doi:10.1182/blood-2013-03-489369Prepublished online September 12, 2013;2013 122: 2978-2986   

 Beau R. Webber, Michelina Iacovino, Si Ho Choi, Jakub Tolar, Michael Kyba and Bruce R. Blazar from primitive to definitive hematopoietic potential in vitro and in vivo

regulatory regions correlates with transitionRunx1DNA methylation of

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Regular Article

HEMATOPOIESIS AND STEM CELLS

DNA methylation of Runx1 regulatory regions correlates with transitionfrom primitive to definitive hematopoietic potential in vitro and in vivoBeau R. Webber,1 Michelina Iacovino,2 Si Ho Choi,2 Jakub Tolar,1 Michael Kyba,2 and Bruce R. Blazar1

1Division of Hematology-Oncology, Blood and Marrow Transplantation, and 2Lillehei Heart Institute, Department of Pediatrics, University of Minnesota,

Minneapolis, MN

Key Points

• DNA methylation profile ofRunx1 locus correlates withtranscriptional activity andpromoter usage during blooddevelopment.

• Distal promoterhypomethylation is a novelsignature of definitivehematopoiesis and ispromoted in vitro by directinteraction with HoxB4.

The transcription factor Runx1 (AML1) is a central regulator of hematopoiesis and is

required for the formation of definitive hematopoietic stem cells (HSCs). Runx1 is

alternatively expressed from two promoters: the proximal (P2) prevails during prim-

itive hematopoiesis, while the distal (P1) dominates in definitive HSCs. Although

some transcription factor binding sites and cis-regulatory elements have been

identified, a mechanistic explanation for the alternative promoter usage remains

elusive. We investigated DNA methylation of known Runx1 cis-elements at stages of

hematopoietic development in vivo and during differentiation of murine embryonic

stem cells (ESCs) in vitro. In vivo, we find loss of methylation correlated with the

primitive to definitive transition at the P1 promoter. In vitro, hypomethylation,

acquisition of active chromatin modifications, and increased transcriptional activity

at P1 are promoted by direct interaction with HOXB4, a transcription factor that

confers definitive repopulation status on primitive hematopoietic progenitors. These

data demonstrate a novel role for DNA methylation in the alternative promoter usage

at the Runx1 locus and identify HOXB4 as a direct activator of the P1 promoter. This

epigenetic signature should serve as a novel biomarker of HSC potential in vivo, and during ESC differentiation in vitro. (Blood.

2013;122(17):2978-2986)

Introduction

Understanding the molecular pathways governing the developmentof mammalian hematopoietic stem cells (HSCs) is crucial to iden-tifying methods for their derivation from pluripotent cell sources. Ofthe key players involved in the formation of HSCs, the runt-relatedtranscription factor Runx1 plays an essential role. Without Runx1,hemogenic endothelium of the aorta gonad mesonephros does notundergo hematopoietic transition, and Runx1 knockout embryos failto survive past 12.5 days gestation with a complete loss of definitivehematopoiesis.1-4 This defect is mirrored during hematopoieticdifferentiation of embryonic stem cells (ESCs) in vitro, underscoringthe role of Runx1 as a fundamental regulator of the hematopoieticprogram during development.5,6

Runx1 serves as the a subunit of the core-binding factor complexand is the most common translocation in acute myeloid leukemiain humans.7,8 Defining regulation of the Runx1 locus is necessaryfor understanding the complex functional roles and tightly regulatedactivity of Runx1 during HSC development and hematopoieticmalignancy. Runx1 transcription is controlled by two developmen-tally regulated alternative promoters and an intronic enhancer (123)element.9-12 Intriguingly, promoter usage follows a pattern wherebythe proximal (P2) promoter initiates early in primitive hematopoiesis,while the distal (P1) promoter–driven transcription appears later in

definitive hematopoietic cells.13,14 Strikingly, distal promoter pre-dominance is temporally associated with peak HSC expansion in thefetal liver (FL) and continues into adult marrowwhere purified HSCsuse the distal promoter almost exclusively.12,14-16 While Runx1 corepromoter elements fail to restrict reporter expression to the hema-topoietic lineage, the intronic 123 enhancer is sufficient to drivehematopoietic specificity via interaction with Gata2, Ets factors, andthe SCL/Lmo2/Ldb1 complex.9,10 Although much has been learnedabout Runx1 promoter and enhancer usage, the precise mechanismsresponsible for the observed promoter switch are not understood.

