Integration of Elf-4 into Stem/Progenitor and Erythroid Regulatory
Networks through Locus-Wide Chromatin Studies Coupled with
In Vivo Functional Validation
Aileen M. Smith, Fernando J. Calero-Nieto, Judith Schütte, Sarah Kinston, Richard T. Timms, Nicola K. Wilson, Rebecca L. Hannah,
Josette-Renee Landry, and Berthold Göttgens
University of Cambridge Department of Haematology, Cambridge Institute for Medical Research, Cambridge, United Kingdom
stemcelltranscriptionfactorsshowed Pu.1,Fli-1,andErgwereboundtothe ?10Eelement,andmutationofthreehighlycon-
poietic system (reviewed in references 3 and 31). ETS proteins
regulate transcription in concert with other transcription factors
by binding a core ETS binding motif (GGAW) via their ETS do-
main. The ETS factor Elf-4 (also known as myeloid Elf-1-like fac-
line CMK (36) and is expressed in hematopoietic stem cells
(HSCs), myeloid and lymphoid lineages (24, 25, 36), ovary, pla-
and NK-T cell differentiation, implicating Elf-4 in the regulation
of the innate immune system (24), and more recent studies have
CD8?T cells (25, 58).
In addition to regulating the entry of HSCs into the cell cycle,
Elf-4 also promotes the transition of cells from G1to S phase (35)
and has been shown to be a potent activator at the granulocyte-
macrophage colony-stimulating factor, interleukin 3 (IL-3), IL-8,
and lysozyme promoters (14, 18, 36). Moreover, Elf-4 has been
implicated in the development of cancer, as it has been identi-
fied as a recurrent site of retroviral integration (30, 34) and has
AML-ETO and PML-retinoic acid receptor ? (RAR?) fusion
oncogenes repress the expression of ELF-4, and analysis of
acute myeloid leukemia (AML) patient samples with these
translocations showed diminished levels of ELF-4 (1, 10). In ad-
ETS factor, ERG, has been described in a patient with AML (37).
Elf-4 is therefore an important regulator of hematopoiesis with
critical functions both in the stem cell compartment and mature
characterized, very little work has focused on the transcriptional
TS transcription factors are known to be important for the
proliferation and differentiation of cells within the hemato-
ies of Elf-4 regulation have been reported to date. Here we have
taken a locus-wide approach to analyze the regulation of the
mouse Elf-4 gene, which allowed us to identify 5 distinct regula-
tory regions, functionally validated using both in vitro and in vivo
experiments. A tissue-specific enhancer active in blood progeni-
tors and endothelium in transgenic mice was found to be depen-
the transcriptional repressor Gfi1b bound to and repressed one of
three Elf-4 promoters and, furthermore, we found that Elf-4
critical roles played by ETS factors in blood progenitors and en-
Elf-4 as an important component of regulatory networks control-
ling blood progenitor function and erythroid differentiation.
MATERIALS AND METHODS
Expression analysis. RNA was isolated using TRI reagent (Sigma-
Aldrich) according to the manufacturer’s instructions. Prior to cDNA
synthesis, genomic DNA was removed using Turbo DNA free (Ambion).
cDNA was generated using oligo(dT) and Moloney murine leukemia vi-
Received 5 June 2011 Returned for modification 9 July 2011
Accepted 23 November 2011
Published ahead of print 12 December 2011
Address correspondence to Aileen M. Smith, email@example.com, or
Berthold Göttgens, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0270-7306/12/$12.00Molecular and Cellular Biology p. 763–773mcb.asm.org
18S mRNA levels were measured using the primers listed in Table S1 at
http:hscl.cimr.cam.ac.uk/genomic_supplementary.html and quantified
calculated relative to that of Gapdh for cell lines and to 18S for the ery-
throid maturation analysis.
Chromatin immunoprecipitation assays. Chromatin immunopre-
cipitation (ChIP) assays were performed as previously described (27).
