Experimental Hematology 35 (2007) 48–55

The g-globin gene promoter progressively demethylatesas the hematopoietic stem progenitor cells differentiate along

the erythroid lineage in baboon fetal liver and adult bone marrow

Mahipal Singha,b, Donald Lavellea,b, Kestis Vaitkusa,b,Nadim Mahmudb, Maria Hankewycha, and Joseph DeSimonea,b

aJesse Brown VA Medical Center, Chicago, Ill., USA;bSection of Hematology/Oncology, Department of Medicine, University of Illinois at Chicago, Chicago, Ill., USA

(Received 9 June 2006; revised 1 September 2006; accepted 5 September 2006)

Objective. To determine whether the difference in g-globin gene promoter methylation interminal erythroblasts at the fetal and adult stages of development is a result of fetal stage-specific demethylation or adult stage-specific de novo methylation during erythropoiesis.

Materials and Methods. Fetal liver- (FL, n [ 2) and adult bone marrow- (ABM, n [ 3) de-rived hematopoietic stem/progenitor cells and mature erythroblasts were purified by passagethrough a Miltenyi Magnetic Column followed by fluorescein-activated cell sorting (FACS)into subpopulations, defined by expression of CD34 and CD36 antigens. CD34+CD36L,CD34+CD36+, and CD34LCD36+ subpopulations were purified by FACS and their degreeof differentiation verified using the colony-forming cell assay. The methylation pattern of 5CpG sites in the g-globin promoter region of these purified cell populations was determinedusing bisulfite sequencing.

Results. The g-globin promoter was highly methylated in the earliest stage of hematopoieticstem progenitor cells (CD34+CD36L) and methylation progressively decreased as erythroiddifferentiation progressed in FL and appears so in ABM as well.

Conclusions. These data support a model in which differences in the methylation pattern ofthe g-globin gene in differentiating erythroblasts at different stages of development is the re-sult of fetal stage-specific demethylation associated with transcriptional activation, ratherthan de novo methylation in the adults. The difference in the extent of g-globin gene demethy-lation in FL and ABM is correlated with the difference in g-globin expression at these devel-opmental stages. � 2007 International Society for Experimental Hematology. Published byElsevier Inc.

DNA methylation is one of the epigenetic modificationsthat has been studied extensively in eukaryotes [1–5] andhas been shown to play a significant role in tissue-specificregulation of eukaryotic genes, including globin genes [6–10]. Inverse correlation between methylation of DNAsequences near globin genes and the transcriptional activityhas been shown [11–16]. Reactivation of fetal hemoglobin(HbF) by inhibitors of DNA methytransferases in adultsstrongly suggests that DNA methylation is a major mecha-nism of g-globin gene silencing in adult stage erythropoie-

Offprint requests to: Mahipal Singh, Ph.D., Jesse Brown VA Medical

Center, MP151C, 820 South Damen Avenue, Chicago, IL 60612;

E-mail: mpsingh@uic.edu; mahipal55@hotmail.com

0301-472X/06 $–see front matter. Copyright � 2007 International Society for

doi: 10.1016/j.exphem.2006.09.001

sis [17,18]. The mechanism of gene silencing by CpGmethylation involves binding of methylated DNA by pro-teins such as MeCP2 [19,20] and/or methyl-CpG-bindingdomains [21–24] and recruitment of a multiprotein complexthat includes histone deacetylases [25,26]. This proteincomplex then induces a closed chromatin structure [27] sur-rounding the promoter regions, ultimately leading to tran-scriptional repression [2,28,29].

Understanding regulation of g-globin gene expressionduring erythropoiesis in fetal and adult stages of develop-ment is important because increasing HbF is an effectivestrategy to treat sickle cell anemia and b-thalassemia. Usingthe baboon as a model system, we earlier showed that mul-tiple CpG residues in the g-globin gene promoter region inerythroblasts are hypomethylated in the fetal stage, but

Experimental Hematology. Published by Elsevier Inc.

