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DENDRITIC CELLS
Decrypting DC development
The NFIL3–ZEB2–ID2 transcription-factor regulatory circuit switches the E protein–dependent +41 kb Irf8 enhancer
in DC progenitors to the BATF3-dependent +32 kb Irf8 enhancer in mature cDC1s. Deletion of the cryptic +41 kb
Irf8 enhancer impedes cDC1 development.
Martin Guilliams and Tomohiko Tamura
T
he decryption of the German Enigma
cipher by the Allies during World War
II was one of the most decisive code-
breaking successes in history. In this issue of
Nature Immunology, two papers by Murphy
and colleagues (Durai et al.1, and Bagadia,
Huang, Liu et al.2) report that an NFIL3–
ZEB2–ID2 transcription-factor regulatory
circuit controls the switch in enhancers of
the gene encoding the transcription factor
IRF8 to ensure the development of classical
type 1 dendritic cells (cDC1s). These two
studies by the Murphy laboratory will no
doubt be recognized as a decisive step in the
decryption of the central mechanisms that
control DC development.
The development of various immune
cells relies on the establishment of
appropriate gene-expression patterns,
which is controlled mainly by regulatory cis
elements called ‘enhancers’ that are bound
by lineage-specific transcription factors. A
single gene often has multiple enhancers
that act either in a cellular context–
dependent manner to allow expression
at appropriate times or in a redundant
manner to ensure robustness of expression3.
cDC1s serve pivotal roles, particularly
in the host defense against tumors and
viruses, due to their unique ability to
‘cross-present’ exogenous antigens to CD8+
cytotoxic T cells4. The mechanism of cDC1
differentiation is thus an important theme
for understanding the nature of immunity
and for establishing effective therapies
against cancers and infections.
cDC1s are derived from bone marrow
hematopoietic stem cells (HSCs) via
intermediate progenitor cells such as
monocyte–DC progenitors (MDPs),
common DC progenitors (CDPs) and pre-
cDC1s5. The transcription factors IRF8,
BATF3, NFIL3 and ID2 have been shown to
be required for the development of cDC1s4,6.
Among these, IRF8 appears to be the
master lineage-determining transcription
factor, and Irf8–/– mice lack cDC1s and their
precursors, including pre-cDC1s. While
the function of IRF8 has been well studied,
the mechanism by which the expression of
Nature Immunology | www.nature.com/natureimmunology
Irf8 is regulated during DC development
has remained obscure. Irf8 expression is
very low in HSCs but increases at the MDP
stage, is further upregulated in CDPs and
is maintained in cDC1s and plasmacytoid
DCs (pDCs), but becomes modest in
monocytes6–8 (Fig. 1a). Interestingly, Irf8
expression is at first independent of BATF3
in DC progenitors and then becomes
dependent on BATF3 in mature cDC1s. The
mechanisms that control this differential
dependency of Irf8 expression is unknown.
Irf8 has been shown to carry three distinct
enhancers at 50 kb upstream (–50 kb) and
32 kb and 41 kb downstream (+32 kb and
+41 kb, respectively) of the transcription
start site (Fig. 1a). However, the functional
requirement for the endogenous Irf8
enhancers during in vivo DC development
has not been assessed.
Durai et al. clarify the lineage- and
differentiation stage–specific roles of
the three Irf8 enhancers in vivo by using
CRISPR–Cas9 genome editing to generate
mouse strains that lack each enhancer1. The
–50 kb Irf8 enhancer, which contains two
elements that bind the transcription factor
PU.1, had been proposed to induce Irf8 in
MDPs by a study of mice with transgenic
expression of a phage artificial chromosome
reporter9. Durai et al. find, however, that
deletion of this enhancer in vivo does not
affect IRF8 expression in MDPs and the
subsequent DC lineage and instead reduces
IRF8 expression in Ly6C+ monocytes and
F4/80+ peritoneal macrophages but not in
red-pulp macrophages1. Thus, the –50 kb
Irf8 enhancer functions in monocytes and
certain types of macrophages.
The Murphy laboratory previously
suggested that the +32 kb Irf8 enhancer,
which contains four transcription factor
AP1–IRF composite elements that bind
BATF3 and IRF8, maintains high expression
of Irf8 via auto-activation8. By performing
ATAC-seq chromatin profiling and assessing
mice that lack all four such elements (called
‘Irf8 +32–/–’ mice), Durai et al. prove that
this +32 kb Irf8 enhancer is active only
in pre-cDC1s and cDC1s and is indeed
absolutely required for cDC1 development1.
Although the number of Irf8 +32–/– pre-
cDC1s remains normal, IRF8 expression
is reduced in these cells. Thus, the +32 kb
Irf8 enhancer directs the transition from
pre-cDC1 to cDC1 by supporting IRF8
expression.
Durai et al. next seek to understand how
pre-cDC1s are specified from CDPs1. The
authors unexpectedly find that the +41 Irf8
enhancer, which they previously showed
is active in pDCs but not in cDC1s8, is at
an open chromatin configuration not only
in pDCs but also in CDPs and pre-cDC1s.
The authors confirm that deletion of the
+41 Irf8 enhancer reduces the expression of
IRF8 in pDCs but, surprisingly, also reveal
that these ‘Irf8 +41–/–’ mice lack pre-cDC1s
and cDC1s. In addition, the rise in IRF8
expression after the transition from MDP
to CDP is abolished in these mice. As for
the possible transcription factors that act
on this enhancer carrying six E-box motifs,
the authors show that the E protein E2A is
expressed in MDPs and CDPs and that its
expression is reduced in pre-cDC1s and
is absent in cDC1s and pDCs. Strikingly,
bone marrow progenitor cells from mice
deficient in Tcf3 (which encodes E2A) have
diminished potential to give rise to cDC1s
and also pDCs in vivo and in vitro. Thus, the
+41 kb Irf8 enhancer, probably activated by
E2A, directs the transition from CDP to pre-
cDC1 by boosting IRF8 expression before
the +32 kb Irf8 enhancer becomes active. In
pDCs, the +41 kb Irf8 enhancer may bind
another E protein, E2-2, that is essential for
pDC development.
In the accompanying paper by Bagadia,
Huang, Liu et al., the authors try to delineate
the regulatory circuits controlling that
essential switch from the +41 kb Irf8
enhancer in DC progenitors to the +32 kb
Irf8 enhancer in mature cDC1s2. NFIL3,
ID2 and ZEB2 are transcription factors
essential for DC development10–12. The
authors use Zbtb46gfp, Id2gfp and Zeb2egfp
reporter mice (with expression of each
gene product reported by green fluorescent
protein (GFP) or enhanced GFP (EGFP))
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a genes (such as Zeb2 and Sirpa) in the

