Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the −2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring −1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the −2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

GATA factors are often expressed in overlapping and reciprocal patterns in hematopoiesis. Accrued evidence supports a model in which GATA factors cross-regulate transcription of their respective genes through a “GATA switch.”1,2  This hypothesis has been investigated at the Gata2 locus, where several conserved GATA motif-containing regions span ∼ 100 kb. GATA-2 has a critical role in the emergence and maintenance of hematopoietic stem/progenitor cells3-10  and appears to positively regulate its own transcription. During erythroid cell differentiation, GATA-1 mediates repression of Gata2 expression. A cis-element located −2.8 kb upstream from the 1S Gata2 promoter has been proposed to mediate GATA-2-dependent positive feedback before GATA-1-instigated repression.11-13  Removal of this site would thus be predicted to abrogate positive feedback, reducing Gata2 expression in hematopoietic stem/progenitor cells. We previously demonstrated that a distinct cis-element −1.8 kb upstream of the Gata2 promoter maintains repression of Gata2 in late-stage erythroblasts, though it is not required to initiate repression.14  As the patterns of GATA-factor binding at the −1.8 kb and −2.8 kb sites are similar,11-13  we addressed whether the −2.8 kb site is also required for GATA-1-mediated Gata2 repression.

We demonstrate that the −2.8 kb site is not required for initiation or maintenance of Gata2 repression during erythroid differentiation. By contrast, the −2.8 kb site confers maximal Gata2 expression in HSCs and progenitors in specific contexts. Comparison of the −2.8 kb and −1.8 kb sites illustrates how GATA motifs function uniquely and collectively to achieve complex and dynamic transcriptional control at the Gata2 locus during hematopoietic development.

Generation of Gata2 Δ-2.8 knockin mice

We replaced a 400 bp region containing 6 GATA motifs with a LoxP-PGKneo-LoxP cassette (supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Details of the cloning strategy are available on request. Gene targeted ES cell clones were identified by Southern blotting (supplemental Figure 1B-C). Confirmation of Cre-mediated neo excision was performed with PCR before blastocyst injection (supplemental Figure 1D). All mouse studies were conducted under guidelines associated with protocols approved by Harvard Medical School.

Gene expression and chromatin occupancy

Sampling of fetal liver and bone marrow cells, flow cytometric analysis and cell sorting, quantitative ChIP assay, and real-time reverse-transcriptase PCR were performed as published.14 

Previous studies demonstrated a critical role for Gata2 in the emergence and maintenance of hematopoietic stem cells (HSCs).3,4  The −2.8 kb region drives exogenous expression of a reporter gene in HSCs in a manner dependent on GATA15  and E-box16  motifs, and has been proposed to be the primary cis-element conferring Gata2 expression in these contexts.

Mice homozygous for deletion of a 400 bp region located −2.8 kb upstream of the Gata2 1S promoter (supplemental Figure 1) were born at expected Mendelian ratios (data not shown). We quantified Gata2 expression in sorted long-term HSCs (LT-HSCs) and multipotent progenitors (MPPs) from embryonic day (E) 12.5 fetal livers. Δ-2.8 LT-HSCs and MPPs exhibited reduced Gata2 expression versus control cells (Figure 1A), but this reduction did not alter the frequency of the populations (Figure 1B). Gata2 serves critical roles in the maintenance and specification of multipotential progenitors.5-10  Expression of Gata2 was reduced in common myeloid progenitors (CMPs) and megakaryocyte-erythroid progenitors (MEPs) of Δ-2.8 fetal livers (Figure 1C), and was associated with a modest reduction in the frequency of these populations (Figure 1D). Quantitative ChIP analysis with enriched progenitor cells from Δ-2.8 and wild-type E14.5 fetal livers revealed a selective reduction of GATA-2 occupancy at the −2.78 kb site (surrogate for measuring occupancy at the deleted −2.8 kb site; supplemental Figure 2). These results indicate that the −2.8 kb site contributes to Gata2 expression in fetal liver-derived multipotent and committed erythroid progenitors in part through GATA-2 binding.

