Epigenetic Modifications and Chromosome Conformationsof the Beta Globin Locus throughout Development

Kai-Hsin Chang & Xiangdong Fang & Hao Wang &

Andy Huang & Hua Cao & Yadong Yang &

Halvard Bonig & John A. Stamatoyannopoulos &

Thalia Papayannopoulou

# Springer Science+Business Media, LLC 2012

Abstract Human embryonic stem cells provide an alterna-tive to using human embryos for studying developmentallyregulated gene expression. The co-expression of high levelsof embryonic ε and fetal γ globin by the hESC-derivederythroblasts allows the interrogation of ε globin regulationat the transcriptional and epigenetic level which could onlybe attained previously by studying cell lines or transgenicmice. In this study, we compared the histone modificationsacross the β globin locus of the undifferentiated hESCs and

hESC-, FL-, and mobilized PB CD34+ cells-derived eryth-roblasts, which have distinct globin expression patternscorresponding to their developmental stages. We demon-strated that the histone codes employed by the β globinlocus are conserved throughout development. Furthermore,in spite of the close proximity of the ε globin promoter, ascompared to the β or γ globin promoter, with the LCR, achromatin loop was also formed between the LCR and theactive ε globin promoter, similar to the loop that formsbetween the β or γ globin promoters and the LCR, incontrary to the previously proposed tracking mechanism.

Keywords Human embryonic stem cells . Erythroidcells . Fetal liver . Peripheral blood . Erythroblasts .

Hemoglobin . Epigenetics . Histone modifications .

Chromatin conformation

Introduction

The human β globin locus, located on chromosome 11 andspanning roughly 100 kb, encodes five functional globingenes: ε, Gγ, Aγ, δ, and β, that are arranged in the orderaccording to their developmental stage-specific expressionwhich involves two hemoglobin switches, in response to thedifferential local oxygen pressure during development(reviewed in [1]). Embryonic ε globin gene is predominantlyexpressed by the transiently circulated, large, mostly nucleatedprimitive erythroblasts originating from the yolk sac. The firsthemoglobin switch entails the emergence of small, enucleateddefinitive erythroid cells from the fetal liver (FL) at approxi-mately 6 to 8 weeks of gestation expressing fetal Gγ and Aγglobin genes with their ε globin gene silenced. The secondhemoglobin switch occurs around birth when the bone marrowbecomes the major hematopoietic tissue and generates

Drs. Kai-Hsin Chang and Xiangdong Fang contributed equally to thiswork.

Electronic supplementary material The online version of this article(doi:10.1007/s12015-012-9355-x) contains supplementary material,which is available to authorized users.

K.-H. Chang : T. Papayannopoulou (*)Department of Medicine, Division of Hematology,University of Washington,NE Pacific St, Box 357710, Seattle, WA 98195, USAe-mail: thalp@u.washington.edu

X. Fang :Y. YangLaboratory of Disease Genomics and Individualized Medicine,Beijing Institute of Genomics, Chinese Academy of Sciences,Beijing 100029, China

X. Fang :A. Huang :H. CaoDepartment of Medicine, Division of Medical Genetics,University of Washington,Seattle, WA 98195, USA

H. Wang : J. A. StamatoyannopoulosDepartment of Genome Sciences, University of Washington,Seattle, WA 98195, USA

H. BonigDepartment of Cellular Therapeutics/Cell Processing (GMP),German Red Cross Blood Service,Frankfurt 60528, Germany

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enucleated erythroid cells expressing mainly adult β globin. Aregulatory domain named the locus control region (LCR) islocated upstream of the ε globin. The LCR, characterized by 5DNase I hypersensitive sites (HSs) each containing multipletranscriptional factor binding motifs, has been shown to par-ticipate in the regulation of hemoglobin expression.

The temporal and tissue-specific expression characteristicsof the β globin locus have made it an attractive experimentalmodel for studying the regulation of mammalian gene expres-sion. An enormous amount of work has been done to elucidatethe regulation of fetal to adult globin switching (reviewed in[2]), partly due to the fact that a better understanding of thesilencing and reactivation of fetal globin may lead to therapeu-tic opportunities for the treatment of hemoglobinopathies suchas beta thalassemia. In comparison, the regulation of embryonicε globin expression has been studied less extensively. Thedifficulties in obtaining a sufficient number of primary cellsexpressing embryonic ε globin have largely limited the relatedresearch to employing K562 cells [3, 4], a human leukemia cellline that express both embryonic and fetal but not adult hemo-globins, and transgenic mice carrying human β globin locus[5, 6]. Recently, the establishment of methods allowing gener-ation of a large quantity of embryonic globin-expressing eryth-roblasts from human embryonic stem cells (hESCs) lead tostudies comparing the epigenetic landscapes of β globin locusof hESC-derived erythroid cells, FL, and bone marrow eryth-roblasts. It has been found that complex developmental patternsof histone modifications as well as the formation of extendedDNA hypomethylation domains are associated with the humanβ-locus globin switch [7, 8]. In this study, we provide furtherevidence that a looping mechanism is associated with theexpression of ε globin in these hESC-derived erythroblasts,just as it is associated with the expression of fetal γ globin inFL cells and the expression of adultβ globin in adult peripheralblood (PB) CD34+ cells derived-erythroid cells.