49M. Singh et al./ Experimental Hematology 35 (2007) 48–55

highly methylated in the adult stage of development [30].However, in that study, only terminally differentiated eryth-roblasts were analyzed. How methylation is modulated dur-ing differentiation from hematopoietic stem progenitor cells(HSPCs) to terminally differentiated erythroblasts in eitherfetal or adult stage is not known. Therefore, it is importantto determine whether the g-globin promoter is methylatedor unmethylated in HSPCs and how the pattern changesas the cells differentiate. In the present study, we analyzedthe pattern of methylation of the g-globin promoter influorescein-activated cell sorting (FACS) purified cellpopulations enriched for cells of different stages of ery-throid differentiation derived from both fetal liver (FL)and adult bone marrow (ABM) to determine how the pat-tern of methylation is established during normal hemato-poietic differentiation and to distinguish whether thedifference in methylation patterns in fetal and adult stagesof development is a result of fetal stage-specific demethyla-tion or adult stage-specific de novo methylation duringerythropoiesis.

Material and methods

Isolation of bone marrow and fetal liver cellsBone marrow (BM) samples were collected by aspiration fromiliac crest of 3- to 5-year-old (weighing 8�9 kg) anemic baboons(Papio anubis). The baboons were bled acutely to maintain a he-matocrit value of 20 by daily removal of 16% to 18% of thepacked cell volume in order to enhance marrow production. BMaspirates (10�20 mL) were obtained after 10 days following thefirst phlebotomy. All procedures were approved by the Institu-tional Animal Care and Use Committee of the University of Illi-nois at Chicago. BM samples were diluted 1:8 in Iscove’smedium containing 1% fetal bovine serum (FBS) and the low-density mononuclear cell fraction was purified by centrifugationon 70% Percoll-gradient (Pharmacia LKB, Uppsala, Sweden) at1600 rpm for 20 minutes at 20�C in a Beckman JE-6B rotor. Fetalliver was obtained from baboon fetuses (n 5 2) at 56 and 58 daysof gestation (full-term 5 184 days). Fetal liver cells were filteredthrough a fine mesh, diluted 1:8 in Iscove’s medium and left for 15minutes on ice to allow hepatocytes to settle in the bottom of thetube. The mononuclear cells were collected from the supernatantafter centrifugation at 1500 rpm for 15 minutes at roomtemperature.

Purification of mature erythroblastsMature erythroblasts were purified as described previously [30].Briefly, mononuclear cells from ABM and FL were washed twicein phosphate-buffered saline (PBS) containing 2 mM EDTA and0.5% bovine serum albumin (BSA). Cells were suspended inPBS (2 � 108 cells/mL) and incubated with human immunoglob-ulin (Ig) G (Sigma Chemical Co., St Louis, MO, USA) for 10 min-utes on ice. Cells were then incubated with purified mousemonoclonal anti-baboon red blood cell antibody (BD Pharmingen,San Diego, CA, USA) at 10�C for 15 minutes at a concentration of4�5 � 106 cells/mg followed by three washes in PBS. Cells werethen incubated with rat anti-mouse IgG microbeads (Miltenyi

Biotech, Auburn, CA, USA) at 10�C for 20 minutes, followedby three washes in PBS. The magnetically labeled cells were sus-pended in PBS and passed through 30-mM mesh preseparation fil-ters mounted on LS columns (Miltenyi Biotech, Auburn, CA,USA). Approximately 1�2 � 108 cells were loaded on each col-umn. The columns were washed three times with 3 mL PBS. Alleluted cells were pooled as an erythroblast-depleted fraction forpurification of hematopoietic stem progenitor cells. The erythro-blast cells retained in LS columns were eluted in 5 mL PBS. Cy-tospin preparations of the eluted cells were stained with Wright’sstain to evaluate purity and were observed to be 95% to 99% pure.

