Mechanism governing a stem cell-generatingcis-regulatory elementRajendran Sanalkumara,b, Kirby D. Johnsona,b, Xin Gaoa,b, Meghan E. Boyera,b, Yuan-I Changb,c, Kyle J. Hewitta,b,Jing Zhangb,c, and Emery H. Bresnicka,b,1

aDepartment of Cell and Regenerative Biology, Wisconsin Institutes for Medical Research, Carbone Cancer Center, University of Wisconsin School of Medicineand Public Health, Madison, WI 53705; cMcArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI53706; and bUniversity of Wisconsin–Madison Blood Research Program, Madison, WI 53705

Edited by Stuart H. Orkin, Children’s Hospital and the Dana–Farber Cancer Institute, Harvard Medical School and Howard Hughes Medical Institute, Boston,MA, and approved February 11, 2014 (received for review January 2, 2014)

The unremitting demand to replenish differentiated cells in tissuesrequires efficient mechanisms to generate and regulate stem andprogenitor cells. Although master regulatory transcription factors,including GATA binding protein-2 (GATA-2), have crucial roles in thesemechanisms, how such factors are controlled in developmentally dy-namic systems is poorly understood. Previously, we described fivedispersed Gata2 locus sequences, termed the −77, −3.9, −2.8, −1.8,and +9.5 GATA switch sites, which contain evolutionarily conservedGATA motifs occupied by GATA-2 and GATA-1 in hematopoieticprecursors and erythroid cells, respectively. Despite commonattributes of transcriptional enhancers, targeted deletions ofthe −2.8, −1.8, and +9.5 sites revealed distinct and unpredictablecontributions to Gata2 expression and hematopoiesis. Herein, wedescribe the targeted deletion of the −3.9 site and mechanisticallycompare the−3.9 site with other GATA switch sites. The−3.9−/− micewere viable and exhibited normal Gata2 expression and steady-statehematopoiesis in the embryo and adult. We established a Gata2repression/reactivation assay, which revealed unique +9.5 siteactivity to mediate GATA factor-dependent chromatin structuraltransitions. Loss-of-function analyses provided evidence for amechanism in which a mediator of long-range transcriptional con-trol [LIM domain binding 1 (LDB1)] and a chromatin remodeler[Brahma related gene 1 (BRG1)] synergize through the +9.5 site,conferring expression of GATA-2, which is known to promote thegenesis and survival of hematopoietic stem cells.

cis element | HSCs

Whereas proximal promoter sequences assemble the basaltranscriptional machinery and RNA polymerase, distant

cis-regulatory elements often confer tissue-specific or context-dependent transcriptional regulation. Enhancer elements residemany kilobases upstream or downstream of a promoter or withinintrons, and extensive efforts have focused on elucidating “ac-tion-at-a-distance” mechanisms (1). Long-range transcriptionalcontrol involves physical interactions between proteins bound atdistal regions and promoter sequences and higher order struc-tural transitions, including subnuclear relocalization of targetloci (2–4). Given the high frequency of long-range mechanismsat mammalian loci and the mutations that disrupt the function ofsuch elements in pathological conditions, elucidating the un-derlying mechanisms in development, tissue homeostasis, anddisease is critically important. In the context of the essentialprocess of hematopoiesis, we have been dissecting long-rangemechanisms controlling the expression and function of the mas-ter regulator GATA binding protein-2 (GATA-2) (5–12).The dual zinc finger transcription factor GATA-2 is expressed

in hematopoietic stem cells (HSCs), select hematopoietic pro-genitors, endothelial cells, neurons, and additional specializedcell types (13–18). Targeted deletion of Gata2 revealed its es-sential function for hematopoiesis. Gata2-nullizygous mouseembryos die from severe anemia at embryonic day (E) 10.5 (13,15), and Gata2+/− HSCs have reduced activity in competitive

transplants (19, 20). Heterozygous mutations of GATA2 underliethe development of a human immunodeficiency syndrome, mon-ocytopenia and mycobacterial infection (MonoMAC), and relateddisorders, which are accompanied by myelodysplastic syndromeand acute myeloid leukemia (21–23). Although the critical roleof GATA-2 in hematopoietic stem/progenitor biology has beenestablished through rigorous genetic studies, many questionsremain unanswered regarding mechanisms underlying Gata2 ex-pression and regulation.Studies in cultured and primary erythroid cells revealed five

GATA-1– and GATA-2–occupied upstream (−77, −3.9, −2.8,and −1.8 kb) and intronic (+9.5 kb) sites of the Gata2 locus (10).Because GATA-2 occupies these prospective regulatory sites inerythroid precursor cells lacking GATA-1, we proposed that thisreflects GATA-2–mediated positive autoregulation (10). Be-cause GATA-1 is expressed during erythropoiesis, it displacesGATA-2, instigating Gata2 repression (24). GATA-1–mediateddisplacement of GATA-2 from chromatin is termed GATAswitching, and the GATA factor-occupied sites are deemedGATA switch sites (10, 24).Despite the compelling biochemical and molecular attributes

of the GATA switch sites, targeted deletion of the −1.8 and −2.8sites individually in the mouse revealed only minor roles in max-imizing Gata2 expression in hematopoietic precursors (6, 7).The −1.8−/− and −2.8−/− mice were born at normal Mendelian ra-tios, and hematopoiesis was largely normal in steady-state and stresscontexts. The −1.8 element is uniquely required to maintain, but

Significance

The continuous replenishment of differentiated cells, for example,those constituting the blood, involves proteins that control thegeneration and function of stem and progenitor cells. Although“master regulators” are implicated in these processes, many ques-tions remain unanswered regarding how their synthesis andactivities are regulated. We describe a mechanism that con-trols the production of the master regulator GATA bindingprotein-2 (GATA-2) in the context of blood stem and progenitorcells. Thousands of GATA-2 binding sites exist in the genome,and genetic analyses indicate that they differ greatly and un-predictably in functional importance. The parameters involvedin endowing sites with functional activity are not established.We describe unique insights into ascertaining functionally im-portant GATA-2 binding sites within chromosomes.