Materials and Methods

hESC Culture

hESC line H1 (NIH code WA01) was cultured as previouslydescribed [9]. Briefly, H1 was propagated on irradiated mu-rine embryonic fibroblasts (MEFs) on gelatin coated tissueculture plates (BD falcon, San Jose, CA) in ES mediumconsisted of Dulbecco modified Eagle medium/F12 mediumsupplemented with 15% knock-out serum replacement, 1 mMsodium pyruvate (all 3 from Invitrogen, Carlsbad, CA),0.1 mM β-mercaptoethanol (Sigma, St Louis, MO), 0.1 mMminimum essential media (MEM) nonessential amino acids(Mediatech, Herndon, VA), 1 × penicillin/streptomycin(Mediatech), and 2 ng/mL basic fibroblast growth factor(bFGF) (Peprotech, Rocky Hill, NJ). For MEF-free cultures,

hESCs were transferred to tissue culture plates coated withmatrigel (BD Biosciences, San Jose, CA) diluted 1:50 withPBS (Thermo Fisher Scientific, Waltham, MA) and culturedwith MEF-conditioned ES medium (MEF-CM) for 3 gener-ations. MEF-CM was prepared by incubating 50 mL of ESmedium per 6×106 irradiated MEFs at 37°C, 5% CO2, in ahumidified incubator for 3 days. bFGF was added to a finalconcentration of 4 ng/mL. These MEF-free hESCs were usedfor chromatin immunoprecipitation (ChIP) assays.

Erythroid Differentiation

hESCs were induced to undergo hematopoietic differentiationvia embryoid body (EB) formation [10]. Briefly, hESCs cul-tured withMEFs were harvested by gentle scraping and seededonto ultra-low attachment plates (Corning, Acton, MA) in EBmedium consisting of Iscove MEM (IMDM) (Mediatech),15% ES-qualified FBS (Invitrogen), 10% protein-free hybrid-oma medium II (PFHM-II) (Invitrogen), 300 μg/mL iron-saturated human transferrin (Sigma), 50 μg/mL ascorbic acid(Sigma), 0.1 mM β-mercaptoethanol, 1 × penicillin/strepto-mycin, and 2 mM L-glutamine (Mediatech). After 7 days, EBswere dissociated into single cells and cultured in an erythroid-inducing medium composed of Stemline hematopoietic stemcell growth and expansion medium (Sigma) supplementedwith 2 mML-glutamine, 1× penicillin/streptomycin, 0.1 mMMEM nonessential amino acids, 0.1 mM β-mercaptoethanol,200 μg/ml iron-saturated transferrin,10 μM ethanolamine,10 μg/ml insulin (all from Sigma), 6 U/ml erythropoietin,50 ng/ml stem cell factor (both fromAmgen, Thousand Oakes,CA), 20 ng/ml interleukin-3, 20 ng/ml interleukin-6, 40 ng/mlinsulin-like growth factor-1, 10 ng/ml vascular endothelialgrowth factor (all from Peprotech, Rocky Hill, NJ), 5% proteinfree hybridoma medium, 1× insulin-transferrin-selenium (bothfrom Invitrogen), and 1× EX-CYTE (Millipore, Billerica,MA). for 14 days. The purity of cells was confirmed usingflow cytometry by staining cells with phycoerythrin (PE)conjugated anti-glycophorin-A (Gly-A) antibody (DAKO,Glostrup, Denmark). When the erythroid population com-prised less than 93% of total cells, they were subjected tofurther enrichment using theMACS system per manufacturer’sinstruction (Miltenyi Biotech, Auburn, CA).

CD34+ cells from healthy volunteer-donor mobilized PBand dissociated FL cells (50–100-day gestation) were cul-tured in the same erythroid-inducing medium to generateadult- and fetal-type erythroid cells. FL cells were obtainedfrom the fetal tissue repository (University of WashingtonBirth Defects Research Laboratory) and PB-derived CD34+

cells were obtained from German Red Cross Blood Service,or from the NIH repository (Fred Hutchinson Cancer Re-search Center) with permission of the University of Wash-ington Institutional Review Board. Cell purity was alsoconfirmed and further enriched if necessary as described

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above. β-locus globin expression patterns were determinedby RNase protection assay as previously described [11] andby RNA-sequencing.

