modulated by accessory cells or the stromal milieu in general.
Bcl11a Is Required for Cytokine-Induced pDC Differentiation from
Fetal Liver Progenitors in Vitro. The capacity of Bcl11a-deficient
fetal liver cells to differentiate into pDCs was further inves-
tigated. Bcl11a−/−fetal liver cells were cultured in defined cy-
tokine-supplemented media to reveal whether pDC precursors,
which might persist in vivo, could be differentiated ex vivo. The
development of pDCs and cDCs depends crucially on the he-
matopoietic cytokines Flt3-ligand (Flt3L) and GM-CSF in both
human and mouse (10). Flt3L alone induces the growth of cDC
and pDC in vitro from progenitor cells, and moreover, these are
the only cell populations generated in Flt3L-supplemented bone
marrow cultures (33). In contrast, GM-CSF exclusively promotes
the differentiation of cDC from early hematopoietic progenitors
and induces a single population of Cd11c+Cd11b+B220–cDCs,
but not Cd11c+Cd11b–B220+pDCs, in culture (33).
In agreement with the observations made in vivo using KO
mice, the ex vivo differentiation of Bcl11a−/−hematopoietic fetal-
liver precursors failed to generate prototypical Cd11c+Cd11b–
Ly6c+B220+pDCs in Flt3L-supplemented cultures (Fig. 1 A and
B). Bcl11a−/−KO cultures consisted almost entirely of Cd11c+/−
Cd11b+B220–myeloid cells and cDCs at all three time-points
examined (days 3, 7, and 10). pDC and pDC-like cells that per-
sisted were on average reduced >10-fold compared with cultures
derived from littermate controls. In contrast to the profound im-
pedance of pDC differentiation, Bcl11a−/−cDCs in Flt3L cultures
appeared to differentiate fully, although clearly reduced in their
relative percentage (Fig. 1 A and B). As reported in a recent study
(9), cDC generation from Bcl11a−/−KO fetal precursors in control
experiments using GM-CSF is abundant and unperturbed. The
expression of PDCA1 was low among all Flt3L-derived pDCs, as
previously documented (34), so the additional surface-marker
Ly6c was included along with Cd11b, Cd11c, and B220 to track the
outgrowth of cells using flow cytometry analysis. These cellular
phenotypic profiles were confirmed in three independent experi-
ments. Moreover, similar to RT-PCR of KO whole fetal liver,
Siglec-H transcripts were undetectable among fetal liver pre-
cursors cultured in Flt3L for 3 d (Fig. 1C), underscoring the ab-
sence of this pDC-specific molecular program. The lack of a
functional pDC phenotype in Bcl11a−/−mice was also supported
by the lack of IFN-α production subsequent to stimulation of
cultured cells with the type A CpG oligodeoxynucleotide D19, as
determined by ELISA (n = 1) of culture supernatants (Fig. 1D).
Bcl11a Is Required for Cytokine-Induced pDC Differentiation from
Adult Bone Marrow in Vitro. To determine whether the defect in
fetal pDC development observed among Bcl11a-deficient con-
ventional knockout mice is retained in adults, we constructed
a floxed (F) allele of Bcl11a (Fig. S2 and SI Text). Our strategy
results in deletion of the first exon, which is included in all
previously characterized Bcl11a transcripts in mouse and man (2,
4, 35, 36). We crossed these mice to transgenic mice in which Cre
recombinase is driven by the promoter of the Mx-1 gene (37).
The Mx1-cre transgene is expressed in hematopoietic stem cells
(HSCs) and in all downstream lineages after induction with type
I IFN or poly(I):poly(C) (pI:pC), allowing reliable deletion
throughout the entire adult hematopoietic compartment. These
mice were also crossed to a floxed STOP-YFP reporter mouse to
positively identify the cells in which Mx1-Cre recombinase is
Young adult (5 wk) Bcl11aF/FMx1-CreYFP+mice underwent
pI:pC induction, and bone marrow was collected at 8 wk for
Flt3L and GM-CSF–supplemented cultures. In agreement with
the observations made in vivo and ex vivo using KO mice, YFP+
bone marrow precursors failed to generate Cd11c+Cd11b–YFP+
pDCs in Flt3L-supplemented cultures (Fig. 1E). To confirm phe-
notype, ∼75% of the Cd11c+CD11b–pDCs in the control cultures
were weakly positive for both PDCA-1 and Siglec-H. Interestingly,
Bcl11aF/FMx1-CreYFP+cultures consisted of substantial numbers
of Cd11c+/−Cd11b+MHC IIhiYFP+cDCs at day 10 (Fig. 1F),
indicating a difference in cDC development between fetal and
adult precursors. The cDC to pDC ratio was significantly altered in
YFP+cells compared with total bone marrow in Flt3L supple-
mented cultures (Fig. 1J; 207.6 ± 87.7 to 3.05 ± 0.63, respectively,
P = 0.018). In GM-CSF–supplemented cultures, cDCs differenti-
ated normally and abundantly from YFP+bone marrow (Fig. 1 G
and H). These cellular phenotypic profiles were confirmed in trip-
licate and in n = 3 independent experiments. Though total cell
numbers were greatly reduced in Flt3L-supplemented cultures
[15.8 × 106vs. 3.43 × 106in control and conditional KO (cKO),
respectively], there was also mild reduction of total cells in GM-
CSF–supplemented cultures (51.1 × 106vs. 35.7 × 106in control and
cKO, respectively; Fig. 1I), indicating either a slight reduction in
cDC development efficiency or possibly a reduction in cell survival.
