Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation

Manna, Sugata; Kim, Jong Kyong; Baugé, Catherine; Cam, Margaret; Zhao, Yongmei; Shetty, Jyoti; Vacchio, Melanie S.; Castro, Ehydel; Tran, Bao; Tessarollo, Lino; Bosselut, Rémy

doi:10.1038/ncomms9152

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Published: 02 September 2015

Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation

Sugata Manna¹^na1,
Jong Kyong Kim¹^nAff6,
Catherine Baugé¹^nAff7,
Margaret Cam²,
Yongmei Zhao³,
Jyoti Shetty³,
Melanie S. Vacchio¹,
Ehydel Castro¹,
Bao Tran³,
Lino Tessarollo⁴ &
…
Rémy Bosselut¹

Nature Communications volume 6, Article number: 8152 (2015) Cite this article

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Abstract

Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4⁺ T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation.

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Introduction

Lineage differentiation during metazoan development is mediated by cell-specific transcriptional programs directing gene expression. The control of such transcriptional ‘circuits’ involves transcription factors that directly or indirectly bind specific DNA sequences, and locus-specific modifications of DNA or histones. Although much progress has been made in identifying transcription factors driving cell differentiation, including during T-cell development in the thymus^1,2, the role of DNA or histone modifiers is less well understood.

Much attention has focused on histone post-translational modifications, including methylation, acetylation and ubiquitination^3,4, which affect DNA accessibility and recruitment of specific transcriptional activators or repressors. The trimethylation of histone H3 lysine 27 (H3K27Me3) is of specific interest because it promotes lineage-specific gene silencing in embryonic stem cells and is therefore thought to be important to maintain multipotency^5,6,7,8. H3 K27 is methylated by Ezh2, a methyl transferase part of Polycomb-complex 2 (PRC2), or the related molecule Ezh1. H3K27Me3 accumulation at promoter sites promotes silencing at least in part through recruitment of Polycomb-complex 1 (PRC1) components^5,6,7,8.

Even though gene-targeted clearance of H3K27Me3 is thought to be essential for the emergence of lineage-specific gene expression during development, the mechanisms governing the removal of the mark are not well understood. The discovery of H3K27 demethylases Jmjd3 and Utx has suggested that active demethylation contributes to the depletion of H3K27Me3 and to initiation of lineage-specific gene expression^{9,10,11,12,13}. However, whereas both molecules are necessary during mouse embryonic or postnatal development^{14,15,16,17,18,19}, it has remained unclear whether such biological functions involve their demethylase activity. Indeed, a Y-chromosome-encoded homolog of Utx, Uty, which lacks demethylase activity, rescues Utx functions in Utx-deficient male embryonic stem cells and embryos^14,16,17,18, in which its expression largely mirrors that of Utx^16,20. Similarly, a Utx mutant engineered to lack demethylase activity rescues the development of female Utx-deficient embryos^17,18. Strikingly, Uty rescues the development of embryos lacking both Utx and Jmjd3 (ref. 21). Thus, the contribution of Jmjd3 and Utx demethylase activity to their biological functions has remained elusive.

We thought that assessing the role of these molecules in T-cell development would help address this critical question. In developing thymocytes and in effector T-cells, gene-specific levels of H3K27 methylation are developmentally controlled and removal of the tri-methyl mark is associated with induction of gene expression, in keeping with the idea that the mark promotes gene repression^22,23,24,25. In the thymus, H3K27Me3 developmental removal has been observed at the promoter of the gene (Zbtb7b) encoding the CD4⁺-lineage-specific transcription factor Thpok²⁶, a molecule required for the development of CD4⁺ T-cells from CD4⁺CD8⁺ (‘double positive’, DP) thymic precursors²⁷. Furthermore, the differentiation of DP thymocytes into CD4⁺-lineage cells is not associated with cell division, suggesting that an active H3K27Me3 removal mechanism is required to clear the mark; in contrast, rapidly proliferating cells can conceivably ‘dilute’ the mark during cell division.

By examining the role of Jmjd3 and Utx in non-dividing DP and CD4⁺ and CD8⁺ ‘single-positive’ thymocytes, the present study shows that these enzymes are redundantly required for H3K27Me3 homeostasis and gene expression. They are needed for the removal of H3K27Me3 at, and expression of, multiple promoters characteristic of mature thymocytes and T-cells, in particular S1pr1, a gene needed for thymocyte egress. As a result, Jmjd3 and Utx are required for the generation of a normal T-cell repertoire. Importantly, S1pr1 expression was not rescued by Uty in male cells lacking both Jmjd3 and Utx, supporting the idea that it requires Jmjd3- or Utx-catalyzed H3K27Me3 demethylation at its promoter. These findings demonstrate that Jmjd3 and Utx are required for T-cell differentiation.

Results

Jmjd3 and Utx are important for T-cell development

To evaluate the role of Utx and Jmjd3 in T-cell development, we inactivated the genes encoding these enzymes (Kdm6a and Kdm6b, respectively called Utx and Jmjd3 hereafter). Because germline disruption of either gene causes embryonic or neonatal death^{14,16,17,18,28}, we used Cd4-Cre to target DP thymocytes for deletion of Jmjd3^f and Utx^f conditional alleles (Supplementary Fig. 1a,b), in which LoxP sequences flank exons encoding the catalytic JmjC domain²⁹. We verified deletion of floxed sequences in thymocytes from Jmjd3^f/f and Utx^f/f Cd4-Cre mice (called Jmjd3- and Utx-deficient or -KO hereafter) by genomic DNA PCR, and loss of protein expression by immunoblotting (Supplementary Fig. 2a–c).