Isolation of hematopoietic stem progenitor cells by FACSMature erythroblast-depleted FL or ABM cells were suspended inPBS (20 � 106 cells/mL) and incubated with 30% rabbit serum for10 minutes on ice to block the Fc receptors. This was followed bya 60-minute incubation on ice with 12.8 anti-CD34 primary anti-body (a gift from Dr. Robert Andrews, Fred Hutchinson CancerResearch Center, Seattle, WA, USA) at a concentration of 1 mg/106 cells as described previously [31]. Cells were collected bycentrifugation at 1400 rpm for 7 minutes followed by two washesin PBS. The cells were then incubated with goat anti-mouseIgM:phycoerythrin (PE) (Southern Biotechnology AssociatesInc., Birmingham, AL, USA) in the dark for 30 minutes on ice fol-lowed by two washes with PBS. Cells were suspended in 1 to2 mL PBS and incubated with mouse anti-human CD36:fluores-cein isothiocyanate (FITC) (Immunotech, Marseille, France)monoclonal antibody (mAb) for 30 minutes on ice. Cells were re-covered by centrifugation and washed twice with PBS and resus-pended in 0.5 to 1 mL PBS with 10% BSA. Propidium iodide (PI)was added to each tube (1 mg/vial), filtered in 5 mL falcon non ad-herent tubes, and placed in the dark on ice until FACS analysis.The cells were gated on PI-negative viable cells. CD34þCD36�,CD34þCD36þ, and CD34�CD36þ cell populations were sepa-rated using a MoFlow High Performance Cell Sorter (DaKo Cyto-mations, Fort Collins, CO, USA) at the University of Illinois atChicago Flow Cytometry Facility. To assess the specificity of an-tibody binding, IgG1:FITC and IgM:PE doubly labeled isotypecontrol as well as 12.8-IgM:PE and CD36:FITC single colorcontrols were simultaneously processed in each experiment.

Colony-forming cell (CFC) assayThe degree of differentiation and purity of subpopulations of ery-throid progenitors isolated on the basis of CD34 and CD36 expres-sion were determined by CFC assay. A 10� cell suspension (1�5�103 cells/mL) was prepared in Iscove’s modified Dulbecco me-dium (IMDM) with 2% FBS and 0.3 mL of this suspension wasadded to 3 mL of MethocultGF H4434 semisolid medium(StemCell Technologies, Vancouver, BC, Canada) containing1% methylcellulose, 30% FBS, 1% BSA, 10�4M 2-mercaptoe-thanol, 2 mM L-glutamine and a cocktail of recombinant humancytokines, erythropoietin (3 U/mL), interleukin-3 (10 ng/mL),stem cell factor (50 ng/mL), and colony-stimulating factor-gran-ulocyte macrophage (10 ng/mL) in IMDM. Each plate con-tained 100 cells (FL) or 500 cells (ABM). The cultures wereplated in duplicate. The culture dishes were placed in a fullyhumidified (O95%) atmosphere at 5% CO2 and incubated for12 to 14 days at 37�C, prior to counting colonies.

50 M. Singh et al. / Experimental Hematology 35 (2007) 48–55

Preparation of genomic DNAGenomic DNAwas isolated from erythroblasts using Qiagen BloodMini Kit according to the procedure specified by manufacturer(Qiagen, Valencia, MA, USA) and 500 ng of this genomic DNAwas used directly for bisulfite treatment. DNA was isolated fromFACS purified progenitor cells using the Proteinase K/sodium do-decyl-sulfate method [32]. Briefly, purified cells (500�10,000)were suspended in 2 mL PBS. Cells were lysed in 18 mL of a solutioncontaining 1 mM SDS, 2 mg Escherichia coli tRNA, 5.6 mg protein-ase K for 60 minutes at 37�C followed by 15-minute incubation at98�C. DNA was stored at �20�C until used for bisulfite treatment.

