RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element

ArticleinThe EMBO Journal 30(19):4059-70 · August 2011with38 Reads
Impact Factor: 10.43 · DOI: 10.1038/emboj.2011.285 · Source: PubMed
Abstract

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

Full-text

Available from: Constanze Bonifer
RUNX1 regulates the CD34 gene in
haematopoietic stem cells by mediating
interactions with a distal regulatory element
Elena Levantini
1,2,
*, Sanghoon Lee
3
,
Hanna S Radomska
1,2,11
,ChristopherJ
Hetherington
1,2
, Meritxell Alberich-Jorda
1,2
,
Giovanni Amabile
1,2
,PuZhang
1,2
,
David A Gonzalez
1,2
, Junyan Zhang
1,2
,
Daniela S Basseres
4
,NicolaKWilson
5
,
Steffen Koschmieder
6
, Gang Huang
7
,
Dong-Er Zhang
8
, Alexander K Ebralidze
1,2
,
Constanze Bonifer
9
, Yutaka Okuno
10
,
Bertie Gottgens
5
and Daniel G Tenen
2,3,
*
1
Division of Hematology/Oncology, Beth Israel Deaconess Medical
Center, Center for Life Science, Boston, MA, USA,
2
Harvard Stem Cell
Institute, Harvard Medical School, Boston, MA, USA,
3
Cancer Science
Institute, National University of Singapore, Singapore,
4
Department of
Biochemistry, Chemistry Institute, University of Sao Paulo, Sao Paulo,
Brasil,
5
Department of Haematology, Cambridge Institute for Medical
Research, University of Cambridge, Cambridge, UK,
6
Department of
Medicine A (Hematology, Oncology and Pneumology), Muenster
University, Muenster, Germany,
7
Division of Pathology, Experimental
Hematology and Cancer Biology, Children’s Hospital Medical Center,
Cincinnati, OH, USA,
8
Department of Pathology, University of
California, San Diego School of Medicine, San Diego, CA, USA,
9
Department of Experimental Haematology, Leeds Institute of Molecular
Medicine, University of Leeds, St James’s University Hospital, Leeds, UK
and
10
Department of Hematology, Kumamoto University of Medicine,
Kumamoto, Japan
The transcription factor RUNX1 is essential to establish the
haematopoietic gene expression programme; however, the
mechanism of how it activates transcription of haemato-
poietic stem cell (HSC) genes is still elusive. Here, we
obtained novel insights into RUNX1 function by studying
regulation of the human CD34 gene, which is expressed in
HSCs. Using transgenic mice carrying human CD34 PAC
constructs, we identified a novel downstream regulatory
element (DRE), which is bound by RUNX1 and is neces-
sary for human CD34 expression in long-term (LT)-HSCs.
Conditional deletion of Runx1 in mice harbouring human
CD34 promoter–DRE constructs abrogates human CD34
expression. We demonstrate by chromosome conformation
capture assays in LT-HSCs that the DRE physically inter-
acts with the human CD34 promoter. Targeted mutagen-
esis of RUNX binding sites leads to perturbation of this
interaction and decreased human CD34 expression in
LT-HSCs. Overall, our in vivo data provide novel evidence
about the role of RUNX1 in mediating interactions be-
tween distal and proximal elements of the HSC gene CD34.
The EMBO Journal (2011) 30, 4059–4070. doi:10.1038/
emboj.2011.285; Published online 26 August 2011
Subject Categories : chromatin & transcription; development
Keywords: CD34; gene regulation; haematopoietic stem cell;
long-range chromatin looping; RUNX proteins
Introduction
Understanding how transcription factors and cis-regulatory
elements set up long-range interactions to orchestrate gene
expression is a key issue in genome biology. Since the initial
observation reporting chromatin looping at the b-globin gene
locus (Carter et al, 2002; Tolhuis et al, 2002), similar inter-
actions have been shown to occur between promoters and 5
0
and/or 3
0
UTRs of many genes, and even trans-interactions
between loci on different chromosomes have been reported
(Chen et al, 1998; Brown et al, 2002; Ling et al, 2006;
Marenduzzo et al, 2007; Vernimmen et al, 2007; Barnett
et al, 2008; Chavanas et al, 2008; Liu et al, 2009; Theo
Sijtse Palstra, 2009; Boney-Montoya et al, 2010). A model in
which intervening inactive chromatin lying between distant
elements is looped out, and forms an active chromatin hub,
has been proposed (Patrinos et al, 2004; Theo Sijtse Palstra,
2009). Structural proteins, transcription factors, or compo-
nents of the preinitiation complex have been implicated as
candidate molecules mediating chromatin looping (Kim et al,
2007, 2009; Marenduzzo et al, 2007; Williams et al, 2007;
Liu et al, 2009; Theo Sijtse Palstra, 2009; Deshane et al, 2010).
However, while roles for individual factors in long-range
interactions have been identified, little is known about the
role of individual factor binding sites on the stability of these
interactions and how the lack of such binding sites would
impact on gene expression. In addition, it is still unknown
how these mechanisms operate in stem cells.
The best-characterized adult stem cells are haematopoietic
stem cells (HSCs), which are capable of life-long self-renewal
and generation of the full spectrum of mature blood cells.
It was recently revealed that a specific combination of trans-
cription factor binding sites (GATA, ETS and SCL/TAL1) was
able to mediate HSC-specific expression (Gottgens et al, 2002;
Pimanda et al, 2007). One additional factor that is essential
for the formation of HSCs and the establishment of a blood-
cell-specific gene expression programme in the embryo is
RUNX1 (Okuda et al, 1996; Okada et al, 1998; Cai et al, 2000;
Ichikawa et al, 2004a, b, 2008; Growney et al, 2005; Putz et al,
2006; Chen et al, 2009; Hoogenkamp et al, 2009; Jacob and
Osato, 2009). However, while the importance of RUNX1 in
the establishment of the expression of haematopoietic genes
Received: 22 April 2011; accepted: 19 July 2011; published online:
26 August 2011
*Corresponding authors. E Levantini, Beth Israel Deaconess Medical
Center, Center for Life Science, Room 428, 3 Blackfan Circle, Boston,
MA 02115, USA. Tel.: þ 1 617 735 2233; Fax: þ 1 617 735 2222;
E-mail: elevanti@bidmc.harvard.edu or
DG Tenen, Center for Life Science, Room 437, 3 Blackfan Circle, Boston,
MA 02115, USA. Tel.: þ 1 617 735 2235; Fax: þ 1 617 735 2222;
E-mail: dtenen@bidmc.harvard.edu
11
Present address: Comprehensive Cancer Center, Ohio State University,
Medical Center, Columbus, OH, USA
The EMBO Journal (2011) 30, 4059–4070
|
&
2011 Europe an Molecular Biology Organization
|
All Rights Reserved 0261-4189/11
www.embojournal.org
& 2011 European Molecular Biology Organization The EMBO Journal VOL 30
|
NO 19
|
2011
EMBO
THE
EMBO
JOURNAL
THE
EMBO
JOURNAL
4059
Page 1
is well characterized, little is known about the mechanistic
details of its role in activating the actual transcription of these
genes, particularly at the HSC level. To gain further insights
into the role of specific transcription factors regulating HSC-
specific gene expression, we studied the regulation of the
human CD34 (hCD34) gene locus. The hCD34 antigen is
specifically expressed on human HSCs and early progenitors
(Krause et al, 1996), and has been successfully used as a
marker to identify, enrich and purify HSCs in clinical trans-
plantation settings (Dunbar et al , 1995; Galy et al, 1995;
Stella et al, 1995; Link et al, 1996; Michallet et al, 2000).
Identification of the hCD34 promoter revealed the presence of
functional binding sites for c-myb, ets-2, MZF-1, Sp1, Sp3 and
NFY (Melotti and Calabretta, 1994; Morris et al, 1995; Perrotti
et al, 1995; Radomska et al, 1999). However, neither the
promoter alone or in combination with a later-identified 3
0
enhancer is sufficient to drive hCD34 expression in cell
lines and/or transgenic mice (Radomska et al, 1998, 2002).
In contrast, PAC clones carrying the entire hCD34 gene on a
PvuI fragment with 18.3 kb of 5
0
- and 25.6 kb of 3
0
-flanking
regions contain the complete set of critical control elements
necessary to direct hCD34 expression in functional HSCs
(Okuno et al, 2002a, b). We have previously shown that
murine LT-HSCs are highly enriched in the murine CD34
/low
hCD34
þ
fraction of the LSK population (lineage
; Sca1
þ
;
c-kit
þ
cells), suggesting that the human and murine CD34
genes are differently regulated in LT-HSCs (Okuno et al,
2002b).
To identify the critical regulatory elements required for
hCD34 expression in LT-HSCs, we generated different
transgenic mouse lines carrying various combinations of
hCD34 genomic elements. The work described here identifies
a novel regulatory element located at þ 19 kb, the down-
stream regulatory element (DRE), which is necessary for
hCD34 expression in LT-HSCs. The DRE contains four
binding sites for RUNX together with other binding sites for
factors known to be active in stem cells. Experiments
with conditional Runx1 knockout mice demonstrate that
the presence of RUNX1 is essential for the activity of this
element. By chromosome conformation capture (3C) analysis
performed in LT-HSCs, we demonstrate that the DRE
physically interacts with the hCD34 promoter through the
RUNX binding sites.