DNA methylation is an epigenetic regulatory mechanism thatcontributes prominently to embryonic development and lineagecommitment in the hematopoietic system.17-19 Recent advances ingenome-wide analysis of methylation have identified tissue-specificdifferentially methylated regions (TDMRs) as dynamic regulators ofgene expression during development and disease.20 Further evidencesuggests that intronic enhancers are frequently TDMRs involved inlineage commitment and can influence the usage of alternativepromoters.20,21 In spite of these observations, methylation of theRunx1 regulatory elements has not yet been assessed in the context ofhematopoietic development, and relatively little is known about themechanisms underlying the Runx1 promoter switch.

Submitted March 11, 2013; accepted September 3, 2013. Prepublished online

as Blood First Edition paper, September 12, 2013; DOI 10.1182/blood-2013-

03-489369.

J.T., M.K., and B.R.B. contributed equally to this study.

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.

© 2013 by The American Society of Hematology

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Here, we examine the DNA methylation status of Runx1 reg-ulatory elements at stages of hematopoietic development in vivo andduring hematopoietic differentiation of ESCs in vitro. We find thatthe P2 promoter is unmethylated in all cell types examined, includingpluripotent murine embryonic stem cells (mESCs). In accordancewith its role as a hematopoietic-specific enhancer, a striking loss ofmethylation is observed at the 123 enhancer element upon com-mitment to the hematopoietic lineage in vivo and in vitro. We showthat hypomethylation of the distal promoter region is correlated withdefinitive HSC potential in vivo and can be promoted during in vitrohematopoietic differentiation by HOXB4. Concordantly, HOXB4overexpression promotes P1 transcription and an increased P1/P2messenger RNA (mRNA) ratio. Chromatin immunoprecipitation(chIP) analyses in mESC-derived hematopoietic cells overexpressingHOXB4 show that P1 is preferentially bound by HOXB4, acquireshistone modifications permissive to transcription, and undergoes a de-crease in occupancy by the maintenance methyltransferase DNMT1.Thus, decreased methylation, acquisition of active chromatin modifi-cations, and increased transcriptional activity at the P1 promoter ispromoted in vitro via physical interaction with HOXB4.

Materials and methods

Information concerning cell culture, ESC differentiation, retroviral transduc-tion, embryo dissection, cell isolation and fractionation, bisulfite sequencing,methylation analysis, quantitative reverse transcriptase polymerase chain re-action, and chIP is provided in the supplemental Data.

Statistics

Statistical analysis of methylation between individual populations was per-formed by using the online quantification tool formethylation analysis program(http://quma.cdb.riken.jp/). Differences between grouped methylation datawere determined by using an unpaired 2-tailed Student t test, withP values lessthan .05 considered significant. All other analyses were performed by usinga Student t test, with P values less than .05 considered significant.

Results

Proximal Runx1 P2 promoter is unmethylated regardless of

lineage or developmental status

The exceptionally large size of the Runx1 locus (224 kb) limited ourability to perform locus-wide exploratory bisulfite sequencing. Wethus focused on Runx1 regulatory elements previously confirmed toexhibit hematopoietic activity10 (Figure 1A; supplemental Table 1).The Runx1 P2 promoter is the most CpG dense (63% GC, 1.03 CpGobserved [Obs]/global expected [Exp]) of the three Runx1 elementsanalyzed and the only one to contain a classic CpG island (GC.50%,Obs/Exp P . .6). To determine whether the proximal Runx1 pro-moter is differentially methylated during hematopoietic devel-opment in vivo, we analyzed bisulfite-treated genomic DNA fromE14.5 mouse embryonic fibroblast (MEF) cells, E8.5 yolk sac (YS)CD411, E14.5 FL Lin-Sca-11CD482CD1501, and adult marrowLin-c-Kit1Sca-11CD1501CD482 (KLS1 SLAM) cells representingnonhematopoietic, primitive hematopoietic, and two stages of defini-tive HSCs, respectively (Figure 1B; supplemental Figure 1A-B). Toparallel this analysis in vitro using mESCs, we used an established2-stage differentiation process (Figure 1B). mESCs were first differ-entiated as embryoid bodies (EBs) for 6 days to obtain primitivec-Kit1CD411 progenitors (supplemental Figure 2A). Since HOXB4overexpression is an efficient method of obtaining ESC-derivedhematopoietic progenitors capable of robust long-term reconstitutionposttransplant,22 we used overexpression of HOXB4 and cocultureon hematopoietic supportiveOP9 stromal cells to obtainmESC-deriveddefinitive HSCs. Day 6 EB cells were infected with MSCV-HOXB4-IRES-GFP or MSCV-IRES-GFP control virus and cocultured on OP9cells. The GFP1c-Kit1CD451 fraction was isolated from HOXB4 andcontrol cocultures after 6 days (6 1 6 days) and from HOXB4 co-cultures at 11 days (61 11 days) at which point no hematopoietic cellswere observed in the control cocultures (supplemental Figure 2B).