CD4 and CD8 SP thymocytes developed in mice with Cd4-Cre-mediated deletion of Jmjd3, Utx or both (dKO thereafter) (Fig. 1a). However, we noted increased numbers of CD4 SP thymocytes; most of them were mature post-selection cells, characterized by high-level T-cell antigen receptor (TCR) and low-level CD24 expression (TCR^hi CD24^lo, Fig. 1a,b). Increased thymocyte numbers contrasted with reduced numbers of total and naive CD4⁺ T-cells in the spleen (Fig. 1a,b and Supplementary Fig. 3a). Because the developmental block was incomplete even in double mutant mice, and because effects on CD8⁺ T-cell differentiation were not as pronounced (Supplementary Fig. 3b,c), we considered that earlier disruption of Jmjd3 and Utx might have a greater impact on thymocyte development. Consequently, we inactivated Jmjd3 and Utx using Lck-Cre, which becomes active in earlier T-cell progenitors than Cd4-Cre. Lck-Cre disruption of both genes resulted in an accumulation of mature (TCR^hi CD24^lo) CD4 SP thymocytes and a CD4⁺ lymphopenia similar to those seen with Cd4-Cre disruption (Supplementary Fig. 3d,e). Of note, the total number of thymocytes was not reduced by Lck-Cre-mediated deletion of Jmjd3 and Utx (Supplementary Fig. 3e, top graph), indicating that neither enzyme was required for cell proliferation during early T-cell development. These experiments suggested that Jmjd3 and Utx are specifically important for thymocyte differentiation beyond the DP stage, and we further explored their function using Cd4-Cre-mediated disruption.

**Figure 1: T-cell development in the absence of H3K27 demethylases.**

To verify that the effects of Jmjd3 and Utx inactivation were cell-intrinsic, we generated mixed bone marrow chimeras, in which dKO and wild-type cells would compete in the same (wild type) environment. We transplanted a 1:1 mix of allelically marked dKO (CD45.2) and wild-type (CD45.1) haematopoietic progenitors into lethally irradiated wild-type (CD45.1/CD45.2) recipients. The dKO component showed accumulation of mature CD4 SP thymocytes and peripheral CD4⁺ lymphopenia, unlike wild-type competitors, indicating that the effect of Jmjd3 and Utx is cell-intrinsic (Fig. 2a). Accordingly, at all levels of chimerism, dKO cells were overrepresented relative to wild-type cells among mature CD4 SP thymocytes but underrepresented among CD4⁺ splenocytes (Fig. 2b,c). Control experiments with wild-type littermate donors showed that these effects were specific of dKO cells (Fig. 2b,c and Supplementary Fig. 4). These experiments demonstrate a cell-intrinsic need for Jmjd3 and Utx during the late stages of CD4⁺ T-cell development in the thymus and for the formation of normal CD4⁺ T-cell populations.

**Figure 2: Jmjd3 and Utx are required for thymocyte maturation.**

Jmjd3 and Utx are needed for S1pr1 and Klf2 expression

The competitive disadvantage of dKO cells in mixed BM chimeras suggested that the effects of Jmjd3 and Utx disruption were partly masked in intact mice by shifts in the TCR repertoire of differentiating thymocytes. To examine this possibility, we fixed TCR specificity using the MHC II-restricted OT-II TCR transgene³⁰. In H-2^b mice, the OT-II TCR promotes positive selection, resulting in the generation of large numbers of CD4 SP thymocytes and CD4⁺ T-cells expressing the transgenic Vα2 TCRα chain. Similar to their wild-type counterparts, dKO OT-II thymi had large populations of Vα2^hi cells, most of which were CD4 SP (Fig. 2d,e). In the Vα2^hi CD4⁺CD8^int subset, which comprises cells undergoing positive selection, surface expression of CD5 or CD69 proteins, both of which serve as indicators of TCR signalling, was similar in wild-type and dKO OT-II thymocytes (Fig. 2f). We conclude from these findings that Jmjd3 and Utx are not required for TCR signalling and positive selection per se.

However, similar to mice with a diverse TCR repertoire, mature (Vα2^hi CD24^lo) CD4 SP thymocytes were in markedly greater numbers in dKO than in wild-type OT-II mice (Fig. 2g,h). In addition, the number of peripheral dKO OT-II CD4⁺ T-cells was drastically reduced (Fig. 2g,h). The contrast between thymus and spleen prompted us to examine expression of S1pr1^31,32, a sphingosine receptor needed for thymic egress. S1pr1 mRNA expression was sharply reduced in mature OT-II dKO CD4 SP thymocytes (Fig. 3a) and the same was true of surface S1pr1 protein (Fig. 3b). Expression of Klf2, encoding a transcription factor important for late thymocyte maturation, including for S1pr1 expression³³, was also diminished (Fig. 3a). Single disruption of Jmjd3 or Utx had partial effects on S1pr1 expression in mature OT-II thymocytes (Fig. 3c,d and Supplementary Fig. 5a), and caused a combination of thymocyte accumulation and lymphopenia, similar to but less marked than in dKO mice (Fig. 2i,j and Supplementary Fig. 5b,c).