Author contributions: R.S., K.D.J., X.G., and E.H.B. designed research; R.S., K.D.J., X.G.,M.E.B., and Y.-I.C. performed research; K.D.J. contributed new reagents/analytic tools;R.S., K.D.J., X.G., K.J.H., J.Z., and E.H.B. analyzed data; and R.S., K.D.J., and E.H.B. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: ehbresni@wisc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1400065111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1400065111 PNAS | Published online March 10, 2014 | E1091–E1100

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not to initiate,Gata2 repression in late-stage erythroblasts, but thismolecular defect was not coupled to major functional deficits (6).In contrast to the −1.8 and −2.8 site deletions, targeted deletionof the +9.5 intronic site is embryonically lethal at E13.5–E14.5(5). The +9.5 site is essential for GATA-2 expression in hema-topoietic stem and progenitor cells (HSPCs) and in endotheliumduring embryogenesis (5, 9, 25, 26). Definitive hematopoiesis isseverely impaired in +9.5−/− mice due to defective HSC produc-tion, as demonstrated by competitive transplants and imaging ofHSC genesis from hemogenic endothelium in the dorsal aorta (25).The +9.5 site contains an E-box–GATA composite element,

which mediates assembly of a complex containing GATA-1 orGATA-2, T-cell acute lymphocytic leukemia 1 (TAL1), LIMdomain binding 1 (LDB1), and LIM domain only 2 (LMO2).The GATA and E-box motifs, and the spacing between the

motifs, are essential for +9.5 site enhancer activity in reporterassays (11). The E-box binding protein TAL1 cooperates withGATA factors in the assembly of a multicomponent complex onE-box–GATA composite elements at genes important for bloodcell development and function (27–33). The TAL1-interactingproteins LDB1 and LMO2 control the development and functionof HSPCs (22, 34–38). In addition to binding sites containingGATA–E-box composite elements, like the +9.5 site, TAL1 occu-pies GATA motif-containing sites lacking a consensus E-box, pre-sumably via recruitment by the GATA factor (28). The LIMdomain binding-1 coregulator LDB1 promotes chromatin loop-ing (39, 40) and facilitates HSC maintenance, primitive hema-topoietic progenitor generation, and erythroid differentiation(37, 41–44). Certain patients with MonoMAC who lack GATA2coding region mutations harbored deletion or point mutations in

Fig. 1. The −3.9 GATA switch site bears hallmarks of an important cis-regulatory element. (A) Sequence alignment of the −3.9 site demonstrates conser-vation among vertebrates. The WGATAR motifs and intervening sequence that were removed by homologous recombination are indicated. (B) ChIP-sequencing profiles for factor occupancy and histone modifications at the Gata2 locus mined from existing datasets (37, 69–72). BM, bone marrow;H3K27a, acetylation of H3 at lysine 27; H3K4m1, monomethylation of histone H3 at lysine 4; MEL, murine erythroleukemia cells. (C) Strategy for targeteddeletion of the −3.9 site. Following NeoR excision, the targeted allele has a 126-bp Xba I-to-Not I fragment containing a single LoxP site substituted for theGATA motifs and intervening sequence. Arrowheads indicate positions of primers used for genotype determination. (D) Representative gel shows PCR-basedstrategy to distinguish WT and targeted alleles following NeoR excision. (E) Genotypes of viable pups from mating −3.9+/− males and females determined atthe time of weaning. Expected numbers of pups based on Mendelian ratios are shown in parenthesis. (F) Representative −3.9+/+ and −3.9−/− embryos at E12.5.

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or near the +9.5 site (5, 45). Thus, mutations of the +9.5 element,an essential mediator of definitive hematopoiesis in the mouse,underlie human hematopoietic pathology.The contributions of the −77 and −3.9 GATA switch sites to

Gata2 regulation in vivo have not been reported. The −3.9 siteharbors two inverted GATA motifs and contains canonicalattributes of cis-regulatory elements, including DNase I hyper-sensitivity and GATA site-dependent enhancer activity in atransfection assay (9, 12). Because these attributes are sharedwith one or more of the −2.8, −1.8, and +9.5 sites, which differgreatly in their importance in vivo, the contribution of the −3.9site can only be ascertained by disruption of this site at the en-dogenous locus. Herein, we describe the consequences of a −3.9site deletion from the endogenous Gata2 locus and a mecha-nistic comparison of the −3.9 site with the −2.8, −1.8, and +9.5sites in biologically relevant contexts. These studies led to a modelto explain the unique importance of the +9.5 site.

ResultsSequence Conservation and Transcription Factor Occupancy Do NotPredict Gata2 Cis-Element Function in Vivo. The −3.9 site containsinverted WGATAR motifs that are well conserved among verte-brates (Fig. 1A). GATA-2 occupancy of the −3.9 site, first de-scribed in GATA-1–null G1E cells (12), occurs in multiple celllines and primary cells, including lineage negative (Lin−) he-matopoietic progenitors from bone marrow (Fig. 1B). Featurescommonly associated with enhancers [DNase hypersensitivity,monomethylation of histone H3 at lysine 4, acetylation of H3 atlysine 27, and p300 occupancy (46, 47)] characterize the −3.9 siteand other GATA switch sites. To assess −3.9 site function, we

deleted 27 nucleotides encompassing the GATA motifs andintervening sequence by homologous recombination and excisedthe NeoR gene (Fig. 1C). Targeted and WT alleles were distin-guishable by PCR with primers flanking the −3.9 site (Fig. 1D).Like −1.8 and −2.8 mice, genotypes of −3.9−/− and −3.9+/− mutantsconformed to Mendelian genetics, indicating the −3.9 site is dis-pensable for viability (Fig. 1E). The −3.9−/− embryos wereindistinguishable from WT littermates and did not exhibit he-matopoietic or vascular defects characteristic of +9.5−/− embryos(Fig. 1F).Although a singleGata2 allele is sufficient to confer viability in