Figure 1. Runx1 genomic locus and cell populations for bisulfite analysis. (A) Murine Runx1 locus and CpGs analyzed by bisulfite sequencing. For P1 and P2, CpG

number represents distance from transcription start site. For123, CpG number represents distance from the 39 end of the polymerase chain reaction (PCR) amplicon. (B) Cell

populations isolated in vivo and from ESC differentiation strategy for bisulfite analysis.

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In vivo, we find the P2 promoter to be unmethylated in MEFs,a nonhematopoietic control (0.8%6 0.79%) (Figure 2A). No changein P2 promoter methylation was associated with primitive hemato-poiesis because P2 was also unmethylated in E8.5 YS CD411 cells(1.7% 6 0.85%) (Figure 2A). This absence of P2 promoter meth-ylation continues in E14.5 FL Lin-Sca-11CD482CD1501 cells(3.1% 6 1.63%), and in adult marrow KLS 1 SLAM (0% 6 0%)(Figure 2A), indicating that the Runx1 P2 promoter does not exhibitdifferential methylation between lineages or during hematopoieticdevelopment in vivo.Although previous reports indicate a P1 bias inFL and adult HSCs,12,15,16 our data indicate that the methylationstatus of the P2 promoter does not establish this bias.

The absence of P2 promoter methylation was mirrored in vitro inundifferentiated mESCs (1.4%6 0.91%) and did not change in day6 EB c-Kit1CD411 cells (2.1% 6 1.02%) (Figure 2B). P2 re-mained unmethylated in day 61 6 OP9 cocultures in both controlc-Kit1CD451 cells (2.2% 6 1.79%) and HOXB4 overexpressingcells at day 61 6 (0.6%6 0.56%) and day 61 11 (1.2%6 0.82%)(Figure 2B). These observations are consistent with previous reportsthat CpG-dense core promoter regions are predominantly unmethy-lated,23,24 and that P2 expression exhibits a lower degree of lineage-restricted expression than P1 during development.25 Indeed, globalDNA methylation data released by the ENCODE Consortium con-firms a foci of hypomethylation at the P2 promoter in multiple celllineages.26

Hypomethylation of the 123 enhancer correlates with the

hematopoietic lineage

The recently described intronic123 enhancer element has been shownto drive hematopoietic-specific transcription.10 Sequence analysis ofthis region shows low CpG density and does not specify a classic CpGisland (58%GC, 0.31 Obs/Exp). In vivo,we find the123 enhancer ishighly methylated in MEFs (79.4% 6 4.66%), but methylation issignificantly decreased in E8.5 YS CD411 cells (1.7% 6 1.22%),E14.5 FL Lin-Sca-11CD482CD1501 (18.8% 6 8.29%), andadult KLS 1 SLAM (4.2% 6 2.54%) (Figure 3A-B). These re-sults demonstrate that 123 enhancer hypomethylation occursduring embryonic hematopoiesis and continues into fetal and adulthematopoiesis.

This correlation of 123 hypomethylation and the hematopoieticlineage also occur during hematopoietic differentiation of mESCs.The123 enhancer is methylated to a high degree in undifferentiatedmESCs (84.7%6 4.23%), followed by a significant decrease in day6 EB c-Kit1CD411 hematopoietic cells (6.9% 6 1.81%) that ismaintained inOP9 cocultured control c-Kit1CD411 cells at day 61 6(11.4% 6 11.1%) and in HOXB4 overexpressing cells at both day61 6 (2.5%6 1.81%) and day 6111 (5%6 1.64%) (Figure 3C-D).To further define the hematopoietic specificity of 123 hypo-methylation, we compared the 123 methylation profile of day 6EB c-Kit1CD412 cells to that of day 6 c-Kit1CD411 cells, sinceCD41 is the earliest known marker of hematopoiesis.27,28 We foundthat 123 hypomethylation strongly correlated with the acquisitionof hematopoietic fate as defined by CD41 (70.9% 6 6.1% vs6.9% 6 1.81%; P 5 .0002) (Figure 4A). In contrast, there is nostatistically significant difference in the methylation of the distalpromoter between these populations (Figure 4B).