Phenotypic Analysis of Erythroblasts

The expression of erythroid marker glycophorin-A and leuko-cyte common antigen CD45 was examined using flow cytom-etry analysis. Antibodies used were glycophorin-A–PE(DAKO) and allophycocyanin (APC) conjugated CD45 (BDBiosciences). Cells were washed once with PBS-0.5% BSAand then incubated with antibodies for 30 min at 4°C, washed,resuspended in PBS-0.5% BSA containing 100 ng/mL propi-dium iodide (Sigma) for dead cell exclusion. Cells were ac-quired using FACScalibur (BD Biosciences), and results wereanalyzed with Cell Quest Pro (BD Biosciences). Gatings wereset according to isotype controls.

Chromatin Immunoprecipitation (ChIP) Assays

Two ChIP protocols were used. For RNA polymerase II(Pol II), transcription factor IID (TFIID), TFIIB, and mono-methylated H3 lysine 27 (H3K27me1), ChIP assays wereperformed as described by Yin et al. [12]. For trimethylatedH3 lysine 4 (H3K4me3) and trimethylated H3 lysine 27(H3K27me3), ChIP assays were performed as described byKimura et al. [13] as part of ChIP sequencing procedure. Forboth procedures, chromatin was fragmented to roughly 200–500 bp in size by sonication with a microtip (Fisher SonicDismembrator 500) (Thermo Fisher Scientific). Antibodiesused were: Pol II (SC-899), TFIID (SC-273X), TFIIB (SC-225), (all three were from Santa Cruz, CA), AcH3 (Upstate/Millipore, 06-599, Billerica, MA), H3K27me1 (Upstate/Milli-pore, 07-448), H3K27me3 (Upstate/Millipore, 07-449), andH3K4me3 (Cell signaling, 9751, Danvers, MA). For PolII,TFIID, TFIIB, and H3K27me1, the relative enrichment offragments was determined by quantitative real-time polymer-ase chain reactions (PCR) using FastStart SYBR Green Master(Roche Applied Science, Indianapolis, IN) in the MJ ResearchDNA Engine Opticon 2 (Bio-rad, Hercules, CA) and analyzedwith MJ Opticon Monitor Analysis Software Version 2.02.Human genomic DNA (Bioline, Taunton, MA) was used togenerate standard curves. The PCR conditions were: 10 min at95°C, followed by 40 cycles of 40 s at 95°C, 1min at 60°C, and1 min at 72°C, and then 5 min at 72°C, followed by meltingcurve analysis. Primer sequences are provided in Supplemen-tary Table 1. One way analysis of variance (ANOVA) followedby Tukey’s Post Hoc Test was performed using GraphPadInState version 3.10 (GraphPad Software, Inc. La Jolla, CA)to determine whether the differences between means of eachgroup were statistically significant. When 2 bars are labeledwith the same letter, it indicates that these 2 sets of data are notstatistically significantly different. For H3K4me3, H3K27me2,

and AcH3, ChIP sequencing was performed. The libraries wereconstructed according to the Illumina (San Diego, CA) proto-col. Briefly, purified ChIP DNAwas end-repaired using End-ItDNA end-repair kit (Epicentre,Madison,WI.) and adenine wasadded to the 3′ ends using Klenow fragment (3′-5′ exo-) (NewEngland Biolabs (NEB), Ipswich, MA). Adapters (Illumina)were ligated to the DNA fragments using T4 DNA ligase(NEB) and the products were PCR amplified (Phusion, NEB)and size-selected on 2% agarose gels. The libraries were se-quenced on Genome Analyzer IIX or Hi-Seq 2000 platforms(both from Illumina) by the High-througput Genomics Center(University of Washington, Seattle, WA). Regions of localenrichment of short-read sequence tags mapped to the genomewere identified using HotSpot algorithm (http://www.uwencode.org/proj/hotspot-ptih/). One percent false discovery ratethresholds (FDR 0.01) were computed for each cell type byapplying the HotSpot algorithm to an equivalent number ofrandom uniquely mapping 36mers. Data alignment was per-formed using Bowtie aligner (version 0.12.7) (http://bowtie-bio.sourceforge.net/index.shtml). The raw data have been de-posited to NCBI Gene Expression Omnibus with accessionnumber GSE35375.

