Epigenetic Thpok silencing limits the time window to choose CD4+ helper-lineage fate in the thymus
2013; Springer Nature; Volume: 32; Issue: 8 Linguagem: Inglês
10.1038/emboj.2013.47
ISSN1460-2075
AutoresHirokazu Tanaka, Taku Naito, Sawako Muroi, Wooseok Seo, Risa Chihara, Chizuko Miyamoto, Ryo Kominami, Ichiro Taniuchi,
Tópico(s)Immunotherapy and Immune Responses
ResumoArticle12 March 2013free access Source Data Epigenetic Thpok silencing limits the time window to choose CD4+ helper-lineage fate in the thymus Hirokazu Tanaka Hirokazu Tanaka Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Taku Naito Taku Naito Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, JapanPresent address: Department of Molecular Immunology, Toho University School of Medicine, Tokyo, Japan. Search for more papers by this author Sawako Muroi Sawako Muroi Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Wooseok Seo Wooseok Seo Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Risa Chihara Risa Chihara Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Chizuko Miyamoto Chizuko Miyamoto Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Ryo Kominami Ryo Kominami Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan Search for more papers by this author Ichiro Taniuchi Corresponding Author Ichiro Taniuchi Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Hirokazu Tanaka Hirokazu Tanaka Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Taku Naito Taku Naito Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, JapanPresent address: Department of Molecular Immunology, Toho University School of Medicine, Tokyo, Japan. Search for more papers by this author Sawako Muroi Sawako Muroi Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Wooseok Seo Wooseok Seo Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Risa Chihara Risa Chihara Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Chizuko Miyamoto Chizuko Miyamoto Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Ryo Kominami Ryo Kominami Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan Search for more papers by this author Ichiro Taniuchi Corresponding Author Ichiro Taniuchi Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Author Information Hirokazu Tanaka1,‡, Taku Naito1,‡, Sawako Muroi1, Wooseok Seo1, Risa Chihara1, Chizuko Miyamoto1, Ryo Kominami2 and Ichiro Taniuchi 1 1Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan 2Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan ‡These authors contributed equally to this work. *Corresponding author. Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. Tel.:+81 45 503 7044; Fax:+81 45 503 7043; E-mail: [email protected] The EMBO Journal (2013)32:1183-1194https://doi.org/10.1038/emboj.2013.47 Present address: Department of Molecular Immunology, Toho University School of Medicine, Tokyo, Japan. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info CD4+ helper and CD8+ cytotoxic T cells differentiate from common precursors in the thymus after T-cell receptor (TCR)-mediated selection. Commitment to the helper lineage depends on persistent TCR signals and expression of the ThPOK transcription factor, whereas a ThPOK cis-regulatory element, ThPOK silencer, represses Thpok gene expression during commitment to the cytotoxic lineage. Here, we show that silencer-mediated alterations of chromatin structures in cytotoxic-lineage thymocytes establish a repressive state that is epigenetically inherited in peripheral CD8+ T cells even after removal of the silencer. When silencer activity is enhanced in helper-lineage cells, by increasing its copy number, a similar heritable Thpok silencing occurs. Epigenetic locking of the Thpok locus may therefore be an independent event from commitment to the cytotoxic lineage. These findings imply that long-lasting TCR signals are needed to establish stable Thpok expression activity to commit to helper T-cell fate and that full commitment to the helper lineage requires persistent reversal of silencer activity during a particular time window. Introduction Differentiation of multi-potent precursors into a particular lineage upon exposure to stimuli from external environmental cues is accompanied by the expression of lineage-specific genes together with repression of alternative lineages, a process termed as lineage specification. As a consequence of sequential exposures to differentiation signals, specific gene expression signatures that confer unique cellular functions to differentiated cells are established (lineage commitment) (Cantor and Orkin, 2001; Murphy and Reiner, 2002; Rothenberg and Dionne, 2002). Mechanisms that guarantee the stable inheritance of established gene signatures