**Figure 3: Jmjd3 and Utx are needed for *S1pr1* expression.**

Expression of S1pr1 was impaired, with a trend towards reduced Klf2 expression, in mature (TCR^hi CD24^lo) dKO or Jmjd3-KO CD4 SP thymocytes carrying an endogenous TCR repertoire (Supplementary Fig. 6a,b) indicating that the impact of Jmjd3 and Utx on S1pr1 was not unique to the OT-II system. The same was true in mature (Vα2^hi CD24^lo) CD8 SP thymocytes of mice carrying the MHC I-restricted P14 TCR transgene, which normally promotes positive selection of Vα2^hi CD8 SP thymocytes and T-cells (Supplementary Fig. 6c). Accordingly, P14-transgenic dKO mice had increased numbers of transgene-specific mature thymocytes and reduced numbers of CD8⁺ T-cells (Supplementary Fig. 6d–f).

We next examined how Jmjd3 and Utx disruption affected S1pr1 expression. We first noted persistent expression of surface CD69 on mature Vα2^hi CD24^lo dKO OT-II CD4 SP thymocytes, but not on their wild-type counterparts which downregulated CD69 as they matured (Fig. 3e). Because surface CD69 is internalized upon association with S1pr1(ref. 34), it was possible that persistent CD69 surface expression on dKO cells resulted from reduced S1pr1 expression. Alternatively, it could reflect persistent high-level Cd69 gene expression. Because Cd69 is induced by TCR signalling, the latter hypothesis would suggest that mature dKO thymocytes had greater responsiveness to TCR engagements, which, in turn, would impair S1pr1 expression. Indeed, TCR engagement repressed S1pr1 in mature thymocytes (Supplementary Fig. 7), as previously shown in peripheral T-cells³¹, and the same was true of Klf2 (Supplementary Fig. 7). To determine if disruption of Jmjd3 and Utx caused increased TCR signalling in mature thymocytes, we assessed Vα2^hi CD24^lo wild-type and dKO thymocytes for expression of the TCR-induced mRNAs encoding CD69 and the transcription factor Nur77 (ref. 35) (Nr4a1). Both were expressed at similar levels in wild-type and dKO cells (Fig. 3f), indicating that persistent expression of CD69 in mutant T-cells was post-transcriptionally controlled.

In addition, we reasoned that, if the reduced expression of S1pr1 in mutant T-cells was caused by increased responsiveness to intrathymic signals, from TCR or other receptors, disrupting intrathymic interactions by placing mutant thymocytes in suspension culture should raise their S1pr1 expression to wild-type levels. Validating this reasoning, expression of S1pr1 in wild-type mature CD4 SP cells increased after in vitro culture, whereas that of Nr4a1 decreased, attesting disrupted TCR signalling (Fig. 3g). However, expression of S1pr1 in dKO mature cells was not significantly increased by in vitro culture, despite a reduction of their Nr4a1 expression similar to wild-type thymocytes (Fig. 3g). We conclude from these experiments that Jmjd3 and Utx do not promote S1pr1 expression by restraining thymocyte sensitivity to intrathymic signals.

Jmjd3 and Utx control H3K27 methylation at S1pr1 and Klf2

To further investigate the mechanisms of reduced S1pr1 expression, we performed H3K27Me3 chromatin immunoprecipitation (ChIP) at the S1pr1 promoter. ChIP signals were higher in dKO than wild-type mature OT-II CD4 SP cells, indicating that Jmjd3 and Utx controlled H3K27 demethylation at S1pr1 (Fig. 4a). To define the footprint of these enzymes on H3K27Me3 homeostasis and gene expression, we performed both ChIP followed by deep sequencing (ChIPseq) and microarray analyses on wild-type and dKO OT-II mature Vα2^hi CD24^lo CD4 SP thymocytes. ChIPseq confirmed the greater H3K27 trimethylation at S1pr1 in dKO than in wild-type cells, and the same was true at Klf2 (Fig. 4b). This indicated that reduced expression of both genes was associated with H3K27Me3 accumulation. In contrast, Jmjd3 and Utx disruption did not affect H3K27Me3 decoration at multiple other loci, including Cd5 or Rag1 and Rag2.

**Figure 4: Jmjd3 and Utx redundantly promote H3K27Me3 demethylation.**

Genome-wide impact of Jmjd3 and Utx

We next assessed the 20218 unique genes included in ChIPseq and array analyses for ChIPseq signals (normalized read numbers) at promoters, defined as 4 kbp segments centred over a transcription start site. Using the peak-finding algorithm SICER, we found 7730 promoters overlapping an H3K27Me3 peak in either wild-type or dKO cells (Fig. 5a). At most of these 7730 ‘Peak’ loci, there was little or no difference between wild-type and dKO H3K27Me3 signals (Fig. 5b and Supplementary Fig. 8a; examples in Supplementary Fig. 8b,c).

Among these 7730 ‘peak’ loci, we found 55 ‘overdecorated’ genes characterized by a 2-fold or greater H3K27Me3 signals in dKO than wild-type mature OT-II CD4 SP cells, accepting a 5% false discovery rate (FDR) (Fig. 5b,c and Supplementary Table 1; examples in Figs 4b and 5b and Supplementary Fig. 8d). Analyses of wild-type and dKO mature CD4 SP thymocytes expressing an endogenous TCR repertoire found increased H3K27Me3 signals in dKO cells at the vast majority of these 55 overdecorated loci, including S1pr1 and Klf2 (Supplementary Fig. 8d–f). Analyses in Jmjd3 or Utx single-deficient mature thymocytes suggested that Jmjd3 was primarily responsible for this effect (Supplementary Fig. 8d–f). We also identified 14 ‘underdecorated’ genes, with paradoxically lower H3K27Me3 signals in dKO than in wild-type cells (Fig. 5b,c and Supplementary Fig. 8a). Since H3K27Me3 signals at most of these loci were low even in wild-type cells (Fig. 5c and Supplementary Fig. 8a), and there was no change in their expression (Supplementary Fig. 8g), we did not investigate them further.