Irf8 expression HSC transition from ZEB2-EGFPhi, ID2-GFPlo or
ZBTB46-GFPlo CDP to ZEB2-EGFPlo, ID2-
pDC GFPhi or ZBTB46-GFPhi cDC1-committed
Low High
CDP to previously defined pre-cDC18.
Subsequently, the authors demonstrate that
this ZEB2-EGFPlo, ID2-GFPhi, ZBTB46-
MDP
GFPhi cDC1-committed CDP subset is
? severely diminished in the absence of Id2
or Nfil3 but is greatly increased in absence
cDC1
of Zeb2.
MФ Mo
Pre-cDC1 CDP But how does one define the precise
regulatory relationship among NFIL3,
ID2, and ZEB2 in the circuit controlling
cDC1 development? To crack this ‘cipher’,
Murphy and colleagues apply an ingenious
genetic epistasis strategy they achieve by
–50 Irf8 +32 +41 crossing Nfil3-, Id2- or Zeb2-deficient mice
to one another to determine the regulatory
PU.1 BATF3 E2A
IRF8 E2-2
hierarchy of these transcription factors
(Fig. 1b). The result is a regulatory circuit
in which Id2 is repressed by ZEB2, and
Zeb2 in turn is repressed by NFIL3. The
b Which genetic regulatory circuit controls Irf8 expression during cDC1 development? expression of NFIL3 therefore enforces Id2
Conventional DC expression by suppressing Zeb2. Finally,
+32 +41
development NFIL3 CDP the authors demonstrate that ID2
cipher E2A
ZEB2
E2-2
terminates the E2A activity at the +41 kb
ID2 Irf8 enhancer, which indicates that NFIL3 is
involved in the switch of the E2A-controlled
A
2

E2A
ID2
E2
ZEB

Zeb2 hiId2 lo E2A/E2-2 NFIL3

NF
3

+41 kb Irf8 enhancer to the BATF3-

BA
NFIL

IL3
BATF

ZEB

TF3

controlled +32 kb Irf8 enhancer during

ID2 ZEB2
2

cDC1 development (Fig. 1b).