Briefly, cells were treated with formaldehyde, and the cross-linked chro-
matin was sonicated (average size, 300 bp) prior to immunoprecipitation
with anti-acetyl H3K9 antibody (06-599; Millipore) or anti-H3Me3K4
antibody (04-745; Millipore; a gift from L. O’Neill, Birmingham, United
Kingdom). Oligonucleotides used to generate the Elf-4 tiling array were
designed on a repeat masked sequence across the Elf-4 locus (ChrX:
43492286-43571850, mm7). Oligonucleotides were spotted in triplicate
by using the BioRobotics MicroGrid II total array system. The rolling
were plotted using the variable-width bar graph drawer (http://hscl.cimr
and sonicated into fragments of about 150 to 400 bp prior to immuno-
precipitation with anti-Gfi1b antibody (sc-8559X; Santa Cruz Biotech-
nology). Samples were amplified using the ChIP-Seq DNA sample prep
kit from Illumina, following the manufacturer’s instructions, and se-
quenced using the Illumina 2G genome analyzer. Sequencing reads were
mapped to the mouse reference genome by using Bowtie (http://bowtie
-bio.sourceforge.net/index.shtml) (28), converted to a density plot, and
displayed with UCSC Genome Browser (http://genome.ucsc.edu/index
erated by amplifying regions of the Elf-4 locus from bacterial artificial
chromosome clone RP23-99C8 (BACPAC Resources, Oakland, CA) and
cloning into the XhoI/HindIII sites of pGL2 basic or pGL2 promoter
reporter constructs (Promega, United Kingdom). Primers are listed in
Table S1 at http://hscl.cimr.cam.ac.uk/genomic_supplementary.html,
and comparative sequence alignments are shown in Fig. S1 at the same
website. Numbering of the cloned regions is relative to the ATG of
ENSEMBL transcript Elf-4-001. Mutation constructs were made as pre-
viously described (13) and verified by sequencing. The primers used are
listed in Table S1 at http://hscl.cimr.cam.ac.uk/genomic_supplementary
transient transfections, cells were electroporated with 10 ?g of plasmid
and 2 ?g or 5 ?g (HPC7 and BW5147) of the control plasmid pEFBOS
LacZ and assayed as previously described (12). Data were normalized to
the control plasmid and are expressed relative to pGL2 basic expression.
Stable transfections were performed as previously described (27).
Transgenic mouse analysis. The transgenic beta-galactosidase re-
porter constructs were generated by cloning the Elf-4 ?39P, ?30P, and
?2P fragments into the XhoI/HindIII site of the pGLac LacZ plasmid
(GenBank accession number U19930). The Elf-4 ?10E, ?10EmETS1-2,
were generated by pronuclear injection of the beta-galactosidase reporter
sciences (Guangzhou) Inc. (Guangzhou, China). Whole-mount embryos
were stained with 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside
(X-Gal) for beta-galactosidase expression and photographed using a
Nikon digital sight DS-FL1 camera attached to a Nikon SM7800 micro-
scope for magnification. Images of sections were acquired with a Zeiss
AxioCam MRc5 camera attached to a Zeiss Axioscope2plus microscope.
All images were processed using Adobe Photoshop (Adobe Systems, San
were stained with Ter119-allophycocyanin and CD71-phycoerythrin
(BD, Oxford, United Kingdom) antibodies, and dead cells were excluded
using 7-aminoactinoycin (7AAD). Flow cytometry analysis was per-
formed using a FACSCalibur apparatus (BD), and cell sorting was per-
formed using a Dakocytomation MoFlo cell sorter. In vitro erythroid dif-
ferentiation assays were performed as previously described (57, 59).
extracted by blunt dissection and homogenized. Lineage-negative hema-
topoietic progenitor cells were isolated using the magnetically activated
cell sorting murine lineage cell depletion kit (Miltenyi Biotec GmbH,
Gladbach, Germany) according to the manufacturer’s instructions. A to-
tal of 2 ? 105lineage-negative cells were plated onto fibronectin-coated
12-well plates (BD Discovery Labware, Bedford, MA) and grown in ery-
throid differentiation medium (57, 59).
Retroviral transfection. Elf-4 cDNA from Riken mouse FANTOM
clone F630208F07 was cloned into a retroviral overexpression construct
containing murine stem cell virus pGKprom-Puro-IRES-EGFP (PIG).
Retrovirus production was carried out using the pCL-Eco retrovirus
packaging vector (Imgenex, San Diego, CA) in the 293T cell line. Murine
retroviral supernatant was replaced with erythroid differentiation me-
Microarray sequence accession number. The microarray sequence
data derived from our experiments have been deposited in ArrayExpress
under accession number E-MEXP-2908.
been successfully used to predict functional regulatory elements
for many genes (6, 26, 27, 43, 56). An 80-kb region encompassing
//hscl.cimr.cam.ac.uk/genomic_supplementary.html). To iden-
tify potential candidate promoter regions, we combined the se-
quence conservation analysis with Refseq (46) and ENSEMBL
(17) gene structures based on experimentally observed tran-
scriptional start sites within the Elf-4 gene locus (see Fig. S2A at
feature indicative of promoters.