Deletion of Bcl11a in Hematopoietic Precursors Results in a Robust
Loss of pDCs but Spares Lin−Sca-1+c-kit+Progenitors, T cells, and
cDCs. The floxed (F) allele of Bcl11a was crossed to transgenic
mice in which Cre recombinase is driven by the promoter of the
Vav gene (38). The iVav-cre transgene is expressed in HSCs and in
all downstream lineages (39), allowing reliable deletion through-
out the entire embryonic and adult hematopoietic compartment.
Young adult (6 wk) Bcl11aF/FVav-Cre mice consistently dis-
played a radical loss of B cells and pDCs in bone marrow, with
a relative 10- to 50-fold reduction compared with control litter-
mates (n = 5 independent experiments) as determined by PDCA1,
CD11c, and B220 staining (Fig. 2A). The typical loss of B cells in
any given experiment was observed to be proportional to the loss of
pDCs, and the approximate B:pDC ratio of 10:1 typically observed
in controls was also maintained in Bcl11aF/FVav-Cre knockout mice
irrespective of the degree of cellular deletion in the bone marrow.
An examination of the periphery resulted in a similar pairwise
reduction of B cells and pDCs in the spleen (Fig. 2A); however,
here the reduction was not as pronounced as in bone marrow.
Importantly, splenic cDCs (B220–CD11chighCD11b+MHCIIhigh)
were unaffected by Bcl11a deletion (Fig. 2B), as were CD8+or
CD8−cDC subsets (CD11c+CD8+CD11b−MHCII+B220−;
CD11c+CD8+CD11b−MHCIIhighB220−, respectively; Fig. 2C).
To determine whether dendritic cell precursors were impaired,
bone marrow was stained for various lineage markers, including
those that discriminate common myeloid progenitors (CMPs; Lin−
Flt3+Sca-1−c-kithighCD115+) and CDPs (Lin−Flt3+Sca-1−c-kitlo
| www.pnas.org/cgi/doi/10.1073/pnas.1319228111Ippolito et al.
CD115+; Fig. 2D). Consistent with a recent report (6), we observed
an ∼twofold reduction of Lin−Sca-1+c-kit+cells (LSK; Fig. 3D) in
Bcl11aF/FVav-Cre bone marrow. However, neither macrophage-DC
progenitor (MDP) nor CDP progenitor populations were sub-
stantially impaired in their relative percentages (Fig. 3D). Further,
statistically similar relative percentages of cDCs (CD11c+CD11b+
B220−), granulocytes (Gr-1+CD11b+), macrophages (CD11b+
F4/80+), and T cells (CD3e+CD4CD8 DP and SP) were observed
in bone marrow and spleen of cKO and control littermate mice
(Fig. 2E). These observations are consistent with the original
report (5) of conventional embryonic deletion of Bcl11a wherein
B cells were selectively abolished yet T cells as well as myeloid and
erythroid cells were preserved.
We confirmed these observations in the aforementioned
Bcl11aF/FMx1-Cre adults following pI:pC-induced deletion (37).
Here, too, B cells and pDCs were significantly reduced with little
alteration of other cell populations (Fig. 2F). Except for the pe-
culiar observation that T-cell absolute numbers are reduced ex-
clusively among c-kit+precursor T cells when tamoxifen-inducible
CreERT2 is used to induce a global deletion of Bcl11a exon 4 (6),
our results are consistent with all prior data derived from con-
ventional embryonic Bcl11a-deleted mice wherein approximately
normal T-cell development occurs. Furthermore the Mx1-Cre data
eliminate the possibility that this difference with our results de-
rived from sparing of T cells of embryonic origin.