Our data are the first to demonstrate a role of a specific
transcription factor in establishing chromatin looping in
primary stem cells; they show that specific transcription
factor binding sites are required for interactions between
distal and proximal regulatory elements in vivo, and expand
the role of RUNX1 in facilitating looping of distant regulatory
elements in HSCs.
Results
A genomic region located between þ 17.4 and þ 19.6 kb
of the hCD34 gene is necessary for its expression
in LT-HSCs
In order to identify which elements mediate expression of
hCD34 in LT-HSCs, we generated different PAC constructs
containing deletions of the 3
0
-flanking region in the context of
the original B70 kb PvuI fragment containing all necessary
cis-elements, as depicted in Figure 1A. Multiple founder
lines for each transgenic construct were obtained, and
hCD34 antigen expression was quantified on LT-HSCs
(CD48
SLAM/CD150
þ
LSK cells; Kiel et al, 2005) using
flow cytometry analysis of bone marrow cells from transgenic
mice. This showed that all the LT-HSCs from mice carrying
either 25.6 kb (construct A) or 19.6 kb (construct B) of the
3
0
-flanking region expressed the hCD34 antigen on the cell
surface (Figure 1B). Mice carrying construct A displayed
99.2
±
0.5% (s.d.) of hCD34
þ
LT-HSCs, with a mean fluores-
cence intensity (MFI) of 8617
±
125 (s.d.); mice transgenic for
construct B displayed 99.1
±
0.8% (s.d.) of hCD34
þ
LT-HSCs,
with an MFI of 8611
±
95 (s.d.). These values were obtained
with all three founder lines transgenic for construct
A (copy number ranged 3–5 per genome) and in the eight
founder lines carrying construct B (copy number ¼ 2–6).
An additional deletion of 2.2 kb achieved in mice carrying
17.4 kb of 3
0
-flanking region (construct C) was sufficient to
completely abolish hCD34 expression in vivo in SLAM
þ
LSKs, in the three lines transgenic for construct C (copy
number ¼ 2–7; Figure 1A and B). These studies demonstrated
that critical cis-regulatory element(s) are located between
þ 17.4 and þ 19.6 kb. By conducting a computational se-
quence analysis of the 2.2-kb region, we identified multiple
putative ‘stem cell-related’ transcription factor binding sites,
such as two potential E-Box/GATA paired motifs, known
to bind SCL/LMO2/GATA2 (Wadman et al, 1997), and four
potential RUNX binding sites (Figure 1C). These sites were
located in a 0.8-kb region, spanning from þ 18.8 to
þ 19.6 kb, which we named the DRE (Figure 1C).
The DRE is necessary and sufficient for hCD34
gene expression in SLAM
þ
LSKs
In order to assess the functional role of the DRE in LT-HSCs,
we generated construct D (Figure 2A), which contains a
deletion of the DRE in the context of the 25.6-kb 3
0
-flanking
sequence (construct A). All transgenic lines carrying this
construct failed to express hCD34 in SLAM
þ
LSKs in vivo
(Figure 2B), demonstrating that the DRE is necessary for
expression of hCD34 in LT-HSCs. Three independent founder
lines were obtained for construct D, with copy numbers
per genome ranging between 3 and 7. To test whether the
DRE is sufficient for hCD34 expression in vivo, we placed it as
the sole regulatory element 3
0
of the hCD34 gene (construct E;
Figure 2C). Majority of SLAM
þ
LSKs from mice carrying
construct E exhibited high levels of hCD34 expression
(98.8
±
0.4% (s.d.)), with an MFI of 8603
±
68 (s.d.;
Figure 2D). These values, obtained in nine independent
transgenic lines (copy number ¼ 3–7), were not statistically
different from the percentages observed in LT-HSCs from
mice carrying construct A (P ¼ 0.15). Therefore, the DRE is
required for expression in LT-HSC.
We next investigated whether expression of hCD34
in phenotypic SLAM
þ
LSKs correlated with long-term (LT)
haematopoietic reconstitution capacity. Bone marrow trans-
plantation assays demonstrated the LT-reconstitution poten-
tial of FACS-purified hCD34
þ
SLAM
þ
LSKs from hCD34
þ
transgenic mice carrying either 25.6 kb (construct A), 19.6 kb
(construct B) or the sole DRE as 3
0
element (construct E),
indicating that sequences containing the DRE are sufficient
to drive hCD34 expression in functional LT-HSCs
(Supplementary Table S1). Lethally irradiated mice that did
not receive any transplanted bone marrow cells succumbed
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
The EMBO Journal VOL 30
|
NO 19
|
2011 & 2011 European Molecular Biology Organization4060
Page 2
few weeks after irradiation, as expected. Therefore, the
DRE is capable of mediating expression of hCD34 in func-
tional LT-HSC.
RUNX1 binds to the DRE
RUNX proteins are transcription factors whose importance
has been well established in haematopoiesis. In particular,
Construct
name
Founder
lines
A
3
PvuI
PvuI
18.3 kb 24.8 kb 25.6 kb
AscII
Bsi WI
19.6 kb
B
8
C
3
PvuI
Fsp I
17.4 kb
18.3 kb 24.8 kb
18.3 kb
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
24.8 kb
hCD34 in
SLAM
+
LSKs
Yes
Yes
No
A
A
C
LSK
SLAM
+
250K
200K
150K
100K
50K
250K
200K
150K
100K
50K
250K
200K
150K
100K
50K
hCD34 in SLAM
+
B
sca-1
c-kit
CD150
CD48
hCD34
FSC
99.3
0.1
1.9
98.6
9.2
9.6
1.4
B
1.2
7.9
+19.6 kb
+18.8 kb
+17.4 kb
R
U
NX
RUNX
RUNX
RUNX
E
-
Box/GATA
E
-
Box/GATA
+19.6 kb
DRE
3241
0.8 kb
C
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
Figure 1 A genomic region located between þ 17.4 and þ 19.6 kb of the human CD34 gene is necessary for its expression in SLAM
þ
LSKs.
(A) Diagram of human CD34 genomic fragments used in transgenic mice. All fragments (A–C) were derived from PAC54A19 and digested as
indicated. Thick vertical lines represent the eight hCD34 coding exons. (B) Flow cytometry was performed on bone marrow cells from
transgenic mice carrying constructs A–C. Cells were stained with anti-mouse c-kit and sca-1 antibodies to identify the LSK (lineage
; Sca1
þ
;
and c-kit
þ
population), as indicated in the left panels; anti-murine CD48 and CD150/SLAM antibodies were used to identify the CD150 single
positive population (SLAM
þ
cells) contained within the LSK compartment, as indicated in the middle panels; and anti-hCD34 antibody was
used to quantify the amount of hCD34
þ
cells contained within the SLAM
þ
population (right panel). The percentage of the gated populations is
noted. All plots display a representative example from each of the three different transgenic lines. T-test (two-tailed; type 3) showed that the
data, obtained in three independent transgenic lines from construct A (n ¼ 3 per founder, a part from one founder in which we did not observe
germ line transgene transmission), were not statistically different (P ¼ 0.55) from the percentages observed in eight independent transgenic
mice for construct B (n ¼ 3 per founder, a part from 1 founder that did not display germ line transmission) and were instead statistically
different (P ¼ 2.2, E22) from three independent transgenic lines from construct C (n ¼ 3 per founder). (C) In silico sequence analysis was
carried out to identify regions of interest contained in the 2.2-kb genomic region spanning from þ 17.4 and þ 19.6 kb. The most 3
0
0.8 kb,
identified here as the downstream regulatory element (DRE), contains four RUNX sites and two E-Box/GATA motifs.
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
& 2011 European Molecular Biology Organization The EMBO Journal VOL 30
|
NO 19
|
2011 4061
Page 3
RUNX1 is required for HSC generation (Cai et al, 2000; Chen
et al, 2009), and inactivation of RUNX1 has been indicated to
affect the HSC pool (Ichikawa et al, 2008; Jacob and Osato,
2009). Furthermore, RUNX1 is expressed in LT-HSCs
(Supplementary Figure S1), leading us to hypothesize that
RUNX1 could be a mediator of hCD34 expression in those
cells. Therefore, we tested the ability of RUNX1 to bind
to the predicted consensus sites identified within the DRE
(Figure 1C). Quantitative ChIP assay performed on human
primary umbilical cord blood cells (495% CD34
þ
) showed
that RUNX1 binds to a genomic DNA region containing RUNX
consensus sites #1–3 and site #4, whereas we did not detect
RUNX1 binding to the corresponding sequences in the
hCD34
cell line HL60 (Figure 3A). In addition, we detected
histone H3 acetylated at lysines 9 and 14 of histone
H3 (aH3-Ac) at sites #1–3 and #4 in cord blood cells,
(Figure 3B). As histone acetylation is regarded as one of the
epigenetic marks accompanying active chromatin (Roth and
Sweatt, 2009), these data provide functional evidence that
these sites are involved in an open chromatin complex in
cord blood cells.
