Cell-type-specific epigenetic marking of the IL2 gene at a distal cis-
http://www.100md.com
《核酸研究医学期刊》
Division of Biology 156-29, California Institute of Technology Pasadena, CA 91125, USA
*To whom correspondence should be addressed. Tel: +1 626 395 4992; Fax: +1 626 449 0756; Email: evroth@its.caltech.edu
ABSTRACT
T-cells retain cell-type-specific programming for IL-2 inducibility through many rounds of division without being stimulated to transcribe the locus. To understand the layering of controls needed to poise this gene heritably for activation, we have used chromatin immunoprecipitation to map histone modifications across the murine IL2 locus, from –10.2 through +0.25 kb, in induction-competent and incompetent cells. In highly inducible EL4 T-lineage cells, stimulation with PMA/A23187 induced strong acetylation of histone H3 and H4, in parallel with transcriptional activation, from –4.6 through +0.25 kb. However, dimethylation of histone H3/K4 was already fully elevated across the same restricted domain before stimulation, with little change after stimulation. RNA polymerase II binding, in contrast, was only found at the known promoter region after stimulation. Similar patterns of histone modifications were seen also in normal IL-2-inducible T-lineage cells. However, neither acetylated histone H3, H4 nor dimethylated histone H3/K4 marking was detected, with or without stimulation, in expression-incompetent cells (NIH/3T3 or Scid.adh). These results identify a discrete new domain of IL2 regulatory sequence marked by dimethylated histone H3/K4 in expression-permissive T-cells even when they are not transcribing IL2, setting boundaries for histone H3 and H4 acetylation when the IL2 gene is transcriptionally activated.
INTRODUCTION
Mammalian development makes extensive use of stem-like cells for different tissues, in which a specific range of developmental potentials is programmed for future use without being overtly expressed, a condition that can persist for long periods of time (1). Epigenetic chromatin marking could be involved in establishment and long-term maintenance of the latent gene expression capabilities that must exist in these cells. However, for most mammalian systems of developmental gene regulation in which chromatin configurations have been studied closely, changes in potential for expression of a gene have been tightly linked to changes in actual transcriptional activation of that gene, making the mechanisms difficult to disentangle. Chromatin modifications, such as acetylation of histones H3 and H4, and methylation of H3 on Lys-4 (H3/K4), are known to be correlated with ‘open’ chromatin, but in most cases studied this is also transcriptionally active chromatin . Epigenetic marking strictly correlated with latent activity has been analyzed only rarely (4–6). Here, we propose that the murine IL2 gene can shed light on this more general problem in developmental molecular biology.
IL-2 is one of the important cytokines produced by mature T-cells in response to antigenic stimuli. IL2 gene regulation is subject to dual control, since it acutely depends on combinatorial signaling through particular combinations of pathways, and can only be activated in a very limited spectrum of cell types . Although strongly induced by combinations of Ca2+, Ras/MAP kinase and costimulatory signals, IL-2 transcription is transient and returns to an ‘off’ state in expression-competent cells between bouts of stimulation. For the most part, competence to express IL2 is restricted to T-lymphoid cells and a few other hematopoietic cell types, and its expression is observed in certain immature stages (10,11) as well as in the antigen responses of mature T-cells. The regulatory basis for the activation dependence of IL2 gene expression in T-cells is well-understood, with key roles for NF-AT, NF-B/c-Rel and AP-1 factors interacting with constitutive regulators such as Oct-1 in a 300 bp promoter-proximal region . Additional regulators such as HMG box factors and the Ets factor GABP may also play a role, through highly conserved sites just 100–200 bp further upstream (14,15). The binding of these factors in response to activation is correlated with changes in DNA methylation at a series of sites extending from the promoter to about –800 bp (16). However, the basis for IL2 cell-type specificity is still obscure, since all of the known transcription factors used for its induction are expressed broadly in many cell types that cannot express the gene. Meanwhile, the great majority of na?ve T-cells retain the potential to activate IL2 expression even after long periods when the gene is transcriptionally silent. The cell-type-specific regulation of IL-2 presents one of the clearest known cases of dissociation between developmentally programmed competence to express a gene, and actual expression of that gene for the great majority of a cell's lifetime.
Transgenic studies have shown that the highly conserved 600 bp region that contains the proximal promoter-enhancer is not enough for IL2 regulatory sequence activity in transgenic mice (17,18), raising the possibility that additional positive regulatory functions may be encoded elsewhere. Addition of less-characterized sequences to –2.0 to –2.7 kb (with respect to the IL2 transcriptional start site) permit correct expression in a fraction of transgenic founder lines (19,20). While ectopic expression of these constructs is rare, the locus appears to require additional sequences to counteract silencing. Experiments with hybrid regulatory sequence constructs show that one of the functions needed can be supplied by the locus control region of the human CD2 gene (21). We have therefore hypothesized that the basis of cell-type specificity might reside in a discrete regulatory function that controls IL2 chromatin accessibility (7,14). If so, then the sequences through which this effect is mediated could be expected to improve the frequency and efficiency of expression of IL2 regulatory sequence transgenes if these sequences were included.