Both S1pr1 and Klf2 promoters normally ‘clear’ H3K27Me3 during the differentiation of DP into CD4 SP thymocytes (Fig. 4a,b), suggesting that the biological function of Jmjd3 and Utx could be to remove the mark at promoters induced during this developmental step. To evaluate this possibility, we performed H3K27Me3 Chipseq in wild-type CD69^lo DP thymocytes, the cell subset that contains precursors of positive selection that have not undergone TCR signalling. We used DP thymocytes carrying an endogenous TCR repertoire, as all OT-II transgenic thymocytes are subject to TCR engagement. At most of the 55 ‘overdecorated’ promoters, wild-type H3K27Me3 signals were higher in DP than mature CD4 SP thymocytes (Fig. 5d). Reciprocally, we found that 449 genes normally undergo ‘clearance’ of H3K27Me3 (defined as a 50% or greater drop in H3K27Me3 ChIPseq signals) during the differentiation of DP into CD4 SP thymocytes. Within that gene subset, average H3K27Me3 signals were significantly higher in dKO than wild-type mature CD4 SP thymocytes (P<2 × 10⁻¹⁶, paired t-test), with a twofold or greater increase in H3K27Me3 signal at 162 loci (36%) (Fig. 5e). We conclude from these findings that Jmjd3 and Utx are important for H3K27Me3 clearance during DP to CD4 SP thymocyte differentiation.

H3K27Me3 accumulation impairs gene expression

Microarray analyses showed a significant (P<0.05) reduction in expression at 40 of the 55 overdecorated genes in dKO mature thymocytes, and the reduction was >50% at 14 of them, including S1pr1 and Klf2 (Fig. 6a, and Supplementary Table 1). Although the correlation was not perfect, the effect on gene expression was significantly correlated with H3K27Me3 signals in dKO cells (Fig. 6a, P<10⁻⁴, Pearson’s r=−0.54). The greater impact on genes with high H3K27Me3 signals presumably reflected that low H3K27Me3 signals in that overdecorated gene subset indicate methylation changes in a minor fraction of cells. RT-PCR analyses confirmed microarrays findings at Ccnd2, Irf4, Il6ra, Foxo1 and Lfng (Fig. 6b), in addition to S1pr1 and Klf2 (Fig. 3a). Of note, Zbtb7b expression was not reduced in dKO cells (Supplementary Table 1), consistent with the presence of mature CD4 SP thymocytes. Accordingly, H3K27Me3 decoration at the Zbtb7b promoter remaining substantially lower in dKO CD4 SP than in DP thymocytes (Fig. 4b and Supplementary Table 1). We conclude from these analyses that Kdm6-family demethylases are needed for H3K27Me3 homeostasis and expression of a limited gene subset in CD4 SP thymocytes.

**Figure 6: Impact of Jmjd3 and Utx on gene expression in mature CD4 SP thymocytes.**

Because Jmjd3 and Utx have functions independent from their demethylase activities^{14,16,17,18,36,37}, we examined if all changes in gene expression in dKO cells were associated with changes in H3K27Me3 signals. On the full 20218-gene set, microarray analyses identified 115 ‘underexpressed’ genes, with a 50% or greater drop in expression in dKO cells (with FDR<0.05) (Fig. 6c and Supplementary Table 2). Among these, most (70) had promoters overlapping with an H3K27Me3 peak, of which 68 had greater H3K27Me3 signals in dKO than in wild-type thymocytes (Fig. 6d, filled symbols). This reinforces the concept that effects of Jmjd3 and Utx on gene expression were mediated at least in part by their demethylase activity.

However, the additional 45 genes underexpressed in dKO cells did not overlap with H3K27Me3 peaks (Fig. 6d, open symbols), including Ccr7 and Sell (encoding CD62L), which were previously shown to be Klf2-dependent^33,38, and Ccr4 (Supplementary Fig. 9a). Conversely, using the same FDR stringency (<0.05), 41 ‘overexpressed’ genes had at least twofold greater expression in dKO than wild-type cells, although the change was typically modest (Fig. 6c). Athough a few of these overlapped with an H3K27Me3 peak, increased expression was not associated with changes in H3K27Me3 decoration (Supplementary Fig. 9b). These findings are consistent with the idea that Jmjd3 or Utx can affect gene expression indirectly, or through demethylase-independent activities.

Demethylase-dead Uty fails to rescue S1pr1 expression

The evidence for demethylase-independent effects of Jmjd3 and Utx prompted us to investigate further whether their effect on S1pr1 was mediated by H3K27Me3 accumulation. To this end, we took advantage from the fact that the Y-chromosome homolog of Utx, Uty, encodes a protein with little if any demethylase activity, yet carries demethylase-independent functions of Utx^{11,14,16,17,18,36}. Thus, if demethylase-independent activities of Utx were sufficient to promote S1pr1 expression, Uty should promote S1pr1 expression in male mice lacking Jmjd3 and Utx. This was not the case, as expression of S1pr1 was similarly low in OT-II dKO mice of both genders (Fig. 7a). Furthermore, Uty minimally increased the numbers of CD4 SP thymocytes and spleen CD4⁺ T-cells (Fig. 7b,c), the latter remaining well below that in OT-II Jmjd3-deficient mice (6.6±1.6 × 10⁶ versus 15.41±1.7 × 10⁶, mean±s.e.m., Figs 2j and 7c). This supports the conclusion that H3K27Me3 demethylation at S1pr1 is needed for its expression.