RUNX1 regulates hCD34 expression in LT-HSCs
through the DRE
To specifically address whether RUNX1 is involved in vivo in
regulating hCD34 expression in LT-HSCs, we conditionally
B
0
sca-1
c-kit
hCD34
FSC
CD150
CD48
LSK SLAM
+
A
D
Construct
name
hCD34 in
SLAM
+
LSKs
No
Founder
lines
3
Pvu I
Pvu I
18.3 kb 24.8 kb
25.6 kb
V
DRE
D
D
sca-1
c-kit
1.2
10.2
hCD34
FSC
CD150
CD48
C
AscII
Bsi WI
Construct
name
Founder
lines
E
9
0.8 kb18.3 kb 24.8 kb
hCD34 in
SLAM
+
LSKs
Yes
DRE
1.9
7.5
98.6
hCD34 in SLAM
+
LSK
SLAM
+
hCD34 in SLAM
+
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
250K
200K
150K
100K
50K
10
1
10
2
10
3
10
4
10
5
10
5
10
4
10
3
10
2
10
1
10
1
10
2
10
3
10
4
10
5
10
5
10
4
10
3
10
2
10
1
10
1
250K
200K
150K
100K
50K
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
10
1
10
2
10
3
10
4
10
5
Figure 2 The DRE is necessary and sufficient for human CD34 expression in SLAM
þ
LSKs. (A) Construct A (described in Figure 1) was
modified to specifically delete the DRE (construct D). (B) Bone marrow cells from mice carrying construct D have been analysed by flow
cytometry, performed as described in Figure 1. Deletion of the DRE is sufficient to abolish hCD34 expression in SLAM
þ
LSKs (right panel).
A representative FACS plot from one of the three founder lines is shown. T-test (two-tailed; type 3) indicated that the data obtained in three
independent transgenic lines from construct D (n ¼ 3 per founder) were statistically different (P ¼ 2.8, E21) from the percentages observed in
nine independent transgenic mice for construct A (Figure 1). (C) Construct A was engineered to contain the DRE as the sole sequences 3
0
of the
human CD34 gene (construct E). (D) Flow cytometry of bone marrow cells from mice carrying construct E. The DRE is sufficient as a sole
3
0
element to drive hCD34 expression in SLAM
þ
LSKs. A representative FACS plot from one of the nine founder lines is shown. T-test (two-
tailed; type 3) showed that the percentages observed in nine independent mice transgenic for construct E (n ¼ 3 per founder, a part from two
founders in which we did not observe germ line transgene transmission) were not statistically different from the data obtained in mice carrying
construct A (P ¼ 0.15). See also Supplementary Table S1.
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
The EMBO Journal VOL 30
|
NO 19
|
2011 & 2011 European Molecular Biology Organization4062
Page 4
inactivated it in adult haematopoietic cells. hCD34 transgenic
mice carrying construct E (Figure 2C) were bred to condi-
tional Runx1 knockout mice (Runx1
F/F
; Growney et al, 2005)
and Mx1-Cre mice (Kuhn et al, 1995) to obtain interferon-
inducible Runx1 gene excision (Figure 4A). One month after
administration of seven doses of polyinosinic-polycytidylic
acid (PIPC), FACS analysis was conducted on PIPC-treated
Mx1-Cre
þ
/hCD34
þ
/Runx1
F/F
mice (hCD34
þ
Runx1 KO mice)
and control littermates (either Mx1-Cre
/hCD34
þ
/Runx1
F/F
mice or Mx1-Cre
þ
/hCD34
þ
/Runx1
wt/wt
mice, both referred to
as hCD34
þ
Runx1 WT mice). Flow cytometry showed almost
complete abolishment of hCD34 expression in SLAM
þ
LSKs
of hCD34
þ
Runx1 KO mice as compared with PIPC-treated
hCD34
þ
Runx1 WT mice (Figure 4B). Only 1.8
±
0.7% (s.d.)
of Runx1 KO LT-HSCs were positive for hCD34 (MFI 286
±
25
(s.d.)) compared with 98.3
±
0.5% (s.d.) of LT-HSCs from
hCD34
þ
Runx1 WT mice (MFI 8569
±
112 (s.d.)). Percentages
observed in either Mx1-Cre
/hCD34
þ
/Runx1
F/F
mice or Mx1-
Cre
þ
/hCD34
þ
/Runx1
wt/wt
mice were not statistically differ-
ent (P ¼ 0.4). Absence of RUNX1 expression in KO mice was
verified by Q-RT–PCR (Supplementary Figure S2). As disrup-
tion of RUNX1 resulted in loss of hCD34 expression, we
conclude that RUNX1 is a critical transcription factor acting
through the DRE to regulate hCD34 gene expression in
LT-HSCs.
The DRE physically interacts with the hCD34 promoter
The results described above point to a crucial role of RUNX1
with respect to driving hCD34 transcription in LT-HSCs. It has
been previously shown that high-level transcription requires
the interaction of enhancers with promoter elements.
Expression of transcription factors such as EKLF is required
to mediate these interactions (Chen et al, 1998; Brown et al,
2002; Pilon et al, 2006; Rathke et al, 2007; Chan et al , 2008;
Gavrilov and Razin, 2008). Moreover, recent experiments in T
cells indicate that RUNX is required to mediate the close
proximity of Cd4 and Cd8 genes within the intranuclear space
(Collins et al, 2011). However, this study did not address the
question of chromatin looping. To test directly whether
RUNX1 is involved in hCD34 promoter–DRE communication,
we performed 3C assays (Figure 5A) and assessed chromatin
looping in two human haematopoietic cell lines, KG1a
(CD34
þ
) and HL60 (CD34
). In parallel, we assessed
hCD34 and Runx1 expression status (Supplementary Figure
S3a–c). The frequency of interaction was enhanced
6.18
±
0.02 (s.d.) times in hCD34
þ
cells versus hCD34
cells (Figure 5B). By performing 3C assays on FACS-purified
SLAM
þ
LSKs (hCD34
þ
) and lin
þ
cells (hCD34
) from mice
carrying construct A (Figure 1), whose high purity was
verified by resorting analysis (Supplementary Figure S3d),
we demonstrated that the frequency of the DRE–promoter
interaction correlates with the levels of hCD34 expression
(Figure 5C). In particular, hCD34
þ
SLAM
þ
LSKs showed a
10-fold increase in the DRE–promoter interaction as com-
pared with hCD34
lin
þ
cells (Figure 5C). In primary umbi-
lical cord blood cells, the frequency of the DRE–promoter
interaction follows a similar pattern, being 8.7-fold higher in
FACS-purified hCD34
þ
cells compared with hCD34
cells
(Figure 5D and Supplementary Figure S3e). Overall, our
in vivo data indicate that the DRE physically interacts with
the hCD34 promoter in LT-HSCs and in primary hCD34
þ
cord
blood cells, and that the DRE is a critical element required
for hCD34 gene expression in these cells.
RUNX binding sites within the DRE are critical
for hCD34 expression in SLAM þ LSKs
To determine in vivo whether the activity of the DRE in
LT-HSCs is mediated through the RUNX binding sites, we
specifically mutated these sites (construct F; Figure 6A).
Abrogation of RUNX1 binding to the mutated sites has
been verified by electrophoretic mobility shift assays on
293T cells transiently overexpressing FLAG-tagged RUNX1
(Supplementary Figure S4a and b). Mice carrying construct
F showed a decreased percentage of hCD34 expression in
SLAM
þ
LSKs and decreased MFI (Figure 6B), pointing to the
importance of the RUNX1 binding sites for hCD34 expression
in LT-HSCs. In particular, 38.1
±
4.2% (s.d.) of LT-HSCs
expressed hCD34 (versus 99.2
±
0.5% of LT-HSCs from mice
carrying construct A), with an MFI of 2342
±
189 (versus
8617
±
125 in LT-HSCs from mice carrying construct A). Six
independent lines were generated (copy number ¼ 2–6).
In contrast, when we deleted the neighbouring E-Box/GATA
motifs (construct G; Supplementary Figure S4c), none of the
transgenic lines was affected in its ability to express hCD34 in
LT-HSCs (Supplementary Figure S4d). Values obtained on the
11 transgenic lines (copy number ¼ 2–9) showed that
98.9
±
0.4% of LT-HSCs express hCD34 with an MFI of
Fold enrichment
normalized to input
1
2
3
4
P=0.0003 P =0.0001
P =0.3
Runx1
IgG
Runx1
IgG
CB
HL60
P =0.000001
P=0.000006
P =0.02
H3-Ac
IgG
H3-Ac
IgG
CB
HL60
Fold enrichment
normalized to input
20
30
40
50
10
DRE 1–3
B
A
Myf-5 DRE 4
DRE 1–3 Myf-5 DRE 4
Figure 3 RUNX1 binds to the RUNX sites contained within the DRE.
(A) Quantitative ChIP analysis was performed on primary human
cord blood (CB) cells (495% hCD34
þ
) and HL60 cells (hCD34
).
Primers and probes encompassing RUNX sites 1–3, and RUNX site
4, were used to amplify input genomic DNA, and DNA precipitated
by antibodies against either normal IgG or Runx1 or histone 3
acetylated at lysines 9 and 14 (aH3-Ac). The Myf5 promoter was
used as a negative control sequence. Values are normalized to input
genomic DNA. The panel shows the values of fold enrichment
obtained with anti-Runx1 antibodies (black columns for cord
blood & and dotted columns for HL60
) compared with IgG
(white columns for cord blood & and diagonally barred columns
for HL60
), used as a negative control. Values obtained for the fold
enrichment in cord blood cells are statistically different, as shown
(bars indicate standard deviations). (B) The panel shows the
values of fold enrichment obtained with an antibody against aH3-
Ac (black columns for cord blood & and dotted columns for HL60
); IgG (white columns for cord blood & and diagonally barred
columns for HL60
) served as control for the assay specificity.