Previously, we reported that a GFP transgene driven with 8.4 kb of IL2 5'-flanking sequence (8.4 kb IL2-GFP) displayed efficient, lineage-specific expression in transgenic mice, much improved in frequency and fidelity as compared to a 2.0 kb IL2-GFP transgene (20). Thus, the additional 6.4 kb upstream sequence contains important regulatory regions for conferring accessibility to induction on the IL2 gene. Here, we use analyses of the chromatin structure in these IL2 upstream regions to investigate the nature of the regulatory function that they confer.
To analyze features of the distal sequence, we performed chromatin immunoprecipitation (ChIP) assays to define the nature and extent of any associated histone modifications. Here, we report significant levels of histone acetylation (AcH3, AcH4) and histone H3 lys-4 dimethylation (diMeH3/K4) in a discrete region from –4.6 kb through the promoter and first exon of the IL2 gene in IL-2 producing T-cells. Whereas acetylation was seen only in cells transcribing the gene in response to stimulation, diMeH3/K4 is maintained at a high level across the same region in cells competent to express IL2, even before stimulation. These results indicate the presence of transcriptionally permissive state which is marked by diMeH3/K4 without histone acetylation over a discrete 5'-flanking domain of the IL2 gene, and which is restricted to competent IL-2-inducible cells.
MATERIALS AND METHODS
Cell culture and stimulations
EL4.F4 cells (22,23), Scid.adh cells (24), NIH/3T3 cells and splenocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone, South Logan, UT), 50 μM 2-mercaptoethanol, 0.1 mM MEM non-essential amino acids (Invitrogen, Carlsbad, CA), 10 mM HEPES, 1 mM sodium pyruvate (Sigma–Aldrich, St Louis, MO), 2 mM L-glutamine and penicillin/streptomycin.
Cells were stimulated for 3 h with 10 nM PMA and 180 nM A23187 (Sigma–Aldrich). Cell concentrations were 106 cells/ml of non-adherent cells and were semi-confluent of adherent cells. For stimulations of TCR-negative SCID thymocytes, IL-1 (10 ng/ml; Biosource, Camarillo, CA) was also required in the stimulation cocktail (25–27), but IL-1 has no effect on responses of CD4+ CD8+ cortical thymocytes, as previously described (28).
Purification of B220– splenocytes and thymocytes from SCID and MHC-deficient mice
Red-blood cell depleted splenocytes from C57BL/6 mice were stained with biotin-conjugated anti-B220 Ab (Immunotech, Marseille, France), and then incubated with streptavidin-conjugated MACS microbeads (Miltenyi Biotech, Auburn, CA). Cells were passed through MACS columns to remove B220+ cells. Purity of separated cells was determined by staining with FITC-conjugated anti-B220 Ab (BD PharMingen, San Diego, CA). Thymocytes were prepared by mincing the tissue followed by filtration through a wire mesh.
Chromatin immunoprecipitation assay
Antibodies for ChIP assays were purchased from the following companies. Anti-acetyl-histone H3 (06-599), anti-acetyl-histone H4 (06-866) and anti-dimethyl-histone H3 (Lys-4) (07-030) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Pol II (N-20) (sc-899) and normal rabbit IgG (sc-2027) were from Santa Cruz Biotechnology (Santa Cruz, CA).
ChIP assays were performed by the protocol of Upstate Biotechnology with modifications. Briefly, samples of 2 x 107 cells were subjected to protein–DNA crosslinking with formaldehyde at a final concentration of 0.4% for 10 min at room temperature. Cells were lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris–HCl, pH 8.0) and DNA was sheared by sonication (Labsonic 1510, B. Braun, Melsungen, Germany) at 100 W for 1 min, to lengths between 200 and 1000 bp.
One hundred microliters of sonicated sample was kept as an input sample, and the remainder was diluted with 8.1 ml of dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.0 and 167 mM NaCl). Diluted samples were precleared with 100 μl of salmon sperm DNA/protein A agarose (Upstate Biotechnology) for 1 h, then divided into three tubes (3 ml each).