Bisulfite modification of CpG dinucleotidesBisulfite modification of CpG dinucleotides was carried out as de-scribed [33] with slight modifications. Briefly, genomic DNA in18 mL volume was denatured by adding 2 mL freshly prepared 3M NaOH at 37�C for 15 minutes. Denatured DNA was treatedwith 208 mL freshly prepared bisulfite solution (2.3 M sodiummetabisulfite; 0.14 M hydroquinone, pH 5.0). The mixture was

overlaid with 30 mL mineral oil and incubated at 50�C for 4 to5 hours in dark. Bisulfite-treated DNA was purified using the Wiz-ard DNA Clean Up System (Promega, Madison, WI, USA) andeluted in 90 mL of water. DNA was denatured by adding 10 mL3 M NaOH and incubating at 37�C for 15 minutes. DNA wasthen precipitated by adding 10 mL 3 M sodium acetate (pH 7.0),2 mg of glycogen and 333 mL ethanol at �20�C overnight. DNAwas recovered by centrifugation at 14,000 rpm, 10 minutes at4�C in a Beckman microfuge. The DNA was washed twice with70% ethanol, dried in a vacuum desiccator for 20 minutes, resus-pended in 30�40 mL H2O and stored at �20�C until used for poly-merase chain reaction (PCR) amplification. Using this protocol wehave observed O98% efficiency of bisulfite induced C to T residueconversion in different samples studied.

PCR amplification of bisulfiteconverted DNA, cloning and sequence analysisBaboon g-globin gene promoter specific primers were used to am-plify the bisulfite converted DNA [34]. Two rounds of PCR

Figure 1. Fluorescein-activated cell sorting (FACS) of subpopulations of erythroid progenitor cells on the basis of CD34 and CD36 expression. Cells were

depleted of the erythroblasts before initiating the FACS sorting. Gated live cells are shown in histogram for fetal liver (A) and adult bone marrow (B) prior to

sorting. Live cells were gated on the basis of propidium iodide staining. Gates used for sorting of live CD34þCD36�, CD34þCD36þ and CD34�CD36þ

subpopulations are shown by squares. Percentage of each type of progenitor cells in both fetal liver (A) and adult bone marrow (B) is shown in respective

quadrants. (C) Purity of post sort analysis of FACS sorted subpopulations from adult bone marrow (ABM) samples. Panels show a representative flow

cytometric profile of CD34/CD36 expression prior to and following sorting of fetal liver and ABM samples. FITC 5 fluorescein isothiocyanate;

PE 5 phycoerythrin.

51M. Singh et al./ Experimental Hematology 35 (2007) 48–55

amplifications were performed. The initial round of PCR wasperformed using the forward primer BG1 (50-TATGGTGGGA-GAAGAAATTAGTTAAAGG-30) and the reverse primer BG2(50-AATAACCTTATCCTCCTCTATAAAATAACC-30). The sec-ond round of PCR was performed using a semi-nested forwardprimer BG5 (50-GGTTGGTTAGTTTTGTTTTGATTAATAG-30)and the reverse primer BG2. A typical 50 mL reaction contained5 mL of bisulfite treated genomic DNA, 5 mL of 10� PCR buffer,5 mL of 25-mM MgCl2 , 200 mM dNTPs, 1 mM each of the for-ward and the reverse primers and 2.5 units of recombinant TaqDNA Polymerase (Invitrogen Inc., Carlsbad, CA, USA). The fol-lowing PCR conditions were used: preheating for 5 minutes at95�C followed by two cycles at 94�C /2 min, 50�C /2 minutes,72�C /3 minutes. The reaction was further carried out for 25 cyclesat 94�C /2 minutes, 50�C /2 minutes, 72�C /2 minutes followed byan extension cycle of 72�C for 7 minutes. PCR products were col-umn purified using Wizard PCR Preps DNA Purification System(Promega). Purified products were cloned in the pCR4-TOPOvector using a TOPO-TA cloning kit (Invitrogen Inc.) and trans-formed in E. coli TOP-10 cells. Clones were selected on ampicil-lin (50 mg/mL) containing LB plates. Individual clones weresequenced using the ABI PRISM 3100 Genetic Analyzer atthe University of Illinois at Chicago Core Genomic Facility.Five CpG sites within the g-globin gene promoter region(�54, �51, þ5, þ16, and þ48) were analyzed for the presenceor absence of cytosine methylation.