To investigate the ability of Bcl11aF/FVav-cre mice to produce
type I IFN upon viral infection, we analyzed peripheral blood
mononucleated cells of Cre-positive and Cre-negative littermates to
assure expected phenotypes before delivering 1 × 107pfu of human
HSV strain 17 i.v. by tail-vein injection. As shown in Fig. 2G, serum
Cre+ F/F Cre- F/F
cDC - Spleen
Cells in Bone Marrow
Cre+ F/F Cre- F/F
Cre+ F/F Cre- F/F
Progenitors - Bone Marrow
Cre+ F/F pI:pC
Cells in Spleen
conditionally deleted adult mice. Homozygous Bcl11a cKO mice bearing loxP-flanked (F) targeted alleles (Bcl11aF/F) were intercrossed with the Vav-Cre deleter
C57/BL6 strain (Vav-Cre+F/F), which expresses Cre recombinase in HSC. Dot plot of B220+PDCA1+cells (Left) or Cd11cintPDCA+cells (Right) shows a near
complete loss of pDCs in the bone marrow of Vav-Cre+F/F mutants compared with littermate controls (10-fold to 50-fold loss; n = 5 independent experiments).
The simultaneous loss of B220+PDCA1–B cells served as an internal control for Cre-mediated deletion efficiency. A similar loss of pDCs was confirmed in the
spleen (Center), although the fold reduction (∼fivefold) was not as pronounced as in bone marrow. (B) The generation of cDC (B220–MHC IIhiCD11chiCD11b+)
was unaffected by Vav-Cre–mediated knockout of Bcl11a. (C) The frequency of CD8+and CD8−cDC subsets in the spleen were statistically indistinguishable
between Vav-Cre+F/F and controls. (D) Although fewer overall LSK progenitors were detected in Vav-Cre+F/F mice, both MDP and CDP progenitor pop-
ulations persisted within the Flt3+compartment in cKO mice at percentages similar to controls. MDP (Lin–Flt3+Sca-1−CD115+c-kithi); CDP (Lin–Flt3+Sca-1−
CD115+c-kitlo). (E) pDC are also depleted in Bcl11aF/FMx1-Cre mice following induction of Cre-mediated deletion in HSC by treatment with pI:pC, but CDP and
cDC are spared, analogous to results obtained with Vav-Cre deletion. (F) Hematopoietic lineages other than B and pDC were statistically unaffected in Vav-
Cre+F/F mice relative to Vav-Cre–controls across independent experiments (n = 3; four per genotype) plotted as mean ± SEM (*P < 0.05; **P < 0.001; Student
t test). (G) Vav-Cre+F/F mice are significantly impaired in type I IFN response to viral infection with ∼1 × 107pfu of human HSV strain 17 i.v. by tail-vein
injection (n = 1; four per genotype) as measured by ELISA (232 ± 44.4 to 518 ± 144 pg/mL; data as means ± SEM; P < 0.05, Mann–Whitney, two-tailed test).
Conditional elimination of Bcl11a in vivo leads to loss of pDC cellularity and function without perturbation of cDCs. (A) pDC are depleted in Bcl11a
Ippolito et al.PNAS
| Published online March 3, 2014
levels of IFN-α were markedly reduced 12 h postinjection in
Bcl11aF/FVav-Cre mice compared with Cre-negative littermate
controls (232 ± 88.9 to 518 ± 288 pg/mL, respectively). Thus,
Bcl11a-deficient mice have a reduced capacity to generate this
critical pDC mediator of the innate immune response.
Genome-Wide BCL11A Target Genes Include Previously Established
Regulators of pDC Biology. ChIP-seq of the human CAL-1 cell
line was used to identify genome-wide binding sites of BCL11A
in pDCs. We found that BCL11A is recruited to the loci of
several previously established pDC factors, including SPI1, SPIB,
E2-2/TCF4, IRF4, MTG16, and ID2, and, interestingly, to its own
proximal promoter region (Fig. 3A). The occupancy pattern of
BCL11A in CAL-1 cells (within putative proximal promoters and
intronic or intergenic loci) bore a striking resemblance to its binding
distribution in the human EBV-transformed lymphoblastoid cell
line (LCL) GM12878, based on published ENCODE Consortium
data (www.factorbook.org/; Fig. 3B). Additionally, the top-ranked
DNA binding motifs for the top CAL-1 and GM12878 binding
sites were identical (Fig. 3 D and E and Fig. S4). Our data further
suggest that some targets are bound in a cell-context dependent
fashion (Fig. 3C). For example, whereas BCL11A was recruited to
the same location in the ID3 locus in both the B and pDC cell
lines, its recruitment to the ID2 locus was differential across the
two lines. Ongoing studies of these and other cell-contextual
patterns of BCL11A genomic binding are being confirmed in
additional B-cell lines.