**Figure 7: Demethylase-inactive Uty fails to support *S1pr1* expression.**

Enforced S1pr1 expression prevents thymocyte accumulation

The requirement for Jmjd3 and Utx for expression of Klf2 and chemokine receptors involved in intrathymic migration, including CCR4 and CCR7, raised the possibility that the accumulation of dKO thymocytes was not caused by impaired S1pr1 expression. To address this possibility, we enforced S1pr1 expression in OT-II thymocytes lacking both Jmjd3 and Utx. Expression of an S1pr1 transgene resulted in a marked drop in Vα2^hi CD24^lo CD4 SP thymocytes (Fig. 8a), whereas it had a much more modest effect on the DP subset (Fig. 8b). Furthermore, expression of the transgene increased the numbers of Vα2^hi spleen CD4⁺ T-cells (Fig. 8c). We conclude from these experiments that impaired S1pr1 expression is a key factor limiting the egress of dKO thymocytes.

**Figure 8: Enforced S1pr1 expression rescues egress of dKO thymocytes.**

In summary, we demonstrate that Jmjd3 and Utx demethylases are redundantly required for proper T-cell development. We further show that these molecules promote the normal developmental clearance of H3K27Me3 at and expression of specific genes, including S1pr1.

Discussion

The present study demonstrates that Jmjd3 and Utx H3K27Me3 demethylases are redundantly required for the terminal steps of T-cell development, and notably for expression of S1pr1, a gene needed for thymic egress. As a result, Jmjd3 and Utx are needed for the generation of a normal T-cell repertoire. In the immune system, genetic analyses had shown that Jmjd3 contributes to macrophage and T-cell effector differentiation^28,39, and to the differentiation of invariant natural killer T-cells⁴⁰, a population of innate-like lymphocytes recognizing lipid antigens bound to CD1d molecules⁴¹. However, the full impact of Jmjd3 and Utx on gene expression and cell differentiation has remained unclear, as none of these reports had assessed cells lacking both enzymes. The present study demonstrates essential and partly overlapping functions of Jmjd3 and Utx in developing T-cells.

Previous studies have highlighted the importance of Jmjd3 and Utx, the two molecules with H3K27Me3 demethylase activity, in development and disease. These molecules are involved at multiple steps of mouse embryogenesis and during cell differentiation^{14,15,16,17,18,19,28,36,39}. In addition, UTX mutations were found in a subset of children with Kabuki syndrome, a congenital disorder associating multiple developmental defects⁴². Furthermore, Jmjd3 and Utx serve opposite functions during T-cell leukemogenesis, the former promoting tumorogenesis whereas the latter acts as a tumour suppressor gene^43,44.

While Jmjd3 and Utx both have catalytic demethylase activities, the contribution of such activity to their biological functions has remained elusive. Most mechanistic studies addressing this question have focused on early embryonic differentiation, in which Utx is the key physiological player^{14,16,17,18,36}. Utx is notably required for mesoderm formation and expression of the T gene, encoding the key mesoderm inducer Brachyury¹⁷. Utx molecules bind the T promoter in vivo, suggesting a direct effect of Utx on T transcription. However, neither mesoderm formation nor T expression require Utx demethylase activity: both are rescued by Uty, which lacks demethylase activity, or by a Utx mutant lacking demethylase activity^{14,16,17,18,36}. Strikingly, Uty supports the development of embryos harbouring disruptions of both Utx and Jmjd3, questioning the importance of Utx and Jmjd3 demethylase activity for cell differentiation²¹. Although it has been reported that a demethylase-inactive mutant of Jmjd3 fails to rescue the postnatal death of Jmjd3-deficient mice¹⁵, that report did not document expression of the mutant Jmjd3 protein.

Contrasting with T expression and early embryonic development, our study supports the concept that the catalytic activity of Jmjd3 and Utx is crucial for their impact on gene expression and cell differentiation, by showing that Uty does not support expression of S1pr1 in male thymocytes lacking both Jmjd3 and Utx. Of note, similar to the broad distribution of Utx and Uty in developing embryos (with limited tissue specificity in the expression of either gene)^16,20, data from the Immgen data base indicates widespread expression of both genes in immune cells, including throughout T-cell development (www.immgen.org and Mingueneau et al.⁴⁵). Thus, although the generation of conditional alleles encoding catalytically inactive versions of Jmjd3 and Utx will be needed to formally demonstrate that H3K27Me3 demethylase activity is required for expression of S1pr1 in mature thymocytes, our results support such a conclusion, and the broader notion that the full impact of Jmjd3 and Utx on transcription requires their demethylase activity.

In non-proliferating but actively differentiating thymocytes, in which H3K27Me3 cannot be diluted during cell division, the footprint of Jmjd3 and Utx on H3K27Me3 homeostasis is highly specific. Even though a 5% FDR cut-off presumably eliminates authentic functional targets, we estimate that the combined activity of Jmjd3 and Utx is required for H3K27Me3 homeostasis at less than 1% of gene promoters. This suggests that the activity of H3K27 methylases Ezh1 and Ezh2 is tightly controlled and acts as the rate-limiting factor for the propagation of H3K27Me3 in the genome. Most of the effect on H3K27Me3 in mature thymocytes appears attributable to Jmjd3, as disruption of Utx per se only minimally affects promoter decoration.