Elf-4 has previously been shown to be expressed at different
levels in a variety of hematopoietic and nonhematopoietic tissues
tial Elf-4 regulatory elements, levels of Elf-4 expression were as-
sessed by quantitative real-time PCR in a panel of hematopoietic
and endothelial cell lines (see Fig. S2B at http://hscl.cimr.cam.ac
.uk/genomic_supplementary.html). Elf-4 expression levels rela-
tive to the housekeeping gene Gapdh were similar in the 416B
itor), BW5147 (T-cell), and MS1 (endothelial) cell lines (see Fig.
S2B at the URL above). In comparison, the expression levels of
Elf-4 in the erythroid J2E cell line were virtually undetectable (see
Fig. S2B at the URL above).
To identify candidate Elf-4 regulatory elements, we next ana-
lyzed chromatin modification profiles by ChIP with microarray
technology (ChIP-chip). Chromatin was prepared from the four
cell lines expressing Elf-4 (416B, HPC7, BW5147, and MS1) and
Smith et al.
mcb.asm.orgMolecular and Cellular Biology
immunoprecipitated with antibodies against the trimethylated
form of lysine 4 of histone H3 (H3Me3K4) and acetylated lysine
9 of histone H3 (H3AcK9). Modification of chromatin by
regions, while H3AcK9 marks regions of accessible chromatin
across both enhancers and promoters of active genes. However, a
recent comprehensive analysis of multiple histone marks across a
panel of human cell types demonstrated that the H3Me3K4 chro-
characterizes regions with strong enhancer activity (9), and this
notion was further supported by a recent functional analysis of T
cell gene enhancers, where loss of H3K4me3 at enhancer regions
ing T cell development (42). Immunoprecipitated chromatin
was interrogated using an 80-kb tiling array that encompassed
the Elf-4 locus and reached into each of its flanking genes.
Histone H3 trimethylation was observed at each of the three
potential transcriptional start sites, but different patterns were
observed in each of the cell lines tested (Fig. 1A). In all four cell
lines, the highest level of enrichment was observed at the kb
?30 region, which corresponds to the start of the predicted
ensemble transcript Elf-4-001 and the region with the highest
GC percentage across the Elf-4 locus (Fig. 1A). At the most-5=
region (kb ?39), H3Me3K4 was enriched in the 416B myeloid
progenitor cell line and in the MS1 endothelial cell line, with
very low levels of enrichment in the BW5147 T cell line. No
enrichment could be detected at this region for the HPC7 pro-
genitor cell line (Fig. 1A). In comparison, low levels of enrich-
was performed in hematopoietic and endothelial cell lines. The UCSC Genome Browser track (see also Fig. S2 at http://hscl.cimr.cam.ac.uk/genomic
_supplementary.html) is shown for reference, and the y axis represents the fold enrichment over the median intensity across the whole locus and is expressed as
log base 2 values. The light gray bars highlight regions that were strongly enriched for both H3Me3K4 and H3acK9 and correspond to potential transcriptional
start sites. The dark gray bars highlight the regions that showed low-level enrichment for H3Me3K4 and were more highly enriched for H3acK9. Numbering
reflects the distance (in kb) from the ATG of the ENSEMBL transcript Elf-4-001.
Transcriptional Regulation of Elf-4
February 2012 Volume 32 Number 4mcb.asm.org 765
ment for H3Me3K4 were detected at the kb ?2 region in the
416B and BW5147 cell lines, with no enrichment observed in
the HPC7 progenitor cells or the MS1 cell line (Fig. 1A).
Histone H3 acetylation profiles across the Elf-4 locus largely
confirmed the previous results obtained with the H3Me3K4 anti-
the kb ?30 region in each of the four cell lines (Fig. 1). Similarly,
the kb ?39 region and the kb ?2 regions were acetylated in the
416B, MS1, and BW5147 cell lines and displayed a pattern of en-
to the H3Me3K4 profiles, low levels of acetylation were also de-
itor cells (Fig. 1B). In addition to the kb ?39, ?30, and ?2 re-
gions, the 416B, HPC7, and BW5147 cell lines were highly
enriched at the kb ?10 region and had low levels of acetylation at
the kb ?16 region (Fig. 1B). In comparison, the endothelial cell
line MS1 showed a low level of acetylation at the kb ?16 region,
with an absence of acetylation at the kb ?10 region, as seen in the
other cell lines tested (Fig. 1).