even after multiple cell divisions enable differentiated cells to preserve their cell identity. Studies in the last decade have highlighted the crucial contributions of epigenetics in maintenance of gene expression status (Jenuwein and Allis, 2001; Probst et al, 2009; Bonasio et al, 2010). For instance, heterochromatin has been shown to be involved in heritable and stable gene repression (Henikoff, 2000), referred to as gene silencing, in part via relocating gene loci into specialized nuclear compartments such as the peri-nuclear lamina (Schneider and Grosschedl, 2007; Reddy et al, 2008). It is thus conceivable that lineage commitment would involve epigenetic changes towards heterochromatin-like structures at many developmentally regulated genes to assure their subsequent silent state. However, how the epigenetic machinery that delivers lineage-specific epigenetic marks is linked with lineage commitment remains uncharacterized. Specifically, it is not clear whether such epigenetic machinery becomes functional upon lineage commitment or acts in parallel with lineage specification. In order to better understand an epigenetic link between environmental cues and establishment of cell identity, it is important to unravel how specific epigenetic modifications are accumulated on the relevant genes. There are two major lineages of T lymphocytes, the CD4+ helper and the CD8+ cytotoxic cells, and they differentiate from a common precursor, the CD4+CD8+ double-positive (DP) thymocyte, after positive selection. During this selection process, only DP thymocytes that receive optimal TCR-signal strength as a result of TCR engagement with peptide–MHC complexes are allowed to further differentiate (Germain, 2002; Starr et al, 2003; Singer et al, 2008). When CD4+CD8+ DP thymocytes are positively selected through MHC class II molecules (MHC class II-selected thymocytes), they develop into CD4+CD8− single-positive (SP) thymocytes that are committed to the helper lineage. On the contrary, DP thymocytes that undergo selection via MHC class I molecules with a help of CD8 co-receptors terminate CD4 expression and differentiate into CD4−CD8+ SP thymocytes committed to the cytotoxic lineage (Ellmeier et al, 1999). It has been proposed that differences in TCR-signal length during positive selection instruct distinct fates in post-selection thymocytes. Several lines of evidence have shown that persistent TCR signalling is essential for development of CD4+ helper T cells (Sarafova et al, 2005; Singer et al, 2008; Adoro et al, 2012). On the other hand, temporary termination of Cd8 gene expression in post-selection thymocytes (Brugnera et al, 2000) results in a disruption of TCR signals specifically in MHC class I-selected thymocytes, instructing them to become cytotoxic-lineage cells (Singer et al, 2008). However, it remains obscure why long-lasting TCR signals are necessary for commitment to the helper lineage. Recently, it has been recognized that appropriate linkage of TCR specificity to MHC class during helper/cytotoxic lineage choice requires input from the zing finger transcription factor ThPOK (also known as cKrox), which is encoded by the Zbtb7b gene (hereafter referred to as the Thpok gene in this manuscript) (He et al, 2005, 2010; Sun et al, 2005). Gain and loss of function studies of ThPOK demonstrated a dominant role of ThPOK in acquiring a CD4+CD8− phenotype that is independent of TCR specificity to MHC class. Thus, both MHC class I-selected and class II-selected thymocytes are redirected to alternative CD4+CD8− helper and CD4−CD8+ cytotoxic lineages by ectopic induction or loss of ThPOK function, respectively (He et al, 2005; Sun et al, 2005). Because of its potent activity, expression of the Thpok gene from its two promoters, distal P1 and proximal P2, must be strictly regulated during thymocyte differentiation. During thymocyte maturation, ThPOK first appears after positive selection, increases in MHC class II-selected thymocytes, but then disappears in MHC class I-selected cells (He et al, 2005; Muroi et al, 2008). In sorting out mechanisms that control lineage- and stage-specific Thpok expression, previous studies have identified two essential cis-regulatory elements. A transcriptional silencer (referred to as the Thpok silencer in this manuscript) is essential to control helper lineage-specific expression of Thpok (He et al, 2008; Setoguchi et al, 2008). On the other hand, a transcriptional enhancer in the proximal region of the gene plays an essential role in increasing Thpok expression at later maturation stages in MHC class II-selected thymocytes (Muroi et al, 2008). Interestingly, conditional ablation of ThPOK function from