An epigenetic profile of early T-cell development from multipotent progenitors to committed T-cell...

Eur. J. Immunol. 2014. 44: 1181–1193 Molecular immunologyDOI: 10.1002/eji.201344022 1181

An epigenetic profile of early T-cell developmentfrom multipotent progenitors to committed T-celldescendants

Maria Alessandra Vigano1, Robert Ivanek2,3, Piotr Balwierz4,5,Philipp Berninger4, Erik van Nimwegen3,4, Klaus Karjalainen6

and Antonius Rolink1

1 Developmental and Molecular Immunology Group, Department of Biomedicine, University ofBasel, Basel, Switzerland

2 Department of Biomedicine, University of Basel, Basel, Switzerland3 Swiss Institute of Bioinformatics, Basel, Switzerland4 Biozentrum, University of Basel, Basel, Switzerland5 Institute of Clinical Sciences, Imperial College London and MRC Clinical Science Centre,

London, UK6 School of Biological Sciences, Nanyang Technological University, Singapore

Cellular differentiation of the T-cell branch of the immune system begins with the HSC,which undergoes a series of stages characterized by progressive restriction in multi-potency and acquisition of specific lineage identity At the molecular level, the restric-tion of cell potential, commitment, and differentiation to a specific lineage is achievedthrough the coordinated control of gene expression and epigenetic mechanisms. Here,we analyzed and compared the gene expression profiles and the genome-wide histonemodification marks H3K4me3 (H3 lysine 4 trimethylation) and H3K27me3 (H3 lysine 27trimethylation) in (i) in vitro propagated HSCs, (ii) in vitro generated and propagatedpro-T cells derived from these stem cells, and (iii) double-positive thymocytes derivedfrom these pro-T cells after injection into Rag-deficient mice. The combined analyses ofthe different datasets in this unique experimental system highlighted the importanceof both transcriptional and epigenetic repression in shaping the early phases of T-celldevelopment.

Keywords: Development � Epigenetics � Genome-wide � T cells

� Additional supporting information may be found in the online version of this article at thepublisher’s web-site

Introduction

The generation of mature cells of the hematopoietic lineages isa very dynamic process and due to their limited life span has tocontinue lifelong. All cells of the hematopoietic system develop

Correspondence: Prof. Antonius Rolinke-mail: [email protected]

from pluripotent HSCs, which are very rare cells found in adultbone marrow (BM). The differentiation of HSCs into the vari-ous hematopoietic lineages is usually pictured in a hierarchicalfashion in which these cells develop first into progenitors andthen into precursors, with decreasing pluripotency and increasingcommitment to single differentiation pathways [1–3]. All adulthematopoietic lineages develop in the BM, except T cells, whichmature in the thymus. Since the thymus does not contain undiffer-entiated BM cells (hereafter HSCs), it has to be constantly seeded

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1182 M. Alessandra Vigano et al. Eur. J. Immunol. 2014. 44: 1181–1193

by progenitor cells from the BM. Although the precise nature ofthese progenitors is still under debate, it is well accepted that theyhave multiple-lineage developmental potential upon entrance intothe thymus [4–6]. Under the influence of signals from the thymicenvironment, which include Notch, IL-7, stem cell factor (SCF),and cell intrinsic signals, including the pre-T-cell receptor (TCR),these progenitors lose their multipotency and differentiate into theT-cell lineage [7–10]. At the CD4 and CD8 double-positive (DP)stage, these cells undergo positive selection and subsequent nega-tive selection. Those T cells that survive these selection processesare then allowed to migrate into the periphery.

At the molecular level, the restriction of cell potential, com-mitment, and differentiation to a specific lineage is achievedthrough the coordinate control of gene expression and epigeneticmechanisms. The epigenetic control relies mostly on DNA methy-lation, posttranslation modification of histones, and chromatinremodeling [11]. Nucleosomes are the fundamental unit of chro-matin and are composed of two copies of each of the four corehistones H2A, H2B, H3, and H4. Posttranslational modificationsof the N-terminal tails of the histones, which are more exposed andaccessible to enzymatic activity, include acetylation, methylation,phosphorylation, ubiquitination, and sumoylation and directly orindirectly influence the structure of the nucleosome [12]. Forexample, histone H3 lysine 4 trimethylation (H3K4me3), mostlyobserved on the nucleosomes closest to the transcriptional startsite (TSS), provides docking sites for effector complexes contain-ing histone acetylase activities, which, in turn, introduce a neg-ative charge and, therefore, relax the nucleosome structure. Theexposed underlying DNA sequences are then more accessible todifferent DNA binding proteins. Hence, H3K4me3 marks activechromatin [13] including active transcriptional enhancers in earlystages of T-cell development [14].

Another lysine methylation, histone H3 lysine 27 trimethyla-tion (H3K27me3) has been associated with transcriptional repres-sion, since it is established by the Polycomb repressive complex2 (PRC2), an important multiprotein complex that maintains spe-cific patterns of gene expression during development [11]. Partic-ular attention has recently been given to the so-called “bivalentdomains” identified in ES cells [13]. These promoters are charac-terized by the presence of both active (H3K4me3) and repressive(H3K27me3) marks and occur mostly at promoters of transcriptionfactors (TFs) important in directing cellular fate. In ES cells, thesegenes are generally silent but can be rapidly induced or perma-nently repressed according to the developmental pathway under-taken, concomitantly resolving the bivalent status in the terminaldifferentiated state—keeping H3K4me3 and losing H3K27me3 ifactivated, or vice versa, if completely repressed [15].