To complement our ChIP-chip analysis, we also took advan-
tage of publicly available ChIP-Seq data sets produced as part of
the ENCODE project (48). Analysis of the Elf-4 locus showed
H3K4me3 enrichment at each of the candidate Elf-4 regulatory
regions in K562 cells (see Fig. S3 at http://hscl.cimr.cam.ac.uk
/genomic_supplementary.html) and the ?39P, ?30P, ?2P, and
?10E regions in mouse bone marrow (see Fig. S4 at the URL
above). The ?30P promoter region was the most highly enriched
with our ChIP-chip data in hematopoietic cell lines (see Fig. S3
clearly enriched at each of the three candidate Elf-4 promoters in
the murine erythroleukemia (MEL) cell line (see Fig. S5 at the
showed a broad peak of enrichment over the ?16E and the ?10E
enhancer regions in the Elf-4 locus in both K562 and mouse bone
marrow (see Fig. S3 and S4 the URL above). Importantly, two
marks of active regulatory regions, H3K27ac and DNase I, both
showed enrichment of each of the candidate Elf-4 regulatory re-
gions identified by our ChIP-chip analysis (see Fig. S3 and S5 at
with the data from the ENCODE project were used to generate
epigenetic profiles for H3Me3K4, H3acK9, H3K4me1, H3K27ac,
that there are five potential regulatory regions for Elf-4 in hema-
topoietic and endothelial cell lines and in mouse bone marrow.
of H3Me3K4 and Pol II enrichment at the kb ?39, ?30, and ?2
regions, is consistent with three promoter regions for Elf-4, with
the kb ?16 and ?10 regions likely to represent transcriptional
Transcriptional analysis of Elf-4 regulatory elements re-
date Elf-4 promoter regions identified by ChIP-chip, promoter
elements were tested using luciferase transient-transfection as-
says. Each candidate promoter region (?39P, ?30P, and ?2P)
was cloned upstream of luciferase in the pGL2 basic vector and
tested relative to the empty vector control in the 416B, HPC7,
BW5147, and the MS1 cell lines. In each of the four cell lines, the
promoter region with the greatest activity was the Elf-4 ?30P
region, suggesting that this is the dominant promoter in hemato-
poietic and endothelial cell lines. Similar levels of activity for the
Elf-4 ?30P region were observed in the hematopoietic 416B and
lial MS1 cell line Elf-4 ?30P has particularly strong promoter
activity (Fig. 2A), which is entirely consistent with our ChIP-chip
analysis data, which showed the highest level of enrichment of
H3Me3K4 and H3acK9 at this region in this cell line (Fig. 1). The
(Fig. 2A). The pattern of activity was similar to that seen for the
?30P region, with the 416B and BW5147 cell lines showing sim-
ilar levels of activity and the HPC7 cell line showing much lower
endothelial cell lines. (A) Candidate Elf-4 promoter elements (?39P, ?30P,
and ?2P) were assayed by transient transfection in the 416B myeloid, HPC7
multipotent, BW5147 T cell, and MS1 endothelial cell lines. Promoter ele-
ments upstream of luciferase were tested, and the data were normalized rela-
tive to the pGL2 promoterless luciferase control vector. The means and the
standard errors of the means are shown for three experiments performed in
triplicate. (B) The candidate ?10E and ?16E Elf-4 enhancer elements were
was tested using either the heterologous SV40 promoter or the ?39P, ?30P,
and ?2P promoter elements, and results are plotted relative to the promoter-
shown for three experiments performed in triplicate.
Smith et al.
mcb.asm.orgMolecular and Cellular Biology
activity. However, unlike the ?30P region, the ?2P region has
very little activity in the MS1 cell line, again consistent with the
The Elf-4 ?39P candidate promoter element showed no signifi-
cant activity in any of the four cell lines tested (Fig. 2A).
In addition to the three promoter elements enriched for both
H3Me3K4 and H3acK9, two other regulatory elements (?10E
.ac.uk/genomic_supplementary.html) and also did not corre-
fore considered that these two elements may represent candidate
enhancers for Elf-4. To assess enhancer activities, the ?10E and
?16E elements were cloned downstream of a luciferase reporter
cassette driven by either the heterologous SV40 promoter or the
?39P, ?30P, and ?2P promoter elements and tested in stable
transfection assays using the 416B cell line (Fig. 2B). The ?10E
region displayed modest enhancer activity when tested with the
ger activity when tested in conjunction with the ?30P promoter
tested with all four promoters. Overall, our analysis of the tran-
scriptional activity of potential Elf-4 candidate regulatory ele-
ments has identified three promoters and two enhancers.
data set containing the genome-wide binding profiles for 10 dif-
ferent hematopoietic transcription factors in the multipotent he-
this data set to gain insight into potential upstream regulators of
Elf-4. There were no significant peaks for Scl/Tal-1, Lyl-1, and
Lmo2 across the Elf-4 locus. However, ChIP-Seq analysis of the
remaining 7 transcription factors (Gata2, Runx1, Meis1, Pu.1,
Fli1, Erg, and Gfi1b) showed clear binding of each of the factors
across the different Elf-4 regulatory elements identified in this
study except for the ?39P element, which did not bind any of the
factors tested in the HPC7 cell line (see Fig. 5, below; see also Fig.