peripheral CD4+ T cells indicated that extra-thymic expression of ThPOK is still necessary to maintain CD4+ helper T-cell identity (Wang et al, 2008). Furthermore, retroviral transduction of ThPOK into fully differentiated peripheral CD8+ T cells can activate helper lineage-related genes as well as repress cytotoxic-related genes, albeit only to limited extent (Jenkinson et al, 2007). These findings indicate that the helper lineage-specific Thpok expression established in the thymus must be sustained in the peripheral T cells to maintain their lineage identity. However, although roles of cis-regulatory elements in regulating Thpok expression during lineage commitment in thymus have begun to be characterized (Taniuchi, 2009), it remains unknown how the established state of the Thpok gene, either active or repressive, is stably maintained in differentiated T cells. To address this point, we used genetic approaches that enabled us to modify Thpok silencer activity. We show that a silencer-dependent deposition of repressive epigenetic marks establishes a repressive state that is inherited independently of the silencer in peripheral CD8+ T cells via inhibiting the two promoters by different mechanisms. Furthermore, we provide evidence showing that such epigenetic Thpok silencing can also occur in CD4+ helper-lineage cells when silencer activity persists after positive selection. These findings suggest that epigenetic mechanisms that lock the Thpok locus can take place even in MHC class II signalled cells if the silencer activity is not properly terminated. Thus, our findings strongly suggest that, for appropriate helper-lineage choice by MHC class II-selected cells, reversal of the Thpok silencer upon receipt of TCR signals must persist for a certain time interval to avoid epigenetic sealing of the Thpok gene. We thus propose that such continuous counter-silencing by long-lasting TCR signals is necessary to establish stable Thpok expression, hence to fully commit to helper T-cell fate. Results Epigenetic modifications in the Thpok gene There is now compelling evidence that developmentally regulated genes receive dynamic modifications of local chromatin structure (Bonasio et al, 2010), and in particular, heterochromatin-like structures are known to be involved in stable gene repression (Henikoff, 2000). We therefore examined how the chromatin structure at the Thpok locus is altered during T-cell development. Since tri-methylation at two distinct histone H3 lysine residues, lysine 4 and lysine 27 (H3K4me3 and H3K27me3), has been shown to mark active and repressive states, respectively (Bernstein et al, 2005), of the target genes, we performed chromatin immunoprecipitation (ChIP)-on-chip analysis of distinct T-cell subsets. Accumulation of the repressive H3K27me3 mark at the distal P1 promoter and its upstream region was detected from immature CD4−CD8− DN thymocytes onward in cell subsets that do not express Thpok, while it was not present at a significant level at the proximal P2 promoter (Figure 1A and C). In contrast, H3K27me3 deposition at the distal P1 promoter was replaced by active H3K4me3 marks in cells expressing Thpok, such as CD4+CD8− SP thymocytes, along with accumulation of H3K4me3 at the proximal P2 promoter (Figure 1A). Interestingly, a comparison of the Thpok silencer-deficient and wild-type Thpok loci by analytical ChIP assays revealed that the loss of the Thpok silencer resulted in not only a decrease in H3K27me3 marks but also an increase in H3K4me3 marks around the P1 promoter in DP thymocytes (Figure 1B and C). Similar changes resulting from loss of the silencer were also observed at the P2 promoter, albeit to a lesser extent (Figure 1B and C). These results indicate that the silencer is involved in the initial deposition of H3K27me3 marks as well as in preventing premature H3K4me3 loading prior to positive selection. Figure 1.Changes of histone H3 modifications pattern in the Thpok gene during T-cell development. (A) Association of histone H3 K4 tri-methylation (H3K4me3) and histone H3 K27 tri-methylation (H3K27me3) in the Thpok locus. The signal intensities of individual oligonucleotide probe as revealed in a ChIP-on-chip assay in the indicated T-cell subsets are shown along with a schematic structure of the Thpok gene (top). Positions of the silencer (S), proximal enhancer (PE), distal P1 promoter (P1) and proximal P2 promoter (P2) are indicated. One representative result of two experiments is shown. (B, C) Comparison of H3K4me3 (B) and H3K27me3 (C) patterns at the region around the distal P1 and proximal P2 promoters between silencer-sufficient (Thpok+/+) and -deficient (ThpokΔS/ΔS) DP thymocytes