Although Jmjd3 or Utx ChIP attempts were not successful with our current antibodies, it was reported that Jmjd3 binds the S1pr1 promoter in the HL60 cell line⁴⁶, supporting the idea of a direct Jmjd3 involvement. Of note, that study documented Jmjd3 binding to more than 8,000 genes, of which only a minor subset (fewer than 200) depended on Jmjd3 for their expression⁴⁶. Conversely, Utx binds the T promoter, yet promotes its activity through demethylase-independent mechanisms¹⁷. Thus, for both Jmjd3 and Utx, promoter recruitment does not appear as a proper predictor of involvement in H3K27Me3 removal.

Although we found a clear link between H3K27Me3 retention and reduced gene expression in cells both lacking Jmjd3 and Utx, the correlation was not absolute. In fact, a perfect correlation between the impact of these enzymes on H3K27Me3 decoration and transcription was not expected, for two complementary set of reasons. First, Jmjd3 and Utx can promote gene-specific transcription without a corresponding impact on local H3K27Me3 decoration. Indeed, we found that these enzymes affected expression of genes with no H3K27Me3 decoration. Such effects presumably involve two non-mutually exclusive mechanisms. Jmjd3 and Utx can affect locus-specific transcription indirectly, through their impact on the expression of trans-acting transcription factors, or directly, through demethylase-independent mechanisms. Illustrating the first possibility, Jmjd3 and Utx control of Klf2 expression may account for their impact on Klf2 targets Ccr7 and Sell (encoding CD62L). Direct, demethylase-independent effects have been identified in multiple contexts for both Jmjd3 and Utx. In addition to those of Utx in ES cells, such functions of Jmjd3 promote transcriptional activation by T-bet³⁷, a transcription factor required for the differentiation of Th1 effector CD4⁺ T-cells⁴⁷. Multiple mechanisms appear to support such functions. Jmjd3 links T-bet to chromatin remodelling complexes at a specific subset of T-bet target genes, whereas both Jmjd3 and Utx have been reported to associate with the MLL enzymatic complexes involved in H3K4 methylation and promoting gene expression^{10,13,43,48,49}.

Conversely, Jmjd3 and Utx-dependent clearance of H3K27Me3 was not strictly required for gene expression. This points to the existence of additional levels of control on transcriptional repression mechanisms downstream of H3K27Me3. H3K27Me3-mediated gene repression is thought to primarily involve H3K27Me3-bound PRC1 complexes, including H3K27Me3-binding subunits (Cbx proteins) and components responsible for transcription repression. Despite the similarity among isoforms of Cbx proteins, recent studies have highlighted unique functional and biochemical properties specific of each variant^8,50,51. Similarly, there is much plasticity in the association of silencing-mediating subunits to PRC1 (ref. 8). In addition, Polycomb-independent mechanisms have been proposed to contribute to the repressive effects of H3 K27 trimethylation⁸, including interference with the ‘activating’ acetylation of H3 K27 and effects on nucleosome packaging or DNA opening for transcriptional elongation^46,52. Our findings suggest that such ‘effector’ mechanisms of H3K27Me3-mediated transcriptional repression are subject to control, possibly by extracellular signals.

Our study also documents that Jmjd3 and Utx are not absolutely required for gene-specific clearance of H3K27Me3 during the differentiation of DP into CD4 SP thymocytes. Surprisingly, H3K27Me3 removal at Zbtb7b, encoding the CD4-differentiating factor Thpok, was only modestly affected by Jmjd3 and Utx disruption; accordingly, neither enzyme was needed for Zbtb7b expression and for the differentiation of CD4⁺-lineage thymocytes. Such observations in non-dividing thymocytes imply the existence of Jmjd3- and Utx-independent H3K27Me3 removal mechanisms. These may involve some degree of promiscuity in substrate specificity among JmjC-domain demethylases, as recently suggested⁵³. Alternatively, it is possible that demethylase-independent mechanisms contribute to remove H3K27Me3 at select genes. One particularly interesting possibility is the transcription-associated substitution of conventional histone H3 by variant H3.3 molecules⁵⁴; this mechanism could conceivably replace methylated histone H3 by newly synthesized variant molecules that had not been subject to K27 methylation.

Many of the genes dependent on Jmjd3 and Utx for H3K27Me3 clearance and expression, including S1pr1, Klf2, Ccnd2, Il6ra, Foxo1 or Lfng, are characteristic of mature T-cells and induced during late thymocyte differentiation. Although it is possible that the impact of Jmjd3 and Utx disruption on S1pr1 is amplified by a ‘sampling bias’, as cells that fail to express S1pr1 accumulate in the thymus, this would not explain the impact on other genes. The preferential impact of Jmjd3 and Utx on such ‘late maturation’ genes is consistent with a role for these enzymes in integrating extracellular cues at promoters activated during terminal thymocyte differentiation. The demethylase activity of these enzymes requires oxygen and α-ketoglutarate, produced from glucose or glutamine⁵⁵, which control their function in embryonic stem cells. Thus, it is possible that Jmjd3 and Utx activation, and action at S1pr1, serves as a postselection licensing signal for thymocytes having undergone positive selection and increased their metabolic activity.