Figure 1

−2.8 kb GATA switch site regulates Gata2 expression in hematopoietic stem and progenitor cells, but is dispensible in erythroid cells. Gata2 expression within LT-HSC and MPP populations (LT-HSC defined as Lindimckit+Sca1+CD150+ and MPP defined as Lindimckit+Sca1+CD34+ or Lindimckit+Sca1+CD150) from E12.5 fetal livers normalized to β-actin (A). Frequencies of LT-HSCs and MPPs in wild-type and Δ-2.8 fetal livers (B). Gata2 expression within CMP and MEP populations (with CMP defined as LindimSca1,c-kit+CD34+, FcγR and MEP defined as Lindim Sca1c-kit+CD34, FcγR) from E12.5 fetal livers normalized to β-actin (C). Frequencies of CMPs and MEPs from wild-type and Δ-2.8 fetal livers (D). Gata2 expression was assessed by qPCR in Stage I through Stage IV sorted erythroid cells, corresponding to CD71loTer119 (committed erythroid progenitors, Stage I), CD71hiTer119 (proerythroblasts, Stage II), CD71hiTer119+ (basophilic erythroblasts, Stage III), and CD71loTer119+ (late erythroblasts, Stage IV) from wild-type and Δ-2.8 embryos. (E). Relative number of cells in Stage I-IV was determined in wild-type and Δ-2.8 E13.5 fetal liver cells, based on CD71 and Ter119 expression (F). Mean ± SEM. Statistical significance was assessed by 2-sided Student t test. *P ≤ .05 and **P ≤ .01.

Figure 1

−2.8 kb GATA switch site regulates Gata2 expression in hematopoietic stem and progenitor cells, but is dispensible in erythroid cells. Gata2 expression within LT-HSC and MPP populations (LT-HSC defined as Lindimckit+Sca1+CD150+ and MPP defined as Lindimckit+Sca1+CD34+ or Lindimckit+Sca1+CD150) from E12.5 fetal livers normalized to β-actin (A). Frequencies of LT-HSCs and MPPs in wild-type and Δ-2.8 fetal livers (B). Gata2 expression within CMP and MEP populations (with CMP defined as LindimSca1,c-kit+CD34+, FcγR and MEP defined as Lindim Sca1c-kit+CD34, FcγR) from E12.5 fetal livers normalized to β-actin (C). Frequencies of CMPs and MEPs from wild-type and Δ-2.8 fetal livers (D). Gata2 expression was assessed by qPCR in Stage I through Stage IV sorted erythroid cells, corresponding to CD71loTer119 (committed erythroid progenitors, Stage I), CD71hiTer119 (proerythroblasts, Stage II), CD71hiTer119+ (basophilic erythroblasts, Stage III), and CD71loTer119+ (late erythroblasts, Stage IV) from wild-type and Δ-2.8 embryos. (E). Relative number of cells in Stage I-IV was determined in wild-type and Δ-2.8 E13.5 fetal liver cells, based on CD71 and Ter119 expression (F). Mean ± SEM. Statistical significance was assessed by 2-sided Student t test. *P ≤ .05 and **P ≤ .01.

To examine the function of this site at a different developmental stage, we isolated LT-HSC and MPP populations from adult Δ-2.8 and wild-type bone marrow. Gata2 expression was reduced in adult MPPs, but not LT-HSC (supplemental Figure 3A). We observed a similar reduction in Gata2 in MPP from Δ-1.8 mice (supplemental Figure 3A), implying some functional overlap. Loss of either site did not alter LT-HSC, MPP, CMP, GMP, or MEP numbers in adult mice (supplemental Figure 3B-C). No differences in hematopoietic colony formation or peripheral blood parameters were apparent in the Δ-2.8 or −1.8 adult mice (supplemental Table 1 and supplemental Figure 3D). Competitive primary and secondary transplant experiments revealed no statistically significant differences in the ability of Δ-2.8 or Δ-1.8 HSCs to contribute to hematopoiesis in these assays (supplemental Figure 3). Thus, while both sites confer maximal Gata2 expression in certain contexts, they are not essential for hematopoiesis in the mouse.

Our prior work demonstrated that the −1.8 kb site maintains GATA-1-mediated repression of Gata2 during erythroid differentiation.14  As the 2 sites have similar patterns of GATA-factor binding,11-13  we investigated Gata2 repression during erythroid differentiation in Δ-2.8 mice. Gata2 expression was reduced in Stage I of erythroid differentiation in the Δ-2.8 mice (Figure 1E). Stage I contains MEP activity,17  consistent with of the findings of Figure 1C. Despite this reduction, Gata2 expression was normal in Stages II-IV in the Δ-2.8 fetal livers, indicating that the −2.8 kb site is not required for initiation or maintenance of GATA-1-mediated repression (Figure 1E). Only modest differences in the relative abundance of cells from each stage were apparent in Δ-2.8 versus wild-type mice (Figure 1F).