determined by analytical ChIP assays. Positions of each region analysed are shown as distance from the transcriptional start site in the P1 or P2 promoter-derived Thpok mRNA. In the panel shown at the right, the P1 promoter is also included as a reference. Data are from one of two independent experiments. Download figure Download PowerPoint Another known epigenetic modification involved in gene silencing is DNA methylation (Jones, 2012). We therefore next compared the DNA methylation status of the Thpok gene in CD4+ and CD8+ peripheral T cells. We tested 21 CpG islands derived from the Thpok silencer, distal P1 promoter, proximal P2 promoter and proximal enhancer (PE) and only three regions (S-4, 1-4 and 1-5) exhibited slightly increased DNA methylation in CD8+ T cells compared to CD4+ T cells (Figure 2A). Thus, our analyses did not detect any clear differences in the DNA methylation status of active versus repressed Thpok genes, although they could not formally exclude involvement of DNA methylation at other sites in repression of the Thpok gene. Figure 2.Comparison of DNA methylation status of the Thpok locus in CD4+ and CD8+ T cells. (A) DNA methylation status in CD4+ and CD8+ T cells analysed by methylation-specific PCR at selected CpG islands located within the silencer (S), distal P1 promoter (P1), proximal P2 promoter (P2) and proximal enhancer (PE) are shown as percentages. (B) Effects of chemical inhibitors of histone deacetylases, Trichostatin A (TSA), and DNA methyltransferase, 5-aza-2′-cytidine (5-AzaC), on the Thpok repression. Histograms showing Thpok-GFP expression from the Thpokgfp allele in activated CD8+ T cells 3 days after treatment with either inhibitor in culture. Expression of GFP in control CD4+ T cells is shown as a thin line for reference. (C) Semi-quantitative RT–PCR for the P1 promoter- and the P2 promoter-derived Thpok mRNA after inhibitor treatment is shown with the β-actin mRNA as control. Wedges indicate 1:3 dilution of template. Data are from one of two independent experiments.Source data for this figure is available on the online supplementary information page. Source Data for Fig 2C [embj201347-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint To further examine roles of epigenetic modifications in Thpok repression, we treated CD8+ T cells with Trichostatin A (TSA) or 5-aza-2′-deoxycytidine (5-AzaC), inhibitors of histone deacetylases (HDAC) and DNA methyltransferase, respectively. Three days after TSA treatment, Thpok expression in activated CD8+ T cells was significantly increased specifically from the P1 promoter, albeit at a level still well below that in CD4+ T cells. On the other hand, 5-AzaC induced a slight derepression of the Thpok gene mainly from the P2 promoter in a variegated manner (Figure 2B and C). Although it was not clear whether these inhibitors induced Thpok de-repression directly or indirectly, these results, along with the distinct H3K27me3 deposition pattern, suggested that different mechanisms are likely to be involved in preventing the activity of the two promoters for stable repression of the Thpok gene in CD8+ T cells. Conditional deletion of the Thpok silencer from CD8-lineage cells Our result showed that lineage-specific chromatin structures are established at the Thpok locus and that this correlates with the presence of the Thpok silencer. To further investigate the relevance of such epigenetic modifications for the regulation of Thpok expression, we wished to test whether the repressive state is maintained independently of the silencer in differentiated CD8+ T cells, as was observed at the Cd4 locus (Zou et al, 2001). To this end, we have generated a ThpokSfl allele in which an 800-bp region including the entire Thpok silencer sequence was flanked by two loxP sites (Supplementary Figure 1). The excision of the silencer in precursor DP thymocytes by Cd4-Cre transgene resulted in the loss of peripheral CD8+ T cells, as was previously observed in the case of germline deletion of the silencer (Figure 3A; Setoguchi et al, 2008). On the contrary, deletion of the Thpok silencer in developing CD4−CD8+ SP thymocytes by E8I-Cre transgene (Maekawa et al, 2008), which is expressed after the cells have downregulated the CD24 surface marker, did not impair development of CD8+ T cells (Figure 3A). Genotype analyses by Southern blot confirmed that the excision of the silencer by E8I-Cre was not at a detectable level in total thymocytes, but was almost complete in and restricted to CD8+ cells among splenic T cells (Figure 3B). The level of Thpok mRNA in these silencer-deficient CD8+ T cells was slightly elevated, but still <20% of that in CD4+ T cells (Figure 