Epigenetic profiling is crucial for understanding development,and a growing number of datasets that describe various differ-entiation processes is now available. In the hematopoietic system,these genome-wide analyses are constrained to more mature com-partments, due to very limited numbers of HSCs and multipotentprogenitors. With the exception of the report of Adli et al., inwhich they performed epigenome profiling of ex vivo LSK cells,by adapting the ChIP-seq procedure to a low number of cells, the

majority of datasets are from in vitro expanded populations orcell lines or are representative of more differentiated populations[16–19]. Very recently, Zhang et al. produced a detailed genomicand epigenomic analysis of early T-cell differentiation, using thewell-known in vitro model of OP-9 DL1 stromal-cell culture tosupport the growth of early T-cell progenitors [20].

In this study, we employ a different cell culture model system,which combines an in vitro expansion of BM hematopoi-etic multipotent progenitors [21] and a stromal-cell-free cellculture system to induce T-cell commitment [22, 23]. Using thissystem, we explore the transcriptional and epigenetic changesoccurring during the earliest stages of T-cell development fromBM-derived hematopoetic stem cells. We characterize the genome-wide H3K4me3 and H3K27me3 profiles together with a transcrip-tome analysis of three important stages of early T-cell develop-ment. We follow the dynamic molecular changes accompanyingdifferentiation of HSCs via T-cell-precursor state into committedDP T-cells in a stromal-cell-free culture. Our results highlighted theimportance of epigenetic regulation of transcriptional repressionfor this developmental pathway.

Results

Generation of early T cells

In order to characterize the transcriptional and epigenetic land-scapes of early stages of T-cell differentiation, we exploited theimmortalization method of multipotent hematopoietic progenitorsas recently described by Ruedl et al. [21]. BM cells derived from5-fluorouracil-treated C57BL/6 (B6) mice were transduced with aretrovirus encoding an NUP98–HOXB4 fusion protein. Transducedcells were then propagated in medium containing SCF and IL-6for prolonged periods of time (up to 3 months). Like HSCs, thesecultured cells express CD117 and Sca1 (Supporting InformationFig. 1A.2). Moreover, by 4 weeks after injection into sub-lethally irradiated Rag2-deficient BALB/c mice (Rag2−/−), theseB6-derived cells were fully able to reconstitute the thymus T-cell populations (Supporting Information Fig. 1B) and the BMwith B cells and B-cell progenitors (Supporting Information Fig.1C.1), myeloid cells (Supporting Information Fig. 1C.2), erythroidcells (Supporting Information Fig. 1C.3), and NK cells (Support-ing Information Fig. 1C.4). Based on these findings, the NUP98–HOXB4 transduced cells have stem-cell properties and there-fore will hereafter be called HSCs. Recently, we (Rolink et al.,manuscript in preparation) [23] and others [22] have establisheda stromal-cell-free culture system that efficiently allows the dif-ferentiation and growth of lineage-negative (Cd3−, Cd4−, Cd3−,Cd19−, Cd11b−, Gr1−, Nk1.1−, and Ter119−), Sca1-positive(Ly6a+), and CD117-positive (Kit+), LSKs into pro-T cells. Thus,LSKs cultured on plate-bound recombinant DLL4 (delta-like 4)in the presence of IL-7 and SCF differentiated into DN2-likepro-T cells as indicated by the expression of CD44 and CD25(Supporting Information Fig. 2D). Also, NUP98–HOXB4 trans-duced HSCs cultured under these conditions differentiated into


Eur. J. Immunol. 2014. 44: 1181–1193 Molecular immunology 1183

DN2-like cells. Thus, NUP98–HOXB4 transduced HSCs whencultured in the presence of SCF and IL-6 express CD44 but notCD25 (Supporting Information Fig. 2A). However, when culturedfor 14 days on plate-bound recombinant DLL4 in the presence ofIL-7 and SCF, around 10% of these gained CD25 expression (Sup-porting Information Fig. 2B). Upon sorting and subsequently con-tinued culture on plate-bound recombinant DLL4, in the presenceof IL-7 and SCF, these cells kept their CD44- and CD25-positivephenotype (Supporting Information Fig. 2C) and, in fact, wereindistinguishable from LSK-derived DN2-like pro-T cells (Support-ing Information Fig. 2C vs. Supporting Information Fig. 2D). More-over, upon injection of these NUP98–HOXB4 transduced HSC-derived pro T cells into Rag2−/− mice, a complete reconstitutionof the thymus was observed at 3–4 weeks after injection.

In this study, gene expression analysis and epigenetic profil-ing with two opposing histone marks, H3K4me3 and H3K27me3,by chromatin immunoprecipitation coupled with high-throughputsequencing (ChIP-seq) were performed on the NUP98–HOXB4transduced HSCs; the NUP98–HOXB4 transduced HSCs derivedpro-T cells and the pro-T cells derived DP thymocytes, as illustratedin Figure 1A. DP thymocytes were enriched (over 95% purity) byFACS sorting.