Enhancer methylation has been shown to influence gene ex-pression.29 We examined the expression of the P1 and P2 mRNAin day 6 EB c-Kit1CD412 and c-Kit1CD411 cells and found thatboth P1 and P2 transcriptional activities are increased in c-Kit1

CD411 cells (Figure 4C). Thus, 123 methylation influences theactivity of both Runx1 promoters in a manner that correlates withCD41 acquisition.

Distal Runx1 P1 promoter is hypomethylated in HSCs in vivo

Several studies have observed that expression of the P1 Runx1mRNAisoform is specifically upregulated in definitive hematopoietic cellsduring development and comprises the majority of Runx1 transcript inHSCs.12,15,16 Although the P2 promoter is CpG dense and structurallysimilar to housekeeping promoter elements,9 the P1 promoter is morecomplex in terms of transcription factor binding sites and compar-atively CpG poor (46% GC, 0.25 Obs/Exp). Consistent with thedefinitive hematopoiesis-specific activity of the P1 promoter, a highdegree of methylation is observed in MEFs (86.3% 6 3.76%)(Figure 5A-B). Compared with MEFs, there is a modest yet statis-tically significant decrease in P1 promoter methylation in E8.5 YSCD411 cells (48.6% 6 5.45) (Figure 5A-B). However, an evengreater degree of P1 hypomethylation is observed in definitive HSCs,

Figure 2. Bisulfite analysis of proximalRunx1promoter. (A)Methylation patterns of theRunx1P2 promoter in cells derived fromE14.5MEFs, E8.5 YSCD411 (E8.5 YS), E14.5 FL

Lin-Sca-11CD482CD1501 (E14.5 FL), and adultmarrowLin-c-Kit1Sca-11CD1501CD482 (KLS1SLAM). Sequencing reactions of individual amplicons are represented by each row of

circles. Open circles denote unmethylated CpGs, and solid circles represent methylated CpGs. (B) Methylation patterns in mESCs, day 6 EB c-Kit1CD411 (D6 EB), and from OP9

cocultures:GFP1c-Kit1CD451cells isolated from IRES-GFPcontrol groupat day6 (D616control) andHOXB4-IRES-GFPgroupat day6 (D616HoxB4)andday11 (D6111HoxB4).

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where E14.5 FL Lin-Sca-11CD482CD1501 (8.1% 6 3.89%) andKLS1 SLAM (9%6 5.65%) cells exhibit a significantly lower levelof P1methylation comparedwithE8.5YSCD411 cells (Figure 5A-B).

HoxB4 alters P1 promoter methylation and transcriptional

activity in vitro

We next determined whether this in vivo epigenetic signature ofdefinitive HSCs is replicated during mESC differentiation. Like the123 enhancer, P1 is highly methylated in undifferentiated mESCs(90%6 3.3%) (Figure 5C-D). Paralleling our results in vivo, a modestdecrease is observed in day 6EB c-Kit1CD411 cells (68.9%65.65%)and in OP9 cocultured control c-Kit1CD451 cells at day 6 1 6(71.6% 6 7.06%) (Figure 5C-D). However, in OP9 coculturedc-Kit1CD451 cells overexpressingHOXB4,we observe a significantdecrease in P1 methylation compared with control cells at day 61 6(47.8% 6 5.4%), followed by a further decrease by day 6 1 11(27%6 5.08%), corresponding to a population shown to possessmore robust hematopoietic repopulating potential after adoptivetransfer in vivo (Figure 5C-D). These data demonstrate that theRunx1 P1 promoter is methylated in pluripotent mESCs and remainsmethylated in the first wave of c-Kit1CD411 hematopoietic pro-genitors. Maturation to c-Kit1CD451 progenitors on OP9 alone doesnot change this methylation profile, whereas overexpression ofHOXB4 during this process results in decreased P1 methylation.

To determine whether P1 hypomethylation is linked to promoterswitching during differentiation, we examined relative P1 vs P2mRNA levels using isoform-specific quantitative reverse transcriptase

polymerase chain reaction. As expected, P2 dominates in primitive EB-derived cell populations and in OP9 control cocultures (Figure 5E).However, the P1:P2 ratio is higher in HOXB4-overexpressing OP9cocultures, and even higher in purified c-Kit1CD451 hematopoieticcells overexpressing HOXB4 (Figure 5E). These data suggest thatHOXB4-induced P1 hypomethylation is associated with a consequentincrease in the P1:P2 mRNA ratio in vitro.