Evidence for a BCL11A-Regulated ID/E–Protein Interplay in Control of
pDC Development. Recent findings have linked Id2 and Id3 regu-
lation to differentiation of cDCs and pDCs via transcriptional in-
hibition of E2-2/Tcf4 (24, 26, 40). Among our CAL-1 genome-wide
CAL1 (2,288) GM12878 (5,418)
E = 9.7e-1103
the CAL-1 human pDC (black, peak score ≥10) were determined and aligned with ENCODE Consortium ChIP-seq data for the human B lymphoblastoid cell line
GM12878 (red, peak score ≥10). ID4 and the T-cell–specific paralogue, BCL11B, were chosen as negative controls because of their similarity to BCL11A and
other IDs that contained highly correlated peaks. CAL-1 ChiP-seq resulted in smaller and fewer peaks than the ENCODE data (Fig. S3). (B) A pie chart rep-
resentation of the distribution of BCL11A binding sites in six different genomic regions. Core promoters are within ±2 kb from the transcriptional start site
(TSS); upstream is from 2 to 20 kb upstream from the TSS; and intergenic is a region not included as a promoter, upstream region, intron, or exon. The CAL-1
ChIP-seq peak distribution shows a striking similarity to that of the ENCODE Consortium’s GM12878 cell line. (C) Overlap of BCL11A target genes between the
two cell lines. A target gene was defined by a binding site occurring within 50 kb upstream through the intron of that gene. Analysis of the false discovery
rate and associated Q-values were performed using Benjamini–Hochberg statistics. (D) De novo motif analysis from BCL11A ChIP-seq in CAL-1 cells. Alignment
of the top 500 and top 1,000 CAL-1 ChIP-seq peaks show strong similarity to the top-ranked BCL11A ChIP-seq motif found in GM12878 cells, an EICE consensus
site. (E) EICE motifs comprise the top-ranked BCL11A binding motifs in the GM12878 cell line. EICE is a previously identified composite DNA binding site for
the Ets factor PU.1/SPI1 and for the IFN regulatory factor 4 (IRF4) that mediates cooperative binding of these factors to DNA. MEME analysis was used to
calculate the expectation E-value for the occurrence of this motif within the GM12878 coincident peak sequences of BCL11A, PU.1/SPI1, and IRF4.
ChIP-seq analysis of genome-wide BCL11A DNA binding in vivo. (A) Peak mapping along the loci of multiple pDC-related genes. ChIP-seq peaks from
| www.pnas.org/cgi/doi/10.1073/pnas.1319228111Ippolito et al.
target genes we observed strong BCL11A binding to ID3 (Fig. 3A).
However, ID2 peaks were weak and did not align with the rela-
tively strong ID2 peaks observed in GM12878 B cells (Fig. 3A).
Using ChIP followed by endpoint PCR, we confirmed (n = 3)
robust recruitment of BCL11A to the proximal promoter of ID3 in
chromatin prepared from the CAL-1 cell line, but not from an
amplicon 10 kB upstream or from a cell line (Hodgkin lymphoma
L428) negative for BCL11A expression (Fig. 4B). Additionally,
the proximal promoter of E2-2/TCF4 was also efficiently PCR
amplified (n = 2) from CAL-1 chromatin (Fig. 4B). Within the
ChIP regions of ID3 and E2-2 (base pairs –679 and –3762 upstream
of their respective transcriptional start sites) reside evolutionarily
conserved sequences (Fig. S4) that match the BCL11A binding
consensus of Fig. 3D. In ID3 this putative BCL11A binding site is
positioned <100 bp downstream of a MspI/HpaII restriction site
whose differential methylation has been correlated with myeloid vs.
relative luciferase activity
ID3 Luciferase Reporter
6 Hours 12 Hours 24 Hours
BJAB Burkitt’s lymphoma B-cell lines altered transcription (average threefold) of the HLH proteins ID2 and ID3 compared with mock-transduced control cells.
Multiple (n > 4) independent experiments tracked by multiple probe elements spotted per LymphoChip cDNA microarray. (B) Inducible shRNA silencing of
BCL11A down-regulates transcription of ID3 and E2-2. (Left) CAL-1 pDC cell line stably transduced with inducible shRNA targeted to exon 2 of Bcl11a under
the control of a doxycycline (DOX)-inducible H1 promoter containing TETR binding sites (59). RT-PCR with optimal cycling conditions in the CAL-1 pDC cell line
revealed strong knockdown of BCL11A transcripts beginning 6 h postinduction and continuing for >24 h. ID3 and E2-2 transcripts were correspondingly
reduced throughout the 24-h experiment, whereas ID2 was consistently, although transiently, induced; the irrelevant target gene CD37 was unaffected.