In summary, we demonstrate extensive H3K27Me3 changes during the differentiation of CD4⁺ T-cells from DP thymocytes and show that enzymatic removal of H3K27Me3 is required for the proper outcome of this differentiation step. Our unique approach, in non-proliferating cells, demonstrates an in vivo requirement for H3K27Me3 demethylase activity in gene expression during cell differentiation.

Methods

Generation of conditional Jmjd3- or Utx-deficient mice

The targeting vector for each conditional knockout mouse included an inserted neomycin (Neo) cassette flanked by Frt, and three loxP sites flanking the Neo cassette and Jmjd3 exons 14–20 or Utx exon 24 (covering exon(s) encoding the JmjC domain). After transfection of the targeting vector into embryonic stem (ES) cells, recombinant ES cells were selected by G418 and FIAU (1–2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil), screened by Southern blot analyses, and injected into blastocysts derived from C57BL/6 mice. Deletion of Frt-flanked Neo cassette was achieved by crossing of mice heterozygous for the recombinant allele to mice expressing Flpe recombinase driven by β-actin promoter, resulting in the floxed allele (Jmjd3^f or Utx^f). Similarly, the deleted allele (Jmjd3⁻ or Utx⁻) was obtained by Cre-mediated deletion of the floxed sequence through mating of mice. The genotype of recombinant mice was verified by Southern blot analysis for the first two generations and subsequently determined by PCR analysis. We used Cre-negative Jmjd3^f/f or Utx^f/f littermates as ‘wild-type’ controls in this study. For the generation of OT-II or P14-transgenic mutant mice or S1pr1 transgenic mice, Jmjd3 and Utx mutant lines were crossed to OT-II³⁰ or P14 (ref. 56) TCR transgenic, or S1pr1 (ref. 57) transgenic mice. Primers for genotyping are listed in Supplementary Methods.

Mice

CD45.1 and CD45.2 C57BL/6 mice were obtained from the National Cancer Institute Animal Production Facility. All transgenic mice were maintained heterozygous for the transgene. Experiments were performed on female mice except when otherwise indicated. Mice were housed in specific pathogen-free facilities and analysed between 6 and 16 weeks of age. Animal procedures were approved by the NCI Animal Care and Use Committee.

Antibodies

The following antibodies were purchased from either BD Biosciences or eBioscience (clone name and final concentration used in parenthesis): CD4 (RM4.5, 1 μg ml⁻¹), CD8α (53-6.7, 2.5 μg ml⁻¹), CD8β (53-5.8, 2.5 μg ml⁻¹), TCR (H57–597, 1 μg ml⁻¹), CD24 (M1/69, 2.5 μg ml⁻¹), CD69 (H1.2F3, 1 μg ml⁻¹), CD44 (IM7, 1 μg ml⁻¹), CD45.2 (104, 0.5 μg ml⁻¹), anti-CD45.1 (A20, 1 μg ml⁻¹), CD5 (53-7.3, 1 μg ml⁻¹), MHC II I-A/I-E (M5/114.15.2, 1 μg ml⁻¹) and Vα2 (B20.1, 1 μg ml⁻¹). The rat anti-mouse S1pr1 antibody was kindly provided by Dr Jason Cyster (University of California, San Francisco) and subsequently purchased from R&D Systems (MAB7089). The Jmjd3 antibody for immunoblot analysis was generated in rabbits using a peptide (amino acids 211–230 of mouse Jmjd3) as an epitope, and affinity-purified; the Utx antibody was purchased from Bethyl laboratories (A302–374A). Final concentrations for Jmjd3 and Utx antibodies were 2 and 0.2 μg ml⁻¹ for immunoprecipitation and immunoblotting, respectively. Anti-H3K27Me3 (#17–622) antibodies for ChIPseq were purchased from Millipore.

Cell preparation and flow cytometry

Thymocytes and splenocytes were isolated from mice (6–10 weeks of age) and analysed by immunostaining and flow cytometry as described⁵⁸. Data were acquired on a LSR Fortessa cytometer (BD Biosciences) and analysed with Flowjo software. The S1pr1 staining was performed by incubating with the rat anti-mouse S1pr1 antibody (2 μg/10⁶ cells), followed by staining with anti-rat IgG PE (eBioscience, 12-4822-82, 10 μg ml⁻¹). Cells were then washed and blocked with 2% normal rat serum before being incubated with additional antibodies. For sorting of mature CD4 SP thymocytes (TCR^hi CD24^lo), CD4 SP thymocytes were pre-enriched by using a Dynal mouse CD4 negative isolation kit (Invitrogen), stained with antibodies, and sorted on a BD FACSAria cell sorting system (BD Biosciences).

Bone marrow chimeras

T-cell-depleted (Pan T Dynal kit, Invitrogen) bone marrow was isolated from CD45 disparate animals, mixed at a 1:1 ratio, and injected into lethally irradiated (900 rads) recipients heterozygous for CD45.1 and CD45.2, which were analysed 8 weeks after transplantation.

In vitro thymocyte culture

Sorted mature thymocytes were resuspended in RPMI-1640 medium (supplemented with 10% FCS, 1% Pen/Strep/Glu) and incubated overnight in presence or absence of plate bound anti-CD3 (145-2C11; BD Biosciences, coated with 1 μg ml⁻¹ solution) at 37 °C, 5% CO₂.