ResultsTo understand how methylation of the g-globin promoterchanges during differentiation of normal erythroid progen-itor cells in vivo at both the fetal and adult stages of devel-opment, the g-globin gene promoter methylation patternwas analyzed in subpopulations of cells enriched for dif-ferent stages of erythroid differentiation. Cells analyzedincluded an erythroblast fraction and FACS purifiedCD34þCD36�; CD34þCD36þ and CD34�CD36þ subpop-ulations (Fig. 1A, B). Post sort analysis of FACS-purifiedsubpopulations revealed a purity of 85% to 98% of eachsubpopulation in different experiments (Fig. 1C). Morpho-logical examination of these four subpopulations is consis-tent with the CD34þCD36� as the most immature fractionand the erythroblasts as the most mature fraction. TheCD34þCD36þ and CD34�CD36þ cells were morphologi-cally indistinguishable and appeared to represent intermedi-ate stage of differentiation (Fig. 2).

Clonal analysis of purified erythroid subpopulationsIn order to assess the degree of differentiation of purifiederythroid progenitor cell populations, the CFC assay wasperformed. CFC analysis of CD34þCD36�, CD34þCD36þ

and CD34�CD36þ cells from FL revealed a cloning

Figure 2. Wright’s Giemsa-stained cytospin preparations of subpopulations purified from adult bone marrow samples. 1, 2, 3, and 4 represent CD34þCD36�,

CD34þCD36þ, CD34�CD36þ and mature erythroblast cell populations, respectively.

52 M. Singh et al. / Experimental Hematology 35 (2007) 48–55

efficiency of 89%, 38.5%, and 12%, respectively. This re-sult shows that CD34þCD36� cells produced about twofold more CFCs as compared to CD34þCD36þ cell popula-tion. Moreover, the colony size appeared to be significantlysmaller as the erythroid progenitor cells progressed towardmaturation, i.e., from CD34þCD36� to CD34þCD36þ

cells. The CD34þCD36� and CD34þCD36þ cell popu-lations generated mostly burst-forming unit erythroid(BFU-E) colonies (96.6% and 100%, respectively)(Fig. 3A), although, their cloning efficiencies were differ-ent. On the other hand, the relatively mature CD34�CD36þ

cell population generated mostly colony-forming unit ery-throid (CFU-E; 75%) colonies. Our data suggest that theCD34þCD36� subpopulation is enriched for early BFU-Es, the CD34þCD36þ subpopulation is enriched for lateBFU-Es and the CD34�CD36þ subpopulation is enrichedfor CFU-Es. The clonogenic potential of ABM-derived ery-throid progenitor subpopulations was lower than FL. Acloning efficiency of 1.13%, 1.20%, and 2.33% CFCs wasobserved in ABM-derived CD34þCD36�, CD34þCD36þ,

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Figure 3. Colony-forming cell (CFC) pattern of fluorescein-activated cell

sorting-purified subpopulations of cells from fetal liver (A) and adult bone

marrow (B). Results are shown as percent of colony types from total num-

ber of colonies generated in each subpopulation. The colonies were as-

sayed in duplicate plates. The results are from one fetal liver (A) and

mean and standard deviation of percentage of three adult bone marrow

samples (B). BFU-E 5 burst-forming unit erythroid; CFU-E 5 colony-

forming unit erythroid; CFU-GM 5 colony-forming unit granulocyte

macrophage.

and CD34�CD36þ cell populations (n 5 3), respectively.Similar to FL, ABM-derived CD34þCD36� andCD34þCD36þ cell populations also generated mostlyBFU-Es (45.19% 6 8.98% and 56.64% 6 17.36%, respec-tively) whereas the relatively mature CD34�CD36þ cellsproduced mostly CFU-E (87.35% 6 18.05%) colonies(Fig. 3B). Unlike FL, 43% of the CD34þCD36� cellsalso generated CFU-granulocyte macrophage colonies.