HoxB4 directly activates the Runx1 P1 promoter

It was unclear whether the mechanism underlying HOXB4-mediatedactivation of P1 was direct or indirect. To answer this question, weperformed chIP analysis in ESC-derived hematopoietic cells duringdifferentiation to examine the level of HOXB4 binding at Runx1regulatory regions. We find that HOXB4 preferentially binds the P1promoter upon overexpression at day 6 1 6 and day 6 1 11, thusidentifying a direct role for HOXB4 in the modulation of P1transcriptional activity (Figure 6A).

We next explored changes in chromatin organization over the timecourse of hematopoietic differentiation. Bivalency of lineage-specificgenes is a hallmark of pluripotency and involves the co-localizationof active (H3K4me3) and repressive (H3K27me3) histone modifica-tions, which subsequently resolve to the presence of one or the otherduring lineage commitment.30 We find that H3K27me3 is uniformlyabsent at P1,123, and P2 as compared with a silenced control region(Pitx1) at all time points, demonstrating that theRunx1 locus is primedfor activation early in the hematopoietic lineage and is not altered byHOXB4 overexpression (Figure 6B). Consistent with our expression

Figure 3. Bisulfite analysis of 123 enhancer methylation. (A) Methylation patterns of the Runx1 123 enhancer in cells from in vivo–derived E14.5 MEFs, E8.5 YS CD411

(E8.5 YS), E14.5 FL Lin-Sca-11CD482CD1501 (E14.5 FL), and adult marrow Lin-c-Kit1Sca-11CD1501CD482 (KLS 1 SLAM). Sequencing reactions of individual amplicons

are represented by each row of circles. Open circles denote unmethylated CpGs, and solid circles represent methylated CpGs. (B) Quantification of percent CpG methylation

at123 in hematopoietic populations derived in vivo. ***P, .001; **P, .01. (C) Methylation patterns in mESC day 6 EB c-Kit1CD411 (D6 EB) and from OP9 cocultures: GFP1

c-Kit1CD451 cells isolated from IRES-GFP control group at day 6 (D6 1 6 control) and HOXB4-IRES-GFP group at day 6 (D6 1 6 HoxB4) and day 11 (D6 1 11 HoxB4). (D)

Quantification of percent CpG methylation at 123 in cell populations isolated during hematopoietic differentiation of mESCs. ***P , .001.

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data, H3K4me3 is enriched at the P2 promoter and an active controllocus (Gapdh) over the course of differentiation and is not altered byHOXB4 (Figure 6B). Conversely, H3K4me3 presence at the P1promoter is significantly increased upon overexpression of HOXB4by day 6 1 6 and is maintained through day 6 1 11 (Figure 6B).Finally, we find that H3K9Ac—a mark of actively transcribedpromoters—is present throughout differentiation at P2 but issignificantly increased at P1 only in day 6 1 11 HoxB4 over-expressing cells (Figure 6C), a finding that is consistent with both ourmethylation and expression data in that this time point coincides withthe highest degree of P1 hypomethylation and maximal level of P1expression in vitro.31 From these collective results, we conclude thatHOXB4preferentially binds to theRunx1P1 promoter and stimulatestranscription via decreased methylation and establishment of a per-missive chromatin state in ESC-derived hematopoietic cells.

DNMT1 occupancy at P1 is decreased in HOXB4

overexpressing cells

DNA methylation patterns are either maintained during replicationvia the activity of the maintenance methyltransferase DNMT1, orestablished de novo by DNMT3a and DNMT3b.32-34 To determinewhether the loss of methylation at P1 is accompanied by decreasedinteraction with members of the DNA methyltransferase family, wemeasured the level of DNMT1, DNMT3a, and DNMT3b occupancyat the Runx1 promoters. We did not detect binding of DNMT3a orDNMT3b to P1 (data not shown); however, we found that DNMT1did interact with P1, and this interaction was significantly decreasedat day 6 1 11 in HOXB4-overexpressing cells (Figure 6D). Asexpected, occupancy was universally low at the hypomethylatedP2 promoter at all time points (Figure 6D). These data are consistent

with a mechanism in which DNMT1 is occluded from accessing theP1 promoter in the presence of HOXB4, resulting in a gradual lossof established methylation patterns over subsequent cell divisions.