Gata2+/− and +9.5+/− mouse strains, heterozygosity in thesemice reduces Gata2 expression, as well as HSC generation andfunction (5, 19, 20, 25). To assess the influence of the −3.9mutation on HSC genesis, we conducted whole-mount 3D em-bryo imaging to visualize c-Kit+ hematopoietic clusters con-taining HSCs in E10.5 aorta gonad mesonephros (AGM) of−3.9+/+ and −3.9−/− littermates. Whereas +9.5−/− AGM is al-most devoid of c-Kit staining (25), −3.9−/− AGM hematopoieticclusters were indistinguishable in size and number vs. those ofWT littermates (Fig. 2A). Quantitation of Gata2 mRNA levels inE13.5 fetal livers and brains revealed no differences between−3.9+/+, −3.9+/−, and −3.9−/− littermates (Fig. 2B).In adult −3.9+/+, −3.9+/−, and −3.9−/− mice, complete blood

cell count (CBC) measurements were compared at 2 and 6 mo ofage (Tables S1 and S2). The quantities of circulating blood celltypes were indistinguishable. Because GATA-2 haploinsufficiencyincreases quiescence and apoptosis in primitive bone marrow cellswithout changes to circulating blood cells (19), the −3.9 mutationmight alter GATA-2 levels in HSPCs without significantly affect-

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Fig. 2. The −3.9 site is dispensable for Gata2 expression during hematopoiesis. (A) Whole-mount immunostaining of CD31+ cells (magenta) and c-Kit+ cells(green) within the aorta region of E10.5 embryos. The −3.9−/− embryos were compared with WT littermates and with +9.5−/− embryos that almost entirely lackc-Kit+ HSCs (25). (Scale bars, 100 μM.) Quantitation of the number of c-Kit+ cells per dorsal aorta (DA) (four embryos each for −3.9+/+ and −3.9−/−; two embryosfor +9.5−/−) (mean ± SEM). (B) Quantitative analysis of Gata2mRNA in E13.5 livers [six litters: −3.9+/+ (n = 12), −3.9+/− (n = 20), −3.9−/− (n = 18)] and brains [fourlitters: −3.9+/+ (n = 9), −3.9+/− (n = 14), −3.9−/− (n = 11)] (mean ± SEM). −RT, no reverse transcriptase. (C) Ter119+ and Lin− populations were sequentiallyisolated from bone marrow via magnetic bead separation. Enrichment of the distinct populations was confirmed in −3.9+/+ bone marrow samples bymeasuring the expression of the lineage-restricted genesMpl (Lin−) and Hbb-b1 (Ter119+). (D) Comparison of Gata2mRNA expression in Lin− and Ter119+ cellsfrom three independent isolations (mean ± SEM). Fluorescence-activated cell sorting of Sca-1+ and c-Kit+ double-positive cells from Lin− cells of −3.9+/+ and−3.9−/− mice (E) and comparison of Gata2 expression in Lin−Sca+Kit+ cells from two independent biological replicates (mean ± SD) (F) are shown. ***P < 0.001(two-tailed unpaired Student t test).

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ing differentiated cell types. Gata2 mRNA levels were quantitatedin Lin− progenitors and Ter119+ erythroid cells isolated frombone marrow of −3.9+/+ and −3.9−/− mice. The purity of eachpopulation was confirmed by quantitating expression of lineage-specific markers Mpl and Hbb-b1 (Fig. 2C). Although highlyexpressed in the Lin− population, Gata2 expression levels wereindistinguishable between −3.9+/+ and −3.9−/− mice (Fig. 2D).In Ter119+ cells, Gata2 expression was not greater than in thecontrol lacking reverse transcriptase. Sca-1 and c-Kit double-pos-itive cells were sorted from the Lin− population (Lin−Sca+Kit+)of −3.9+/+ and −3.9−/− bone marrow to enrich for HSPCs (Fig.2E). Gata2 expression in these cells was not influenced signifi-cantly by the −3.9 mutation (Fig. 2F).Despite certain shared molecular attributes of the −3.9 and

+9.5 sites, the conserved GATA motifs of the −3.9 site weredispensable for Gata2 expression in the embryo and adult, steady-state hematopoiesis, and embryogenesis. To elucidate the uniquemolecular underpinnings of the critical +9.5 site activity, wemechanistically compared the four GATA switch sites (−3.9, −2.8,−1.8, and +9.5) that have been functionally analyzed in vivo.

Requirements for Assembly of an Intronic Enhancer ComplexContaining Master Regulators of Hematopoiesis. The −3.9 and +9.5GATA switch sites are DNaseI-hypersensitive in murine eryth-roleukemia cells (Fig. 1B) and E14.5 fetal liver (48) (Fig. 3A),indicative of an open chromatin configuration. As an alternativeapproach to evaluate chromatin accessibility at these sites, weconducted formaldehyde-assisted isolation of regulatory elements(FAIRE) analysis (49). Genome-wide analyses indicated thatFAIRE peaks overlap with multiple open chromatin parameters,and FAIRE can be conducted with fewer cells than conventionalChIP or DNase I hypersensitivity and/or sensitivity assays (50, 51).FAIRE analysis of E12.5 fetal liver cells demonstrated en-

hanced accessibility at the −3.9 and +9.5 sites (Fig. 3B), with theopen chromatin restricted to the GATA switch sites (Fig. 3C).Allele-specific FAIRE analysis was conducted on −3.9+/− and