Expression analysis

Gene expression profiling of HSCs, pro-T cells, and DP thymocyteswas performed through Affymetrix GeneChip Mouse Gene 1.0 STarrays and the subsequent analysis (see Materials and methods)identified 3797 genes with more than twofold change in expres-sion in at least one pairwise comparison, and with an FDR <0.05.In particular, as shown in Figure 1B, 708 genes were upregulatedand 492 were downregulated from HSCs to pro-T cells, while 1494genes were downregulated and 978 upregulated from pro-T cellsto DP thymocytes. Also, when comparing the two more distantpopulations (DP thymocytes vs. HSCs), we found more down-regulated than upregulated genes (1797 and 1158, respectively),indicating that the process of differentiation is regulated more byrepression than activation of genes. A list with the differentiallyexpressed genes in the three populations is provided in SupportingInformation Table 1.

Hierarchical clustering performed with the most variable genesrevealed that the DP population formed the most distant cluster(Fig. 1C), suggesting that DP cells are more distant transcription-ally from the other two populations, reflecting their more exten-sive molecular differentiation. This clear separation is in agree-ment with the recently published profiles of a similar experimen-tal model of T-cell differentiation [20] and with the clusteringprofiles recently published by the Immunological Genome Con-sortium [24].

A direct comparison of our transcriptional profile with boththe dataset of the Immunological Genome Consortium [24],Figure 2A, and the dataset of Zhang et al. [20], Figure 2B, con-firmed that our cell populations are bona fide, reflecting the earlydevelopmental stages of T-cell differentiation. In fact, our in vitro

expanded HSCs are more similar to the progenitor populations(MPP, ST-HSC, and CLP) of the first dataset (Fig. 2, lower lefttable) and to the FLDN1 of the latter (Fig. 2, lower right table).Our in vitro pro-T cells are closer to the in vivo population firstseeding the thymus (DN2a, ETP, and DN2b) or to equivalent pop-ulations of the in vitro system (FLDN2a and FLDN2b), placing ourpro-T cells during the commitment stage toward the T lineage.Finally, the DP thymocytes generated from the pro-T cells are alsovery close to the other DP populations of both datasets.

We also performed a Gene Ontology analysis of the differen-tially expressed genes. As expected, genes involved in T-cell activa-tion and differentiation were enriched in HSCs-to-pro-T-cell tran-sition, indicating the commitment toward the T-cell fate, while inthe cohort of downregulated genes we found enrichment in genesinvolved in inflammatory and defense responses, chemotaxis, andcarbohydrate binding, suggesting repression of functions that aremore specific of other hematopoietic lineages (Fig. 3A). Signifi-cant enrichment of genes involved in T-cell activation and differ-entiation, TCR, and VDJ recombination was observed among theupregulated genes in pro-T-to-DP transition. Interestingly, genesregulating lipids and sterol biosynthetic pathways and monosac-charide metabolic processes were enriched among downregulatedgenes from pro-T-to-DP transition (Fig. 3A), suggesting a shift tolower metabolic activity by nonproliferating DP cells.

Overall, the expression profiles gave confidence in our experi-mental system in reflecting the actual physiological steps of earlyT-cell differentiation. In particular, genes belonging to the TCRand pre-TCR complexes were activated in the pro-T cells stage(Cd3d, Cd3e, Cd3g, Ptcra), while the coreceptor components Cd4,Cd8a, and Cd8b1 were actually highly induced in the DP popula-tion. (Fig. 3B). Activation of genes involved in the signaling path-way of the TCR/pre-TCR also occurred in pro-T cells, while genesbelonging to the “naıve T-cell fingerprint” according to Chamberset al. [25] were induced mainly at the more differentiated DP thy-moctytes’ stage. Expression of large numbers of TFs fluctuates asexpected for important regulators of development, in very goodagreement with the results of Zhang et al. [20]. Moreover, TFs,which are important for other lineage specification and differen-tiation [26], were downregulated either at the pro-T or DP stage.Cebpα,β,δ and Lmo1 were reduced in both pro-T and DP stages,while expression of Bcl11a, Gfi1b, Hhex, Lmo2–3, Lyl1, Runx2–3,and Sfpi1 was only decreased in DP stage, suggesting that pro-Tcells are not yet fully committed, in analogy of thymic ex vivoisolated DN2 subpopulation [5,6,20,27].

We also modeled the dynamics of expression levels inour system by using TF binding sites prediction at gene pro-moters. The MARA tool (Motif Activity Response Analysis,http://ismara.unibas.ch/fcgi/mara) predicts the key TFs and theiractivity based on the expression changes of their target genes. Thetop-scoring motifs included binding sites for TFs with a clear rolein T-cell development such as Id1, Runx1, Elk4, Egr1–3, Ahr, Myb,and Tcf4 [28,29]. Some of these TFs are also marked with an aster-isk in Figure 3B and their activity profiles are shown in SupportingInformation Fig. 3. A complete list of the binding sites and theirrelative scores is provided in Supporting Information Table 2.


http://ismara.unibas.ch/fcgi/mara


Figure 1. Gene expression analysis of early T-cell development. (A) Schematic representation of the three T-cell populations used in this work:HSC, pro-T, and DP. (B) Gene expression changes in pairwise comparison. Genes upregulated greater than twofold are depicted in red, genesdownregulated greater than twofold are in green. (C) Hierarchical clustering with the most variable genes in the three populations. The tree of thesix samples is depicted on top of the heatmap. The color scale represents the log2 fluorescence intensity of each gene from the microarrays. Thetwo RNA replicates of the three populations were all derived from independent experiments.