Microarray and quantitative RT-PCR analyses

Total RNA from sorted Vα2^hi CD24^lo (OT-II) or TCR^hi CD24^lo (endogenous repertoire) CD4 SP thymocytes or cultured thymocytes was isolated using RNeasy Plus Mini Kit (Qiagen). Microarray analyses (Affymetrix Mouse Gene 1.0 ST array) were performed by the NCI microarray facility following the manufacturer’s recommendation (Affymetrix). For quantitative RT-PCR analysis, cDNA was synthesized from total RNA using ThermoScript RT-PCR system (Invitrogen), and the PCR was performed on a 7500 Real time PCR system (Applied Biosciences). The mRNA levels of target genes were normalized with 18S rRNA, and represented as the fold change over wild-type (or unstimulated control, Supplementary Fig. 7) values (set arbitrarily as 1). Primer sequences will be provided upon request. Microarray data were analysed using Partek Genomic Suite from Affymetrix ‘cel’ files generated at the NCI microarray facility. Primers for RT-PCR are listed in Supplementary Methods.

ChIP

Sorted mature (TCR^hi or Vα2^hi CD24^lo) thymocytes were digested with Mnase (Sigma-Aldrich, N3755, 0.4 U ml⁻¹), followed by sonication to generate mainly mononucleosomes with minor fraction of dinucleosomes. Sonicated supernatants were pre-cleared for 1 h with protein G beads at room temperature and then immunoprecipitated with anti-H3K27Me3 (10 μg ml⁻¹, pre-adsorbed on Protein G beads) or control mouse monoclonal IgG (10 μg ml⁻¹) overnight. Immunoprecipitated complexes were collected on protein G beads and DNA was purified by PCR Purification Kit (Qiagen) and analysed by quantitative (Sybr green) PCR (see Supplementary Methods for primer sequence). Data is presented as a percent input of each IP sample relative to input chromatin.

ChIPseq and data analysis

The fastq files were aligned against the genome reference mm9 and promoter reads (±2 kb from the transcription start site) detected for each gene using the Rsubread package in R⁵⁹. Promoter counts were normalized by loess using csaw package⁶⁰. Broad peaks were detected using SICER⁶¹, and promoter reads overlapping with SICER peaks in two biological replicates per group (dKO and WT) further analysed for differential binding using the glmQLFit function in EdgeR^62,63. Offsets were used to generate normalized bedgraph files for visualization in IGV. Entries redundant for gene name and boundaries were eliminated, leaving a total of 20218 unique entries processed for combined ChIPseq-microarray analyses.

Statistical analyses

Statistical analyses were performed using Prism software except as noted above for microarrays and ChIPseq data. Bars in graphs indicate average±s.d. Except where otherwise indicated, comparisons were performed by two-tailed unpaired Student’s t-test. Two-tailed paired t-test was used where biologically appropriate (for example, competitive bone marrow chimeras). Significance levels (P-values) are indicated on figures. For statistical comparisons, sample size was >3 and was determined empirically based on pilot analyses. We used neither randomization nor blinding; animals were excluded from analyses only on the basis of criteria (for example, age or poor health status) unrelated to the experiment result.

Additional information

Accession codes. Microarray and ChIPseq data were deposited in the GEO database (accession mumbers GSE70363 and GSE70795, respectively).

How to cite this article: Manna, S. et al. Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation. Nat. Commun. 6:8152 doi: 10.1038/ncomms9152 (2015).

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Gene Expression Omnibus

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Acknowledgements

We thank J. Cyster for the anti-S1pr1 antibody, H. Chi and R. Flavell for the S1pr1 transgenic mouse, S. Reid and E. Southon for assistance in generating the targeted alleles, B. Taylor, K. Wolcott and S. Banerjee for cell sorting, X. Wu for microarray analyses, and J. Ashwell, A. Bhandoola, A. Gégonne, P. Love, J. O’Shea and D. Singer for reading the manuscript. This work utilized the computational resources of the NIH HPC Biowulf cluster and was supported by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research.

Author information

Jong Kyong Kim
Present address: Present address: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China,
Catherine Baugé
Present address: Present address: EA4652 Microenvironnement Cellulaire et Pathologies, UFR de médecine, Université de Caen Basse-Normandie, Caen, France,
Sugata Manna: These authors equally contributed to this work

Authors and Affiliations

Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA
Sugata Manna, Jong Kyong Kim, Catherine Baugé, Melanie S. Vacchio, Ehydel Castro & Rémy Bosselut
Collaborative Bioinformatics Resource, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA
Margaret Cam
Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, 21702, Maryland, USA
Yongmei Zhao, Jyoti Shetty & Bao Tran
Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, 21702, Maryland, USA
Lino Tessarollo

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Sugata Manna
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Contributions

S.M. and J.K. contributed to research design, performed and analysed experiments and contributed to manuscript writing. C.B. contributed to research design, constructed homologous recombination vectors and characterized recombinant ES cell lines. M.V. and E.C. designed, performed and analysed experiments. Y.Z., J.S. and B.T. performed and M.C. analysed deep sequencing studies. L.T. supervised ES cell work and provided advice with homologous recombination procedures. R.B. contributed to research design and data analysis, supervised the research, and wrote the manuscript.

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Correspondence to Rémy Bosselut.

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Supplementary Figures 1-9, Supplementary Tables 1-2 and Supplementary Methods (PDF 2797 kb)

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Manna, S., Kim, J., Baugé, C. et al. Histone H3 Lysine 27 demethylases Jmjd3 and Utx are required for T-cell differentiation. Nat Commun 6, 8152 (2015). https://doi.org/10.1038/ncomms9152

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Received: 01 May 2015
Accepted: 23 July 2015
Published: 02 September 2015
DOI: https://doi.org/10.1038/ncomms9152

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