Ten micrograms of antibodies were added to each tube and incubated for 3–4 h. After that, 60 μl of salmon sperm DNA/protein A agarose was added and incubated overnight at 4°C. Agarose was washed once with 1 ml of low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0 and 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0 and 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate and 1 mM EDTA), and twice with TE buffer (pH 8.0). Immune complexes were eluted twice with 250 μl of elution buffer (1% SDS and 0.1 M NaHCO3). Protein–DNA crosslinking was reversed by incubation at 65°C for 4 h with 0.2 M NaCl, and then protein was digested at 45°C for 1 h in the presence of 10 mM EDTA (pH 8.0), 40 mM Tris–HCl (pH 6.5) and 40 μM proteinase K. The DNA was purified by phenol–chloroform extraction followed by ethanol precipitation.
Quantitative PCR analysis
Quantitations of chromatin immune precipitates were performed by real-time PCR using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Primers were designed by the Primer3 website (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (29) to amplify 100–250 bp amplicons. PCR analyses were performed in 25 or 30 μl reactions with SYBR Green (PE Applied Biosystems). All primer sets used gave no signal (CT > 40) in control reactions without template. Dissociation curve analyses showed that single products with expected Tm values were generated by each primer set. A second peak of lower Tm was detected only in products of one primer set (–8.9 to –8.7), or as a shoulder in products of one other set (–3.2 to –3.0).
Standard curves were determined for each primer set by dilution of input DNA in a 0.1–100 ng range, and only primer sets yielding slopes close to the theoretical values for 1.9- to 2.0-fold amplification per cycle were used. The amounts of each sequence in the input and precipitated DNA were calculated from the cycle thresholds (CT) for each primer set by using the standard curves. Relative units recovered for each primer set were determined by dividing the calculated amounts of precipitated sequence by the amount of that sequence in the input DNA. Due to lower cell recovery of primary thymocytes, results in Figure 7 were further normalized by the results of the S16 ribosomal protein gene.
Figure 7 Dimethylated histone H3/K4 association with IL2 5'-flanking region is correlated with IL-2 inducibility in immature thymocytes: ChIP analysis of the IL2 locus in thymocytes from SCID and MHC-deficient mice. Cells from MHC-deficient mice, >95% CD4+ CD8+ TCR-low, were cultured with or without PMA/A23187 for 3 h. Cells from SCID mice, >95% CD4– CD8– TCR-negative, were cultured with or without stimulation for 2 h, and IL-1 was also added as indicated. To correct for differences in efficiency of immune precipitation between these samples due to the extensive differences in cell physiology, ChIP analysis results for diMeH3/K4 at the IL2 locus for each sample were normalized by results at the S16 ribosomal protein locus. Two independent experiments were done with similar results.
RNA purification and real-time quantitative RT–PCR
Total RNA was extracted from the cells by RNAzol (Leedo Medical, Houston, TX) following the manufacturer's instructions. One microgram of RNA was treated with RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37°C. After that, DNase was inactivated by the incubation for 5 min at 75°C. Then, first-strand synthesis reactions were performed with Super-script II reverse transcriptase (Invitrogen, Carlsbad, CA) and 500 ng oligo dT primers (Pharmacia, Uppsala, Sweden) following standard protocols.
Real-time PCR was performed by using ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). Taqman probes and primers for murine IL-2 (FAM labeled) and GAPDH (VIC labeled) were purchased from PE Applied Biosystems. To normalize the samples, control GAPDH CT values were subtracted from IL-2 CT values of each sample (CT). Then, CT of the unstimulated NIH/3T3 sample (reference sample) was subtracted from CT of each sample (CT). Relative levels of IL-2 mRNA were calculated as 2–CT.
RESULTS
Stimulation with PMA/A23187 induces histone acetylation at the IL2 locus in IL-2 producing cells only
Several lines of evidence suggested that regulatory elements important for cell-type-specific accessibility of the IL2 gene might reside on the 5' side of the well-characterized promoter-enhancer region. First, a series of T-cell-specific DNase I hypersensitive sites were found between –10 and –2 kb from the transcriptional start site of the IL2 gene, including a cluster of cell-type-specific sites from about –2.8 to –4.5 kb which were hypersensitive even in resting cells (20). Second, a GFP reporter transgene controlled by an extended 8.4 kb upstream region of the IL2 gene, which includes most of the new hypersensitive sites, showed more efficient, lineage-specific expression in transgenic mice than a GFP transgene controlled by a 2.0 kb IL2 regulatory region, which does not include any of the new hypersensitive sites (20). Most recently, comparative genomics have made it possible to identify three islands of mouse–human sequence similarity upstream of the IL2 gene, as indicated in Figure 1 (for full three-way alignment of murine, human and rat sequences, see Supplementary Figure 1). Two of these islands lie within the region included in the 8.4 kb IL2 promoter transgene and one lies just beyond it.