Values obtained for the fold enrichment in cord blood cells are
statistically different, as indicated. All the data are representative of
three independent experiments performed in duplicate. See also
Supplementary Figure S1.
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
& 2011 European Molecular Biology Organization The EMBO Journal VOL 30
|
NO 19
|
2011 4063
Page 5
8615
±
130, indicating that these binding sites are not
required for hCD34 expression.
To address the effect of mutations in RUNX binding sites
at the chromatin level, we FACS purified the LSK-HSCs into
hCD34
low
and hCD34 intermediate (hCD34
int
) populations
from mice carrying construct F, according to the gating
strategy utilized throughout this study to define hCD34
and hCD34
þ
cells (see Figures 1B, 2B, D and 4B), and
performed 3C assays (Figure 6C). A 5.2-fold decrease in the
frequency of the DRE–promoter interaction was observed in
hCD34
low
HSCs versus hCD34
int
HSCs. These data show that
the levels of hCD34 expression parallel the frequency of the
promoter–DRE interaction (Figure 6C), which is affected
when RUNX binding sites are mutated (Figure 6A). Levels
of hCD34 expression in hCD34
low
and hCD34
int
HSCs from
mice carrying construct F have been normalized to the
expression observed in LSK-HSCs from mice transgenic for
construct A (Figure 6C), in which the RUNX binding sites are
intact. In particular, hCD34
low
LSK-HSCs from mice carrying
construct F express 8%, and hCD34
int
LSK-HSCs express 25%
of the levels measured in LSK-HSCs from mice carrying
construct A (Figure 6C).
Overall, these in vivo data show that to achieve hCD34
expression, an optimal promoter–DRE interaction is required
to occur through intact RUNX binding sites.
Discussion
To define genomic regulatory elements required to achieve
gene expression in LT-HSCs, we focused on the regulation
of the human CD34 gene, which encodes a surface marker for
HSCs (Krause et al, 1996). Previously, we identified an
element just downstream from exon 8 of the CD34 gene,
which exhibited enhancing activity in CD34
þ
cell lines in
in vitro assays (Burn et al, 1992). However, subsequent
studies showed that this 3
0
enhancer in combination
with the CD34 gene promoter lacked activity when stably
integrated into the chromatin (Radomska et al, 1998), and
furthermore, additional long-range element(s) were found to
be critical for tissue-specific human CD34 gene expression
in vivo (Radomska et al, 1998; Okuno et al, 2002a). To map
these elements precisely, we generated a series of transgenic
mice carrying the hCD34 genomic locus with different
combinations of flanking sequences. The transgenic mouse
experiments described here identified a 3
0
distal cis-regulatory
element (DRE) that is necessary to drive hCD34 expression in
LT-HSCs and that depends on RUNX1 for its activity. When
comparing the human DRE sequence with corresponding
sequences from other vertebrates, such as rhesus and
mouse, of the four RUNX1 binding sites present on the
human sequence, only site 2 is conserved between these
species, whereas the remaining sites are only present in
humans and rhesus, and not in mouse (Supplementary
Figure S6). Interestingly, the entire set of four RUNX1 binding
sites is only present in those species in which CD34 is
expressed on LT-HSCs (Supplementary Figure S6), hinting
to the possibility that differences in regulatory elements
might account for the differential expression of CD34 on
human versus murine HSCs. Despite intensive investigation,
the function of the CD34 molecule on HSCs is still obscure,
and the possibility that CD34
cells are capable of
LT reconstitution of the haematopoietic system has been
LSK SLAM
+
B
hCD34
+
Runx1 WT
hCD34
+
Runx1 KO
sca -1
c-kit
hCD34 in SLAM
+
CD150
CD48
4.4
7.8
1.9
7.8
98.6
1.3
hCD34
FSC
X
Tg E hCD34
18.3 kb 5/DRE3
Mx1-Cre-Runx1
F/F
PIPC (0.8 mg)
A
7 Injections every
other day
1 month
FACS
analysis
10
5
10
5
10
4
10
4
10
3
10
3
10
2
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
250K
200K
150K
100K
50K
250K
200K
150K
100K
50K
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
Figure 4 RUNX1 regulates human CD34 expression in SLAM
þ
LSKs. (A) Schematic of the breeding strategy used to obtain hCD34
þ
mice
(hCD34
18.3kb5
0
/DRE3
0
) with conditional deletion of Runx1. hCD34 transgenic mice carrying construct E were bred to conditional Runx1 knockout
(KO) mice (Runx1
F/F
) and Mx1-Cre mice, to obtain interferon-inducible Runx1 gene excision. Mice have been treated with seven injections of
PIPC and analysed 1 month later. (B) Flow cytometry analysis on bone marrow cells from PIPC-treated hCD34
þ
Runx1 WT or hCD34
þ
Runx1
KO mice. Representative FACS plots obtained from one WT and one KO mouse are shown. T-test (two-tailed; type 3) showed that
the percentages observed in hCD34
þ
Runx1 WT (n ¼ 6) were statistically different (P ¼ 6.7, E19) from the data obtained in hCD34
þ
Runx1 KO mice (n ¼ 6). See also Supplementary Figure S2.
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
The EMBO Journal VOL 30
|
NO 19
|
2011 & 2011 European Molecular Biology Organization4064
Page 6
C
hCD34
lin
+
cellshCD34
+
SLAM
+
LSKs
Promoter–DRE relative
cross-linking frequency
normalized to hCD34
+
SLAM
+
LSKs
1
0.5
10 ×
hCD34 expression
normalized to the levels
detected in hCD34
+
SLAM
+
LSKs
1
0.5
D
hCD34
cord bloodhCD34
+
cord blood
Promoter–DRE relative
cross-linking frequency
normalized to hCD34
+
cord blood cells
1
0.5
8.7 ×
hCD34 expression
normalized to the levels
detected in hCD34
+
cord blood cells
1
0.5
Pr–6Pr–5Pr–4Pr–3Pr–2Pr–1
1
2
3
4
5
6
KG1a (hCD34
+
)
HL60 (hCD34
)
Relative cross-linking
frequencies
B
6.2 ×
Pr
12.8 kb
11.3 kb
10.4 kb
7.3 kb
4.1 kb
H3 H3 H3 H3 H3 H3
3.1 kb
H3
34 5621
H3
12.9 kb
A
H3H3
4.7 kb 24.8 kb 20.7 kb
DRE
7.4 kb
4.9 kb
Pr–DRE
Figure 5 The DRE interacts with the human CD34 promoter in SLAM
þ
LSKs. (A) Diagram showing the genomic position of the hCD34
promoter (Pr); the eight coding exons (thick vertical lines); the DRE; and location of the HindIII (H3) restriction enzyme sites (vertical arrows)
used to perform the 3C assay. The block arrows represent the interaction of the Pr fragment (7.4 kb) with the DRE fragment (4.9 kb) and with
six other fragments located in the 3
0
-flanking region and upstream of the DRE (block arrows 1–6). The distance of each H3 fragment from the
DRE is indicated in kb (vertical numbers). (B) Relative crosslinking frequency of the Pr with the DRE and six other 3
0
fragments located
upstream of the DRE. Crosslinking frequency indicates how frequently distal genomic elements interact. Interaction of the 3
0
fragments and the
DRE with the Pr are rare in HL60 cells (hCD34
), whereas they are increased in frequency in KG1a cells (hCD34
þ
), with the DRE showing a
6.2-fold enrichment in the relative crosslinking frequency over the hCD34
HL60 cell line (n ¼ 4 per cell line; two independent 3C
experiments). (C) On the left panel, the relative crosslinking frequency of the Pr and DRE in hCD34
þ
SLAM
þ
LSKs (n ¼ 4; black column; &)
and hCD34
lin
þ
cells (n ¼ 4; white column; &) from mice carrying construct A, as normalized to the levels in hCD34
þ
SLAM
þ
LSKs, is
indicated. Interaction of the DRE with the Pr is rare in hCD34
cells, whereas a 10-fold enrichment is observed in hCD34
þ
LT-HSCs. Only the
relative crosslinking frequency between the Pr and the DRE has been analysed, given the small number of HSCs per mouse. On the right panel,
Q-RT–PCR data verifying the expression levels of hCD34 in the SLAM
þ
LSKs and the lin
þ
cells utilized for the 3C assay are shown. Sorted
SLAM
þ
LSKs have been pooled from a total of 25 mice transgenic for construct A. (D) On the left panel, the relative crosslinking frequency of
the Pr and DRE in hCD34
þ
cord blood cells (n ¼ 4; black column; &) and hCD34
cord blood cells (n ¼ 4; white column; &), as normalized to
the levels in hCD34
þ
cord blood cells, is indicated. Interaction of the DRE with the Pr is weaker in hCD34
cells, whereas an 8.7-fold
enrichment is observed in hCD34
þ
cells. On the right panel, Q-RT–PCR data verifying the expression levels of hCD34 in the cord blood
populations utilized for the 3C assay are shown. See also Supplementary Figure S3.