+9.5+/− E12.5 fetal livers using primers specific for the WT ormutant −3.9 and +9.5 alleles. Chromatin accessibility was con-siderably reduced at the mutant alleles (Fig. 3D). Despite thereduced accessibility resulting from the −3.9 mutation, Gata2expression in −3.9−/− fetal livers was indistinguishable from thatof −3.9+/+ controls (Fig. 2B). Thus, the −3.9 site confers ac-cessibility at this site but is dispensable for Gata2 expression.Because +9.5−/− mice are deficient in fetal liver HSPCs (5), wetook advantage of the intronic location of the +9.5 site to con-duct allele-specific measurements of Gata2 primary transcriptsgenerated from WT and mutant alleles in primary cells from+9.5+/− mice. In E12.5 fetal liver and adult bone marrow,transcription of the mutant allele was substantially reduced (P <0.001) (Fig. 3E), whereas both alleles were expressed equiva-lently in E12.5 fetal brain (Fig. 3E), consistent with our priorfindings (5). In summary, although mutations of the −3.9 and +9.5sites abrogate local chromatin accessibility, this altered molecularattribute is only linked to loss ofGata2 transcription with the +9.5site mutation.Of the Gata2 GATA switch sites analyzed in vivo, only the

+9.5 site contains a conserved E-box in proximity to the GATAmotif. GATA-2, TAL1, and LDB1 ChIP-sequencing analysis inLin− bone marrow (37) revealed the +9.5 site as the sole regionof the Gata2 locus occupied by all of these factors (Fig. 4A).Quantitative ChIP analysis in E12.5 fetal liver revealed TAL1and LDB1 occupancy at the +9.5 site but not at other GATAswitch sites (Fig. 4B). The +9.5 site deletion abrogated TAL1 andLDB1 occupancy (P < 0.001) at the mutant, but not WT, allelesin +9.5+/− fetal liver cells (Fig. 4C). The analyses with primaryfetal liver cells from +9.5+/− mice described above revealed +9.5site-dependent chromatin accessibility and regulatory complexassembly at the Gata2 locus.

Molecular Attributes That Distinguish the +9.5 Site from Other GATASwitch Sites. Because targeted deletion of the −3.9, −2.8, or −1.8site individually did not evoke major biological phenotypes, the

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Fig. 3. GATA switch site mutations abrogate chromatin accessibility at −3.9 and +9.5 sites. (A) DNaseI hypersensitivity at the Gata2 locus in fetal liver mined frommouse Encyclopedia of DNA Elements data (48). (B) Quantitative FAIRE analysis of GATA switch sites in WT fetal liver (n = 4, mean ± SEM). The promoters of theactively transcribed RNA Polymerase II (RPII215) and inactive Keratin 5 (Krt5) genes were used as positive and negative controls, respectively. (C) Quantitative FAIREanalysis of chromatin accessibility at and surrounding the −3.9 and +9.5 sites (n = 4, mean ± SEM). The dashed line illustrates the average FAIRE signal at the Krt5promoter. (D) Allele-specific FAIRE analysis of WT and mutated (Mt) alleles in fetal liver cells from −3.9+/− (n = 4) and +9.5+/− (n = 5) E13.5 embryos (mean ± SEM).Primers used for the allele-specific FAIRE analysis are indicated in Table S3. (E) Allele-specific analysis of Gata2 primary transcripts fromWT and Mt alleles in +9.5+/−

E13.5 fetal liver and brain (n = 8) and adult bone marrow (n = 3) samples (mean ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed unpaired Student t test).

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+9.5 element uniquely endows hemogenic endothelium of theAGM with the capacity to generate long-term repopulatingHSCs (LT-HSCs) that populate the fetal liver (5, 25). In addi-tion, the small number of HSCs generated from +9.5−/− AGMundergo apoptosis and lack LT activity, indicating that the +9.5element also confers LT-HSC survival (25); a similar conclusionemerged from analysis of a conditional Gata2 KO mouse (52).The +9.5 site confers maximal Gata2 expression in the AGMand definitive hematopoietic precursors in the fetal liver (5, 25).A critical question is what molecular attributes underlie theuniquely important +9.5 site activity.To identify molecular attributes that distinguish the +9.5

site from the other GATA switch sites, we developed a Gata2repression/reactivation system using GATA-1–null G1E pro-erythroblast-like cells (53). G1E cells express GATA-2 (53), andectopic expression of GATA-1 represses GATA-2 and overcomesan erythroid maturation blockade (10, 54). Using a conditionallyactive GATA-1 allele, in which GATA-1 is fused to the estrogenreceptor ligand binding domain (ER–GATA-1), β-estradiol treat-ment of G1E–ER–GATA-1 cells rapidly induces Gata2 repression(10). We reasoned that removing β-estradiol from the culturemedia subsequent to Gata2 repression would induce time-dependent loss of ER–GATA-1 activity, ER–GATA-1 dissocia-tion from chromatin, and reversion of ER–GATA-1 influenceson gene expression (Fig. 5A). β-Estradiol treatment of G1E–ER–