3C). Thus, the conditional removal of the Thpok silencer from post-selection thymocytes differentiating into the cytotoxic lineage did not cause a massive de-repression of the Thpok gene. Interestingly, only P1-derived Thpok transcripts were detected in the CD8+ T cells from ThpokSfl/Sfl;E8I mice (Figure 3D), whereas germline deletion of the silencer from a Thpokgfp reporter allele led to de-repression of the gfp gene from both the P1 and P2 promoters in CD8+ T cells (Figure 3E). Figure 3.Effects of conditional removal of the Thpok silencer in CD8-lineage cells on Thpok expression. (A) Dot plots showing CD4 and CD8 expression in total thymocytes and lymph node TCRβ+ T cells from mice of the indicated genotypes. Numbers in dot plots indicate the percentage of cells in each quadrant. (B–D) Genotype analyses by Southern blot (B), relative Thpok mRNA amounts (C) and semi-quantitative RT-PCR for the P1 promoter- and the P2 promoter-derived Thpok mRNA (D) in CD4+ and CD8+ T cells from the indicated mice. (E, G) Semi-quantitative RT–PCR for the P1 promoter- and the P2 promoter-derived gfp mRNA from the Thpokgfp and the silencer-deficient Thpokgfp:ΔS allele in CD4+ and CD8+ T cells (E), and for the P1- and P2-derived Thpok mRNA in activated CD4+ and CD8+ T cells (G). (F) Histogram showing Thpok-GFP expression in the indicated activated T cells of indicated genotypes. (H–J) Genotype analyses by Southern blot (H), relative Thpok mRNA amount (I) and semi-quantitative RT–PCR for the P1 promoter- and the P2 promoter-derived Thpok mRNA (J) in activated CD4+ and CD8+ T cells from ThpokSfl/Sfl mice transduced with control retroviral vector (GFP) or vector encoding Cre recombinase (Cre). Wedges in semi-quantitative RT–PCR assay indicate 1:3 dilution of template, except in (G) (1:5 dilutions). Data are from one of at least two independent experiments.Source data for this figure is available on the online supplementary information page. Source Data for Fig 3G [embj201347-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint We next examined the effect of deleting the Thpok silencer from fully differentiated CD8+ T cells prepared from spleen. As previously reported (Setoguchi et al, 2009), Thpok-GFP expression was detected in activated CD8+ T cells from the Thpokgfp allele, but not the Thpokgfp:ΔPE allele lacking the PE, after in vitro TCR stimulation (Figure 3F), indicating that the PE is necessary for Thpok expression in activated CD8+ T cells. Both P1 and P2 promoter-derived Thpok mRNA was detected in activated CD4+ T cells, however, activated CD8+ T cells contained only the P1-derived Thpok mRNA (Figure 3G). When these CD4+ and CD8+ T cells from ThpokSfl/Sfl mice were transduced with retroviral vectors encoding Cre recombinase, the silencer region was efficiently excised within 48 h (Figure 3H). However, the amount of Thpok mRNA in activated CD8+ T cells was not elevated after removal of the silencer (Figure 3I) and the P2 promoter-derived Thpok mRNA remained undetectable (Figure 3J). These results demonstrate that sequential epigenetic modifications at the Thpok locus during commitment to the CD8+ cytotoxic lineage in the thymus established an epigenetically repressed state that is maintained in peripheral CD8+ T cells independently of the silencer. Effect of enhanced Thpok silencer activity by increasing its copy number It is known that increasing the copy number of a cis-regulatory region can enhance its function (Herr and Gluzman, 1985). Indeed, three copies of the Thpok silencer in a reporter plasmid repressed luciferase reporter expression more efficiently than one copy in a transfection assay (Supplementary Figure 2). We then wished to examine whether and how an increase in silencer copy number at the endogenous Thpok gene would affect its expression. To this end, we have generated a Thpokgfp:3S allele, in which three tandem copies of 202–401 core silencer sequences were inserted into the Thpokgfp reporter allele by sequential gene targeting into ES cells of the Thpok+/gfp genotype (Supplementary Figure 3). Expression of GFP from the Thpokgfp:3S allele was analysed in Thpok+/gfp:3S mice, in which normal CD4+ T-cell development is supported by the half dosage of ThPOK produced from the wild-type allele. The induction of GFP in the CD69+ TCRβhi thymocyte subset from the Thpokgfp:3S allele was lower than from the control Thpokgfp allele with respect to both expression levels and the percentage of GFP+ cells (Figure 4A). Interestingly, GFP expression was lost in some CD4+ T cells but was only reduced in other CD4+ cells, thus exhibiting variegated repression rather than the uniform repression that was observed with the Thpokgfp:ΔPE