Epigenetic marks

In order to characterize the epigenetic state of our cell populations,we generated ChIP-seq profiles for two distinct histone modifica-tion marks H3K4me3, associated mainly to open and active pro-moter regions, and H3K27me3, a repressive mark associated withPolycomb recruitment.

Promoters

The first level of analysis was performed on promoter regions,defined as −1 to +1 kb around TSS, identified by deepCAGE[30] and RefSeq (NCBI/2010). This method estimates 33 916

promoters. High correlation between the enrichment levels amongreplicates, ranging from 0.87 to 0.98 (Supporting InformationFig. 4), enabled us to classify each promoter as “enriched,” thatis positive for the histone mark, or “not enriched,” allowing 1%of false positives, as described in the Materials and methods.Enrichment values for each histone mark at all promoter regionsare provided in Supporting Information Table 3.

H3K4me3 was enriched at 15 154 promoters in HSCs, 17 528in pro-T cells, and 16 221 in DP thymocytes, with more gain ofthis mark from HSCs to pro-T cells (2750) than from pro-T to DPcells (492), as illustrated in Figure 4A. Conversely, there was lessloss from HSCs to pro-T cells (376) than from pro-T to DP cells(1799). On average, changes of H3K4me3 occurred on 16% ofpromoters enriched in each stage.



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HSC MPP (0.759) ST-HSC (0.755) CLP (0.723)ProT DN2a (0.759) ETP (0.734) DN2b (0.726)DP DN4 (0.804) DPsm (0.799) DP69+ (0.738)

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HSC FLDN1 (0.681) FLDN2a (0.645) FLDN2b (0.495)ProT FLDN2a (0.698) FLDN1 (0.694) FLDN2b (0.631)DP ThyDP (0.741) ThyDN3 (0.573) FLDN2b (0.541)

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Figure 2. Correlation of transcriptional profiles with published datasets. Pearson’s correlation coefficients (color scheme is indicated at the upper-left corner of each plot) were calculated using average expression values per group for 10% most variable genes. Coefficients were clustered usingthe average linkage method. (A) Heatmap with samples from the dataset of [24]. (B) Heatmap with samples from the dataset of [20]. The populationnames are the same of the cited publications. The cell populations of this study are indicated with red text. The tables at the bottom of each plotlist in descending order the closest populations to HSCs, pro-T, and DP cells, according to the correlation coefficient, indicated in parenthesis.Lower correlation coefficients with the dataset of [20] are most likely due to the comparison of microarrays versus RNA-seq.

On the other hand, changes in H3K27me3 enrichment affected40 to 50% of promoters in the different stages, with a higher gainof this mark from pro-T to DP cells (2374) than from HSCs topro-T cells (674) and higher loss from HSCs to pro-T cells (2005)than from pro-T to DP cells (541, Fig. 4A). Overall, the H3K27me3mark at promoters was more dynamic, while the H3K4me3 wasmore stable, similar to what has been shown for the developmentof the neuronal progenitors and differentiated neurons from EScells [31]. Of note, using a lower threshold for the enrichment (e.g.5% false positives), the number of promoters positively markedwith each modification increased, especially for the H3K27me3promoters (10% average increase of H3K4me3 vs. 25% averageincrease of H3K27me3), but the transition dynamics were notaffected. We think that this difference is mainly due to the lowerperformance of the H3K27me3 antibody.

Bivalent promoters

With the threshold defined previously (see Materials andmethods), enrichment of both marks could be identified in allthree populations: 702 in HSCs, 993 in pro-T cells, and 1577

in DP thymocytes (Fig. 4A). In concordance with previous studies[16,18,32,33], this set of promoters was defined “bivalent." Thesebivalent promoters were highly dynamic, as only 189 remainedbivalent through all three stages (Fig. 4B), while more than 50%changed their status in each population. It should be noted thatthese bivalent promoters showed only weak enrichment for bothmarks. The fact that only very few bivalent promoters exhibitedhigh enrichment is also supported by clear anticorrelation of thesemarks in all three populations (Supporting Information Fig. 4).

A closer look into the dynamics of epigenetic changes, asschematically depicted in Figure 4C revealed that 58% of thebivalent promoters in HSCs lost the bivalency in pro-T cells, mostly(73%) by losing H3K27me3, but 57% of them regained H3K27me3in DP. On the other hand, 58% of the bivalent promoters inpro-T cells lost bivalency in DP thymocytes, mostly (85%) by los-ing H3K4me3. The resolution of bivalent domains seems to bemore regulated by repressive mechanisms during differentiation.Cui et al. showed that the resolution of bivalent genes from humanCD133+ to CD36+ cells was also regulated by loss of H3K4me3in 53% of the cases [34]. Moreover, the new bivalent promotersof DP mostly became bivalent by acquisition of the H3K27me3, asshown in Supporting Information Fig. 5B.