GATA-1 cells for 24 h strongly reduces Gata2 mRNA andprotein (10). β-Estradiol washout after 24 h induced reactivation

of Gata2 primary transcripts (Fig. 5B) and GATA-2 protein(Fig. 5C), concomitant with reduced ER–GATA-1 levels (Fig. 5C).Because we are unaware of a system that allows one to study theconversion of the repressed Gata2 locus to an active locus, thissystem has unique utility for elucidating GATA switch sitefunction.GATA-1–mediated Gata2 repression is associated with GATA-1

replacement of GATA-2 at GATA switch sites (10). To inves-tigate the impact of the Gata2 reactivation on the GATA switch,ChIP analysis was used to quantitate changes in ER–GATA-1and GATA-2 occupancy during Gata2 repression and reacti-vation (Fig. 5D). As expected (24), 24 h of β-estradiol treatmentinduced replacement of GATA-2 by ER–GATA-1 at all sitestested, although ER–GATA-1 occupancy of the −1.8 site was low,consistent with our prior findings. Surprisingly, 24 h after washingout β-estradiol, when GATA-2 is readily detected by Westernblotting, GATA-2 occupancy was only restored at the +9.5 and−3.9 sites (54% and 57% reoccupancy, respectively), despite lossof GATA-1 from all sites (Fig. 5D).Given that GATA-2 occupies all of the Gata2 GATA switch

sites before repression but only reoccupies +9.5 and −3.9 sitesfollowing Gata2 reactivation, we tested whether ER–GATA-1–induced repression creates inaccessible chromatin at the −1.8and −2.8 sites that persists after ER–GATA-1 dissociation fromchromatin and prevents subsequent GATA-2 occupancy. We usedquantitative FAIRE to analyze chromatin accessibility in theGATA-2 repression/reactivation system. In uninduced proliferating

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Fig. 4. Unique propensity of TAL1 and LDB1 to occupy the +9.5 site. (A) ChIP-sequencing profiles of factor occupancy at the Gata2 locus in lineage-negativebone marrow cells mined from existing datasets (37). (B) Quantitative ChIP analysis of TAL1 and LDB1 occupancy at GATA switch sites of the Gata2 locus inE13.5 fetal liver (n = 3, mean ± SEM). ***P < 0.001. The Necdin promoter was used as a negative control. (C) Allele-specific ChIP analysis of TAL1 and LDB1occupancy at WT and Mt alleles in fetal liver from E13.5 +9.5+/− embryos (n = 4, mean ± SEM). ***P < 0.001 (two-tailed unpaired Student t test). PI,preimmune.

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G1E-ER-GATA-1 cells expressing Gata2, chromatin accessibil-ity of the GATA switch sites was significantly greater than in theinactive Necdin promoter (Fig. 6A). ER–GATA-1–mediatedGATA-2 displacement reduced FAIRE signals at GATA switchsites 2.8- to 14-fold (Fig. 6B, 24 h). Upon Gata2 reactivation,chromatin accessibility was restored (108% of 0 h) only at the+9.5 site (Fig. 6B, 48 h; P < 0.001); accessibility of −1.8, −2.8,and −3.9 sites remained low (Fig. 6B, 48 h). The unique tripartitechromatin signature of the +9.5 site, in which accessibility is lostupon repression and restored upon reactivation, reflects +9.5site activity to mediate dynamic chromatin transitions duringGata2 activation. Because this tripartite chromatin signaturedistinguishes the +9.5 site from other switch sites, we evaluatedmechanisms underlying this unique behavior.In principle, certain factors might be selectively retained at the

+9.5 site, thus explaining its unique capacity to reestablish ac-cessible chromatin. Based on the +9.5 site-restricted TAL1 andLDB1 occupancy in fetal liver (Fig. 4B), we asked whether TAL1and LDB1 occupancy uniquely characterizes the +9.5 site. Usingthe repression/reactivation system, we quantitated TAL1 andLDB1 occupancy at the active (0 h, uninduced), repressed (24 h,estradiol-induced), and reactivated (48 h, washout) Gata2 locus.Twenty-four hours after β-estradiol treatment, when GATA-2

protein is undetectable, TAL1 and LDB1 levels were unchangedor increased slightly (Fig. 6C). Both factors occupied the −2.8,−1.8, and +9.5 sites of the active Gata2 locus (Fig. 6D). UponGata2 repression, TAL1 and LDB1 occupancy decreased at the−2.8 and −1.8 sites but was partially retained at the +9.5 site.Brahma related gene 1 (BRG1) occupied the active and re-pressed loci (Fig. 6E). The TAL1 and LDB1 levels retained atthe +9.5 site were comparable to the highly active βmajor pro-moter and were considerably higher (P < 0.001) than those at the−1.8 and −2.8 sites (Fig. 6D); ER–GATA-1 is known to increaseTAL1 recruitment to the βmajor promoter (40). It is attractive topropose that TAL1/LDB1/BRG1 retention at the +9.5 site ofthe repressed Gata2 locus reflects a priming mechanism thatcreates epigenetic memory to ensure a rapid increase in thechromatin accessibility and factor occupancy required for sub-sequent locus reactivation.

Establishing and Maintaining Physiological GATA-2 Levels: DualRequirement for a Chromatin Looping Factor and a ChromatinRemodeler. Of the −3.9, −2.8, −1.8, and +9.5 GATA switchsites analyzed in mutant mouse strains, TAL1/LDB1 chromatinoccupancy is unique to the +9.5 site. We conducted loss-of-function analyses to establish the importance of the +9.5 site-occupied components. Regarding the possibility of priming the+9.5 site to ensure rapid chromatin remodeling as a requisitestep in reactivation, because TAL1 and GATA-1 can localize tochromatin sites with and interact functionally with the ATPasecomponent (BRG1) of the SWI/SNF chromatin remodelingcomplex (55–57), BRG1 might mediate chromatin remodeling atthe +9.5 site. To assess the contributions of LDB1 and BRG1 tomaintenance of Gata2 expression, uninduced G1E–ER–GATA-1cells were treated twice with siRNA and harvested 24 h after thesecond treatment (Fig. 7A). When knocked down individually,loss of LDB1 or BRG1 did not influence GATA-2 levels (Fig.7B), whereas simultaneous loss of LDB1 and BRG1 substantiallyreduced GATA-2. LDB1 and BRG1 activity to establish Gata2expression was assessed with the reactivation system (Fig. 7C).Although knocking down LDB1 or BRG1 individually did notprevent Gata2 reactivation, GATA-2 levels were partially re-duced (Fig. 7D, Left and Fig. S1). Knocking down LDB1 didnot affect GATA-2 chromatin occupancy (Fig. S2). Resemblingthe results with uninduced cells, knocking down LDB1 andBRG1 simultaneously greatly reduced reactivation (Fig. 7D,Right). Thus, LDB1 and BRG1 establish and maintain GATA-2expression.In uninduced cells and reactivation contexts, the LDB1 knock-