GAPDH amplification shows equivalence of mRNA amplification and loading. (Right) At 24 h, shRNA-mediated BCL11A knockdown resulted in ID3 tran-
scriptional inhibition >80%, and E2-2 of 60%, when normalized to the GAPDH housekeeping control gene. Mean ± SD depicted; n ≥ 3 experiments. (C)
BCL11A is recruited to 5′ regulatory regions of ID3, E2-2, and itself. ChIP from CAL-1 human pDC cells using anti-BCL11A or control IgG antibodies was
analyzed by endpoint PCR. Primers annealing 10 kb upstream of the ID3 TSS failed to amplify and thus served as a negative control. (D) Overexpression of
BCL11A (XL) up-regulates ID3 promoter-driven reporter transcription in CAL1 pDCs and Raji B cells. (Upper) Schematic of the ID3 proximal promoter indicating
the species-conserved EICE-like BCL11A DNA binding motif (Fig. S4); the location of BLIMP-1 and E-box motifs; and the HpaII methylation site. An 820-bp
fragment spanning this region (indicated by arrows) that was consistently PCR amplified in ChIP experiments (indicated by wedges) was inserted upstream of
luciferase in the pGL2 luciferase vector. (Lower) Dual luciferase activity was determined following transient transfection with increasing DNA levels (in
nanograms) of the reporter into CAL1 human pDC cells or Raji B cells. Mean relative luciferase activity ± SEM obtained depicted; n = 2 independent
experiments read in triplicate. (E) Knockdown of BCL11A results in MTG16 knockdown 24 h post-DOX induction in CAL-1 pDC cells.
BCL11A regulates ID2, ID3, MTG16, and E2-2/TCF4 in human B and pDC cell lines. (A) BCL11A isoforms (XS or XL) retrovirally overexpressed in Raji and
Ippolito et al. PNAS
| Published online March 3, 2014
lymphoid lineage choice (23) (Fig. 4C). Consistent with its
absence in the CAL-1 ChIP-seq target list, we found no evidence
for BCL11A recruitment to ID2. However, we confirmed binding
of BCL11A to its own locus (Fig. 4B), raising the possibility that
BCL11A is autoregulatory.
As an initial test of function, we overexpressed the major
BCL11A isoform (XL) or a smaller, less well-characterized (4)
isoform (XS) as retroviruses in Raji B cells. We observed that
both BCL11A-XL and -XS strongly up-regulated ID3 (average of
2.8- to 3.0-fold, respectively; Fig. 4A), whereas ID2 was uniformly
repressed (2.6- to 3.0-fold) by BCL11A-XL and -XS. Next,
inducible short-hairpin RNA interference in CAL-1 pDC cells
was used to specifically target a region (exon 2) common to all
BCL11A isoforms. We observed a robust knockdown of BCL11A
transcripts as well as a corresponding reduction in both ID3 and
E2-2 transcripts at select time points (Fig. 4C). Knockdown of
BCL11A was nearly absolute by the end of the first 24 h of shRNA
induction. Assayed at the 24-h time point and normalized to the
housekeeping control gene GAPDH, shRNA-mediated BCL11A
knockdown resulted in an ID3 transcriptional inhibition of over
80%, and E2-2 of 60%, whereas the irrelevant target CD37 was
broadly unchanged. Direct ID3 regulation was confirmed by
BCL11A up-regulation of ID3 promoter-driven luciferase activ-
ity in CAL1 and Raji (two- to 10-fold; Fig. 4D) transient trans-
fections. Measurement at the earliest time point (6 h) showed, as
expected, down-regulation of the direct E2-2 target gene, ID2
(19). However, at subsequent time points (12 h, 24 h), ID2
consistently and gradually returned to baseline levels, suggesting
an indirect affect by BCL11A knockdown. As a potential mech-
anism, the ETO family corepressor MTG16, an established (41)
negative regulator of ID2 (41) and a BCL11A target gene (Fig. 3A),
was virtually depleted upon BCL11A shRNA knockdown (Fig. 4E).
Cell viability was similar among experimental and control groups at
all time points. These results collectively support a model for ID/E–
protein interplay regulated by BCL11A through its direct tran-
scriptional activation of the pDC-essential gene E2-2 and the E-
protein antagonist ID3, while indirectly suppressing ID2 expression
through direct activation of MTG16.
BCL11A has an essential role in B-cell development (5) and
B-cell malignancy (2). Recent reports have suggested a broader
role for Bcl11a in HSC and lymphoid development (6). An ad-
ditional role for BCL11A in pDC development had been implied
only indirectly by microarray gene expression profiling (42, 43)
and by the abundant presence of BCL11A protein in this cell
type (44). Here, we establish experimentally a regulatory role for
BCL11A in pDCs pivotal to their development and differentia-
tion. We show that Bcl11a deficiency in vivo results in loss of
both fetal and adult pDC, while sparing other lineages down-
stream of the HSC. We identify BCL11A-regulated target genes
previously implicated as critical for pDC development (18, 27).