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
& 2011 European Molecular Biology Organization The EMBO Journal VOL 30
|
NO 19
|
2011 4065
Page 7
reported in the mouse (Zanjani et al, 1998, 1999, 2003).
In addition, the activation status of the cells appears to
influence the expression of the CD34 molecule (Sato et al ,
1999), implying that identifying the molecular mechanisms
regulating CD34 expression may contribute to understand
how HSC gene regulation is achieved and modulated during
homoeostasis, activation and stress-induced status.
Our data suggest that RUNX1 is crucially required for
hCD34 promoter–DRE interactions, and that other transcrip-
tion factors in the DRE cannot fully compensate for its
absence. The specific requirement of RUNX1 in the context
of the hCD34 DRE is also underlined by lack of any significant
effect on hCD34 gene expression when neighbouring GATA
and SCL/TAL1 binding sites were deleted in the DRE,
hCD34 in SLAM
+
B
LSK SLAM
+
2.7
F
7.8
sca-1
c-kit
CD150
CD48
hCD34
FSC
hCD34 in LSKs
41.1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
250K
200K
150K
100K
50K
hCD34
int
LSK-HSCs
hCD34
low
LSK-HSCs
0.5
0.25
Promoter–DRE relative cross-
linking frequency normalized to
hCD34
+
LSK-HSCs from mice
carrying construct A
C
hCD34
FSC
hCD34
FSC
hCD34 expression
normalized to hCD34
+
LSK-HSCs from mice
carrying construct A
250K
200K
150K
100K
50K
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
10
5
10
4
10
3
10
2
10
1
250K
200K
150K
100K
50K
99.8 99.6
0.5
0.25
5.2 ×
A
Construct
name
hCD34 in
SLAM
+
LSKs
Founder
lines
TGTAGG
TGTA
GA
TGTA
GG
TGTGTA
GGG
6 Yes
(strongly
decreased in
percentage
and intensity)
F
PvuI
PvuI
18.3 kb 24.8 kb 25.6 kb
X XX X
4321
Figure 6 Intact RUNX sites within the DRE are necessary for human CD34 expression in SLAM
þ
LSKs and to maintain the promoter–DRE
interaction. (A) Construct A (described in Figure 1) was modified to contain mutations in the Runx sites located in the DRE (construct F).
The expanded section shows relative location of the mutated Runx sites in the DRE, and within the framed box the specific mutations are
indicated relative to the wild-type sequence (Supplementary Figure S5). (B) Flow cytometry of bone marrow cells from mice carrying construct
F. A representative example from one of the six founder lines is shown (three mice per each founder were analysed, a part from two founders in
which we did not detect germ line transgene transmission). Mutation in the Runx sites strongly decreases hCD34 expression in SLAM
þ
LSKs in
comparison with SLAM
þ
LSKs from mice carrying construct A, in terms of percentage (from 99.2
±
0.5% (s.d.) to 38.1
±
4.2% (s.d.),
respectively; P ¼ 3.44, E17) and mean fluorescence intensity (from 8617
±
125 (s.d.) to 2342
±
189 (s.d.), respectively; P ¼ 2.1, E24).
(C) LSK-HSCs from mice carrying construct F have been subdivided into a hCD34
low
and a hCD34 intermediate (hCD34
int
) population, in
accordance to the hCD34 gating strategy adopted throughout this study, to perform 3C assays. In the upper panel, post-sorting analysis
indicates the purity of the sorted population. The lower left panel shows Q-RT–PCR data, indicating the levels of hCD34 expression in the
hCD34
low
(black column; &) and the hCD34
int
population (white column; &), normalized to the levels of hCD34 expression detected in LSK-
HSCs from mice carrying construct A. The lower right panel indicates the relative crosslinking frequency of the promoter and DRE in the same
hCD34
low
(black column; &) and hCD34
int
(white column; &) FACS-purified cells, as normalized to crosslinking frequency of LSK-HSCs from
mice transgenic for construct A. Interaction of the DRE with the Pr fragment is less frequent in hCD34
low
cells (n ¼ 4) than in hCD34
int
LSKs
(n ¼ 4), showing a 5.2-fold enrichment in the relative crosslinking frequency in cells with higher hCD34 expression. Sorted populations have
been pooled from 18 mice transgenic for construct F. See also Supplementary Figure S4.
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
The EMBO Journal VOL 30
|
NO 19
|
2011 & 2011 European Molecular Biology Organization4066
Page 8
although this combination of elements has been shown to be
sufficient to mediate HSC-specific expression in the mouse
(Gottgens et al, 2002; Pimanda et al, 2007).
It was recently shown that RUNX proteins regulate the
intranuclear positioning of the Cd4 and Cd8 loci (Collins et al,
2011). An interesting hypothesis is therefore that RUNX1 may
be required to bring together promoter-distal regulatory ele-
ments at nuclear ‘transcription factories’ (Jackson et al, 1993;
Osborne et al, 2004), where genes are dynamically recruited
during activation and abatement of their transcription
(Schoenfelder et al, 2010). Moreover, RUNX1 proteins localize
in defined subnuclear domains (Berezney et al, 1996; Stein
et al, 1999; Li et al, 2005), and a unique intranuclear
trafficking sequence that targets RUNX1 to these sites has
been identified (Zeng et al, 1997, 1998; Zaidi et al, 2002).
Interestingly, molecular alterations that cause misrouting
of RUNX1 in the nucleus result in abnormal synergism with
other transcription factors (Li et al, 2005), aberrant gene
expression and development of disease (Jackson, 1997;
Stein et al, 2000a, b), suggesting that the correct subnuclear
targeting of RUNX1 is an integral component of multifactor
interaction to control cell-specific gene expression.
We previously reported that the PU.1 promoter directly
interacts with a critical upstream regulatory element (URE)
(Ebralidze et al, 2008). Similarly to what we observed with
the hCD34 DRE, we detected decreased interaction between
the PU.1 promoter and the URE in primary haematopoietic
stem/progenitor cells when RUNX binding sites in the PU.1
URE were mutated (PZ, AKE, Annalisa Di Ruscio, DGT,
unpublished observations). These results suggest a more
general role of RUNX proteins in mediating interactions of
distal regulatory elements at its target genes.
Overall, our work contributes to elucidate the mechanism
leading to gene regulation in the rare population of HSCs by
identifying trans-acting regulatory proteins that establish the
three-dimensional molecular organization of long-range
regulatory elements. Our data open a new field of investiga-
tion aimed at identifying specific genetic mutations that lead
to abnormal gene expression, therefore contributing to HSC
defects and specific disease onset.
Materials and methods
Detailed protocols in the Supplementary data.
Mice
hCD34 transgenic mice contained hCD34 PAC constructs derived
from clone 54A19 (Radomska et al, 1998; Okuno et al, 2002b).
To obtain the transgenic constructs, a combination of both homo-
logous recombination and enzymatic restriction was used (Supple-
mentary Figure S5) (Datsenko and Wanner, 2000). Transgenic mice
carrying construct E were bred with Runx1
F/F
(Growney et al, 2005)
and Mx-Cre1 mice (Kuhn et al, 1995), and treated with PIPC to
achieve excision of the Runx1 locus. Seven doses of 0.8 mg/mouse
were given every other day. All animal experiments were performed
by using procedures described in the protocol approved by our
Institutional Animal Care and Use Committee.
Genotyping protocols
DNA was extracted from snip-tails, phenol/chloroform purified, and
analysed by Southern blot and PCR, as described in Supplementary
data.
Bone marrow transplantation
Fifty hCD34
þ
LT-HSCs (lineage
; Sca1
þ
; c-kit
þ
; CD150
þ
;
and CD48
cells) isolated from bone marrow of transgenic mice
(FVB/N, Ly5.1
þ
) were tail vein injected into congenic FVB/N mice
(Ly5.2
þ
), with 5 10
5
supporting bone marrow cells from
Ly5.1/Ly5.2 heterozygous mice. Recipient mice were irradiated
(10.1 Gray), and reconstitution assessed 5 months after transplanta-
tion by FACS analysis, as described in the Supplementary data.
RNA isolation and quantitative real-time PCR analysis
RNA was extracted using the RNeasy Mini Kit (Qiagen). Quantita-
tive real-time PCR analysis was performed on a Corbett Rotor Gene
6000. Primers and probes were synthesized as follows and gene
expression was compared with 18S expression (Eukaryotic 18S
rRNA endogenous control, Applied Biosystems): hCD34 sense: 5
0
-
AACTACAACACCTAGTACCCTTGGAA-3
0
, antisense: 5
0
-GAATTTGA
CTGTCGTTTCTGTGATG-3 and probe: 5
0
FAM-CCCTGTGTCTCAA
CATGG-CAATGAGGCC-TAMRA-3
0
. Runx1 sense: 5
0
-CGAGATTCAAC
GACCTCAGGTT-3
0
, antisense: 5
0
-AAGACGGT-GATGGTCAGAGTGA-3
0
and probe: 5
0
-FAM-TCGGGCGGAGCGGTAGAGGC-BHQ1-3
0
.
Flow cytometric analysis and cell sorting
Bone marrow cells were stained (see Supplementary data) and
sorted on MOFlo (MOFlo-MLS, Cytomation) or FACS Aria. Data
were analysed with FlowJo software (Treestar, Inc.).
