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
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