Coupled with the recent finding (20) that Bcl11a is significantly
up-regulated in CDPs, which exclusively give rise to pDCs and
cDCs, our results strongly support a model for “pDC priming”
(20, 45) in which differentiation into pDCs represents the default
pathway for CDPs (readdressed below).
At present, all current evidence (6, 20) indicates that Bcl11a is
expressed uniformly in the HSC compartment, even though this
compartment comprises a heterogeneous collection of cells. There
is no evidence for Bcl11a differential expression between lym-
phoid-primed vs. myeloid-primed stem cells or progenitors, so it is
unexpected that Bcl11a deletion in HSCs would result in a pan–
lymphoid-specific defect, while leaving myeloid cells unaffected.
That hypothesis conflicts with the data obtained here and in the
original report of embryonic germ-line Bcl11a deletion (5) in
which only B cells (specifically B220+cells) were grossly affected.
Conditional KO models produced by our group (promoter and
exon 1 targeted deletion) and by Yu et al. (6) (exon 4 targeted
deletion) reveal an approximate twofold reduction in LSKs, but, in
the case of our model using hematopoietic-specific deletion, the
relative fraction of GMP, MDP, and CDP are not affected; CD8+
and CD8−cDC subsets are spared; and only B cells and pDCs are
abolished. So clearly in addition to an effect on LSK hematopoi-
esis in general, there is a selective effect on CDP to pDC lineage
development. In support of this selective effect, recent functional
analyses (46) have provided compelling evidence that although
BCL11A is expressed broadly among human HSCs and multi-
lymphoid progenitors (MLPs)—which exhibit hybrid transcrip-
tional states resembling stem, myeloid, and lymphoid programs
extending across lineage-specific boundaries—the phenotype of
silencing BCL11A in single-cell MLPs is consistent precisely with
the phenotype of mice deficient in Bcl11a in which there is no
B-cell development. The authors concluded that Bcl11a directs
MLP commitment exclusively to the B-cell lineage and does not
have a pan-lymphoid effect (46). Analogously, our data suggest
that Bcl11a selectively directs CDP commitment to the pDC lineage.
The field of pDC biology has generally focused on adult
progenitors isolated from bone-derived and adult-derived pDC
progenitors. We and others have used fetal liver isolated from
perinatally lethal Bcl11a null mice (5, 9) to confirm an absolute
cellular, molecular, and functional absence of pDCs in the em-
bryo. As with these observations, Esashi et al. (34) in their
studies of the signal transducer STAT5 found that fetal liver
pDCs were responsive to Flt3-L, supporting the hypothesis that
fetal and adult pDC progenitors share a common developmental
pathway. HSCs isolated from human fetal liver also give rise to
pDC (47), suggesting that the same ontogenetic pathway is
conserved in humans. This conclusion is further buttressed by the
work here showing that Bcl11a, when eliminated conditionally in
bone marrow HSCs, is equally critical to adult pDC development
and function. In contrast to pDC development, an odd, merely
partial defect in Bcl11a−/−cDC differentiation can be observed
in vitro but only when fetal liver precursors are cultured with
Flt3L (not GM-CSF; ref. 9; our data), whereas their development
in vivo is unimpeded and persists normally whether cDC are de-
rived from fetal precursors transplanted to adult chimeras (9) or
from conditionally deleted adult precursors (our data). This
finding indicates that cDC development is Bcl11a-independent.
Microarray profiling of the common lymphoid progenitor
(CLP) or CMPs illustrated how, among 218 differentially ex-
pressed genes, BCL11A and E2-2 are two key elements dis-
tinguishing the gene expression profile of human pDCs from cDCs
(48). Furthermore, prior genome-wide analysis highlighted
BCL11A and E2-2 as key genes for pDC vs. cDC lineage dis-
crimination conserved between mouse and human (13). Our
results advance a model in which BCL11A regulates E2-2 activity
by both direct and indirect mechanisms, suggesting a network
through which BCL11A-driven pDC development might occur.
We observed BCL11A-mediated modulation of both ID2 and ID3
transcription levels, and the recruitment of BCL11A to consensus
DNA binding sites upstream of the promoters of ID3 and of the
pDC essential and specific transcription factor E2-2. Bcl11a-
directed shRNA interference strongly reduced ID3, MTG16, and
E2-2 expression, whereas ID2 expression was derepressed, albeit
transiently. That ID2 modulation was observed in the absence of
BCL11A is consistent with its direct repression by E2-2 (19).