Western blot
A quantity of 60 mg of nuclear extracts was diluted with 1 PBS to
20 ml total volume, mixed with equal volume of 2 Laemmli
Sample Buffer and boiled at 1001C for 10 min. Samples were loaded
on 7.5% SDS–PAGE gels and proteins transferred to nitrocellulose
membranes. Following blocking in 5% milk/TBST (TBST: 25 mM
Tris–HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl and 0.1% Tween 20),
membranes were stained overnight at 4 1C with anti-FLAG M2
monoclonal antibody (Sigma, no. F3165) diluted 1:10 000 in 5%
BSA/TBST/0.1% sodium azide. FLAG-tagged proteins were
detected following the staining with horseradish peroxidase-
conjugated anti-mouse secondary antibody (1:3000 dilution; Santa
Cruz, no. sc-2055) at room temperature for 1 h. Signals were
detected by enhanced chemiluminescence and quantified by
ImageQuant software (Molecular Dynamics).
ChIP assay
Cells (10
6
for each antibody) were used to crosslink chromatin using
the protocol from Millipore (Milton Keynes, UK), with minor
modifications. HL60 cells were grown in RPMI supplemented with
10% FBS. Umbilical cord blood cells have been kept in culture
(QBSF-60 Stem Cell Medium (Quality Biologicals Inc), supplemen-
ted with 20% FBS, 100 ng/ml SCF, 100 ng/ml Flt3 ligand, 50 ng/ml
TPO and 50 ng/ml IL6) for eight passages. Briefly, cells were
incubated in 1% formaldehyde for 10 min at room temperature.
Glycine (0.125 M) was added to stop crosslinking. Crosslinked
chromatin was sonicated for 150 s at 30% amplitude (Branson
Digital Sonifer, Danbury, CT). Polyclonal antibodies raised against
H3 acetylated at lysines 9 and 14 (aH3-Ac) from Millipore were
used, whereas the RUNX1 antibody was purchased from Abcam
and the IgG control from Sigma. DNA was purified by phenol–
chloroform extraction, and specific regions were amplified by
Q-PCR. Primers and probes used were the following: for the DRE
Runx sites 1–3 region, sense 5
0
-CTCTCAGGTCACGCAGACAC-3
0
;
antisense 5
0
-TAGGTTCACCCACAGGCTTC-3
0
; probe 5
0
-FAM-TGTCC
GTGTGGGAGGCAGGA-BHQ1-3
0
; for the DRE Runx site 4 region,
sense 5
0
-CTCTGCCTTTGAGGAGCAAG-3
0
; antisense 5
0
-TACACCCTT
CCCTGACCATC-3
0
; probe 5
0
-FAM-ATTTGGAGCAGGCCTGGGGC-B
HQ1-3
0
; for the Myf5 promoter region, sense 5
0
-CGAAAACTGGG
CTTCTTCTG-3
0
; antisense 5
0
-GAGCACCTTCTCCTTTGTGC-3
0
; and
probe 5
0
-FAM-TGCAGGTCTTTGGCCTGCTCA-BHQ1-3
0
.
Chromosome conformation capture
3C assay was used to detect genomic loci interactions (Dekker et al,
2002). Genomic DNA was uniformly digested, with an efficiency
490%, calculated following a published formula (Hagege et al,
2007). Oligonucleotides and probes for Q-RT–PCR to test for loci
interactions and restriction enzyme efficiency were as described in
the Supplementary data.
Statistical analysis
All values are presented either as mean
±
s.d. or as representative
images of at least three independent experiments. In the case
of animal studies, three mice of each different founder line were
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
& 2011 European Molecular Biology Organization The EMBO Journal VOL 30
|
NO 19
|
2011 4067
Page 9
analysed. All comparisons were made using the unpaired Student’s
t-test for samples with unequal variance. Differences were
considered statistically significant at Po0.05 (indicated by
asterisks).
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
These studies were supported by NIH Grant CA41456 (to DGT),
Flight Attendant Medical Research Institute (to EL), DFG Grants
KO2155/1-1, 2-1 and 2-2 (to SK) and Leukaemia and Lymphoma
Research UK (to BG). GA is an American Italian Cancer Foundation
Fellow. We thank the BIDMC Animal Transgenic facility directed by
Joel Lawitts, and the DFCI and BIDMC Flow Cytometry Facilities.
We are grateful to Frank Rosenbauer, Li Chai and members of the
Tenen lab, in particular Gottfried Von Keudell, Annalisa Di Ruscio,
Min Ye, Hong Zhang, Akos Czibere, Rob Welner and Philipp Staber
for discussion during preparation of the manuscript.
Author contributions: EL and DGT designed the study; EL, SL,
HSR, GA, DSB and YO performed and planned research; EL, HSR,
CJH, GA, MAJ, PZ, DEZ, AKE, CB, BG and DGT analysed data;
CJH, MAJ, DG, JZ, NKW, SK and GH performed research; and EL
and DGT wrote the paper.
Conflict of interest
The authors declare that they have no conflict of interest.
References
Barnett DH, Sheng S, Charn TH, Waheed A, Sly WS, Lin CY, Liu ET,
Katzenellenbogen BS (2008) Estrogen receptor regulation
of carbonic anhydrase XII through a distal enhancer in breast
cancer. Cancer Res 68: 3505–3515
Berezney R, Mortillaro M, Ma H, Meng C, Samarabandu J, Wei X,
Somanathan S, Liou WS, Pan SJ, Cheng PC (1996) Connecting
nuclear architecture and genomic function. J Cell Biochem 62:
223–226
Boney-Montoya J, Ziegler YS, Curtis CD, Montoya JA, Nardulli AM
(2010) Long-range transcriptional control of progesterone recep-
tor gene expression. Mol Endocrinol 24: 346–358
Brown RC, Pattison S, van Ree J, Coghill E, Perkins A, Jane SM,
Cunningham JM (2002) Distinct domains of erythroid Kruppel-
like factor modulate chromatin remodeling and transactivation
at the endogenous beta-globin gene promoter. Mol Cell Biol 22:
161–170
Burn TC, Satterthwaite AB, Tenen DG (1992) The human CD34
hematopoietic stem cell antigen promoter and a 3
0
enhancer
direct hematopoietic expression in tissue culture. Blood 80:
3051–3059
Cai Z, de Bruijn M, Ma X, Dortland B, Luteijn T, Downing RJ,
Dzierzak E (2000) Haploinsufficiency of AML1 affects the
temporal and spatial generation of hematopoietic stem cells in
the mouse embryo. Immunity 13: 423–431
Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P (2002) Long-
range chromatin regulatory interactions in vivo. Nat Genet 32:
623–626
Chan PK, Wai A, Philipsen S, Tan-Un KC (2008) 5
0
HS5 of the human
beta-globin locus control region is dispensable for the formation
of the beta-globin active chromatin hub. PLoS One 3: e2134
Chavanas S, Adoue V, Mechin MC, Ying S, Dong S, Duplan H,
Charveron M, Takahara H, Serre G, Simon M (2008) Long-range
enhancer associated with chromatin looping allows AP-1 regula-
tion of the peptidylarginine deiminase 3 gene in differentiated
keratinocyte. PLoS One 3: e3408
Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA (2009)
Runx1 is required for the endothelial to haematopoietic cell
transition but not thereafter. Nature 457: 887–891
Chen X, Reitman M, Bieker JJ (1998) Chromatin structure and
transcriptional control elements of the erythroid Kruppel-like
factor (EKLF) gene. J Biol Chem 273: 25031–25040
Collins A, Hewitt SL, Chaumeil J, Sellars M, Micsinai M, Allinne J,
Parisi F, Nora EP, Bolland DJ, Corcoran AE, Kluger Y, Bosselut R,
Ellmeier W, Chong MM, Littman DR, Skok JA (2011) RUNX
transcription factor-mediated association of Cd4 and Cd8 enables
coordinate gene regulation. Immunity 34: 303–314
Datsenko KA, Wanner BL (2000) One-step inactivation of chromo-
somal genes in Escherichia coli K-12 using PCR products.
Proc Natl Acad Sci USA 97: 6640–6645
Dekker J, Rippe K, Dekker M, Kleckner N (2002) Capturing
chromosome conformation. Science 295: 1306–1311
Deshane J, Kim J, Bolisetty S, Hock TD, Hill-Kapturczak N,
Agarwal A (2010) Sp1 regulates chromatin looping between an
intronic enhancer and distal promoter of the human
heme oxygenase-1 gene in renal cells. J Biol Chem 285:
16476–16486
Dunbar CE, Cottler-Fox M, O’Shaughnessy JA, Doren S, Carter C,
Berenson R, Brown S, Moen RC, Greenblatt J, Stewart FM,
Leitman SF, Wilson WH, Cowan K, Young NS, Nienhuis AW
(1995) Retrovirally marked CD34-enriched peripheral blood and
bone marrow cells contribute to long-term engraftment after
autologous transplantation. Blood 85: 3048–3057
Ebralidze AK, Guibal FC, Steidl U, Zhang P, Lee S, Bartholdy B,
Jorda MA, Petkova V, Rosenbauer F, Huang G, Dayaram T,
Klupp J, O’Brien KB, Will B, Hoogenkamp M, Borden KL,
Bonifer C, Tenen DG (2008) PU.1 expression is modulated by
the balance of functional sense and antisense RNAs regulated by
a shared cis-regulatory element. Genes Dev 22: 2085–2092
Galy A, Travis M, Cen D, Chen B (1995) Human T, B, natural killer,
and dendritic cells arise from a common bone marrow progenitor
cell subset. Immunity 3: 459–473
Gavrilov AA, Razin SV (2008) Spatial configuration of the chicken
alpha-globin gene domain: immature and active chromatin hubs.