Figure 1 Diagrams of DNase hypersensitive sites and primer positions on the IL2 locus. DNase hypersensitive sites in stimulated and unstimulated purified splenic T-cells are shown by arrows (). Regions of high homology with an upstream sequence in the human IL2 gene were determined by Blast search (H). Putative matrix attachment region (MAR) and a region of high homology with an intronic sequence in the Bruton's thymidine kinase gene (Btk) are also indicated. For full alignment of these murine IL2 sequences with human and rat orthologues using FamilyRelations and SeqComp software (46), see Supplementary Figure 1. Bottom: the functional importance of these sequences is demonstrated by the distinct behavior of IL2 regulatory sequence transgene constructs previously reported by this group (20). The sequences to –8.4 kb, but not the sequences to –2.0 kb, are sufficient to direct IL2-like expression of the reporter gene in a high frequency of independent integration sites (20).
Although indicating some functional significance for the region between –2.0 and –8.4 kb, the transgenesis results did not determine whether the sequences in this region act as auxiliary stimulation-dependent enhancers, amplifying the response upon induction, or whether they mark the locus stably as accessible to induction by a separate mechanism uncoupled from transcription. To determine which regions of the extended IL2 upstream region might participate in control of IL-2 expression, we used ChIP with quantitative real-time PCR quantitation. Although regions enriched for simple-sequence repeats needed to be excluded from analysis, we designed 17 primer sets to amplify segments from the first transcribed exon up to –10 kb of the IL2 gene (Figure 1 and Table 1), including amplicons bridging all three of the conserved regions. To correlate chromatin structure with competence to express IL-2, the murine thymoma cell line EL4, the immature pro T-cell line Scid.adh and the non-hematopoietic cell line NIH/3T3 were subjected to ChIP assay with or without PMA/A23187 stimulation. Of these, EL4 is the only one which expresses IL-2 in response to stimulation (Figure 2A). However, in unstimulated EL4 cells, the IL2 gene is virtually silent, with levels of RNA >105-fold lower than after 3 h of stimulation (note log scales in Figure 2).
Table 1 Sequences of the primers
Figure 2 Quantitative RT–PCR of IL-2 mRNA induction in different cell types. RNA was extracted from the indicated cell types, either unstimulated or stimulated with PMA/A23187 for 3 h. IL-2 mRNA levels were determined in the samples by real-time PCR following reverse transcription, and normalized to equal GAPDH levels. Units of IL-2 mRNA expression were then calculated as 2–CT relative to an unstimulated NIH/3T3 sample (20 = 1). (A) Cell lines NIH/3T3 (fibroblasts, nonhematopoietic), Scid.adh (pro-T-like thymic lymphoma), and EL4 (mature T-cell thymic lymphoma), compared with primary B220– splenic lymphocytes (mature T-cells). Levels of IL-2 in stimulated EL-4 and B220– splenic T cell samples are within 2- to 3-fold of the levels of GAPDH (data not shown). (B) Primary immature pro-T cells from SCID thymus, compared with primary CD4+ CD8+ TCR-low cortical thymocytes from MHC-deficient thymus. For SCID thymocytes, stimulation was only for 2 h, in the presence or absence of IL-1 as indicated. Levels of IL-2 in maximally stimulated SCID thymocytes are 1% of the level of GAPDH. Note that all results are displayed on a log scale. ND = not determined.
In resting EL4 cells, acetylation of histones H3 and H4 was not substantially elevated across the IL2 locus, and the recovery of all IL2 flanking sequences was low compared to the positive control. However, acetylation was greatly increased after 3 h of stimulation with PMA/A23187, in a defined interval across the sequences from –4.6 through +0.25 kb. By this time point, specific transcription factor occupancy of target sites in the enhancer is already maximal, as we have reported previously (30). The acetylated region included two islands of mouse/rat/human conserved sequence, at about –2.2 and at about –3.9 kb, but excluded the third, more distal conserved region at about –8.6 kb and all other regions tested out to –10.2 kb.
Unexpectedly, within the region from –4.6 through +0.25 kb, the sequence that was least enriched in acetylated H3 and H4 immune precipitates after stimulation was the amplicon from the IL2 proximal promoter region itself (–0.24 to –0.05 kb) (Figure 3A; top). Since the proximal promoter is known to be a core region for IL-2 transcription factor binding which undergoes dramatic changes in nuclease accessibility upon stimulation (14,30–34), the regulatory protein complexes that are assembled there upon induction might exclude nucleosomes or mask epitopes of acetylated histone. A similar result has been reported for active promoters in the ?-globin and prostate-specific antigen loci (6,35). In accordance with our results, a recent report has demonstrated directly that stimulation causes specific loss of histone protein from the proximal promoter region of the IL2 gene (36).