Runx1 P1 methylation as signature of definitive HSCs

Finally, to compare our in vivo and in vitro observations that P1methylation is correlated with definitive HSCs during development,we grouped all cell populations studied on the basis of whether theyare known to possess hematopoietic repopulating capacity, and wecompared mean levels of P1 promoter methylation. This comparisonindicated that P1 promoter hypomethylation correlated with repopulat-ing capacity in a similar fashion in vivo and in vitro (Figure 7A). Toincrease the resolution of P1 promoter demethylation, we applied thesame grouped comparison with the individual CpG dinucleotides in theP1 promoter and found that overall methylation was significantly de-creased for each CpG, with the exception of the CpG located at position2371 relative to the P1 transcription start site. In particular, the CpGslocated at positions 2436 and 2271 were the most significantly dif-ferent between repopulating andnon-repopulating cell types (Figure 7B).

Discussion

Here, we identify previously undescribed changes in DNA methyl-ation at Runx1 regulatory regions during hematopoietic development.These changes are associated with commitment to the hematopoieticlineage, and they distinguish definitive repopulating HSC populationsfrom earlier, primitive non-repopulating cells. We find that the CpG-dense P2 promoter is unmethylated in mESCs and remains unmeth-ylated regardless of lineage and stage of hematopoietic development.Conversely, the 123 intronic enhancer is methylated in mESCs,nonhematopoietic fibroblasts, and c-Kit1CD412 cells fromday 6 EBsbut is dramatically hypomethylated upon acquisition of CD41 andremains unmethylated throughout hematopoietic development. Im-portantly, we demonstrate that hypomethylation of the P1 Runx1promoter is specific to cell populations enriched in definitive repop-ulating capacity in vivo. Moreover, in mES-derived c-Kit1CD411

hematopoietic progenitors, the P1 promoter remains methylated atlevels similar to those observed in E8.5 YS. Overexpression ofHOXB4 results in a significant decrease in P1 methylation con-sistent with its ability to generate mESC-derived hematopoieticprogenitors capable of long-term repopulation in transplantrecipients.22 Thus, our results identify hypomethylation of the distalRunx1 promoter as a novel epigenetic signature of repopulatinghematopoietic cells during development and provide critical insightinto the dynamic epigenetic changes influencing the Runx1 locus.

The developmental processes leading to the formation of definitiveHSCs rely on the properly orchestrated activity of a complex networkof critical transcription factors to guide genetic programs associatedwith differentiation. Runx1 is a critical transcription factor involvedin the development of definitive HSCs1,2,5 and, not surprisingly, isfrequently disregulated in hematopoietic malignancy.7,8 A diversearray of Runx1 mRNA isoforms have been identified35 and arisethrough a combination of alternative splicing as well as alternatepromoter usage.9,12,13,36 As methods for genome-wide analysis ofCpG methylation have improved, a growing number of TDMRsassociated with normal and abnormal development have beenidentified.37 Previously, it was unknownwhether theRunx1 regulatoryregions were TDMRs. In the case of the Runx1 P2 promoter, our datanow indicate it is not a TDMR because P2 is unmethylated in a widearray of cell types. This observation is consistent with previous reports

Figure 4. Bisulfite analysis andRunx1 expression in day 6 EB subpopulations. (A)

Methylation patterns of theRunx1123 enhancer in c-Kit1CD412 and c-Kit1CD411 cells

from day 6 EBs. Sequencing reactions of individual amplicons are represented by each

row of circles. Open circles denote unmethylated CpGs, and filled circles represent

methylatedCpGs. (B)Methylation patterns of theRunx1P1promoter in c-Kit1CD412and

c-Kit1CD411 cells from day 6 EBs. (C) Quantitative reverse transcriptase polymerase

chain reaction analysis of Runx1 P1 and P2 mRNA isoforms in c-Kit1CD412 and c-Kit1

CD411 cells from day 6 EBs. ns, not significant.

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Figure 5. Bisulfite analysis and mRNA expression from Runx1 P1 promoter. (A) Methylation patterns of the Runx1 P1 promoter in cells from in vivo–derived E14.5 MEFs,

E8.5 YS CD411 (E8.5 YS), E14.5 FL Lin-Sca-11CD482CD1501 (E14.5 FL), and adult marrow Lin-c-Kit1Sca-11CD1501CD482 (KLS 1 SLAM). Sequencing reactions of

individual amplicons are represented by each row of circles. Open circles denote unmethylated CpGs, and solid circles represent methylated CpGs. (B) Quantification of

percent CpG methylation at P1 in hematopoietic populations derived in vivo. ***P , .001. (C) Methylation patterns in mESCs, day 6 EB c-Kit1CD411 (D6 EB) and from OP9

cocultures: GFP1c-Kit1CD451 cells isolated from IRES-GFP control group at day 6 (D61 6 control) and HOXB4-IRES-GFP group at day 6 (D61 6 HoxB4) and day 11 (D61 11

HoxB4). (D) Quantification of percent CpG methylation at P1 in cell populations isolated during hematopoietic differentiation of mESCs. ***P , .001; *P , .05. (E) P1/P2 mRNA

levels after normalization to Gapdh over the course of hematopoietic differentiation in vitro.