down decreased TAL1 levels without a concomitant change inGATA-2 levels (Fig. 7B). This result is consistent with our priorTAL1 knockdown in G1E–ER–GATA-1 cells, which did notalter GATA-2 levels (58). LDB1 occupies the TAL1 locus (37)(Fig. S3), and therefore might directly regulate TAL1 expression.Although individually knocking down LDB1 prevented the res-toration of TAL1 occupancy at the +9.5 site to a level com-mensurate with the active Gata2 locus (Fig. 7E), under theseconditions, the washout still restored chromatin accessibility(Fig. 7F). TAL1 occupancy (Fig. 7E) and chromatin accessibility(Fig. 7F) decreased at the −1.8 site upon repression and were notrestored upon Gata2 reactivation. Unlike the individual factorknockdowns, the LDB1/BRG1 double knockdown, which blockedGata2 reactivation, prevented the washout-induced restoration ofopen chromatin at the +9.5 site (Fig. 7E). Because the washoutdecreased GATA-1 occupancy at all Gata2 GATA switch sites(Fig. 7G), the inability to reactivateGata2 when LDB1 and BRG1levels are limiting cannot be explained by GATA-1 retention. Theseresults suggest that the failure to establish an active enhancercomplex at the +9.5 site underlies the reactivation defect.

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Fig. 5. Gata2 repression/reactivation assay. Evidence for distinct functionalproperties of the GATA switch sites is illustrated. (A) Schematic representa-tion of the experimental strategy for Gata2 repression and reactivation inG1E–ER–GATA-1 cells. Treatment of G1E–ER–GATA-1 cells with β-estradiolactivates ER–GATA-1, leading to loss of Gata2 transcripts and protein by 24 h(10). Washout of β-estradiol reverses Gata2 repression, leading to restorationof GATA-2 by 48-h treatment. (B) Quantitative real-time PCR was used tomeasure Gata2 primary transcripts during locus reactivation. After 24 h,β-estradiol treatment (+estradiol) cells were washed in PBS and cultured inmedia without β-estradiol (washout) for an additional 24 h. RNA was iso-lated and analyzed before β-estradiol treatment (0 h); after 24 h of β-es-tradiol treatment; and 2, 6, 12, and 24 h following washout (n = 4, mean ±SEM). (C) Representative Western blots of GATA-2 and ER–GATA-1 fromsamples isolated at the same times as the corresponding RNA samples. (D)Relative chromatin occupancy of ER-GATA-1 (○) and GATA-2 (●) duringGata2 reactivation using quantitative ChIP (n = 3, mean ± SEM).

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DiscussionHow to distill large chromatin occupancy datasets into functionallycritical genomic sites, especially sites distal to genes, represents aformidable problem with far-reaching implications. Our mousestrains lacking GATA switch sites differ grossly in phenotypesand offer a unique opportunity to elucidate mechanisms thatendow GATA factor-bound chromatin sites with nonredundantactivity in vivo. Despite certain shared molecular attributes ofthe −3.9 and +9.5 sites, the conserved GATA motifs of the −3.9site were dispensable for Gata2 expression in the embryo or adult,in steady-state hematopoiesis, and in embryogenesis.Because the coupling of the E-box and GATA motif and

intronic location distinguish the +9.5 site from the −3.9, −2.8,and −1.8 sites, it is attractive to propose that these attributes areimportant determinants of +9.5 site activity in vivo. In Gata2-expressing fetal liver cells, the +9.5 site resided in open chro-matin and assembled a complex containing GATA-2, TAL1, andLDB1 (Fig. 7I). Simultaneous interrogation of the chromatinaccessibility of WT and mutant alleles in +9.5+/− fetal liver cellsand analyses with the repression/reactivation system revealedthat the +9.5 site mediates dynamic chromatin structure tran-sitions. Although ER–GATA-1 converted accessible chromatinat the +9.5 site and other Gata2 GATA switch sites into in-accessible chromatin, concomitant with repression, the washout-induced loss of GATA-1 activity selectively converted +9.5 site-inaccessible chromatin into accessible chromatin. Intriguingly,TAL1 and LDB1 were partially retained at the +9.5 site but notat other Gata2 GATA switch sites, and upon reactivation, chro-matin accessibility was only restored at the +9.5 site. These resultssuggest that TAL1/LDB1 retention creates an epigenetic memorythat ensures reassembly of the functional +9.5 site enhancer and

Gata2 transcriptional activation. This mechanism may be con-ceptually similar to the findings that target gene occupancy bycertain transcription factors is retained in mitotic chromatin andis associated with more rapid transcriptional activation upon entryinto G1 (59, 60).GATA-1 and GATA-2 occupy the TAL1 locus and loci-encoding

components of the TAL1 complex (e.g., the corepressor ETO2)(61, 62). Herein, we demonstrate that TAL1 protein expressionis sensitive to LDB1 protein levels. The relationship betweenLDB1, TAL1, and GATA-2 can be modeled as a coherent type Ifeed-forward loop (Fig. 7H), which is predicted to require per-sistently elevated input signals (e.g., those controlling the LDB1level/activity), although filtering out transiently elevated inputsignals, to achieve a robust output (e.g., enhanced HSC genera-tion) (63). LDB1 and BRG1 synergistically confer +9.5 site-accessible chromatin, enhancer activity, and Gata2 expression.Although BRG1 was reported to interact functionally with TAL1(32), we are unaware of examples of a mechanism requiring bothBRG1 and LDB1. The BRG1 activity may have broad implica-tions in diverse contexts, because BRG1 is required for Pax6-dependent control of neural fate (38), for Olig2 to establish anoligodendrocyte-specific transcriptional program (64), and forcardiovascular development (65, 66).In summary, we describe a mechanism that endows a stem

cell-generating enhancer element with its unique activity anddifferentiates it from other GATA factor-bound chromosomalsites that we have rigorously analyzed in vivo. Intrinsic to thismechanism is synergism between a chromatin looping factor anda chromatin remodeler to generate physiological levels of themaster hematopoietic regulator GATA-2 (Fig. 7I). In the contextof hematopoiesis, it will be particularly instructive to consider