Figure 3 Histone acetylation pattern of the IL-2 gene. (A) Acetylated histone H3 and H4 of the IL-2 gene were analyzed with quantitative ChIP assay. Results of EL4, Scid.adh and NIH/3T3 cells are shown. (B) Acetylated histone H3 and H4 on the promoter of S16 ribosomal protein. The results are shown as mean ± SD of three or four independent experiments. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
Acetylation at the IL2 locus appears to be regulated normally by an equilibrium between histone acetyltransferase and histone deacetylase (HDAC) activities, because exposure of the cells to HDAC inhibitor trichostatin A increases acetylation of H3 and especially H4, even in the absence of stimulation (Supplementary Figure 2A). This increased acetylation is not associated with transcriptional induction; in fact, trichostatin A decreases the induction of IL-2 in response to stimulation under the conditions used here (37) (Supplementary Figure 2B). Even so, most increased acetylation induced by trichostatin A is seen in the region between about –5 kb and the promoter region, where the stimulation-inducible acetylation occurs (Supplementary Figure 2A).
The IL2-nonproducing cell lines, Scid.adh and NIH/3T3, maintained very low levels of acetylation of the IL2 gene flanking sequences with no increase upon stimulation (Figure 3A; middle and bottom); however, the acetylation of a sequence from the S16 ribosomal protein promoter was comparable between all these three cell lines (Figure 3B). Controls without antibody or with normal IgG precipitation did not detectably precipitate IL2 sequences in any of these cell lines (Supplementary Figure 3). Thus, there is a specific IL2 upstream region extending to –4.6 kb, i.e. >4 kb beyond the well-characterized proximal promoter-enhancer, which is subject to histone acetylation only upon stimulation and only in IL-2 induction-competent cells.
Dimethylation of histone H3/K4 at the IL2 upstream region before stimulation in EL4 cells
To assess whether there is any other kind of modification at the IL2 locus that might mark it as accessible, we monitored dimethylation of histone H3/K4, which is also associated with active chromatin and often accompanies acetylation (6,38–41). Like AcH3 and AcH4, diMeH3/K4 was highly associated with sequences from –4.6 through +0.25 kb in EL4 cells after stimulation (Figure 4A; top). However, unlike acetylation, a significant level of diMeH3/K4 was observed from –4.6 through +0.25 kb before stimulation as well, comparable to its level after stimulation. In unstimulated EL4 cells, this modification could also be seen to extend through the proximal promoter (–0.24 to –0.05 kb), although it appeared somewhat decreased after stimulation. In the Scid.adh and NIH/3T3 cells, in contrast, which cannot express IL-2, no association of diMeH3/K4 with the IL2 gene was observed, with or without stimulation (Figure 4A; middle and bottom). These differences were specific to the IL2 gene, because diMeH3/K4 modification of chromatin associated with the S16 ribosomal protein promoter was comparable in the three cell lines (Figure 4B).
Figure 4 Dimethylated histone H3 at lys-4 is observed before stimulation in EL4 cells. (A) ChIP analyses were performed for diMeH3/K4 on the IL2 locus of EL4, Scid.adh and NIH/3T3 cells. DiMeH3/K4 on the promoter of S16 ribosomal protein is shown in (B). The results are shown as mean ± SD of three independent experiments. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
RNA polymerase II is not bound to the distal region of the IL2 gene
Both AcH3, H4 and diMeH3/K4 modifications extend >4 kb from the known promoter-enhancer of the IL2 gene. We considered possible explanations for the relatively sharp 5' border and the broad extension of the modified region all the way through the promoter-enhancer. In some cases, pol II itself is recruited to a distal regulatory region (42), from which it can spread acetylated histone in a 5' to 3' polar fashion as it moves along the gene . Therefore, we analyzed pol II binding on the IL2 gene in EL4 cells by ChIP assay. As expected, pol II immune precipitation of stimulated EL4 cell samples enriched for promoter proximal sequences (amplified with primers from –0.24 to –0.05 kb), with further enrichment of sequences immediately downstream of the transcriptional start site (+0.06 to +0.25 kb). Pol II recruitment was correlated with transcriptional activation, as little or no enrichment was seen in samples from unstimulated cells. However, even in stimulated EL4 cells, only minimal binding was found from –4.6 through +0.25 kb, or in more distal 5' sequences (20% of promoter level) (Figure 5). Thus, no evidence for specific pol II recruitment was found in any of the upstream regions examined.