Chromatin Conformation Capture (3C) Assays

The higher chromatin structure of the erythroblasts of the threedevelopmental stages was analyzed using 3C assays essential-ly as previously described [14]. Briefly, cross-linked DNAwas prepared from the nuclei of erythroblasts and subjected todigestion with restriction enzymeHindIII (New England Biol-abs, Ipswich, MA). Afterward, DNA fragments were ligatedwith T4 DNA ligase (New England Biolabs). The DNAwasconcentrated by ethanol precipitation and subjected to RNaseA treatment and further purification by phenol/chlorophormextraction and ethanol precipitation. Control template wassimilarly prepared from mixed equimolar amounts of BACClone CTD2596M16 containing human β-globin locus andBAC Clone RP11-687D2 containing human glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene. The ligationproducts were quantitated by real time PCR using PowerSYBR Green PCR master mix (Applied Biosystems, FosterCity, CA) or Universal ProbeMaster (Roche Applied Science)with probes (5′ FAM/3′ BHQ-1, Biosearch Technologies,Novato, CA) specific to ε, γ, β globin or GAPDH fragments.The ligation frequencies between the globin site pairs werenormalized and expressed as the percentage of the interactionobserved within GAPDH gene. Primer and probes sequencesare provided in Supplementary Table 2.

RNA-Sequencing

Total RNA was extracted from erythroblasts using TRIzol®Reagent (Invitrogen, Carlsbad, CA) per manufacturer’s

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instruction. 18S and 28S rRNAs were depleted from totalRNA using Ribosomal RNA Depletion Kit (Applied Bio-systems). cDNA library was constructed using SOLiDWhole Transcriptome Analysis Kit (Applied Biosystems).Massively parallel ligation sequencing was carried out to 50bases using Applied Biosystems SOLiD System accordingto manufacturer’s instruction. Corona_lite_plus (version4.2.1) from Applied Biosystems were used to map sequencereads to human reference sequence (hg18) from Universityof California–Santa Cruz. Ribosome RNA reads filteringwas executed at the beginning of reads mapping with fulllength of 50 bp. The remaining reads were mapped to thereference with the length of 45 bp and 40 bp respectively.Five mismatches were allowed at each mapping step. Readsmapped to unique loci and a fraction of those mapped tomultiple loci (2–10) were used for further analysis. To calcu-late gene expression intensity, the read counts were normal-ized to reads per kilobase of exon model per million mappedreads (RPKM). Spots where RPKM were less than 0.01 forgenes in all three libraries were removed from analysis.

Results

The Three Erythroid Populations Represent Three DistinctDevelopmental Stages with Unique Globin ExpressionProfiles

To study the epigenetic regulation of hemoglobin switching indifferent developmental stages, we first obtained nucleatederythroblasts by differentiating hESCs, FL, and adult PBCD34+ cells in the erythroid inducing medium. The expressionof surface marker CD45 and glycophorin-A was examinedbetween day 8 and day 14 after cells were directed to undergoerythroid differentiation (Fig. 1a). Interestingly, while the im-mature erythroid cells from FL and PB CD34+ cells expressedleukocyte common antigenCD45, the immature erythroid cellsfrom hESCs lacked such expression. With further maturation,both FL- and PB-derived erythroblasts lost CD45 expression.RNase protection assays revealed that hESC-derived erythro-blasts expressed high levels of embryonic ε and fetal γ globinwith little adult β globin whereas FL-derived erythroblastsmainly expressed fetal γ globin and PB-derived erythroblastsmainly expressed adult β globin, although occasionally hadelevated fetal γ globin expression (Fig. 1b). The respectiveglobin expression patterns of these three types of erythroblastswere also confirmed using RNA-sequencing (SupplementaryTable 3). These results demonstrated that the three erythroidpopulations under investigation here possessed unique globinexpression patterns associated with embryonic/early fetal,fetal, and adult developmental stages.

ChIP assays were then performed with chromatin obtainedfrom undifferentiated hESCs as well as the three different

erythroid populations to interrogate whether the distinct glo-bin expression patterns were a reflection of the recruitment oftranscriptional complexes to the specific globin regions(Fig. 1c). While little RNA Pol II was found to be associatedwith the entire β globin locus of undifferentiated hESCs,elevated levels of RNA Pol II were found to be associatedwith the LCR, especially the DNaseI HS4 of the three ery-throid populations (Fig. 1c).Much higher levels of RNAPol IIwere detected at the globin promoters and exons in accordancewith the unique globin expression pattern of the specificerythroid population. Specifically, RNA Pol II was associatedwith both embryonic ε and γ globin promoters and exons ofhESC-derived erythroblasts; with only γ globin promotersand exons of FL-derived erythroblasts; and mainly with βglobin promoter and exon of PB-derived erythroblasts. Therecruitment of transcription factor IIB (TFIIB) and TFIID,which are part of the RNA Pol II preinitiation complex,showed similar patterns with elevated LCR recruitment inerythroid cells as compared to undifferentiated hESCs, andglobin gene-specific recruitment that was consistent with theglobins expressed by the individual erythroid populations(Fig. 1d, e). Together, these data show that the unique globinpatterns displayed by these three developmentally distincterythroid populations were a result of targeted recruitment ofRNA polymerase II and its preinitiation complex to the spe-cific globin genes, and not a result of failed elongation by non-expressed genes.