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Fig. 6. Molecular attributes of the +9.5 site revealed by the repression/reactivation assay. (A) Quantitative FAIRE analysis of chromatin accessibility at GATAswitch sites in untreated G1E-ER-GATA-1 cells (0 h). The inactive Necdin locus was used as a negative control (n = 4, mean ± SEM). (B) Quantitative FAIREanalysis of chromatin accessibility at GATA switch sites upon Gata2 repression and reactivation. FAIRE signals for the 0-h times were normalized to 1.0 (n = 4,mean ± SEM). (C) Representative Western blots of GATA-2, TAL1, and LDB1 protein levels at the same times analyzed by ChIP. The asterisk representsa nonspecific band. (D) Quantitative ChIP analysis of TAL1 and LDB1 chromatin occupancy at the active (0 h), repressed (+24 h), and reactivated (WO) Gata2locus. The βmajor promoter was used as a positive control (n = 4, mean ± SEM). **P < 0.01; ***P < 0.001. (E) Quantitative ChIP analysis of BRG1 chromatinoccupancy at the active (0 h) and repressed (+24 h) Gata2 locus and control sites (βmajor and Necdin) (n = 3, mean ± SEM). ***P < 0.001. WO, washout.

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whether this mechanism is quite selective for controlling GATA-2and the GATA-2–dependent genetic network or if it has animpact, more broadly, on the HSPC transcriptome.

Materials and MethodsGeneration of Gata2 Δ-3.9 Mutant Mice. The Gata2 −3.9 site sequence AGA-TAGGAAAATGGCCGCGCGCTATCT containing the inverted WGATAR motifswas replaced with a LoxP-phosphoglycerate kinase neomycin (neo)-LoxPcassette via homologous recombination. Targeting was confirmed bySouthern blotting. Chimeric mice were generated by blastocyst injection,and first filial generation pups were screened for germ-line transmission byPCR. NeoR excision was achieved by mating to CMV-cre strain B6.C-Tg(CMV-

cre)1Cgn/J mice (Jackson Laboratory). Cre-mediated excision of NeoR in theprogeny was confirmed by PCR using primers flanking the targeted se-quence. Primer sequences are provided in Table S3.

Analysis of Mouse Embryos and Tissues. Staged embryos were obtained fromtimed matings of Gata2 −3.9 heterozygotes. Embryo viability was scored bythe presence of a beating heart. E13.5 fetal livers and brains were harvestedinto TRIzol (Invitrogen) for RNA extraction. For CBC analysis, blood samplesfrom 33 anesthetized mice (four litters: 10 −3.9+/+, 14 −3.9+/−, and 9 −3.9−/−

mice) were collected by retroorbital bleeding at 2 and 6 mo of age. CBC mea-surements were collected using a Hemavet CBC Analyzer (Drew Scientific, Inc.).Bone marrow was isolated from femurs and tibias into PBS containing

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Fig. 7. Mechanism underlying +9.5 site function. (A) Knockdown strategy. Cells were transfected with siRNA twice with a 24-h interval and harvested 48 hafter the first transfection. (B) Representative Western blots of GATA-2, TAL1, LDB1, and BRG1 following knockdown of Ldb1 and Brg1 mRNAs individually orin combination. The asterisk represents a nonspecific band. Schematic representation (C) and Western blots (D) of siRNA-mediated factor knockdown strategyduring Gata2 repression/reactivation. G1E–ER–GATA-1 cells were induced with β-estradiol (0 h) and transfected twice with factor-specific or control siRNAs at6 and 24 h postinduction. β-estradiol was washed out at the time of the second transfection. Protein and RNA samples were collected at 24 and 48 h. −,β-estradiol uninduced at 0 h; +, β-estradiol induced at 24 h; WO, β-estradiol washout. All conditions received specific siRNA or nontargeting control siRNA atsame molar concentration. (E) ChIP analysis of TAL1 occupancy at the +9.5 and −1.8 sites quantitated under individual knockdown or LDB1 and BRG1combined knockdown conditions (n = 4, mean ± SEM). **P < 0.01; ***P < 0.001. (F) FAIRE analysis of chromatin accessibility of +9.5 and −1.8 sites followingLDB1 and BRG1 individual or LDB1/BRG1 combined knockdown (n = 3, mean ± SEM). ***P < 0.001. (G) ChIP analysis of GATA-1 occupancy at GATA switch sitesfollowing LDB1/BRG1 combined knockdown during GATA-2 reactivation (n = 4, mean ± SEM). **P < 0.01; ***P < 0.001. (H) Type I coherent feed-forward loopnetwork motif that controls Gata2 expression. (I) Model depicts GATA switch-site chromatin architecture and its relationship to Gata2 expression. The red Xindicates that the motif has been deleted.