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of other CpG-rich core promoters23,24 and is supported by genome-wideDNA methylation profiles released by the ENCODE Consortium.26

That the P2 methylation pattern is established at the pluripotent stageand does not change in differentiated cell types or during hematopoieticdevelopment suggests that methylation of P2 does not influencelineage-specific changes in P2 transcription during development. Itseems unlikely that P2methylation acts as an on/off switch, since the P2isoform is detected in a diverse array of cell types,25 including un-differentiated mESCs,38 and the P2 promoter remains unmethylated in

FL and adult HSCs, even though P1 is the dominant promoter in thesepopulations.12,15,16 Therefore, our observations suggest that P2methylation is not involved in the lineage restriction of P2 duringdevelopment. Intriguingly, intragenic TDMRdownstream of P2 havebeen identified, raising the possibility that methylation at theseregions could have a role in the regulation of P2 activity.17

In contrast to the proximal promoter, the123 intronic enhancer isa TDMR, and while the hematopoietic-specific activity of the Runx1123 enhancer element is documented,10 our data are the first to

Figure 6. Runx1 chIP analysis. (A) Quantitative PCR (qPCR) analysis of chIP performed against HOXB4. Data were normalized to the percent of pre-IP input for each

sample and are expressed as the fold change vs the day 61 6 (D61 6) control population. Data are representative of at least two independent IPs. (B) qPCR analysis of chIP

performed against H3K4me3 and H3K27me3. Data are expressed as the percent of pre-IP input for each sample and are representative of at least 2 independent IPs. **P, .01.

(C) qPCR analysis of chIP performed against H3K9Ac. Data are expressed as the percent of pre-IP input for each sample and are representative of at least 2 independent IPs.

***P, .001. (D) qPCR analysis of chIP performed against Dnmt1. Data are expressed as the fold change vs Gapdh control locus for each sample and are representative of at

least 2 independent IPs. **P , .01.

Figure 7. Comparison of P1 methylation in repopulating and non-repopulating cell types. (A) Percent CpG methylation at P1 promoter in repopulating (R) and non-

repopulating (NR) cell populations. Bar indicates the mean. **P , .01 (unpaired Student t test). (B) Percent methylation at individual CpGs within P1 promoter in repopulating

and non-repopulating cell populations. Bar indicates the mean. **P , .01; *P , .05 (unpaired Student t test). ns, not significant.

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identify changes in CpG methylation at the 123 enhancer elementduring hematopoietic development. Previous data indicating thatmethylation of intronic enhancer elements influences tissue-specificgene expression29 further supports a role for123 enhancer methyla-tion in the transcriptional activity ofRunx1.We confirm this hypothesisby clearly demonstrating that123 hypomethylation strongly correlateswith increased transcription at both Runx1 promoters at the earlieststage of hematopoietic development, although P2 remains thedominant mRNA isoform in these populations. There is evidencethat intragenic DNA methylation influences alternate promoterusage21; however, our observations indicate that 123 hypome-thylation is not a significant factor in the observed P1/P2 switchbut rather acts as an epigenetic rheostat for theRunx1 locus, mediatinghematopoietic-specific amplification of Runx1 expression. We cannotrule out the possibility that 123 hypomethylation facilitates a morepermissive state for P1 transcription by allowing improved mRNAelongation,39 although this does not fit our observation that 123enhancer methylation is lost early in development when P2 stilldominates compared with adult HSCs.12,15 Considering our datathat P1 hypomethylation results in a shift to P1-biased expression,a more probable explanation is that123methylation is a nonbiasedregulator of transcription from both promoters, and the usage biasis determined by the epigenetic status of P1. Studies using clonalcell populations with well-defined mRNA isoform expression profilesor episomal reporters in which methylation can be artificially manip-ulated might help to refine the role of methylation in regulatingpromoter usage. However, the utility of these assays is limited bythe lack of developmental and genomic context. Regardless, ourdata clearly demonstrate that123methylation influences its enhancercapacity and provides a novel epigenetic signature of the hematopoi-etic lineage, which can be applied to optimize methods for directconversion of other lineages to a hematopoietic fate.40