However, the reciprocal modulation of BCL11A (as well as ID2) by
E2-2, as reported by Reizis and coworkers (19), adds a layer of
complexity that seems to defy a simple linear network defining pDC
specification. Nevertheless, our data in Bcl11a-deficient mice clearly
demonstrate that this factor is necessary for the development of all
mature pDCs. cDCs, which derive solely from the CDP, persist,
indicating the lack of a requirement for BCL11A in development in
| www.pnas.org/cgi/doi/10.1073/pnas.1319228111Ippolito et al.
Because ID proteins antagonize E-protein activity, and E-proteins
are essential to the development and differentiation of lymphocytes
and pDCs (11, 18, 21), the identification of transcription factors
capable of modulating ID expression immediately suggests mecha-
nistic models for lineage determination. ID2 and ID3, in particular,
have documented roles in murine and human B-cell, T-cell, and
pDC biology, and modulate the developmental potential of
CDPs and CLPs (23–30). Though both Id2 and Id3 are coex-
pressed in purified CDPs (40), only Id3 is detectable in CLPs
(49), and notably, differential expression is maintained in the
downstream cDC and pDC progeny where cDCs are strongly
positive for Id2 but chiefly negative for Id3, and the opposite is
true for pDC (40). We have uncovered a role for BCL11A in the
transcriptional regulation of ID3, which calls to mind the related
identification of ID2 as a target gene repressed by BCL11B (50),
a highly similar paralogue of BCL11A essential for T-cell de-
velopment (2). The orchestration and maintenance of lymphoid
cell fates through the timing and overall dosage of ID3 (and
ID2) and the resulting antagonism of E2A function has been
previously described (29, 30). Similar observations regarding gene
dosage and dendritic cell commitment have also been previously
described for the PU.1/SPI1 transcription factor (51). Collectively,
these observations provide a paradigm for understanding how
timing, dosage, and the distinct pairing preferences of individual
ID proteins might determine various lineage commitments.
Additionally, it should be noted that the top-ranked BCL11A
consensus DNA binding motif that we and others (ENCODE
Consortium) have discovered is the same motif previously iden-
tified as an Ets transcription factor/interferon regulatory tran-
scription factor (IRF) composite element (EICE). EICE is known
to be dually occupied by one of the two Ets factors, PU.1/SPI1 or
SPIB, in combination with one of the two IRF factors, IRF4 or
IRF8 (51, 52). Other Ets/IRF factors do not interact in this
fashion (53). Each of these four factors is known to be involved in
pDC development or maintenance (45, 54), and we have previously
confirmed that BCL11A drives both IRF4 and IRF8 expression in
mouse pre-B cells (49, 55). That BCL11A is recruited to the loci of
each of these genes in human pDC CAL-1 chromatin and to evo-
lutionarily conserved EICE-like sites within the promoter regions of
itself, ID3, and E2-2/TCF4, places BCL11A near the top of a pDC
gene regulatory hierarchy (Fig. S4).
In conclusion, rather than linear opposition of a default cDC
differentiation pathwayexertedby E2-2, inpart, through BCL11A-
mediated repression (19), we propose instead a feedback loop
between E2-2 and BCL11A that constitutively maintains pDC
identity (Fig. S5). Our data suggest that in this loop, BCL11A is
activating E2-2 transcription and that BCL11A induces transcrip-
tion of ID3, which may in turn heterodimerize with and reduce the
protein activity of E2-2 (or other E-protein family members). In
this way, ID3 and, perhaps, BCL11A autoregulation, provide ho-
meostatic maintenance within pDC by buffering E2-2. Conversely,
Id2−/−mice display no loss of pDC (24), yet ID2 remains ex-
pressed (albeit at modest levels) in CDP but not in pDC (20,
40); this leads us to speculate that within the CDP, BCL11A
fortifies the default pathway by E2-2–mediated down-regulation
of the ID2 repressor while concurrently driving MTG16 expres-
sion, thereby reducing ID2 activity at the protein level (41). For
ID2 to rise to levels that would push CDPs to cDCs, it must down-
regulate E2-2, BCL11A, and the other default pathway genes that
are increased during the commitment of MDP to CDP but not to
monocytes (20). Accordingly, we observed cDC generation in
Bcl11a cKO mice to be unaffected. In our model, loss of BCL11a
reduces E2-2 and MTG16 levels, allowing a sufficient increase of
ID2 mRNA and activity for normal cDC development from the
CDP (Table S5).
Materials and Methods
Conventional Bcl11a−/−Mice. All housing, husbandry, and experimental
procedures with Bcl11a knockout and control mice were approved by the
Institutional Animal Care and Use Committees at The University of Texas at
Austin. The generation of Bcl11a−/−mice has been previously described (5).
Mice were routinely PCR genotyped using tail genomic DNA and two primer
pairs at an annealing temperature of 65 °C (primers in Table S1).