Fig. 1 The unique globin expression patterns and transcriptional ma-chinery recruitment to the β globin locus of three developmentallydistinct erythroid populations. a The CD45 and glycophorin-A expres-sion by immature and mature erythroblasts derived from hESC, FL, orPB-CD34+ cells. Erythroblasts were generated from dissociated day-7embryoid bodies prepared from hESCs, from FL mononuclear cells, orfrom mobilized PB CD34+ cells. Cells suspensions were collected be-tween day 8 and day 14, stained with CD45-APC and glycophorin-A-PE,and enumerated using flow cytometry. Gatings were set based on isotypecontrols. b The β locus globins expression by the three erythroid pop-ulations. RNA was prepared from day-14 erythroblasts and RNase pro-tection assay was carried out to determine the levels of ε, γ, and β globintranscripts. Occasionally, elevated levels of γ globin expression by PBCD34+ cells-derived erythroblasts were detected. A non-specific bandslightly higher than the band for ε globin was detected in all samples.Results from RNase protection were also confirmed using RNA-sequencing (Supplementary Table 3). c Recruitment of RNA polymeraseII to the β globin locus in undifferentiated hESCs and three erythroidpopulations. d Recruitment of TFIIB to theβ globin locus. e Recruitmentof TFIID to the β globin locus. For (c), (d), and (e), glycophorin-A+ FLerythroblasts were enriched usingMACS prior to preparing chromatin forChIP assay. The relative degree of enrichment for each target sequencewas determined by first quantifying against a standard curve generatedusing purified human genomic DNA and then normalizing to the level ofhuman GAPDH promoter present in each individual precipitation. Datashown are mean ± standard error of mean (SEM). One-way ANOVAwasperformed followed by Tukey’s Post Hoc Test to determine whether anytwo means of a specific target are statistically significantly different (p<0.05). When two bars are labeled with the same letter, it denotes that thesemeans are not statistically significantly different

b

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Epigenetic Markings Are Conserved in the Three ErythroidPopulations in Accordance with Their Unique GlobinExpression Profiles

Covalent modifications of the N-terminal tails of histonesalter the association between histones and genomic DNA

and lead to changes in chromatin structure and in the ac-cessibility of the gene to transcriptional machineries. Tostudy whether the same histone codes are employed by theβ globin locus throughout development, we performedChIP assays to study the epigenetic landscapes of β globinlocus in undifferentiated hESCs and the three erythroid

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populations (Fig. 2). Histone H3 hyperacetylation (AcH3)was observed in the LCR of the three erythroid populations

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Fig. 2 Epigenetic landscapes of the β globin locus of undifferentiatedhESCs and erythroblasts derived from hESC, FL, and PB. Quantificationof (a) histone H3 acetylation (AcH3), (b) H3K4 trimethylation(H3K4me3) and (c) H3K27 monomethylation (H3K27me1) distributionacross theβ globin locus using ChIP assays. (a) and (b) were quantified byhigh throughput sequencing using Illumina Hi-seq 2000 (a), or GenomeAnalyzer IIX (b). Regions of local enrichment of short-read sequence tags

mapped to the genome were identified using HotSpot algorithm. Dataalignment was performed using Bowtie aligner. (c) was quantified by realtime PCR. Normalization and statistical analyses were performed asdescribed in Fig. 1. An additional panel for the H3K27me1 enrichmentin the promoter regions is provided to assist side-by-side comparison of therelative distribution of H3K27me1 across the ε, γ, andβ globin promotersin each specific cell type