Derivation of robust numbers of in vivo long-term repopulatingHSCs frompluripotent cell sources such as ES and induced pluripotentstem cells is of therapeutic interest. Critical to the success of theseefforts is the identification of signatures associated with the formationof definitive HSCs during development. Although their precise role inthe regulation of gene transcription is not fully understood, TDMRsand differential DNAmethylation have nonetheless proven useful foridentifying differences in cell populations. This is highlighted by theuse of DNA methylation patterns to determine whether somatic cellshave been successfully reprogrammed to a pluripotent state.41,42 Wehave identified the Runx1 P1 promoter as a novel TDMR and havedemonstrated that hypomethylation of this region differentiates pri-mitive non-repopulating progenitors and definitive repopulatingHSCsduring embryonic development in vivo. Our observation that unmod-ified hematopoietic progenitors derived from mESCs do not undergothis decrease in P1 methylation suggests a failure in the epigenetictransition to adult-type definitive hematopoiesis. By overexpressingHOXB4 in ESC-derived hematopoietic cells, we show that it ispossible to promote epigenetic remodeling of the Runx1 locus duringdifferentiation of ESCs to hematopoietic cells in vitro. The hypometh-ylation of P1 induced byHOXB4 results in an increased P1:P2mRNAratio and thus links P1 hypomethylation to the P2-to-P1 promoterswitch observed during hematopoietic development. Mechanistically,we show that when overexpressed, HOXB4 preferentially binds P1,supporting a model in which the epigenetic remodeling and increasedtranscription of P1 is mediated via physical interaction of HOXB4with this locus. Recent HOXB4 chIP-Seq data support our findings

that HOXB4 interacts with the Runx1 distal promoter, albeit usinga slightly different ESC differentiation method, suggesting that this isa robust biological phenomenon.43 Whether demethylation of P1 inthe presence of HoxB4 is an active or passive process remains unclear.However, our observation that P1 methylation is only slightlydecreased at day 61 6 and becomes more pronounced by day 61 11is congruent with a passive loss of methylation. This is supported byreports finding that active demethylation often occurs rapidly—within minutes or hours—and results in nearly complete demethyl-ation of the region in question.44,45 Since HOXB4 physically bindsRunx1 P1, it is conceivable that HOXB4 or a HOXB4-associatedcomplex could physically or functionally occlude the activity offactors involved in maintaining DNAmethylation, such as Dnmt1.Indeed, our data demonstrating a decrease in Dnmt1 occupancy ofP1 in HOXB4-overexpressing cells are consistent with this model.Over successive rounds of DNA replication, this would result ina loss of Runx1 P1 methylation. Previous reports that differentiation-induced hypomethylation of lineage-specific CpG-poor promoterscorrelates with transcription-factor binding further supports thisexplanation.46

The fact that epigenetic remodeling of P1 could be achieved byoverexpressing HOXB4 demonstrates that this in vivo epigeneticsignature is valid during ESC differentiation and can potentially bereplicated if the appropriate extracellular cues are applied duringthe differentiation process, whether in vivo or in vitro, via the induc-tion of appropriate transcription factor circuits. Our single CpG groupanalysis identifies the CpGs located at 2436 and 2271 from the P1transcription start site as the most significantly hypomethylated indefinitive repopulating cell populations and presents an attractivetarget for high-throughput analysis of changes in P1 methylationduring hematopoietic differentiation of pluripotent cells.

Acknowledgments

We thank Troy Lund,Mark Osborn, and Istvan Szatmari for valuableadvice and technical assistance.

This work was supported in part by the National Institutes of Health(NIH) Research Program Grant, National Cancer Institute P01CA065493 and NIH Research Project Grant Program, National In-stitute of Allergy and Infectious Diseases R01 AI081918 (to B.R.B.);NIH Research Project Cooperative Agreement, National Heart, Lungand Blood Institute U01 HL 100407 (to M.K.); 5T32HD060536-2awarded to the University of Minnesota Stem Cell Institute (toB.R.W.); and a grant awarded to theChildren’s Cancer Research Fund.

Authorship

Contribution: B.R.W. designed and performed experiments, analyzeddata, and wrote the manuscript; M.I. and S.H.C. assisted in designingand performing experiments; and J.T., M.K., and B.R.B. discussedexperiments and results and edited the paper.

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

Corresponding author: BruceR.Blazar, Department of Pediatrics,MMC 109, University of Minnesota, Minneapolis, MN 55455;e-mail: blaza001@umn.edu.

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