S6 at http://hscl.cimr.cam.ac.uk/genomic_supplementary.html).
With the Elf-4 ?10E enhancer being able to target expression in
vivo to the fetal liver and dorsal aorta (see Fig. 4, below), we were
binding profiles for hematopoietic transcription factors at this
element. There was very little binding of Gata2 or Runx1, and
contrast, the ETS transcription factors Pu.1, Fli1, and Erg showed
much stronger binding to this element (see Fig. S6). Nucleotide
sequence alignment of the Elf-4 ?10E element in mouse, human,
URL above). Due to the strong binding of the ETS transcription
binding sites present in the conserved core of the ?10E enhancer
may be responsible for its activity. To investigate the importance
of the ETS motifs, mutations were introduced into the ?10E en-
FIG 3 Conserved ETS binding sites are required for the activity of the Elf-4 ?10E enhancer. (A) Nucleotide sequence alignment of the conserved region of the
the wild-type ?10E enhancer and the ?10E enhancer with different combinations of the ETS binding sites mutated. Luciferase values were normalized relative
to the SV40-luciferase control vector. The means and the standard errors of the means are shown for three experiments performed in triplicate.
Transcriptional Regulation of Elf-4
February 2012 Volume 32 Number 4mcb.asm.org 767
Mutation of the first and second ETS motifs resulted in a partial
decrease in the activity of the ?10E enhancer (Fig. 3B), whereas
mutation of the third, fourth, and fifth ETS motifs was sufficient
to abolish the activity of the ?10E enhancer completely (Fig. 3B).
Taken together, these results show that activity of the ?10E en-
hancer critically depends on ETS motifs present in the conserved
activators of Elf-4 expression.
The Elf-4 ?10E enhancer specifically directs expression to
functionally validate the in vivo activity of the Elf-4 regulatory
elements, we generated beta-galactosidase reporter constructs for
F0transgenic embryos for each of the five constructs. The expres-
sion pattern for each Elf-4 regulatory element was assessed by
X-Gal staining of whole-mount E11.5 embryos. No specific stain-
ing patterns were observed for any of the three promoter regions
(Fig. 4A). In contrast, the Elf-4 enhancer elements showed more
consistent staining patterns. The Elf-4 ?16E enhancer was ex-
pressed in the developing neural tube and one embryo showed
staining in the yolk sac and in the heart (Fig. 4A), but when ana-
lyzed in histological sections we could not detect any consistent
FIG 4 The Elf-4 ?10E enhancer specifically targets blood and endothelium in the developing embryo. Elf-4 regulatory elements were validated in vivo using F0
transgenic embryos. (A) Table summarizing the expression patterns of Elf-4 regulatory elements analyzed by beta-galactosidase staining of E11.5 transgenic
embryos. (B) Representative whole-mount E11.5 embryos stained with X-Gal to determine beta-galactosidase expression and tissue sections of the fetal liver,
taken at 2? magnification, and the tissue section images were taken at 20? magnification.
Smith et al.
mcb.asm.orgMolecular and Cellular Biology
and endothelial tissues in whole-mount embryos (SV/Lac/?10E)
(Fig. 4A and B, panel i). Analysis of histological sections con-
firmed that the ?10E enhancer directs expression to a small pro-
aorta, the endothelial lining of vitelline vessels (data not shown),
the yolk sac, and the endocardium of the heart (SV/Lac/?10E)
the enhancer in hematopoietic and endothelial tissues, we also
tested the mutated ?10E enhancer constructs in F0transgenic
of the heart, but at lower levels than seen with the full ?10E en-
4A and B, panel ii). Mutation of the third, fourth, and fifth ETS
motifs dramatically reduced the in vivo activity of the Elf-4 ?10E
enhancer, with no detectable staining in hematopoietic or endo-
in the blood and endothelium, and it confirmed that the ETS
motifs found in the enhancer are critical for its activity.