Figure 5 Binding of RNA polymerase II on the IL2 locus. ChIP analyses were performed for pol II binding on the IL2 locus of EL4 cells. The results are shown as mean ± SD of three independent experiments. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
Histone modifications at the IL2 locus in primary T-cells resemble those in EL4 cells
To determine whether the distinctive patterns of chromatin modifications found in EL4 cells could be relevant to normal T-cell control of IL-2 expression, we examined three developmentally distinct populations of primary T-lineage cells: mature, peripheral splenic T-cells; cortical TCRlow CD4+ CD8+ thymocytes (>95% of cells in MHC-deficient thymus); and immature TCR– SCID thymocytes. As previously reported (10,25,44), both mature peripheral T-cells and SCID thymocytes can be stimulated to express IL-2 (Figure 2A, ‘B220-’; Figure 2B, ‘scid thy’), but the cortical thymocytes, although developmentally intermediate, cannot (Figure 2B, ‘MHC–/–’).
ChIP assays for AcH3 and diMeH3/K4 were performed on samples of stimulated or unstimulated peripheral T-cells, which were highly enriched from C57BL/6 mouse spleens by magnetic bead depletion of B220+ cells, as shown in Figure 6A. In these cells, AcH3 was increased from –4.6 kb through the IL-2 promoter after stimulation, while diMeH3/K4 was high from –4.6 kb to downstream both before and after stimulation (Figure 6B, B220– cells). These patterns in stimulated and unstimulated primary T-cells closely match those in EL4 cells. The maximum increase of AcH3 upon stimulation in B220– cells was only 4- to 5-fold, as compared to >10-fold in EL4 cells (Figure 3A; top), but this may be attributed to known quantitative differences between the frequencies of IL-2 induction in unstimulated primary T-cells and in EL4 cells (Figure 2). The fraction of primary T-cells expressing IL-2 mRNA in a particular response is never as high as the fraction of EL4 cells, which can approach 80% (E. V. Rothenberg, unpublished data), whereas the background level of IL-2 mRNA expression in unstimulated B220– cells ex vivo is often higher than that of unstimulated EL4 cells, possibly due to a low frequency of cells that received some stimulation in vivo already (45). Thus, these results indicate that chromatin modification of the IL2 locus from –4.6 kb through the promoter region may be a stable characteristic of normal mature T-cells.
Figure 6 Pattern of acetylated histone H3 and dimethylated histone H3 at lys-4 at the IL2 locus in an enriched population of mature splenic T-cells. B220+ cells were depleted from C57BL/6 mouse splenocytes by magnetic columns (A), and the remaining B220– cells (T-enriched) were subjected to ChIP assay (B). Two independent experiments were done with similar results. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
Dimethyl H3/K4 marking correlates with competence to express in immature T-lineage cells
To determine how early in T-lineage development this chromatin modification might be detectable, SCID thymocytes were analyzed. Although SCID thymocytes cannot rearrange or express T-cell receptor genes and thus remain immature, they are inducible for IL-2 expression (Figure 2B) (up to 20% of the cells capable of expressing IL-2 mRNA in immediate response to stimulation in C.B17-SCID) (26,27). In two independent experiments, even without stimulation, SCID thymocytes showed high association of diMeH3/K4 with the region from –4.6 kb through the promoter region (Figure 7, top graph). Thus, both immature and mature T-cells specifically modify chromatin in a defined region upstream of the IL2 promoter.
The shared pattern of epigenetic marking in mature and very immature populations could be strictly correlated with competence to express IL-2, or it could be a lineage marker for all normal T-lineage cells. To distinguish these possibilities, we analyzed intermediate CD4+ CD8+ TCR-low thymocytes, which constitute >95% of the cells in the thymus of major histocompatibility complex-deficient mice (MHC–/–). These cells are more advanced than SCID thymocytes developmentally, but have lost or suspended all competence to express IL-2 (10,28,44). As shown in Figure 7 (bottom graph), these cells show much lower levels of diMeH3/K4 modification over the IL2 flanking sequences, relative to S16 positive control sequences, than EL-4 cells, mature T-cells or SCID thymocytes. The pattern is also flat, with little if any increase in modification over the region from –4.6 kb to the promoter. Thus, diMeH3/K4 modification over the region from –4.6 kb to the IL2 promoter is not universal among all T-lineage cells, but is tightly linked to the ability to express IL-2.
In summary, as shown in Figure 8, both diMeH3/K4 and AcH3 types of modifications of the IL2 flanking sequences are cell-type specific. Both are restricted to T-lineage cells that are competent to express IL-2 and not found in fibroblasts or T-lineage cells that lack this competence. However, histone acetylation is tightly linked with the recruitment of active transcription complexes to the IL2 promoter. Only diMeH3/K4 marking is a stable differentiated characteristic of these competent cells that is retained at the locus as long as they retain their developmental state, in vivo and in vitro, even in the absence of activating signals and in the complete absence (<10–5 of the induced levels) of IL-2 mRNA expression.
Figure 8 Changes of histone modifications at the IL2 gene during T-cell differentiation. The correlation between IL-2 expression and histone modifications from –4.6 through +0.25 kb of the IL2 gene is summarized.