GO/Panther/Kegg TERM adj P valueleukocyte activation 3.70E-09

T cell activation 1.40E-07T cell differentiation 2.80E-05

T-cell mediated immunity 9.70E-09

T cell receptor complex 9.10E-04T cell receptor signaling pathway 3.90E-05

GO/Panther/Kegg TERM adj P valuedefense response 9.30E-09

response to bacterium 1.20E-04chemotaxis 6.50E-04

carbohydrate binding 3.80E-07

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GO/Panther/Kegg TERM adj P valueT cell differentiation 3.80E-10

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V(D)J recombination 1.50E-03Zinc finger transcription factor 4.70E-03T cell receptor signaling pathway 1.50E-07

GO/Panther/Kegg TERM adj P valuelipid biosynthetic process 7.90E-11

sterol biosynthetic process 1.20E-08oxidation reduction 4.40E-07

monosaccharide metabolic process 1.40E-04

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Figure 3. Expression overview of T-cell specific genes. (A) Main selected GO, Panther, or KEGG categories enriched in the different groups ofregulated genes with the respective adjusted (Benjamin) p-value. (B) Expression profiles of groups of genes belonging to the TCR complex, T-cell-activation pathway, specific naıve T-cell signature [25], and selected TFs with a documented role in T-cell differentiation. The colors represent theLog2 fluorescence intensity of the Affymetrix arrays. The blue stars mark the TFs whose binding sites have been identified through MARA analysisas described in the Results section.

Gene ontology analysis of the bivalent genes in the three pop-ulations confirmed enrichment of transcriptional regulators, con-sistent with the model that these genes are poised and ready tobecome more permanently activated or repressed in response toexternal signals and/or developmental program. Finally, the genesmarked with bivalent promoters are expressed at medium or lowlevels in the respective stages, as shown in Supporting Informa-tion Fig. 5A. Tbx21 and Gata3, two TFs essential for the laterspecification of T cells into the different functional subset [35],remained bivalent from the HSC to the DP stage (Supporting Infor-mation Fig. 6), in agreement with the profile shown in Wei et al.[18].

Correlation of expression and promoter marks

Enrichment levels for H3K4me3 at promoters were strongly corre-lated with the expression levels of transcripts originating fromthese promoters. The scatter plots in Figure 5 (upper panels)clearly identify these populations of genes with high expressionand enrichment levels. On the other hand, H3K27me3 enrichment

was associated with low-expressed genes (Fig. 5, lower panels),confirming the more “repressive” nature of this modification.

Considering the pairwise changes in gene expression andchanges in their promoter enrichments for the two epigeneticmarks, shown in Supporting Information Fig. 7 for HSCs ver-sus pro-T and pro-T versus DP, the correlation was very highfor H3K4me3 (Pearson’s correlation coefficient of 0.56 and0.6, respectively), while it was negative (or anticorrelation) forH3K27me3. The majority of genes that were increased in expres-sion from one stage to the other also increased the H3K4me3enrichment at their promoters, while the downregulated genesdemonstrated decreased H3K4me3 enrichment. Moreover, a largeproportion of downregulated genes acquired H3K27me3 on theirpromoters.

The TCR component Cd3e, Cd3g, Cd3d, and Cd4 gene promot-ers illustrate very well this correlation as shown in Figure 6. Thegenes at the Cd3 locus were upregulated in the pro-T stage andtheir promoters acquired H3K4me3 at this step and then main-tained it in DP. In HSCs, these genes were not expressed and theirpromoters were moderately enriched with H3K27me3, this marksubsequently disappeared once the genes were turned on. Cd4 was



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Figure 4. Epigenetic marks at promoters. (A) Upper table: total number of promoters in the three populations enriched for H3K4me3 (red),H3K27me3 (blue), or both (brown). Lower table: gain and loss of promoter enrichment for the histone marks in the three population transitions.Color scheme is the same. (B) Venn diagram of the bivalent promoters in HSCs, pro–T cells, and DP cells. (C) Resolution of the bivalent promotersfrom HSCs to pro-T and DP cells (upper panel) and from pro-T to DP cells (lower panel). Heatmaps were generated after centering the promoterenrichments (see Materials and methods) and the color scale represents levels of enrichments: blue, not enriched; red, enriched. The populationsare indicated on the top of each heatmap, while the histone marks are on the bottom. The lines and numbers on the right represent the numberof promoters that are losing H3K4me3 (blue) or H3K27me3 (red) in the subsequent stage; in brown, the promoters that are regaining the bivalency(upper panel). The Chip-seq experiments were performed with two independent immunoprecipitations for each histone mark on chromatinderived from each cell population. The numerical and graphical representations are based on the average of the replicates.

highly induced in DP and its promoter was enriched in H3K4me3at the same time, while H3K27me3 was gradually lost. The TFGfi1b is a good example of repression correlated to H3K27me3deposition; it was specifically downregulated in DP and its pro-moter lost H3K4me3 and gained H3K27me3.

The epiMARA analysis with the epigenetic modifications [36]identified TFs associated with the deposition of the histone marksat promoters. The top-scoring binding sites for each modificationmostly mirrored those identified with the transcriptional activity:TFs with high activity for H3K4me3 deposition have also a sim-ilar activity profile for the transcriptional regulation; for exam-

ple Hif1a (Supporting Information Table 2). A reverse correlationwas observed for H3K27me3 activity. The list of the epiMARAbinding sites and their scores is provided in Supporting Informa-tion Table 2.