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promoters and exons, confirming that while the LCR hasacquired an open chromatin conformation in erythroid cellsof any developmental stage, only the temporally active globingenes have acquired such structure. The developmentallysilenced globin genes are situated within a closed, hypoace-tylated domain. Furthermore, histone H3 lysine 4 trimethyla-tion (H3K4me3), another mark for an active chromatindomain, was studied (Fig. 2b). Consistent with a closedchromatin conformation, no H3K4me3 peaks were detectedin the β globin locus of undifferentiated hESCs. Interestingly,while active chromatin mark AcH3 encompassed both theLCR and active globin genes of erythroid cells (Fig. 2b), onlychromatins of active globin regions, but not the LCR, weremarked by high levels of H3K4me3 (Fig. 2b). This patternwas consistent in all three erythroid populations studied.Finally, the distribution of histone H3 lysine 27 monomethy-lation (H3K27me1) and trimethylation (H3K27me3) wasexamined in the four cell populations. We were unable todetect specific H3K27me3 enrichment across the β globinlocus of the four cell types with ChIP sequencing although itsenrichment was detected at other loci (data not shown).H3K27me1 was decreased in the HSs 2, 3, and 4 of theLCR of erythroid cells in comparison to undifferentiatedhESCs (Fig. 2c). Such differences among the cell types werenot evident in the promoter or exon regions. However, whenε, γ, and β promoters within individual cell types werecompared, we found that the active globin promoters, asopposed to dormant globin promoters, had the lowest levelof H3K27me1 in the erythroid cells (Fig. 2c insert). Our datacontrast the recent findings by Kim and Kim that the β-globin locus is marked by H3K27me3 in non-erythroid293FT cells and by H3K27me1 in K562 erythroid cells[15]. The reason for these disparities is not clear. However,our data do agree with other groups who find thatH3K27me1 is a repressive chromatin mark associated withthe formation of heterochromatin [16, 17], that H3K27me1removal may be a general prerequisite for the initiation ofhigh-level transcription [18], and that H3K27me1 mark isdepleted at the β globin region as compared to ε and γ globinregions in adult CD36+ erythroid precursors [19]. Overall,our studies show that the β globin locus employs similarepigenetic markings throughout development to form a chro-matin structure that allows the selective expression of devel-opmentally regulated globins.

Physical Proximity of the LCR and Globin PromotersChanges According to the Developmental Stageof the Erythroid Cells

It has been shown in the transgenic mice that an activechromatin hub forms between the developmentally activeglobin genes and the HSs of LCR with the inactive globingenes looped out [20]. Here we interrogated the dynamic

long-range interactions across the β globin locus in the threedevelopmentally distinct erythroid populations using 3Cassays (Fig. 3a). In hESC-derived erythroblasts, the interac-tion frequencies between the ε globin promoter and the HSs1–4 of the LCR were found to be significantly higher than thefrequencies observed in the FL- and PB-derived erythroblasts,suggesting the formation of a chromatin loop between theactive ε promoter and the LCR in the hESC-derived erythro-blasts. Furthermore, in the FL- and PB-derived erythroblasts,the interaction frequencies between ε globin promoter andthe γ globin genes decreased as the distance from the ε globinpromoter increased (Fig. 3a, top panel). In contrast, high levelsof interaction between ε globin promoter and both γ globingenes (Gγ and Aγ) were observed in the hESC-derivederythroblasts. No such increased interaction was found be-tween ε globin promoter and the δ and β globin genes in thehESC-derived erythroblasts as compared to the FL- and PB-derived erythroblasts. When the interaction frequencies be-tween Gγ promoter and the LCR was characterized, signifi-cantly higher levels of interactions were found in the hESC-derived erythroblasts than those in the PB-derived erythro-blasts (Fig. 3a, middle panel). Similar levels of interactionfrequencies between Gγ promoter and the LCR in the FL-derived erythroblasts as compared to the hESC-derived eryth-roblasts were also observed in a separate experiment (Supple-mentary Figure 1). On the contrary, only PB-derivederythroblasts showed significant interactions between the βglobin promoter and the LCR (Fig. 3a, bottom panel), consis-tent with the finding that these cells were the only studied celltype that expressed high levels of β globin. Together, thesedata suggest that in hESC-derived erythroblasts where highlevels of ε and γ globins were expressed with little adult δ orβ globin genes, the active ε, Gγ, Aγ globin genes and theLCR were spatially organized into a physically closely asso-ciated hub while the inactive δ and β globin genes weresituated outside the hub.

It has also been proposed that a tracking or a facilitatedtracking and transcription mechanism is responsible for theexpression of embryonic ε globin in K526 cells [3, 4]. Wenext studied both the genic and intergenic transcriptionsacross the beta globin locus in the three erythroblasts popula-tions using RNA-sequencing to obtain absolute quantifica-tions of transcripts (Fig. 3b, Supplementary Table 3). Ourdata confirm that intergenic transcription is a wide-spreadphenomenon in β globin locus of erythroid cells, as previous-ly described byMiles et al. [21] and not necessarily specific tothe intergenic region leading up to ε globin promoter. WhileRNA PolII did appear to track between the LCR and the εglobin promoter, given the low abundance of HS1-ε tran-scripts relative to other intergenic transcripts, such as Aγ-Ψβ1 and Ψβ1- δ, whether this tracking is responsible forthe ε globin activation in hESC-derived erythroblasts remainsto be determined.