Based on the unexpected observation that the −1.8 kb, but not the −2.8 kb, site mediates repression, we reasoned that comparing their capacities to regulate nucleoprotein architecture would yield mechanistic insights. Quantitative ChIP analysis with Δ-2.8 and wild-type E14.5 fetal liver cells revealed low GATA-1 occupancy at the −2.78 kb site (Figure 2A). ChIP analysis of Pol II occupancy demonstrated no change on loss of the −2.8 kb site (data not shown), contrasting with our demonstration that loss of the −1.8 kb site results in Pol II accumulation.14  We quantified dimeH3K4 and trimeH3K27 marks associated with Gata2 repression.11,13,18  Results from the Δ-1.8 mice demonstrated locus-wide reduction in dimethylH3K4 with no significant reductions in trimethylH3K27 (supplemental Figure 5A-B and Snow et al14 ). By contrast, loss of GATA-1 occupancy on deletion of the −2.8 kb site results in locus-wide reduction in one of the marks associated with repression, trimeH3K27 (Figure 2C), while having minimal effects on dimeH3K4 (Figure 2B), and no influence on repression. Although these data implicate a function of the polycomb repressive complex 2 (PRC2)18  at the −2.8 kb site, reduction of H3K27 trimethylation does not reactivate Gata2 transcription (Figure 1).

Figure 2

The −2.8 kb GATA switch site establishes trimeH3K27 marks at the Gata2 locus. Quantitative ChIP analysis of the Gata2 locus in E14.5 fetal liver cells using antibodies to GATA-1 (A), dimeH3K4 (B), trimeH3K27 (C), and Total H3 (D). Updated model of the molecular regulation of Gata2 repression (E). Mean ± SEM. Statistical significance was assessed by 2-sided Student t test and *P ≤ .05 and **P ≤ .01.

Figure 2

The −2.8 kb GATA switch site establishes trimeH3K27 marks at the Gata2 locus. Quantitative ChIP analysis of the Gata2 locus in E14.5 fetal liver cells using antibodies to GATA-1 (A), dimeH3K4 (B), trimeH3K27 (C), and Total H3 (D). Updated model of the molecular regulation of Gata2 repression (E). Mean ± SEM. Statistical significance was assessed by 2-sided Student t test and *P ≤ .05 and **P ≤ .01.

In summary, our analyses of GATA motif function in vivo establish fundamentally different requirements for the −2.8 and −1.8 kb GATA switch elements. As GATA motifs within these sites conferred unique chromatin states and exerted different activities, these results support a model in which initiation/maintenance of Gata2 transcriptional control requires the collective actions of dispersed GATA factor complexes with qualitatively distinct contributions (Figure 2E). As GATA-1 occupies different permutations of GATA motifs, including canonical, palindromic, composite, and noncanonical,19  our results suggest that these permutations may differ considerably in their functionality in vivo. Individual HSs of the murine β-globin locus function additively to confer maximal expression of the β-like globin genes,20  differing from the differential −1.8 kb and −2.8 kb site activities at Gata2. Qualitatively distinct functions of GATA complexes were apparent at Gata2, highlighting the critical need to expand efforts to elucidate GATA motif function at a spectrum of endogenous loci.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors acknowledge the technical assistance of Yuko Fujiwara, Minh Nguyen, and Mayumi Kaku, Grigoriy Losyev at the Brigham and Women's Flow Cytometry Core, John Daly and Susan Lazo-Kallanian at the Dana-Farber Cancer Institute HemNeo Flow Facility, and the staff of the Animal Resource Children's Hospital Boston. The authors thank Stephen Chan and Marc Kerenyi for critical comments.

This work was funded by National Institutes of Health grants DK068634 (E.H.B.) and HL32259 (S.H.O.) and by grant P30 CA014520 to the University of Wisconsin Paul Carbone Cancer Center from the National Cancer Institute. J.W.S. and J.J.T. were funded in part by Fellowships from the Leukemia & Lymphoma Society, and S.H.O. is an Investigator of the Howard Hughes Medical Institute.

National Institutes of Health

Contribution: E.H.B. and S.H.O. conceived of the project; J.A.G., N.E.E, and J.W.S. built initial constructs, screened clones, and generated mice; J.W.S., T.F., K.D.J., and J.J.T performed experiments; J.W.S. and J.J.T. analyzed data; and J.W.S., J.J.T., S.H.O., and E.H.B wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for J.W.S. is Biology Department, Williams College, Williamstown, MA.

Correspondence: Emery H. Bresnick, Department of Cell and Regenative Biology, University of Wisconsin School of Medicine and Public Health, Wisconsin Institutes for Medical Research, Rm 4009, 1111 Highland Ave, Madison, WI 53705; e-mail: ehbresni@wisc.edu; and Jonathan W. Snow, Biology Department, Williams College, Williamstown, MA 01267, MA; e-mail: jonathan.snow@williams.edu.