Genome-wide analysis

To extend the epigenetic analysis over the entire genome, we alsogenerated a map of regions of specific enrichments (peaks) out-side the promoter regions, as described in Materials and methods.As shown in Figure 7, H3K4me3 preferentially marked promoters



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Figure 5. Correlation of genes expression levels and H3K4me3 (top panels) and H3K27me3 (bottom panels) enrichment levels at the correspondingpromoters in HSC (left), pro-T (middle), and DP cells (right). For each plot, the x-axis represents the log2 enrichment of the indicated histone marks,while the y-axis represents the log2 expression of the genes in the indicated population. The red ovals indicate the populations with high expressionand high H3K4me3 enrichment, the blue ovals the population with low expression and high H3K27 enrichment. The graphical representations arebased on the average of the replicates.

and distal promoters; in pro-T cells, this marking was more pro-nounced. The distribution of H3K27me3 peaks was very similar inall the conditions, with a slightly higher percentage of promotersand distal promoters in DP. Comparison with the genomic distri-bution of the regions revealed that H3K4me3 was enriched specif-ically at promoters in all populations, with higher enrichment inpro-T cells. H3K27me3 peaks were more broadly localized andenriched (at much lower levels) at promoters, distal promoters,and to a far lesser extent at the 3′ ends of genes.

Despite the very similar distribution of peaks in the three cellpopulations analyzed, the correlation coefficients of enrichmentvalues in detected peaks outside the promoters were much smallerthan those in promoters, for both histone modifications, with thehighest differences between HSC and DP (Supporting InformationFig. 8). This indicates that epigenetic dynamics was occurringat a greater extent in regulatory regions, than elsewhere in thegenome, in agreement with similar observations in the stromal-cell culture system utilized by Zhang et al. [20]

The examples of TCR beta (Fig. 8) and alpha (Supporting Infor-mation Fig. 9) loci well illustrate the dynamic of H3K4me3 duringdevelopment. This histone modification is not only required for

opening the locus and promoting transcription but has also beenshown to enhance and possibly target the Rag1/Rag2 recombi-nation complex [19, 37, 38]. H3K4me3 enrichments appeared inthe pro-T stage at the Eβ enhancer, located between the Cβ 2 andthe most Vβ 31 and in the promoter regions of the Dβ 1 (PD β1)and Jβ. (Fig. 8A), correlating with opening of the locus for the firstround of recombination, which occurs between D and J regions[38]. The subsequent recombination between the different Vβ andDJ was also marked by H3K4me3 at the promoters of the differentVβ regions in pro-T cells and these enrichments were maintainedin DP (Fig. 8B). Of note, no H3K27 enrichment was observed inany region of the TCR beta locus in any population. The alphalocus is more complex, since it also contains the entire delta locus,but its chromatin structure also opened by acquiring H3K4me3throughout the Jα regions in DP, where recombination is fullyactive between Vα and Jα (Supporting Information Fig. 9).

Finally, many of the regulatory regions of genes important inT-cell differentiation, such as the Cd3d 3′ and Gata3 3′ enhancers;the Ikzf1 and Ets1 intronic enhancers; the Tcf7, Cd47, Ccl5, Cd8a,and Zcchc2 upstream enhancers [14], are characterized in thisstudy by high dynamics of H3K4me3 marking. A full list of peaks



Cd3 locus

Cd4

Gfi1bChromosome 9

44.78 mb

44.79 mb

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44.81 mb

0102030405060

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Cd3e

Cd3d

Cd3g

Chromosome 2

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Gfi1b

Chromosome 6

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Cd4

H3K

4me3

H3K

27m

e3H

3K4m

e3H

3K27

me3

H3K

4me3

H3K

27m

e3

Figure 6. Epigenetic profiles of Cd3, Cd4, and Gfi1b loci in HSC (tracks 1 and 4), pro-T (tracks 2 and 5), and DP (tracks 3 and 6). H3K4me3 tracks (1–3)and H3K27me3 tracks (4–6) are in different shades of orange and green, respectively. The position of the locus on the chromosome is indicated atthe top of each chart. H3K4me3 and H3K27me3 tracks are indicated with orange and green text on the left, together with the population name inblack in each profile. The Ref-seq transcript or MGI gene tracks are at the bottom of each chart. The graphical representations are based on theaverage of the replicates.

for H3K4me3 and H3K27me3 outside promoter regions is pro-vided in Supporting Information Table 4.

Discussion

Adult T-cell development starts with a still unidentified IL-7R+

population of progenitors derived from the BM HSCs seeding the

thymus [39]. In this organ, through complex interaction withthe stroma and other resident cells, these precursors progres-sively undergo different programs of (i) commitment to the T-celllineage, (ii) massive proliferation, and (iii) specification to thedifferentiated state of T cell. Eventually, these cells will leave thethymus for peripheral secondary lymphoid organs. Several TFswith a key role in the entire process have been identified [28],but little is known about the epigenetic status of the different



Region distribution in the genome

H3K27me3 in HSC

log 2 e

nric

hmen

t rel

ativ

e to

gen

ome

size

log 2 e

nric

hmen

t rel

ativ

e to

gen

ome

size

−1

0

1

2

3

4

H3K27me3 in proT

−1

0

1

2

3

4

H3K27me3 in DP

−1

0

1

2

3

4

H3K4me3 in HSC

−1

0

1

2

3

4

H3K4me3 in proT

−1

0

1

2

3

4

H3K4me3 in DP

−1

0

1

2

3

4

distal promoter

promoter

gene body

after TES

intergenic

distal promoterpromotergene bodyafter TESintergenic

A

B

Figure 7. Distribution of the histone mark enrichments in the genome. (A) The upper pie represents the distribution of different defined regionsin the genome. Promoters are defined as regions encompassing −1 and +1 kb with respect to the TSS (blue); distal promoters are the regionsfrom −10 and −1 kb with respect to the TSS (red); after transcriptional end site are defined as regions +10 kb from the transcriptional end sites(purple); all the remaining regions are classified as intergenic (orange). (B) Enrichment of H3K4me3 (top charts) and H3K27me3 (bottom charts) inthe different regions of the genome in the three populations. Each bar represents one region of the genome, depicted with the same color codeof the upper panels and the y-axis represents the log2 enrichment of the indicated histone marks normalized to the size of the regions in thegenome. The graphical representations are based on the average of the replicates.

progenitor and precursor populations, due to the very limitednumbers of these cells in vivo, rendering high-resolution epige-netic profiling experiments very challenging and demanding. Ourin vitro model of HSC and pro-T-cell expansion has overcomemany of these restrictions and proved to be very useful for genome-wide studies.