DNA methylation pattern of g-globin gene promoterregion in different stages of erythroid differentiationThe methylation profile of the g-globin gene promoter re-gion in purified cell populations was analyzed by bisulfitesequencing. Five CpG sites located within a 105-bp regionof the g-globin promoter (�54, �51, þ5, þ16, and þ48)were assayed. The CpG density of this region is quitehigh compared to other nonrepetitive elements within theglobin gene locus and the functional importance of this re-gion has been demonstrated earlier [35,36]. A total of 30clones for FL and 45 clones for ABM from each purifiedcell population were sequenced and analyzed. The meanand standard deviation of the percentage of methylatedCpG residues for FL (n 5 2) was 95.4% 6 3.96%,66.25% 6 4.17%, 27.3% 6 1.41% and 3.70% 6 5.23%in CD34þCD36�, CD34þCD36þ, CD34�CD36þ anderythroblast cell populations, respectively (Fig. 4A, C).The mean and standard deviation of the percentage ofCpG residues methylated in ABM-derived (n 5 3) cell pop-ulations was 95.72% 6 1.33%, 87.33% 6 6.04%, 78.84% 6

12.44%, and 74.38% 6 8.02% in CD34þCD36�,CD34þCD36þ, CD34�CD36þ and erythroblasts, respec-tively (Fig. 4B, C). Thus, the g-globin gene promoter re-gion in the earliest HSPCs in both FL and ABM washighly methylated. Methylation of the g-globin promotersignificantly decreased as the cells differentiated fromthe early hematopoietic stem/progenitor cell stage (i.e.,CD34þCD36�) to more mature erythroblasts in FL.Methylation also reduced significantly (p ! 0.05) inABM-derived erythroblasts as compared to CD34þCD36�

subpopulation but the reduction was not significant forCD34þCD36þ and CD34�CD36þ subpopulations(Fig. 4C), although a trend is apparent, suggesting thatthe level of g-globin promoter methylation is reduced ina progressive manner during erythroid differentiation inABM. Reduction of methylation was observed to bemuch higher in progressively differentiated subpopulationsderived from FL as compared to ABM, reflecting the dif-ferences in the g-globin expression. No site-specific pat-tern of demethylation was seen in any cell population.

DiscussionThe g-globin promoter in terminally differentiated erythro-blasts is unmethylated in fetal liver but methylated in adults

53M. Singh et al./ Experimental Hematology 35 (2007) 48–55

[30]. Whether it is methylated or unmethylated in HSPCs isnot known. An understanding of how DNA methylation ofthe g-globin gene is modulated during normal erythroid dif-ferentiation would be important in understanding the regu-lation of g-globin gene transcription and to design betterstrategies to reactivate HbF in order to treat sickle cell ane-mia and b-thalassemia. It has been reported earlier that theprimitive hematopoietic stem cells maintain an open

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*P = 0.078**P = 0.080

***P = 0.010C

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chromatin structure [37]. More differentiated cells alongthe hematopoietic hierarchy are thought to characteristi-cally undergo a step-wise progression of epigenetic changesthat control transcriptional events for each stage and classof progenitor cells, and thus, gene sets available for expres-sion in progressively differentiated cells are more and morerestricted [38]. Consistent with this view, we hypothesizedthat the g-globin promoter is unmethylated in HSPCsand remains so during erythroid differentiation in the fetalstage of development, but become methylated de novoduring the erythroid differentiation in adults to silence g-globin expression. An alternate hypothesis is that theg-promoter is methylated in HSPCs and become demeth-ylated during erythroid differentiation in fetal stage ofdevelopment as expression is activated.

To test our hypothesis, we analyzed g-globin promotermethylation in FACS-purified baboon HSPCs from FLand ABM, enriched for various stages of erythroid differen-tiation, based on differential expression of CD34 and CD36markers. The CD34 is a stem and progenitor cell-specificmarker and is absent in the CFU-E stage. The CD36 isthe earliest known erythroid lineage-specific marker, whichappears in late BFU-E stage and its levels progressivelyincrease as the cells differentiate, but diminish in late eryth-roblast stage [39]. The methylation pattern of the g-globinpromoter region was determined in these three purified pop-ulations along with terminally differentiated erythroblasts.Our results demonstrate that the g-globin promoter regionis almost completely methylated in early HSPCs and meth-ylation decreases during erythroid differentiation in bothFL and the ABM (Fig. 4). Demethylation of the g-globingene promoter during erythroid differentiation appears tobe initiated randomly in an incomplete and variegated man-ner with no observed preference for any individual CpG sitewithin individual clones. These results support the hypoth-esis that the g-globin gene promoter is methylated in earlyHSPCs and become demethylated as the cells terminallydifferentiate and express HbF, rather than the alternativethat the g-globin gene promoter is unmethylated in early