moter element, and Elf-4 repression is important for erythroid
regulator of erythroid and megakaryocyte development (11, 49,
54). Further interrogation of our ChIP-Seq data set revealed spe-
line (Fig. 5). Following the generation of a new ChIP-Seq data set
of Gfi1b to the Elf-4 ?30P promoter was also observed in this
erythroid cell model (Fig. 5). Moreover, analysis of ELF-4 and
GFI1B expression across an array of human cell types showed
contrasting expression patterns, particularly in CD71-positive
erythroid progenitors, in which GFI1B expression is very high
compared to ELF-4 (see Fig. S7 at http://hscl.cimr.cam.ac.uk
/genomic_supplementary.html). To determine the relationship
levels of endogenous Elf-4 (see Fig. S2 at http://hscl.cimr.cam.ac
.uk/genomic_supplementary.html). J2E cells were transfected
with the Elf-4 ?30P luciferase reporter plasmid and either the
empty expression vector (MigRI), full-length Gfi1b (MigRI-
Gfi1b), or a truncated version of the protein lacking the SNAG
repressor domain (MigRI-Gfi1b-?SNAG) (Fig. 6A). Overexpres-
sion of Gfi1b repressed the activity of the Elf-4 ?30P promoter by
(P ? 0.05) 30% increase in activity of the Elf-4 ?30P promoter
promoter is mediated through the SNAG domain of Gfi1b (Fig.
of Elf-4 to downregulate its expression.
primitive erythrocytes and a failure to produce definitive enucle-
ated erythrocytes (49). Downregulation of ETS factors is also
known to be important for terminal erythroid differentiation (2,
6, 47). Since Gfi1b is able to repress the activity of the main pro-
moter element in the Elf-4 locus and because Elf-4 expression is
almost absent in the erythroid cell line J2E, we next explored the
possibility of a relationship between these two factors. First, we
ropoiesis. Expression levels of Elf-4 and Gfi1b in E14.5 fetal liver
cells were measured by real-time PCR in CD71?/lowTER119?,
CD71?TER119?, and CD71?TER119?cells, corresponding to
populations of increasing maturity along the erythroid differenti-
ation pathway (59) (Fig. 6B, regions I to III, respectively). The
most mature CD71?TER119?population (region IV) consists
largely of enucleated cells (59) and was therefore excluded from
the real-time PCR analysis. During erythroid maturation, the ex-
in expression (Fig. 6C).
Given the known importance of Gfi1b for erythroid develop-
ment, repression of the Elf-4 promoter by Gfi1b, and reciprocal
expression of Gfi1b and Elf-4 during erythroid maturation, we
tuted an important step during erythroid differentiation. To this
end, we ectopically expressed murine Elf-4 in primary E14.5 fetal
liver erythroblasts by using a retroviral vector (MSCV-Elf-4-
PGKprom-puro-IRES-GFP), which also encodes green fluores-
cent protein (GFP) (see Fig. S8 at http://hscl.cimr.cam.ac.uk
/genomic_supplementary.html) and differentiated transduced
cells in vitro (59). Differentiation of transduced cells was moni-
pared to cells transduced with the empty retroviral vector. Un-
FIG 5 Gfi1b binds to the Elf-4 ?30 promoter in HPC7 and J2E cell lines. Density plots obtained from ChIP-Seq reads for Gfi1b in the HPC7 and J2E cell lines
are displayed as UCSC Genome Browser tracks for the Elf-4 locus. Elf-4 candidate regulatory elements are shown as a reference.
Transcriptional Regulation of Elf-4
February 2012 Volume 32 Number 4mcb.asm.org 769
erythroid differentiation was unaffected by retroviral infection.
Analysis was performed on five populations representing the se-
face expression of CD71 and TER119 (Fig. 6D, regions R1 to R5).
Figure 6E shows the results of a representative experiment. Over-
expression of Elf-4 caused significant changes in fluorescence-
activated cell sorting profiles after just 24 h of differentiation, and
FIG 6 Overexpression of Elf-4 delays erythroid maturation during in vitro differentiation of E14.5 fetal liver cells. (A) Repression of the Elf-4 ?30P promoter by
CD71?/lowTER119?; (II) CD71?TER119?; (III) CD71?TER119?; (IV) CD71?TER119?. (C) Expression of Elf-4 and Gfi1b during in vivo terminal erythroid
indicate standard deviations. (D) Representative FACS plots for TER119 and CD71 expression during in vitro differentiation in lineage-negative E14.5 fetal liver cells
Smith et al.
mcb.asm.orgMolecular and Cellular Biology
those changes were more dramatic after 48 h. Overexpression of
0.01) accumulation of more immature cells in R2 and R3 at the
control (Fig. 6E). Together with the transfection and expression
data, these results suggest that Elf-4 constitutes a key downstream
target gene of Gfi1b during its control of erythroid maturation.
The capability of hematopoietic stem cells to remain quiescent,
on a number of different transcription factors. To understand
how these factors work in concert with each other to form the
be essential to identify both the transcription factors involved as
well as their upstream regulators.