The NUP98–HOXB4 transduced HSCs can be expanded mas-sively in vitro without compromising their differentiation potentialto all blood lineages (Supporting Information Fig. 1 and 2, and[21]). The comparison of the transcriptional profile of our cellswith the published profiles (Fig. 2) placed HSCs very close tothe various progenitor populations. Our pro-T thymocytes, gen-erated in a stromal-cell-free system, but with appropriate Notchsignaling environment, are close to a DN2-like population, whichprogressively commits to the T-cell lineage, by activating theT-cell activation genes (Fig. 3B), key TFs for the cell lineage(Gata3, Bcl11b, Tcf7, Tox) and the Rag genes for starting to rear-range the TCR beta locus. Although we have been able to clonesingle NUP98–HOXB4 transduced cells and reconstitute in vivo allthe hematopoietic compartments (our unpublished observation),

we cannot exclude that some of the observed differences might bethe result of heterogeneity in the HSC population.

The DP thymocytes derived from these in vitro generatedpro-T cells showed a transcriptional profile typical of a committedT cell, with TFs important for other lineage specification such asBcl11a, Gfi1b, and Sfpi1 being down modulated (Fig. 3B). Finally,the enriched categories in the different sets of regulated genes(Fig. 3A) and the binding sites enrichment analysis of their pro-moters (Supporting Information Table 2) further confirmed thecorrect developmental profile. Therefore, we have reproduced invitro the elusive earliest steps of T-cell differentiation from theearliest adult hematopoietic precursor, allowing us to followthe epigenetic changes of a unique starting population, duringthe process of differentiation.

Our genome-wide epigenetic profile confirmed to a large extentwhat had been observed in other developmental systems [12]. TheH3K4me3 mark was significantly enriched at promoters (Fig. 7),mostly localized in a narrow window around the TSS of the genes(Fig. 6 and Supporting Information Fig. 6) and was tightly associ-ated with high expression levels in all three populations (Fig. 4).



TCRbeta D-J-C region TCRbeta V region

PD β1 Jβ

Eβ enhancer

Vβ 12.2 Vβ 16 Vβ 23

Chromosome 6

41.485 mb

41.49 mb

41.495 mb

41.5 mb

41.505 mb

020406080

HS

C

020406080

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DP

02468

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HS

C

02468

10

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T

02468

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DP

283_Trbd1

3284_Trbj1−1

3285_Trbj1−2

03286_Trbj1−3

0103287_Trbj1−4

0103288_Trbj1−5

000103289_Trbj1−6

000103290_Trbj1−7

NSMUST00000103291_Trbc1

ENSMUST00000103292_Trbd2

ENSMUST00000103293_Trbj2−1






ENSMUST00000103299_Trbc2

ENSMUST00000103300_Trbv31

ENSMUST00000169595_Gm17602

Chromosome 6

41.05 mb

41.06 mb

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00103267_Trbv12−1

000103268_Trbv13−1

00000103269_Trbv12−2

T00000103270_Trbv13−2

NSMUST00000103271_Trbv13−3









ENSMUST00000103280_Trbv26 526

H3K

4me3

H3K

27m

e3

H3K

4me3

H3K

27m

e3

Figure 8. Epigenetic profiles of the TCRβ D-J-C (A) and TCRβ V (B) regions of the TCR beta locus. The tracks are as in Figure 6. Selected regions ofthe locus are indicated with arrows. PD, promoter of diversity region; J, joining region; V, variable region. The numbering is according to the IMGTnomenclature (http://www.imgt.org).

Furthermore, changes in enrichment levels at promoters werestatistically correlated with changes in expression (SupportingInformation Fig. 7) confirming the role of this histone modifi-cation in rendering the promoters more accessible to TFs bind-ing and to the transcriptional machinery. This type of analysiscould certainly be extended with other histone modification orRNA polymerase II, more directly linked to transcriptional activ-ity.[40] The H3K27me3 mark was also associated mainly to pro-moter regions and correlated with genes with low expression.Moreover, a group of genes, progressively downregulated duringthe differentiation process, acquired H3K27me3 at their promot-ers. Hence, gene repression is also partially mediated by Polycombrepressive complex 2 recruitment in early T-cell development,as was shown in the T-cell-differentiation model of Zhang et al.[20].

Promoters characterized by the presence of both active(H3K4me3) and repressive (H3K27me3) marks, the so-called“bivalent domains,” were previously identified in ES cells andwere shown to correspond mostly to genes coding TFs involvedin developmental decisions [13]. In line with other reports thatidentified these bivalent domains in more differentiated cells[16, 18, 31–34, 41], we also found a small number of bivalentpromoters in all three populations analyzed. This bivalency washighly dynamic, since more than 50% are changing from onestage to the next (Fig. 4B). Interestingly, DP cells showed the

highest number of bivalent promoters, possibly indicating that DPhave not completed their terminal differentiation program and,more importantly, that they still maintain a high plasticity in termof response to external stimuli once they exit the thymus anddifferentiate further into the various subsets upon antigen stimu-lation, as illustrated by Wei et al. [18].