Generation of Bcl11a cKO Mice. Full details are provided in SI Text.
Flow Cytometry. Analysis was performed on FACSCalibur or Fortessa flow
cytometers (BD Biosciences) and analyzed using CellQuest (BD Biosciences),
WinMDI (Scripps Research Institute),or FlowJo (TreeStar) software. Fetal liver
and spleen were prepared from pups (E15–E18) taken by Cesarean section.
Single-cell suspensions were incubated 15 min on ice with Fc Block (BD
Biosciences) before staining with antigen-specific monoclonal antibodies
(Table S2) in D-PBS/2% (vol/vol) FBS FACS buffer.
RT-PCR. Total RNA was extracted from Bcl11a−/−fetal liver cells (or Bcl11a cKO
spleen cells) using TRIzol reagent (Invitrogen) and oligo-dT cDNA was pre-
pared using SuperScript III First-Strand Synthesis System for RT-PCR (Invi-
trogen). Taq polymerase (New England Biolabs) and a Perkin-Elmer 2700
thermocycler were used to amplify transcripts for the following mouse
genes: Bcl11a, Siglech, Id3, Cd19, β-Actin, Hprt. Primers listed in Table S1.
Culture of Fetal Liver or Bone Marrow Cells. Cells were cultured in 100 ng/mL
Flt3L or 50 ng/mL GM-CSF to induce pDC or cDC expansion, respectively. Time
points are indicated in text and figures. Details in SI Text.
Retroviral Transduction of B-Cell Lines and LymphoChip Gene Expression Profiling.
Phoenix-A (ΦNX-A) amphotropic 293 cells were used to package retroviruses
containing pXY-PURO (negative control vector) or pXY-BCL11A-XS or pXY-
BCL11A-XL using Fugene-6 reagent (Roche). Retroviral supernatant was used to
infect target cells. Following puromycin selection, cells were pelleted by cen-
trifugation, media-aspirated, and the cells lysed in TRIzol reagent for total RNA
extraction. RNA was used for LymphoChip microarray profiling (56) or oligo-
dT cDNA synthesis using a SuperScript III reagent kit (Invitrogen) and for
endpoint RT-PCR experiments. RT-PCR primers and cycling conditions are
listed in Table S1.
ChIP and ChIP Followed by ChIP-seq. ChIP assays were performed as described
(57). More details and the PCR primers used are listed in Table S1. For ChIP-seq,
DNA was analyzed by deep sequencing using Illumina sequencing technology.
Inducible shRNA Knockdown and RT-PCR in the CAL-1 pDC Cell Line. CAL-1 cells
were stably transduced with a retrovirus expressing the bacterial tetracycline
repressor (TETR) and the blasticidin resistance gene, followed by retroviral
transduction with a Phoenix-E packaged pRSMX-PG TETR-inducible vector
containing shRNA targeted to exon 2 of Bcl11a (58). Doxycycline (50 μg/mL)
was applied for induction of shRNA expression. Cells were harvested for
total RNA isolation at multiple time points. RT-PCRs with the listed human
primer pairs (Table S1) were performed to amplify gene transcripts from
induced BCL11A knockdown cells.
HSV Challenge and IFN-α ELISA. Vav-Cre–deleted Bcl11a or cre-negative lit-
termate or age-matched controls were infected with 1 × 107pfu of HSV1
(ATCC) by tail-vein injection. Blood was collected at 6, 12, and 24 h post-
injection by saphenous vein bleed, centrifuged at 10,000 × g for 10 min for
serum collection, and frozen at −80 °C. Detection and quantification of serum
IFN-α was performed by ELISA following manufacturer’s protocol (eBioscience;
Fig. 3G). Before infection, the expected phenotypic B and pDC deficiency in
cre-deleted mice was confirmed by FACS (SI Text).
ACKNOWLEDGMENTS. We thank June V. Harriss, Deborah Surman, and the
late Shanna D. Maika for expert assistance in the generation of Bcl11a con-
ditional knockout mice, and Chhaya Das and Maya Ghosh for help in ChIP
analysis and cell culture. The CAL-1 cell line was kindly provided by
Dr. Takahiro Maeda and Dr. Boris Reizis. Conventional Bcl11a knockout mice
were provided by Dr. Pentao Liu. Support for this work was provided by the
Intramural Research Program of the National Institutes of Health (NIH)
National Cancer Institute, Center for Cancer Research (to L.M.S. and A.L.S.);
NIH Grants F32CA110624 (to G.C.I.) and R01CA31534 (to H.O.T.); Cancer Pre-
vention Research Institute of Texas (CPRIT) Grants RP100612, RP120348; and
the Marie Betzner Morrow Centennial Endowment (H.O.T).
Ippolito et al.PNAS
| Published online March 3, 2014
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