Figure 4. Methylation pattern of the g-globin promoter region in four dif-

ferent cell populations from two fetal liver (FL) (A) and three bone marrow

(B) samples of adult baboon. Red and green colored rectangles represent

methylated and demethylated CpG residues, respectively. Yellow color

represents nonmethylatable residues (a CpG to TpA transition) observed

at positions �51 and þ16 in baboon [44]. 1�15 vertical numbers represent

15 individual plasmid DNA clone sequences. 1,2,3,4 and 5 horizontal num-

bers are five CpG residues located at �54, �51, þ5, þ16, and þ48 base

with respect to transcriptional start of the g-globin gene promoter, respec-

tively. %mC 5 percentage of methylated cytosines in each subpopulation.

(C) Comparative g-globin promoter methylation pattern in fetal liver and

adult bone marrow (ABM). Data represent mean and standard deviation of

percentage of methylation for each of the four cell populations from two

FL and three ABM, respectively, as shown in (A) and (B). *p O 0.05,

**p O 0.05 and ***p ! 0.05 for progressively differentiated cells

compared to the CD34þCD36� subpopulation of adult bone marrow.

=

54 M. Singh et al. / Experimental Hematology 35 (2007) 48–55

HSPCs and becomes methylated de novo in adults.Differential demethylation of g-globin promoter in normalerythropoiesis in FL and ABM, thus, is a developmentalstage-specific phenomenon. In this study, we observedthat CD34þCD36� subpopulation from ABM contained43% myeloid progenitors (Fig. 3B) unlike FL-derivedCD34þCD36� cells, which almost completely lacked mye-loid progenitors. It may be argued that the high methylationof g-globin promoter in CD34þCD36� subpopulation inthe ABM might have partly resulted from the myeloid pro-genitor portion of the CD34þCD36� cells. However, itseems unlikely, because the CD34þCD36þ andCD34�CD36þ subpopulations from the ABM had negligi-ble number of myeloid colonies, but still exhibit highdegree of methylation compared to the erythroblast subpop-ulation and exhibit a trend of progressive reduction in meth-ylation along with differentiation.

The mechanism of progressive demethylation of the g-globin promoter during differentiation is not known. It ispossible that early erythroid progenitor cells lack some pro-tein/transacting factors that may be involved in demethyla-tion process per se. Lineage-specific transacting factors/proteins expressing increasingly during progressive differ-entiation in a developmental stage-specific manner may in-teract with CpG sites and thus prevent access of methyltransferases to these CpG sites, ultimately leading to deme-thylation of the g-globin promoter. This hypothesis isconsistent with earlier studies where Sp1 elements havebeen implicated in prevention of methylation spreading[40,41]. Another explanation could be that active transcrip-tion protects CpG islands from methylation [42]. It is pos-sible that during erythroid differentiation g-globin-specifictranscriptional machinery gradually assembles with a fullcomplement in terminally differentiated cells. It can thenbe argued that progressive demethylation may be the resultof increasing transcriptional activity during erythroid differ-entiation. Whether demethylation is the cause or effect ofg-globin gene expression is, however, not yet clear.

In conclusion, our results show almost complete methyl-ation of g-globin gene promoter region in early HSPCs anda progressive reduction in methylation as the HSPCs differ-entiate toward erythroid-lineage in FL and appear so inABM as well. Furthermore, the difference in the amountof methylation in FL and ABM is consistent with the devel-opmental stage-specific differences in HbF production.

AcknowledgmentThis study was supported by VA Merit Review and National Insti-tutes of Health grant R01 HL73432 (J.D.). We would like to thankDr. Robert Andrews for a gift of 12.8 antibodies. Part of this work[43] was presented in the 47th ASH annual meeting at Atlanta,GA. Thanks are also due to Karen Hagen and Jewell Graves fortheir help in performing and analyzing FACS data.

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