allele (Figure 4A). Since reduced GFP expression was not observed in non-T cells (Figure 4A), the effect of increasing silencer copy number was likely to be CD4-lineage specific rather than a general phenomenon in any types of cells. Figure 4.Redirection of MHC class II-selected cells due to impaired Thpok expression by increase in the silencer copy number from one to three in the Thpok locus. (A) Histograms showing Thpok-GFP expression in the indicated T-cell subsets of Thpok+/gfp, Thpok+/gfp:3S and Thpok+/gfp:ΔPE mice. Numbers in the histogram indicate the percentage of GFP-positive cells, and numbers in parenthesis indicate the mean fluorescence intensity of GFP in GFP-positive cells. Notably, GFP expression in non-T cells of all three genotypes is same. (B) Dot plots showing CD4 and CD8 expression in mature (TCRβhi) thymocytes and lymph node (LN) TCRβ+ cells from mice of the indicated genotypes. Histograms show Thpok-GFP expression from the Thpokgfp allele in TCRβ+ CD8+ LN cells. Numbers in dot plots and histograms indicate the percentage of cells in each quadrant and the percentage of GFP-positive cells. (C) Dot plots showing CD4 and CD8 expression in lymph node TCRβ+ cells from mice of the indicated genotypes in the absence of cell surface expression of MHC class I molecules due to a genetic ablation of β2-microglobulin. Data are one representative of at least three independent experiments. Download figure Download PowerPoint To examine how impaired ThPOK expression due to the insertion of three copies of the Thpok silencer affects T-cell development, we have generated a Thpok3S allele by targeting the same 3S mutation into the Thpok allele (Supplementary Figure 3). In Thpok3S/3S mice, the percentages of CD4+CD8– cells were reduced both in the thymus and in peripheral T-cell pools (Figure 4B). In Thpok3S/gfp mice in which ThPOK protein was produced only from the ThPOK3S allele, the decrease in the CD4+ T-cell subset became more evident with emergence of CD8+ T cells expressing GFP. Given that GFP expression from the Thpokgfp allele is specific to MHC class II-selected cells (Muroi et al, 2008), these CD8+ GFP+ cells are likely to be re-directed MHC class II-selected cells. Indeed, when we examined the differentiation of MHC class II-selected cells in mice with no surface expression of MHC class I molecules, cells with CD4−CD8−, CD4+CD8+ and CD4−CD8+ phenotype emerged in the peripheral lymphoid organs of Thpok3S/3S and Thpok3S/gfp mice whereas those cells were absent in control mice (Figure 4C). These observations indicated that enhanced Thpok silencer activity resulting from an increase in its copy number in the Thpok gene perturbed development of CD4+ T cells via inhibiting Thpok expression in a variegated manner. Possible epigenetic Thpok silencing in CD4-lineage cells Variegated gene expression has been shown to be a characteristic of gene silencing mediated by heterochromatin-like structures (Weiler and Wakimoto, 1995). Given the variegated GFP expression from the Thpokgfp:3S allele in CD4+ T cells and the epigenetic control of the Thpok locus that results in a silencer-independent maintenance of Thpok silencing in CD8-lineage cells, we next addressed whether an epigenetic repressive state, which is maintained independently of the silencers, is similarly established at the Thpokgfp:3S allele in CD4+ helper-lineage cells. To this end, we have generated a Thpokgfp:3SFRT allele in which the three copies of the silencer can be excised upon expression of Flp recombinase (Figure 5A; Supplementary Figure 3). GFP expression from the Thpok+/gfp:3SFRT allele after in vitro TCR stimulation was still low in CD4+ T cells and was not detected in activated CD8+ T cells (Figure 5B). In addition, both P1 and P2 promoter-derived gfp mRNA was reduced in resting CD4+ T cells from Thpok+/gfp:3SFRT mice (Figure 5C). Consistent with these observations, ChIP assays demonstrated an increase and decrease in H3K27me3 and H3K4me3 marks, respectively, at the region nearby the silencer on the Thpokgfp:3SFRT allele, compared to those on the wild-type Thpok allele in the same CD4+ T cells of Thpok+/gfp:3SFRT mice (Figure 5D). Thus, chromatin structures at the Thpokgfp:3SFRT allele in CD4+ T cells were similar to those at the wild-type Thpok allele in CD8+ T cells. To examine whether GFP expression is restored if the three copies of the silencer are removed, we transduced CD4+ T cells with a retroviral vector encoding Flp recombinase to obtain a population of cells in which the FRT-flanked silencers were efficiently excised (Figure 5E). Interestingly, the mean fluorescent intensity of
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