The resolution of bivalency at promoters is mostly due tothe loss of the H3K4me3 mark with concomitant increase inH3K27me3, suggesting that resolution leads most often to repres-sion. A similar picture was shown for the resolution of bivalentdomains in another hematopoietic system from human CD133+

to CD36+ cells [34].In summary, considering the global changes of gene expression

and the epigenetic profile for H3K4me3 and H3K27me3 at promot-ers, it seems that early T-cell differentiation is regulated mostly bygene repression, as it was shown for other lineage-committed cells[41]. An initial wave of high transcriptional activity was observedfrom the HSC to the pro-T stage, with an increase in gene expres-sion, high gain of H3K4me3 and high loss of H3K27me3, andtherefore, more promoters in an “active" configuration. This maybe the cellular response to the sustained Notch and IL-7 signaling.The subsequent step, from pro-T to DP cells, was characterizedby a complete lineage restriction for the other developmentalpathways, leaving only the T-cell specification program in anactive “environment,” and switching off the other alternatives.


http://www.imgt.org


Therefore, many more genes were repressed than activated;H3K27me3 enrichments at promoters was enhanced and reso-lution of some bivalent domain was again mostly toward loss ofactive marks.

In conclusion, our immortalized NUP98–HOXB4 HSCs and invitro differentiated pro-T cells in a stromal-cell-free system providea robust and well-characterized model for studying genome-widegenetic and epigenetic events during the earliest stages T-cell dif-ferentiation.

Materials and methods

Mice, cell culture, and FACS

C57BL/6 and BALB/c Rag2-deficient mice were used. All micewere bred and maintained in our animal facility under specificpathogen-free conditions. All animal experiments were carried outwithin institutional guidelines (authorization 1886 and 1888).

Generation of immortalized HSCs was described in the Resultssection, following the protocol of [21]. The stromal-cell-free con-dition supporting growth and expansion of the pro-T cells weredescribed in [23] and in Rolink et al. (in preparation). FITC-,PE-, allophycocyanin-, or biotin-labeled mAbs specific for CD4,CD8, CD11b, CD11c, CD25, CD44, Ter119, Gr1, and Sca1 werepurchased from BD Biosciences or eBiosciences. Staining of thecells was performed using standard methods. Flow cytometry wasperformed using an FACS Calibur (BD Biosciences) and data wereanalyzed using the Cell Quest Pro Software (BD Biosciences). Forcell sorting, the FACS Aria (BD Biosciences) was used.

ChIP and ChIP-seq

Chromatin immunoprecipitation was performed as described in[42] using approximately 2 × 106 cells and 2 μg of anti-H3K4me3antibody (Abcam ab8580) or 10 μL H3K27me3 (Cell Signaling9733). Individual ChIPs were checked for enrichment at selectedpromoters in standard qPCR reaction. Ten nanograms of ChIPedDNA were processed for Illumina GA sequence analyzer accordingto the manufacturer’s procedure.

Microarray data collection and analysis

Total cellular RNA was extracted from different cultures of HSCs,pro-T, and DP cells with TRIzol reagent (Invitrogen) and fur-ther purified through an RNAeasy column (QIAGEN); 10 ng ofRNA was then used for hybridization to the Affymetrix GeneChipMouse Gene 1.0 ST array, according to the manufacturer’s proto-col. The data analysis was performed with the Partek genomic suitesoftware with standard settings.

Gene ontology classification was performed with DAVID(http://david.abcc.ncifcrf.gov) and the full list of enriched cat-

egories is provided upon request. A detailed analysis of theChIP-seq data is provided in the Supporting Information. Alldatasets have been deposited in GEO with the accession numberGSE47940 (gene expression) and GSE47995 (chip-seq files).

Acknowledgments: We would like to thank Sophie Dessus-Babusand Manuel Kohler for technical assistance in the ChIP-seq exper-iments; Philippe Demougin for the Affymetrix gene expressionfacility; Lukas Burger, Michael Stadler, Patrick Matthias, DirkSchuebeler and Fraser McBlane for the very helpful discussion.Antonius Rolink is the holder of the chair in Immunology endowedby F. Hoffman-La Roche Ltd., Basel. This work was supported bythe SystemsX.ch RTD project Cell Plasticity and Swiss NationalScience Foundation.

Conflict of interest: The authors declare no financial or commer-cial conflicts of interest.

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Abbreviations: DLL4: delta-like 4 · DP: double positive · H3K27me3: H3

lysine 27 trimethylation · H3K4me3: H3 lysine 4 trimethylation · MARA:

motif activity response analysis · TF: transcription factor · TSS: tran-

scriptional start site

Full correspondence: Prof. Antonius Rolink, Developmental andMolecular Immunology Group, Department of Biomedicine, Universityof Basel, Mattenstrasse 28, CH-4058 Basel, SwitzerlandFax: +41-61-695-30 70e-mail: [email protected]

Received: 23/8/2013Revised: 4/12/2013Accepted: 20/12/2013Accepted article online: 27/12/2013


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