1
Bresnick
 
EH
Martowicz
 
ML
Pal
 
S
Johnson
 
KD
Developmental control via GATA factor interplay at chromatin domains.
J Cell Physiol
2005
, vol. 
205
 
1
(pg. 
1
-
9
)
2
Bresnick
 
EH
Lee
 
HY
Fujiwara
 
T
Johnson
 
KD
Keles
 
S
GATA switches as developmental drivers.
J Biol Chem
2010
, vol. 
285
 
41
(pg. 
31087
-
31093
)
3
Ling
 
KW
Ottersbach
 
K
van Hamburg
 
JP
, et al. 
GATA-2 plays two functionally distinct roles during the ontogeny of hematopoietic stem cells.
J Exp Med
2004
, vol. 
200
 
7
(pg. 
871
-
882
)
4
Rodrigues
 
NP
Janzen
 
V
Forkert
 
R
, et al. 
Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis.
Blood
2005
, vol. 
106
 
2
(pg. 
477
-
484
)
5
Tsai
 
FY
Keller
 
G
Kuo
 
FC
, et al. 
An early haematopoietic defect in mice lacking the transcription factor GATA-2.
Nature
1994
, vol. 
371
 
6494
(pg. 
221
-
226
)
6
Tsai
 
FY
Orkin
 
SH
Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation.
Blood
1997
, vol. 
89
 
10
(pg. 
3636
-
3643
)
7
Walsh
 
JC
DeKoter
 
RP
Lee
 
HJ
, et al. 
Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates.
Immunity
2002
, vol. 
17
 
5
(pg. 
665
-
676
)
8
Iwasaki
 
H
Mizuno
 
S
Arinobu
 
Y
, et al. 
The order of expression of transcription factors directs hierarchical specification of hematopoietic lineages.
Genes Dev
2006
, vol. 
20
 
21
(pg. 
3010
-
3021
)
9
Iwasaki
 
H
Akashi
 
K
Myeloid lineage commitment from the hematopoietic stem cell.
Immunity
2007
, vol. 
26
 
6
(pg. 
726
-
740
)
10
Persons
 
DA
Allay
 
JA
Allay
 
ER
, et al. 
Enforced expression of the GATA-2 transcription factor blocks normal hematopoiesis.
Blood
1999
, vol. 
93
 
2
(pg. 
488
-
499
)
11
Grass
 
JA
Boyer
 
ME
Pal
 
S
Wu
 
J
Weiss
 
MJ
Bresnick
 
EH
GATA-1-dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling.
Proc Natl Acad Sci U S A
2003
, vol. 
100
 
15
(pg. 
8811
-
8816
)
12
Martowicz
 
ML
Grass
 
JA
Boyer
 
ME
Guend
 
H
Bresnick
 
EH
Dynamic GATA factor interplay at a multicomponent regulatory region of the GATA-2 locus.
J Biol Chem
2005
, vol. 
280
 
3
(pg. 
1724
-
1732
)
13
Grass
 
JA
Jing
 
H
Kim
 
SI
, et al. 
Distinct functions of dispersed GATA factor complexes at an endogenous gene locus.
Mol Cell Biol
2006
, vol. 
26
 
19
(pg. 
7056
-
7067
)
14
Snow
 
JW
Trowbridge
 
JJ
Fujiwara
 
T
, et al. 
A single cis element maintains repression of the key developmental regulator Gata2.
PLoS Genet
2010
, vol. 
6
 
9
15
Kobayashi-Osaki
 
M
Ohneda
 
O
Suzuki
 
N
, et al. 
GATA motifs regulate early hematopoietic lineage-specific expression of the Gata2 gene.
Mol Cell Biol
2005
, vol. 
25
 
16
(pg. 
7005
-
7020
)
16
Pimanda
 
JE
Ottersbach
 
K
Knezevic
 
K
, et al. 
Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development.
Proc Natl Acad Sci U S A
2007
, vol. 
104
 
45
(pg. 
17692
-
17697
)
17
Rieger
 
MA
Smejkal
 
BM
Schroeder
 
T
Improved prospective identification of megakaryocyte-erythrocyte progenitor cells.
Br J Haematol
2009
, vol. 
144
 
3
(pg. 
448
-
451
)
18
Yu
 
M
Riva
 
L
Xie
 
H
, et al. 
Insights into GATA-1-mediated gene activation versus repression via genome-wide chromatin occupancy analysis.
Mol Cell
2009
, vol. 
36
 
4
(pg. 
682
-
695
)
19
Fujiwara
 
T
O'Geen
 
H
Keles
 
S
, et al. 
Discovering hematopoietic mechanisms through genome-wide analysis of GATA factor chromatin occupancy.
Mol Cell
2009
, vol. 
36
 
4
(pg. 
667
-
681
)
20
Bender
 
MA
Roach
 
JN
Halow
 
J
, et al. 
Targeted deletion of 5′HS1 and 5′HS4 of the beta-globin locus control region reveals additive activity of the DNaseI hypersensitive sites.
Blood
2001
, vol. 
98
 
7
(pg. 
2022
-
2027
)