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2% (vol/vol) FBS and 2 mM EDTA (wash buffer). Dissociated cells werepelleted at 300 × g for 10 min and resuspended in wash buffer at 1 × 108

cells per milliliter. Ter119+ cells were isolated by magnetic bead isolationusing rat anti-mouse Ter-119 Biotin (eBioscience). The Ter-119–depletedpopulation was depleted for additional lineage markers using the EasySepMouse Hematopoietic Progenitor Cell Enrichment Kit (Stemcell Technol-ogies). For isolation of Lin−Sca+Kit+ cells, Sca-1 and c-Kit double-positivecells were sorted from Lin− cells on a FACSAria II cell sorter (BD Biosciences)using rat anti-mouse CD117 allophycocyanin and anti-mouse Ly-6A/E perdi-dine chlorophyll protein-Cyaninine5.5 (eBioscience). Data were analyzed usingFlowJo v9.0.2 software (TreeStar).

Whole-Embryo Confocal Microscopy. Embryos were fixed, stained, and ana-lyzed as described (25, 67). Briefly, E10.5 embryos were stained for c-Kit usingrat anti-mouse c-Kit (BD Biosciences) and Alexa Fluor 647 goat anti-rat IgG(Invitrogen) and then for PECAM1 using biotinylated rat anti-mouse CD31(BD Biosciences) and Cy3-conjugated streptavidin (Jackson ImmunoResearch).Samples were cleared in a 1:2 mix of benzyl alcohol and benzyl benzoate toincrease transparency before imaging with a Nikon A1R Confocal Microscope.Three-dimensional reconstructions were generated from Z-stacks (50–150 op-tical sections) using Fiji software.

Cell Culture. G1E-ER-GATA-1 cells (10, 54) were maintained in Iscove’s mod-ified Dulbecco’s medium (GIBCO) supplemented with 15% FBS (Gemini), 1%penicillin/streptomycin (Gemini), 2 U/mL erythropoietin, 120 nM mono-thioglycerol (Sigma), 0.6% conditioned medium from a Kit ligand-producingCHO cell line, and 1 μg/mL puromycin (Sigma). To induce ER–GATA-1, cellswere treated with 1 μM β-estradiol. For Gata2 reactivation studies, cells wereinduced with β-estradiol for 24 h and then washed with 1× Dulbecco’sphosphate buffered saline to remove β-estradiol. Cells were grown in mediawithout β-estradiol for an additional 24 h, and samples were collected 2, 6,12, and 24 h after washout. Cell cultures were maintained in a 37 °C incubatorwith 5% CO2. siRNA-mediated genetic perturbation was used to knock downfactors in G1E-ER-GATA-1 cells. Specific SMART Pool siRNAs or nontargetingsiRNA pools (240 pmol each; Dharmacon) were transfected into cells usingAmaxa nucleofection kit R. For knockdown analyses in uninduced cells, cellswere transfected twice with a 24-h interval. For analyses in the reactivationparadigm, two transfections were conducted 6 and 24 h after β-estradiolinduction. Samples were harvested at 24 h and/or 48 h.

Quantitative Real-Time PCR Analysis. Total RNA was purified with TRIzol.cDNA was synthesized from 1.5 μg of purified total RNA by Moloney MLV

reverse transcriptase (M-MLV RT). Real-time PCR analysis was conducted withSYBR Green Master Mix (Applied Biosystems). Control reactions lacking M-MLV RT yielded little to no signal. Relative expression was determined froma standard curve of serial dilutions of cDNA samples, and values were nor-malized to 18S RNA expression. Primer sequences are provided in Table S3.

Quantitative FAIRE Assay. FAIRE analysis was conducted as described (50) withminor modifications. Cells were fixed with 1% formaldehyde for 5 min atroom temperature and sonicated to shear the DNA to an average size of200–700 bp. Ten percent of the sonicated chromatin was used as the inputcontrol. Following phenol/chloroform extraction, ethanol-precipitated DNApellets were resuspended in 50 μL of nuclease-free water. For FAIRE analysisof fetal livers, −3.9+/− and +9.5+/− embryos from timed matings were col-lected at E13.5 into ice-cold PBS. Livers were removed and dissociated bypipetting before formaldehyde fixation. An allele-specific quantitative PCRassay was conducted with allele-specific primers that distinguish WT andmutant alleles. Serial dilutions of input samples were used for generatinga standard curve, and relative FAIRE signals were calculated for specific sites.

Western Blot Analysis. Whole-cell lysates were prepared by boiling 1 × 107

cells per milliliter in SDS sample buffer [25 mM Tris (pH 6.8), 2% β-mercap-toethanol, 3% SDS, 0.1% bromophenol blue, 5% glycerol] for 10 min.Samples (10 μL) were resolved by SDS/PAGE and analyzed with specificantibodies. Rabbit anti–GATA-2 (68) and rabbit anti-TAL1 (11) were de-scribed previously. Rat anti–GATA-1 (N-6, sc-265), rabbit anti-BRG1 (H-88,sc10768), and goat anti-LDB1 (N18, sc11198) were from Santa Cruz Bio-technology, and mouse anti–β-actin (3700S) was from Cell Signaling.

Quantitative ChIP Assay.A quantitative ChIP assay was conducted as describedpreviously (8). Cells cross-linked with 1% formaldehyde were sonicated toyield DNA with an average size of 200–700 bp and immunoprecipitated withspecific antibodies [rabbit anti–GATA-1 (68), rabbit anti–GATA-2 (68), andrabbit anti-TAL1 (11)], LDB1 (N18, sc11198), and BRG1 (ab110641). For BRG1ChIP, cells were cross-linked with 1% formaldehyde for 20 min. ChIP sampleswere quantitated relative to the input DNA using real-time PCR analysis.Rabbit preimmune serum or normal IgG was used as a negative control.

ACKNOWLEDGMENTS. This work was supported by Grant R01 DK68634from the National Institutes of Health (NIH) (to E.H.B.), a University ofWisconsin–Madison Stem Cell and Regenerative Medicine Center postdoc-toral fellowship (to R.S.), and an NIH T32 Hematology Training Grant Award(to K.J.H.).

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