DISCUSSION
The basis for competence to induce transcription of the IL2 gene in particular cell types has long been obscure, in contrast to the detailed knowledge that has been accumulated about the mechanism that actually triggers transcription of this locus in competent cells. Previously, we showed that sequences of the IL2 gene between –2 kb and –8.4 kb (extended 5'-flanking sequence) could be involved in such a function, since they greatly enhance the likelihood of expression of an IL2 promoter-GFP transgene in the correct cells (20). The function conferred was not defined, however, and the sequences involved in the function were not mapped. Here, we report that a particular segment of the extended 5'-flanking sequence is a site for epigenetic marking by histone H3/K4 dimethylation that occurs only in IL-2 expression-competent cells. This modification is cell-type specific, tightly linked with competence of the IL2 locus to be induced by stimulation, and coincides with the region in which increased AcH3 and AcH4 modifications occur when transcription is activated (Figures 3, 4 and 6). However, this H3/K4 methylation is uncoupled from detectable IL2 transcription, from pol II binding, and from histone acetylation, and it persists independently of stimulation. Our data provide the first direct evidence for the existence of a discrete, developmentally controlled mechanism that may control cell-type-specific accessibility of the IL2 locus, working through different sequences and different trans-acting factors than those that activate transcription of the gene in response to induction.
The relationship between H3/K4 methylation and IL-2 expression supports the model that sequences between –2 and –8.4 kb can have a discrete role in conferring cell-type-specific accessibility on this gene that is separable from any enhancer activity. Indeed, the 8.4 kb IL2 regulatory region does not drive reporter gene expression any better than the 2.0 kb regulatory region, when tested in transient transfection experiments (20). Two groups of DNase HS sites, centered around different mouse/human homology blocks, suggest that the upstream region may include two types of function. The distal portion of the H3/K4 methylation-marked region, from –4.6 to about –2.8 kb, includes the group of DNase I hypersensitive sites which are most pronounced in resting, not activated, T-cells. Activation of IL-2 transcription induces the appearance of new DNase HS sites, including a cluster around the mouse/human homology block from about –2.2 to –2.3 kb as well as in the promoter-proximal region (14,20). This –2.2 kb region may indeed have cryptic enhancer-like activity. However, activation if anything reduces the prominence of the major DNase hypersensitive site around –4.5 kb (20), at the distal boundary of the MeH3/K4 marked domain. A clear inference would be that any transcription factors binding to regulatory sites in the –2.8 to –4.6 kb upstream region would be distinct from these stimulation-activated transcription factors.
Identification of the critical regulatory elements in this interval is impeded by the remarkably poor sequence conservation between human and murine sequences beyond –2.3 kb in the mouse sequence. When the human, mouse and rat IL2 flanking sequences are aligned (46) (Supplementary Figure 1), the only well-conserved sequence from –2.8 to –4.6 kb is a 64 bp stretch at about –3.9 kb in the murine gene (in the human gene, a LINE element insertion shifts this alignment further upstream). This conserved element should thus provide a useful point of departure for identification of the factors which control the establishment or maintenance of the MeH3/K4 marked domain.
Only minimal histone modifications were seen further upstream of –4.6 kb, with or without stimulation. It remains to be seen whether known boundary-forming transcription factors such as CCCTC-binding factor (CTCF) or the bHLH-ZIP ‘Upstream Factor’ (USF) play a role in setting these limits. Interestingly, there are several E-box motifs which might bind USF between –8.0 and –8.4 kb, and a predicted double binding site around –6.2 kb. A recent article shows that USF binds to the chicken ?-globin 5'HS4 insulator, where it protects the gene from chromosomal silencing by recruitment of histone modification factors (47). It may play a role to keep the IL2 gene accessible by the same mechanisms. Closer to the region where we see discontinuities in IL2 chromatin structure, there is an unusually dense clustering of seven CCCTC motifs between –4.5 and –5.3 kb from the transcription start site. Although functional target sites of CTCF can be difficult to predict based on sequence alone (48), these sites will clearly be of interest for further investigation.