The Elf-4 gene encodes an ETS family transcription factor that
is widely expressed in hematopoietic tissues and is an important
regulator of HSC and CD8?T cell proliferation and the differen-
tiation of NK and NK-T cells (24, 25, 58). Using a locus-wide
ChIP-chip array, we have identified five conserved regulatory re-
gions across the Elf-4 locus. Functional analysis of these regions
showed two promoters and two enhancers to be active in hema-
topoietic and endothelial cell lines. Further analysis of these con-
served regions in transgenic mouse embryos identified a tissue-
specific enhancer that specifically directs Elf-4 expression in the
blood and endothelium. By integration with a genome-wide
ChIP-Seq data set, we identified a number of upstream regulators
for Elf-4, and through functional mutation analysis we identify
ETS family transcription factors Fli1, Erg, and Pu.1 as key media-
onstrated that the transcriptional repressor Gfi-1b specifically re-
critical for the normal maturation of primary erythroid cells.
Comparative sequence analysis has been widely used to iden-
tify key regulatory regions for a number of major hematopoietic
regulators (7, 26, 40, 44, 56). However, in the case of Elf-4, it was
only through combining a computational approach with ChIP-
chip analysis for the presence of specific histone marks that we
gene locus that warranted functional validation. Comparative se-
quence analysis still remains a powerful tool for analyzing gene
regulation, but it is becoming increasingly superceded by the cur-
rent advances in experimental techniques such as ChIP-chip and
ChIP-Seq. As it becomes easier to rapidly perform these genome-
scale studies for a number of different histone markers and/or
transcription factors across several different cell types, the identi-
fication of gene regulatory regions and the tissues they may be
opment found that ChIP-Seq for the enhancer-associated protein
While comparative genomics and/or ChIP-chip/ChIP-Seq
have proven to be extremely accurate methods for the identifica-
any given regulatory element can only be determined through in
may not be enough to produce a specific staining pattern for the
gene of interest. This was indeed the case with Elf-4, for which the
failed to show reproducible staining in E11.5 embryos. This in
itself was not unsurprising, as we and others previously observed
that the promoters alone of other key hematopoietic regulators,
50, 52), have very weak or no activity in transgenic embryos.
Moreover, there is increasing evidence that it is the pattern of
histone markers and transcription factor binding across enhanc-
ers rather than promoters that are responsible for cell-type-
explanations for the lack of staining may be that combinations of
promoters and enhancers are required along with testing at other
time points of development. However, a comprehensive analysis
of all combinations of promoters and enhancers at different time
definitive proof that the ?10E region has enhancer activity with a
tissue-specific pattern that is restricted to the blood and endothe-
lium and thus consistent with the major domains of endogenous
Elf-4 expression. Moreover, analysis of histological sections from
embryos expressing the Elf-4 ?10E region suggests that the en-
hancer is only active in a very small proportion of circulating
primitive red bloods cells. Transcriptional programs in blood
stem/progenitor and endothelial cells have long been recognized
to be highly similar, with many regulatory elements showing ac-
tivity in both lineages (13, 26, 27, 38, 39, 44, 50). In view of the
endothelial staining pattern observed for the ?10E region, we
propose that the hematopoietic activity seen for this element is
most likely in progenitor cells rather than mature blood cells.
However, analysis of the Elf-4 ?10E region in transgenic mouse
express the Elf-4 ?10E region.
Potential upstream regulators of the ?10E enhancer element
were identified by integrating a recently published 10-factor
ChIP-Seq data set to our analysis of the ?10E enhancer. Three
Erg were found to be highly enriched at this region, and we dem-
onstrated that the functionally important motifs in the conserved
region of the ?10E enhancer are indeed ETS motifs. The ETS
family of transcription factors have previously been shown to be
important during hematopoietic and endothelial development.
Two of the ETS transcription factors binding the ?10E enhancer,
Fli1 and Erg, have greater homology to each other than to any of
the other ETS family members. In mouse embryos, Fli1 is ex-
pressed in hematopoietic cells and in the developing vasculature
(33), and Fli?/?embryos die around E12.5 from severe hemor-
rhaging (53). Unlike Fli1, loss of Erg has little effect on the vascu-
lature, but embryos show a failure of definitive hematopoiesis
Fli1 and Erg, both transcription factors have been shown to be
indispensable for normal HSC and megakaryocytic homeostasis
(22). Pu.1, the third ETS family member that binds the ?10E
enhancer, has a critical role in the maintenance and expansion of
HSCs in the fetal liver, and Pu.1?/?mice die at E18.5 from com-
plete hematopoietic failure (20). It is also important to consider
Transcriptional Regulation of Elf-4
February 2012 Volume 32 Number 4 mcb.asm.org 771
that as a member of the ETS family of transcription factors, Elf-4
may autoregulate its own expression. However, we have tested all
commercial Elf-4 antibodies available to us and have so far failed
to detect any enrichment on candidate target regions, including
the ?10E enhancer region, and therefore at this time we cannot
definitively state whether Elf-4 autoregulation occurs.