Collectively, these two manuscripts
NF
ID2 IL3
E

Pre-cDC1
2A
2

elegantly illustrate the non-redundant roles

A

BA

B
E2

ZE use of
ID E2A/E2-2 NFIL3
TF

of Irf8 enhancers during cDC1 development

2
genetic
3

BATF3
epistasis* and reveal the regulatory circuits that
to decrypt ID2 ZEB2
the cDC cipher Zeb2 loId2 hi control the switch from one enhancer to
(*use double KOs) the other during this developmental
E2A/E2-2 NFIL3
pathway. At the same time, these findings

✓ cDC1
ID2 ZEB2
raise several subjects worthy of future
investigation. First, the finding that F4/80+
E2A/E2-2 NFIL3 E2A/E2-2 ZEB2 peritoneal macrophages require the –50 kb
Irf8 enhancer for IRF8 expression, but
red-pulp macrophages do not, raises the
ID2 ZEB2 ID2 NFIL3
+32 +41 question of whether the enhancer usage
Zeb2 loId2 hi BATF3 depends on the cellular origin (i.e., mainly
IRF8 bone marrow HSC origin for peritoneal
macrophages versus mainly embryonic
Fig. 1 | Decrypting DC development through epistasis. a, Overview of the distinct Irf8 enhancers and origin for red-pulp macrophages13) or
their use in myeloid precursors, monocytes (Mo), macrophages (MΦ) and cDC1s. Irf8 expression (key): on the differential tissue imprinting of
HSC <<< MΦ, Mo < MDP < CDP, pre-cDC1, cDC1, pDC. b, Ancient cipher (top left) consisting of four macrophages. Second, identification of
circles from which multiple transcription factors can be chosen: turning each circle results in many the enhancers and transcription factors
combinations of the transcription factors. BATF3, NFIL3, ZEB2, E2A and ID2 were known to be involved that direct Irf8 expression in progenitor
in cDC1 development, but it was not known in which order of hierarchy these transcription factors would cells more upstream than CDPs will be
work. Through their epistasis strategy, Murphy and colleagues have solved the puzzle: NFIL3 inhibits important. This is because IRF8 starts to be
Zeb2, ZEB2 inhibits Id2, and ID2 inhibits the activity of E2A (encoded by Tcf3). This explains the switch expressed at a low level near the HSC stage,
from the E2A-dependent +41 kb Irf8 enhancer to the BATF3-dependent +32 kb Irf8 enhancer. such as in a fraction of lymphoid-primed
multipotent progenitors14,15, and thereby
epigenetically induces early specification
and identify ZBTB46-GFPhi cells within the The authors next perform transcriptomics toward cDC1s15. Moreover, IRF8 has high
CDP population that are already committed analysis and find a stepwise increase in expression in MDPs and common monocyte
to the cDC1 lineage. They also identify the expression of cDC1-associated genes progenitors, establishes their enhancer
very similar cells as ZEB2-EGFPlo or ID2- (including Id2 and Batf3) and a drop in the landscapes and is also required for MDP-to-
GFPhi cells within the CDP population. expression of cDC2- and pDC-associated CDP and common monocyte progenitor–

Nature Immunology | www.nature.com/natureimmunology


to–Ly6C+ monocyte transitions6,7,16. Third,
it is still unknown whether the suppressive
effects of NFIL3 on Zeb2 and of ZEB2 on
Id2 are direct or indirect. Finally, the authors
discuss the possibility that the cDC1–pDC
bifurcation occurs within the CDP or MDP
population via the mutual repression of Id2
(which promotes cDC1 development) and
Zeb2 (which promotes pDC development),
but this appears to not fit with a recent
model17, in which it was proposed that pDCs
develop from lymphoid progenitor cells
independently of the myeloid cDC lineage
and that pDCs could be a subset of innate
lymphoid cells (ILCs)17. Such a stricter and
much earlier split between the lymphoid
pDC lineage and myeloid cDC1 lineage,
however, does not in any way invalidate the
findings about the dependence of cDC1s
and pDCs on specific Irf8 enhancers or the
NFIL3–ZEB2–ID2–E2A circuit featured
here. In fact, NFIL3, ZEB2 and ID2 have
Nature Immunology | www.nature.com/natureimmunology
also been shown to be central players in ILC
development. The same regulatory circuit
could therefore control both DC fate and
ILC fate without the need for a common
cDC1–pDC progenitor. ❐ 3. Schoenfelder, S. & Fraser, P. Nat. Rev. Genet. 20,
Martin Guilliams   1,2 and
Tomohiko Tamura   3,4
1
Laboratory of Myeloid Cell Ontogeny and
Functional Specialization, VIB Center for
Inflammation Research, Ghent, Belgium.
2
Department of Biomedical Molecular Biology,
Ghent University, Ghent, Belgium. 3Department of
Immunology, Yokohama City University Graduate
School of Medicine, Yokohama, Japan. 4Advanced
Medical Research Center, Yokohama City University,
Yokohama, Japan.
e-mail: martin.guilliams@irc.vib-ugent.be;
tamurat@yokohama-cu.ac.jp
Published: xx xx xxxx
https://doi.org/10.1038/s41590-019-0457-3
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References
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019-0449-3 (2019).
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Competing interests
The authors declare no competing interests.