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HindIII cut sites

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Discussion

An extensive body of work has shown that the modificationsof histones, the fundamental constituent of nucleosomes andchromatin fibers, alter gene expression by affecting overallchromatin structure and by modulating the binding of effectormolecules (reviewed in [22]). The vast number of possiblemodifications and the crosstalk between modifications givethe higher organisms tight control and fine tune capabilityrequired for spatially and temporally regulated gene expres-sion. Chromatin profilings in ESCs, which possess both in-definite self-renewal capability and differentiation potential,have revealed bivalency characterized by the co-existence ofrepressive chromatin mark (H3K27me3) and active chromatinmarks including histone acetylation and H3K4me3 at thepromoters of the genes critical for developmental decisions[23–25]. In addition, certain localized epigenetic markings forthe later formation of an active tissue-specific chromatin do-main in differentiated cells, including VpreB1 and λ5 genesfor B cells [26], and the LCR for erythroid cells [27] are foundto be already established in the murine ESCs. Furthermore,epigenetic pre-marking and recruitment of transcriptional fac-tors at the enhancers of silent tissue-specific genes includingliver-specific Alb1, macrophage/dendritic cell-specific Il12b,and thymocyte-specific Ptcra genes at the ESCs stage appearto be important for the transcriptional activation of these genesin differentiated cells [28, 29]. In contrast to these findings, wedid not find evidence of either bivalency or active epigeneticmarks in the entire β globin locus of hESCs. The recent

finding that places hESC and mESC in different developmen-tal stages [30] may have implications on why active LCR isalready formed in the ESCs of murine but not in those ofhuman origin. The heterogeneity of the ESCs population, asdemonstrated by the disappearance of bivalency marks whenESCs are subfractionated [31], may also provide an alternativeexplanation. Nevertheless, the active LCR is formed in thehESC-derived erythroblasts similar to that in the FL-derivedand PB-derived erythroblasts. The activation of the LCR mayhave occurred at the stage of multipotent hematopoietic stemcells during hESC differentiation as CD34+CD38- cells havebeen shown to express embryonic ε and fetal γ globin mRNA[32] and the acquisition of chromatin signatures for LCRactivation has been noted in the multipotent human hemato-poietic stem cells [19].

The characteristic co-expression of high levels of embryonicε and γ globins [9] makes hESC-derived erythroblasts an idealmodel system for studying the transcriptional and epigeneticregulation of embryonic ε globin expression, which previouslyhas been confined to utilizing transgenic mice carrying humanβ globin locus, and K562 cells. Thus far, the studies conductedanalyzing the epigenetic landscapes of hESC-derived erythro-blasts are limited. It was found that while β globin locus genescontain no CpG islands, domains of DNA hypomethylationspanning thousands of base pairs within domains of acetylatedhistones are established around the most highly expressedgenes during each developmental stage when comparinghESC-derived erythroblasts to uncultured FL and bone marrowcells [7, 8]. Consistent with these findings, our study alsoshowed that the LCR is hyperacetylated in erythroblasts of alldevelopmental stages, but such hyperacetylation is confined toonly the actively expressed globin genes of each specific de-velopmental stage. Furthermore, we provided evidence thatH3K4me3 co-localized with AcH3 at the actively transcribedglobin genes and H3K27me1was selectively depleted from theactively transcribed globin promoters. Together with the resultsby Lathrop et al. [8], and Hsu et al. [7], it was revealed thatthroughout development, human primary erythroblasts employthe same histone codes including hypomethylation, histoneacetylation, H3K4me2, H3K4me3, and selective depletion ofH3K27me1 and H3K9me2 to ensure proper temporal expres-sion of specific globin genes. The role of various methyltrans-ferases (reviewed in [33]) and polycomb group (reviewed in[34]) in the establishment and maintenance of these domainsand globin gene regulation requires further studies.

Higher-order chromatin structures, such as chromatin loop-ing, have been implicated in participating in the activation andrepression of genes (reviewed in [35, 36]). The long-rangephysical interactions between the HSs of the LCR and thedevelopmentally active globin genes have been demonstratedin the endogenous mouse β locus, as well as the human βglobin locus in transgenic mice [20, 37, 38] with the potentialinvolvement of BCL11A and Sox6 [39], which may participate