be critical for driving the hematopoietic specificity of several en-
Lmo2 ?75, and the Runx1 ?23 (13, 26, 40). Surprisingly, the
Scl does not bind significantly anywhere across the Elf-4 locus
(data not shown). Recently it has been shown that many
endothelium-specific enhancers contain a FOX:ETS motif that is
synergistically activated by Forkhead and ETS transcription fac-
tors (8). Given that the ?10E enhancer is active in the endothe-
presence of the FOX:ETS motif in the ?10E enhancer, but again,
this motif was absent. Therefore, it appears that the ETS motifs
present in the ?10E enhancer are sufficient to drive the
cannot exclude the possibility that an as-yet-unidentified motif
works in concert with the ETS factors to produce this tissue spec-
Analysis of comprehensive ChIP-Seq data allowed us to iden-
Gfi1b has been shown to be a positive regulator of erythroid dif-
ferentiation and is critical for production of definitive enucleated
expression of Gfi1b in the erythroid cell line K562 induces ery-
throid differentiation that, upon deletion of the SNAG repressor
Gfi1b during erythropoiesis (11) is widely accepted, very little is
allow erythroid differentiation to proceed. Our retroviral expres-
sion experiments now suggest that Elf-4 represents a key Gfi1b
target gene, and it will be interesting to investigate whether Elf-4
also functions as an important target of Gfi1b or its close relative,
Gfi1, in other hematopoietic cell types. Interestingly, following
roblast) stage of fetal liver erythropoiesis, Gfi1b expression itself
decreased as the cells matured toward the RIII (CD71high/
TER119highbasophilic erythroblast) stage of erythroid differenti-
ation, which is consistent with previous observations that Gfi1b
needs to be downregulated for the final maturation of erythroid
cells beyond proerythroblasts (23, 41). In addition to providing
mechanistic insights into Gfi1b control of erythropoiesis, our
tion. Based on the finding that Elf-4?/?HSCs are more quiescent
that Elf-4 functions in a rather generic way to promote prolifera-
ation. Instead, cell numbers were actually reduced in Elf-4-
expressing cultures (data not shown) and showed a pronounced
defect in maturation. Of note, a recent study of T lymphocytes
suggested that Elf-4 suppresses both homeostatic and antigen-
driven proliferation of CD8?T cells (58). Taken together, these
studies highlight the notion that the influence of Elf-4 on the bal-
ance between cell proliferation and quiescence is highly context
dependent. It is likely that these diverse functions of Elf-4 are at
least in part mediated through context-specific protein-protein
target genes. Elucidation of cell-type-specific Elf-4 interaction
partners and of target genes therefore represent promising areas
for future investigation.
Taken together, the data presented here represent not only the
first comprehensive analysis of Elf-4 regulation but also provide
new insights into Elf-4 function within wider hematopoietic reg-
ulatory networks. Using a combination of large-scale experimen-
tal approaches and in vivo functional validation, we have identi-
for Elf-4 expression in hematopoietic progenitor cells and Elf-4
downregulation during erythroid maturation. Moreover, we
etic regulator Elf-4 within the emerging transcriptional networks
some of their more differentiated progeny.
This work was supported by the Leukaemia and Lymphoma Research
Fund (LLR), Cancer Research UK, and the Leukemia and Lymphoma
We thank Richard Auburn at Flychip for printing the custom arrays
ating transgenic embryos. We also thank B. Kee (University of Chicago)
for providing MigRI-Gfi1b and MigRI-Gfi1b-?Snag vectors. The
ENCODE data used in this publication would not have been available
without the work of the members of the ENCODE consortium, and we
data, Michael Snyder (Stanford University), Sherman Weissman (Yale
University) for the RNA polymerase II data, John Stamatoyannopoulos
(University of Washington) for the DNase I data, and Bing Ren (Ludwig
Institute for Cancer Research) for the bone marrow data.
A.M.S., F.J.C.-N., J.S., S.K., R.T.T., N.K.W., R.L.H., and J.-R.L. per-
formed the experiments for our study; A.M.S. and F.J.C-N. analyzed the
results and created the figures; A.M.S., F.J.C.-N., and B.G. designed the
research and wrote the paper.
We declare no competing financial interests.
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