Nucleic Acids Res 36: 4629–4640
Gottgens B, Nastos A, Kinston S, Piltz S, Delabesse EC, Stanley M,
Sanchez MJ, Ciau-Uitz A, Patient R, Green AR (2002) Establishing
the transcriptional programme for blood: the SCL stem cell
enhancer is regulated by a multiprotein complex containing Ets
and GATA factors. Embo J 21: 3039–3050
Growney JD, Shigematsu H, Li Z, Lee BH, Adelsperger J, Rowan R,
Curley DP, Kutok JL, Akashi K, Williams IR, Speck NA, Gilliland DG
(2005) Loss of Runx1 perturbs adult hematopoiesis and is asso-
ciated with a myeloproliferative phenotype. Blood 106: 494–504
Hagege H, Klous P, Braem C, Splinter E, Dekker J, Cathala G, de
Laat W, Forne T (2007) Quantitative analysis of chromosome
conformation capture assays (3C-qPCR). Nat Protoc 2: 1722–1733
Hoogenkamp M, Lichtinger M, Krysinska H, Lancrin C, Clarke D,
Williamson A, Mazzarella L, Ingram R, Jorgensen H, Fisher A,
Tenen DG, Kouskoff V, Lacaud G, Bonifer C (2009) Early chro-
matin unfolding by RUNX1: a molecular explanation for differ-
ential requirements during specification versus maintenance of
the hematopoietic gene expression program. Blood 114: 299–309
Ichikawa M, Asai T, Chiba S, Kurokawa M, Ogawa S (2004a)
Runx1/AML-1 ranks as a master regulator of adult hematopoi-
esis. Cell Cycle 3: 722–724
Ichikawa M, Asai T, Saito T, Seo S, Yamazaki I, Yamagata T,
Mitani K, Chiba S, Ogawa S, Kurokawa M, Hirai H (2004b)
AML-1 is required for megakaryocytic maturation and lympho-
cytic differentiation, but not for maintenance of hematopoietic
stem cells in adult hematopoiesis. Nat Med 10: 299–304
Ichikawa M, Goyama S, Asai T, Kawazu M, Nakagawa M,
Takeshita M, Chiba S, Ogawa S, Kurokawa M (2008) AML1/
Runx1 negatively regulates quiescent hematopoietic stem cells
in adult hematopoiesis. J Immunol 180: 4402–4408
Jackson DA (1997) Chromatin domains and nuclear compartments:
establishing sites of gene expression in eukaryotic nuclei.
Mol Biol Rep 24: 209–220
Jackson DA, Hassan AB, Errington RJ, Cook PR (1993)
Visualization of focal sites of transcription within human nuclei.
EMBO J 12: 1059–1065
Jacob B, Osato M (2009) Stem cell exhaustion and leukemogenesis.
J Cell Biochem 107: 393–399
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
The EMBO Journal VOL 30
|
NO 19
|
2011 & 2011 European Molecular Biology Organization4068
Page 10
Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ
(2005) SLAM family receptors distinguish hematopoietic stem
and progenitor cells and reveal endothelial niches for stem cells.
Cell 121: 1109–1121
Kim SI, Bultman SJ, Jing H, Blobel GA, Bresnick EH (2007)
Dissecting molecular steps in chromatin domain activation dur-
ing hematopoietic differentiation. Mol Cell Biol 27: 4551–4565
Kim SI, Bultman SJ, Kiefer CM, Dean A, Bresnick EH (2009) BRG1
requirement for long-range interaction of a locus control
region with a downstream promoter. Proc Natl Acad Sci USA
106: 2259–2264
Krause DS, Fackler MJ, Civin CI, May WS (1996) CD34: structure,
biology, and clinical utility. Blood 87: 1–13
Kuhn R, Schwenk F, Aguet M, Rajewsky K (1995) Inducible gene
targeting in mice. Science 269: 1427–1429
Li X, Vradii D, Gutierrez S, Lian JB, van Wijnen AJ, Stein JL, Stein
GS, Javed A (2005) Subnuclear targeting of Runx1 is required for
synergistic activation of the myeloid specific M-CSF receptor
promoter by PU.1. J Cell Biochem 96: 795–809
Ling JQ, Li T, Hu JF, Vu TH, Chen HL, Qiu XW, Cherry AM,
Hoffman AR (2006) CTCF mediates interchromosomal colo-
calization between Igf2/H19 and Wsb1/Nf1. Science 312:
269–272
Link H, Arseniev L, Bahre O, Kadar JG, Diedrich H, Poliwoda H
(1996) Transplantation of allogeneic CD34+ blood cells. Blood
87: 4903–4909
Liu Z, Ma Z, Terada LS, Garrard WT (2009) Divergent roles of RelA
and c-Rel in establishing chromosomal loops upon activation of
the Igkappa gene. J Immunol 183: 3819–3830
Marenduzzo D, Faro-Trindade I, Cook PR (2007) What are the
molecular ties that maintain genomic loops? Trends Genet 23:
126–133
Melotti P, Calabretta B (1994) Ets-2 and c-Myb act independently in
regulating expression of the hematopoietic stem cell antigen
CD34. J Biol Chem 269: 25303–25309
Michallet M, Philip T, Philip I, Godinot H, Sebban C, Salles G,
Thiebaut A, Biron P, Lopez F, Mazars P, Roubi N, Leemhuis T,
Hanania E, Reading C, Fine G, Atkinson K, Juttner C, Coiffier B,
Fiere D, Archimbaud E (2000) Transplantation with selected
autologous peripheral blood CD34+Thy1+ hematopoietic stem
cells (HSCs) in multiple myeloma: impact of HSC dose on
engraftment, safety, and immune reconstitution. Exp Hematol
28: 858–870
Morris JF, Rauscher III FJ, Davis B, Klemsz M, Xu D, Tenen D,
Hromas R (1995) The myeloid zinc finger gene, MZF-1, regulates
the CD34 promoter in vitro. Blood 86: 3640–3647
Okada H, Watanabe T, Niki M, Takano H, Chiba N, Yanai N, Tani K,
Hibino H, Asano S, Mucenski ML, Ito Y, Noda T, Satake M (1998)
AML1(/) embryos do not express certain hematopoiesis-re-
lated gene transcripts including those of the PU.1 gene. Oncogene
17: 2287–2293
Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR
(1996) AML1, the target of multiple chromosomal translocations
in human leukemia, is essential for normal fetal liver hematopoi-
esis. Cell 84: 321–330
Okuno Y, Huettner CS, Radomska HS, Petkova V, Iwasaki H,
Akashi K, Tenen DG (2002a) Distal elements are critical for
human CD34 expression in vivo. Blood 100: 4420–4426
Okuno Y, Iwasaki H, Huettner CS, Radomska HS, Gonzalez DA,
Tenen DG, Akashi K (2002b) Differential regulation of the
human and murine CD34 genes in hematopoietic stem cells.
Proc Natl Acad Sci USA 99: 6246–6251
Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand
E, Goyenechea B, Mitchell JA, Lopes S, Reik W, Fraser P (2004)
Active genes dynamically colocalize to shared sites of ongoing
transcription. Nat Genet 36: 1065–1071
Patrinos GP, de Krom M, de Boer E, Langeveld A, Imam AM,
Strouboulis J, de Laat W, Grosveld FG (2004) Multiple interac-
tions between regulatory regions are required to stabilize an
active chromatin hub. Genes Dev 18: 1495–1509
Perrotti D, Melotti P, Skorski T, Casella I, Peschle C, Calabretta B
(1995) Overexpression of the zinc finger protein MZF1 inhibits
hematopoietic development from embryonic stem cells:
correlation with negative regulation of CD34 and c-myb promoter
activity. Mol Cell Biol 15: 6075–6087
Pilon AM, Nilson DG, Zhou D, Sangerman J, Townes TM,
Bodine DM, Gallagher PG (2006) Alterations in expression and
chromatin configuration of the alpha hemoglobin-stabilizing
protein gene in erythroid Kruppel-like factor-deficient mice.
Mol Cell Biol 26: 4368–4377
Pimanda JE, Ottersbach K, Knezevic K, Kinston S, Chan WY, Wilson
NK, Landry JR, Wood AD, Kolb-Kokocinski A, Green AR,
Tannahill D, Lacaud G, Kouskoff V, Gottgens B (2007) Gata2,
Fli1, and Scl form a recursively wired gene-regulatory circuit
during early hematopoietic development. Proc Natl Acad Sci USA
104: 17692–17697
Putz G, Rosner A, Nuesslein I, Schmitz N, Buchholz F (2006) AML1
deletion in adult mice causes splenomegaly and lymphomas.