Fig. 3 Long range interaction between globin genes and the LCR. aPhysical proximity between embryonic ε, fetal γ, or adult β globinpromoters and the other genetic elements of the β globin locus asrevealed by chromatin conformation capture studies. HindIII cut sitesrelative to various genetic elements are shown. Anchor fragmentsencompassing the promoters investigated (thick black lines) are shownin dark gray. Fragments analyzed are shown in light gray. There wasinsufficient amount of FL templates for analyzing the interactionsbetween Gγ promoter and the HSs of the LCR (middle panel) but datafrom an independent experiment are included in Supplementary Fig-ure 1. Data shown are mean ± standard error of mean (SEM). One-wayANOVA followed by Tukey’s Post Hoc Test was performed to analyzewhether the means differ statistically significantly in the experimentswhere ε or β promoters were used as the anchor. For the experimentusing fragment enclosing Gγ promoter as the anchor, Student’s t testwas performed. * denotes that the frequencies found in hESC-derivederythroblasts are significantly different from both the FL- and PB-derived erythroblasts (top panel), or from the PB-derived erythroblasts(middle panel) * in the bottom panel indicates that the interactionfrequencies found in the PB-derived erythroblasts are significantlydifferent from those found in both FL- and hESC-derived erythro-blasts. b Levels of transcripts across the β globin locus determinedby RNA-sequencing. Data shown are genome browser view of RPKMnormalized read counts mapped to the β globin locus. These readcounts were further ln(x+1) transformed to enable viewing of lowabundance transcripts. The untransformed RPKM normalized readcounts are included in Supplementary Table 3

R

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in a large transcriptional interactome [40]. The long-rangeinteractions between LCR and the active β locus globin genes,to our best knowledge, have not been shown in the primaryhuman erythroid cells. While there is little doubt that a loopingmechanism would contribute to the long-range activation of γand β globin genes in primary FL- and PB-derived erythro-blasts, respectively, by the LCR, a different mechanism mightbe involved in the activation of embryonic ε globin, given theclose proximity between ε globin and the LCR. Indeed, atracking mechanism has been proposed to be responsible forthe transfer of RNA polymerase II to the ε globin promoter,based on the continuity of histone modifications, includingAcH3, AcH4, and H3K4me2 and the RNA polymerase IIdistribution from the LCR to the ε globin promoter, and thepresence of intergenic transcripts between HS2 and ε promoter,in the ε globin-expressing K562 cells [3]. While we alsodetected intergenic transcripts, confirming RNA tracking be-tween the LCR and the ε globin gene, and wide distribution ofacetylated H3 in the LCR and ε globin gene, H3K4me3 map-ping clearly showed that the LCR had relatively lowH3K4me3marking as compared to the ε globin gene. Therefore, in the εglobin-expressing hESC-derived erythroblasts, the LCR and εglobin gene were not situated within a continuous chromatindomain. Most importantly, the increased cross-linking frequen-cies between the ε globin promoter and the HSs of the LCR inthe hESC-derived erythroblasts as compared to FL- and PB-derived erythroblasts, revealed by chromatin conformationcapture, suggest a physical proximity between the LCR andthe ε globin promoter closer than otherwise would have beenexpected based solely on the distance, base-pair-wise, betweenthese two genetic elements. Although our results are consistentwith a looping mechanism contributing to the activation of εglobin promoter, we cannot rule out a facilitated tracking andtranscription mechanism that leads to the eventual formation ofa looped active chromatin hub, as proposed by Zhu et al. [4].However, as the levels of HS1-ε intergenic transcripts detectedwere not proportional to the levels of ε globin expressed, howmuch the RNA polymerase II tracking between the LCR andthe ε globin contributes to the eventual ε globin activationremains unclear. Finally, as we have previously demonstratedthat hESC-derived erythroblasts co-express embryonic ε andfetal γ globins at the single cell level, a previously proposedflip-flop mechanism [41] may be at play here. The increasedcross-linking frequencies between the LCR, ε globin and bothGγ and Aγ globin genes in these cells support that ε, Gγ, andAγ globin promoters are held at close physical proximity to theLCR, which can potentially facilitate the co-expression of theseglobin genes.

Overall, our studies provide a comprehensive comparisonof chromatin modifications of the β globin locus of undif-ferentiated hESCs and three erythroblast populations ofdistinct developmental stages. We found that the locus isinactive and without epigenetic pre-marking in the hESCs.

Once activated in the erythroblasts, the locus employs thesame histone codes throughout development to mark activelyexpressed genes and utilizes the looping mechanism to facil-itate transcription.

Acknowledgments The authors express their gratitude towards Drs.Theresa Canfield, Erika Giste, Richard Sandstrom, and R. ScottHansen for assistance with high throughput sequencing. This researchwas supported by the National Institute of Health grants DK077864(K-H.C), HL46557 (T.P.) and 1RC2HG005654 (J.A.S.).

Author Disclosure Statement The authors declare no potential con-flicts of interest.

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