Oncogene 25: 929–939
Radomska HS, Gonzalez DA, Okuno Y, Iwasaki H, Nagy A,
Akashi K, Tenen DG, Huettner CS (2002) Transgenic targeting
with regulatory elements of the human CD34 gene. Blood 100:
4410–4419
Radomska HS, Satterthwaite AB, Burn TC, Oliff IA, Huettner CS,
Tenen DG (1998) Multiple control elements are required
for expression of the human CD34 gene. Gene 222: 305–318
Radomska HS, Satterthwaite AB, Taranenko N, Narravula S,
Krause DS, Tenen DG (1999) A nuclear factor Y (NFY) site
positively regulates the human CD34 stem cell gene. Blood 94:
3772–3780
Rathke C, Baarends WM, Jayaramaiah-Raja S, Bartkuhn M,
Renkawitz R, Renkawitz-Pohl R (2007) Transition from a nucleo-
some-based to a protamine-based chromatin configuration during
spermiogenesis in Drosophila. J Cell Sci 120: 1689–1700
Roth TL, Sweatt JD (2009) Regulation of chromatin structure in
memory formation. Curr Opin Neurobiol 19: 336–342
Sato T, Laver JH, Ogawa M (1999) Reversible expression of CD34 by
murine hematopoietic stem cells. Blood 94: 2548–2554
Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A,
Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimitrova DS,
Eskiw CH, Luo Y, Wei CL, Ruan Y, Bieker JJ, Fraser P (2010)
Preferential associations between co-regulated genes reveal a
transcriptional interactome in erythroid cells. Nat Genet 42: 53–61
Stein GS, van Wijnen AJ, Stein JL, Lian JB, Javed A, McNeil S,
Pockwinse SM (1999) Insight into regulatory factor targeting
to transcriptionally active subnuclear sites. Exp Cell Res 253:
110–116
Stein GS, van Wijnen AJ, Stein JL, Lian JB, Montecino M, Choi J,
Zaidi K, Javed A (2000a) Intranuclear trafficking of transcription
factors: implications for biological control. J Cell Sci 11 3 (Part 14):
2527–2533
Stein GS, van Wijnen AJ, Stein JL, Lian JB, Montecino M, Zaidi K,
Javed A (2000b) Subnuclear organization and trafficking
of regulatory proteins: implications for biological control and
cancer. J Cell Biochem Suppl 79(Suppl 35): 84–92
Stella CC, Cazzola M, De Fabritiis P, De Vincentiis A, Gianni AM,
Lanza F, Lauria F, Lemoli RM, Tarella C, Zanon P, Tura S
(1995) CD34-positive cells: biology and clinical relevance.
Haematologica 80: 367–387
Theo Sijtse Palstra RJ (2009) Close encounters of the 3C kind:
long-range chromatin interactions and transcriptional regulation.
Brief Funct Genomic Proteomic 8: 297–309
Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W (2002)
Looping and interaction between hypersensitive sites in the active
beta-globin locus. Mol Cell 10: 1453–1465
Vernimmen D, De Gobbi M, Sloane-Stanley JA, Wood WG, Higgs DR
(2007) Long-range chromosomal interactions regulate the timing
of the transition between poised and active gene expression.
EMBO J 26: 2041–2051
Wadman IA, Osada H, Grutz GG, Agulnick AD, Westphal H,
Forster A, Rabbitts TH (1997) The LIM-only protein Lmo2 is a
bridging molecule assembling an erythroid, DNA-binding
complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI
proteins. EMBO J 16: 3145–3157
Williams AO, Isaacs RJ, Stowell KM (2007) Down-regulation of
human topoisomerase IIalpha expression correlates with relative
amounts of specificity factors Sp1 and Sp3 bound at proximal and
distal promoter regions. BMC Mol Biol 8: 36
Zaidi SK, Sullivan AJ, van Wijnen AJ, Stein JL, Stein GS, Lian JB
(2002) Integration of Runx and Smad regulatory signals
at transcriptionally active subnuclear sites. Proc Natl Acad Sci
USA 99: 8048–8053
Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa M
(1998) Human bone marrow CD34 cells engraft in vivo and
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
& 2011 European Molecular Biology Organization The EMBO Journal VOL 30
|
NO 19
|
2011 4069
Page 11
undergo multilineage expression that includes giving rise to
CD34+ cells. Exp Hematol 26: 353–360
Zanjani ED, Almeida-Porada G, Livingston AG, Porada CD,
Ogawa M (1999) Engraftment and multilineage expression of
human bone marrow CD34 cells in vivo. Ann NY Acad Sci
872: 220–231; discussion 231–232
Zanjani ED, Almeida-Porada G, Livingston AG, Zeng H, Ogawa M
(2003) Reversible expression of CD34 by adult human bone
marrow long-term engrafting hematopoietic stem cells. Exp
Hematol 31: 406–412
Zeng C, McNeil S, Pockwinse S, Nickerson J, Shopland L,
Lawrence JB, Penman S, Hiebert S, Lian JB, van Wijnen AJ,
Stein JL, Stein GS (1998) Intranuclear targeting of AML/CBFalpha
regulatory factors to nuclear matrix-associated transcriptional
domains. Proc Natl Acad Sci USA 95: 1585–1589
Zeng C, van Wijnen AJ, Stein JL, Meyers S, Sun W, Shopland L,
Lawrence JB, Penman S, Lian JB, Stein GS, Hiebert SW (1997)
Identification of a nuclear matrix targeting signal in the leukemia
and bone-related AML/CBF-alpha transcription factors. Proc Natl
Acad Sci USA 94: 6746–6751
RUNX1 regulates human CD34 expression in HSCs
E Levantini et al
The EMBO Journal VOL 30
|
NO 19
|
2011 & 2011 European Molecular Biology Organization4070
Page 12
    • "This conformation, termed active chromatin hubs (ACHs), is mediated through the binding of transcription factors, and is only present when these genes are expressed. In hematopoietic stem cells (HSCs), the transcription factor RunxI is required for the interaction between the CD34 gene promoter and its downstream regulatory element [Levantini et al., 2011]. Transcription factor-mediated chromatin looping was demonstrated in erythroid cells. "
    [Show abstract] [Hide abstract] ABSTRACT: The organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear-structure building blocks in directly supporting nuclear function in a physiologically responsive manner. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jul 2015 · Journal of Cellular Biochemistry
    0Comments 0Citations
    • "El Omari K reported that lmo2 functions as the scaffold for a DNA-binding transcription regulator complex, on condition knockdown of lmo2 leads to complete loss of PLM primitive hematopoiesis [25]. Using transgenic mice carrying human CD34 PAC gene, Levantini identified a novel downstream regulatory element (DRE) that is bound by runx1 and is necessary for human CD34 in long-term (LT)-HSCs [26]. Recently, Herbomel confirmed that zebrafish HSCs emerge directly from the aortic floor and this process is dependent on Runx1 expression [27]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Amplification of MYCN (N-Myc) oncogene has been reported as a frequent event and a poor prognostic marker in human acute myeloid leukemia (AML). The molecular mechanisms and transcriptional networks by which MYCN exerts its influence in AML are largely unknown. Methodology/Principal Findings We introduced murine MYCN gene into embryonic zebrafish through a heat-shock promoter and established the stable germline Tg(MYCN:HSE:EGFP) zebrafish. N-Myc downstream regulated gene 1 (NDRG1), negatively controlled by MYCN in human and functionally involved in neutrophil maturation, was significantly under-expressed in this model. Using peripheral blood smear detection, histological section and flow cytometric analysis of single cell suspension from kidney and spleen, we found that MYCN overexpression promoted cell proliferation, enhanced the repopulating activity of myeloid cells and the accumulation of immature hematopoietic blast cells. MYCN enhanced primitive hematopoiesis by upregulating scl and lmo2 expression and promoted myelopoiesis by inhibiting gata1 expression and inducing pu.1, mpo expression. Microarray analysis identified that cell cycle, glycolysis/gluconeogenesis, MAPK/Ras, and p53-mediated apoptosis pathways were upregulated. In addition, mismatch repair, transforming and growth factor β (TGFβ) were downregulated in MYCN-overexpressing blood cells (p<0.01). All of these signaling pathways are critical in the proliferation and malignant transformation of blood cells. Conclusion/Significance The above results induced by overexpression of MYCN closely resemble the main aspects of human AML, suggesting that MYCN plays a role in the etiology of AML. MYCN reprograms hematopoietic cell fate by regulating NDRG1 and several lineage-specific hematopoietic transcription factors. Therefore, this MYCN transgenic zebrafish model facilitates dissection of MYCN-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets.
    Full-text · Article · Mar 2013 · PLoS ONE
    0Comments 12Citations
    • "However, these data did not answer the question of whether CTCF binding or other mechanisms would mediate loop formation. Importantly, we recently described that targeted mutations of RUNX binding sites in a downstream regulatory element (DRE) of a human CD34 transgene caused the perturbation of the DRE-promoter interaction in transgenic mice (Levantini et al., 2011). Along with the functional models presented here, specific disruption of PU.1 binding in the URE of the endogenous PU.1 locus can be used to distinguish between correlation and causation of the transcription factor binding and chromosome looping necessary for gene activation. "
    [Show abstract] [Hide abstract] ABSTRACT: To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1's role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation.
    Full-text · Article · Feb 2013 · Molecular cell
    0Comments 31Citations
Show more

Similar publications

Discover cutting-edge research

ResearchGate is where you can find and access the latest publications from your field of research.

Discover more