The mechanism maintaining the MeH3/K4-marked IL2 5'-flanking region in unstimulated cells is clearly distinct from the highly coordinated assemblies of transcription factors, histone acetyltransferases and RNA pol II which are seen associated with ‘open chromatin’ in other sites (42,43,49,50). In the case of the IL2 gene, a stable pattern of H3/K4 methylation appeared to be maintained even in the absence of any substantial pol II binding to the region between –4.6 kb and the promoter. The minimal level of binding that was seen could be consistent with co-precipitation by interaction of distal regulatory regions with the proximal promoter via looping, rather than with direct binding. The IL2 gene thus joins a small number of cases where diMeH3/K4 is associated with accessibility to transcription before transcription itself is induced. In some cases, this modification appears to demarcate the boundaries of a region within which transcription and/or DNA recombination can occur (40,51). Recently, a two-step chromatin modification sequence, with diMeH3/K4 modification preceding histone acetylation, was shown in a variety of genes activated during hybridization between ES cells and somatic cells (52). Even if this sequence is used for regulation of many genes, the regulatory style may differ for each gene. For example, in the activation of HNF-4 gene concomitant with the enterocyte differentiation, diMeH3/K4 is restricted to the promoter region through differentiation while acetylated histones develop from the distal enhancer toward the promoter (49) And at the IgH locus in pro B-cells, peaks of diMeH3/K4 are observed at the borders of active DJ regions and inactive V and C regions (51). For immune regulatory cytokines, such as IL-2, maintaining a transcriptionally permissive stage which is marked by long-range diMeH3/K4 modification alone may be important to preserve the potential for immediate gene activation in response to precisely calibrated external stimuli.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
The authors would like to thank Dr Mary Yui and members of the Rothenberg group for helpful discussions, and the staff of the Caltech Office of Laboratory Animal Research for expert care of the different mouse strains. This work was supported by USPHS grant AG13108. Funding to pay the Open Access publication charges for this article was provided by AG13108.
REFERENCES
Reya, T., Morrison, S.J., Clarke, M.F., Weissman, I.L. (2001) Stem cells, cancer, and cancer stem cells Nature, 414, 105–111 .
Fischle, W., Wang, Y., Allis, C.D. (2003) Binary switches and modification cassettes in histone biology and beyond Nature, 425, 475–479 .
Marmorstein, R. (2001) Protein modules that manipulate histone tails for chromatin regulation Nature Rev. Mol. Cell Biol., 2, 422–432 .
Guo, L., Hu-Li, J., Zhu, J., Watson, C.J., Difilippantonio, M.J., Pannetier, C., Paul, W.E. (2002) In TH2 cells the Il4 gene has a series of accessibility states associated with distinctive probabilities of IL-4 production Proc. Natl Acad. Sci. USA, 99, 10623–10628 .
Yamashita, M., Ukai-Tadenuma, M., Miyamoto, T., Sugaya, K., Hosokawa, H., Hasegawa, A., Kimura, M., Taniguchi, M., DeGregori, J., Nakayama, T. (2004) Essential role of GATA3 for the maintenance of type 2 helper T (Th2) cytokine production and chromatin remodeling at the Th2 cytokine gene loci J. Biol. Chem., 279, 26983–26990 .
Bottardi, S., Aumont, A., Grosveld, F., Milot, E. (2003) Developmental stage-specific epigenetic control of human ?-globin gene expression is potentiated in hematopoietic progenitor cells prior to their transcriptional activation Blood, 102, 3989–3997 .
Rothenberg, E.V. and Ward, S.B. (1996) A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin-2 gene regulation Proc. Natl Acad. Sci. USA, 93, 9358–9365 .
June, C.H., Ledbetter, J.A., Lindsten, T., Thompson, C.B. (1989) Evidence for the involvement of three distinct signals in the induction of IL-2 gene expression in human T lymphocytes J. Immunol., 143, 153–161 .
Powell, J.D., Ragheb, J.A., Kitagawa-Sakakida, S., Schwartz, R.H. (1998) Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy Immunol. Rev., 165, 287–300 .
Chen, D. and Rothenberg, E.V. (1993) Molecular basis for developmental changes in interleukin-2 gene inducibility Mol. Cell. Biol., 13, 228–237 .
Wang, H., Diamond, R.A., Yang-Snyder, J.A., Rothenberg, E.V. (1998) Precocious expression of T-cell functional response genes in vivo in primitive thymocytes before T-lineage commitment Int. Immunol., 10, 1623–1635 .
Jain, J., Loh, C., Rao, A. (1995) Transcriptional regulation of the IL2 gene Curr. Opin. Immunol., 7, 333–342 .
Serfling, E., Avots, A., Neumann, M. (1995) The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation Biochim. Biophys. Acta, 1263, 181–200 .
Ward, S.B. and Rothenberg, E.V. (1998) Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions Nucleic Acids Res., 26, 2923–2934 .
Avots, A., Hoffmeyer, A., Flory, E., Cimanis, A., Rapp, U.R., Serfling, E. (1997) GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction Mol. Cell. Biol., 17, 4381–4389 .
Bruniquel, D. and Schwartz, R.H. (2003) Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process Nature Immunol., 4, 235–240 .
Brombacher, F., Sch?fer, T., Weissenstein, U., Tschopp, C., Andersen, E., Bürki, K., Baumann, G. (1994) IL-2 promoter-driven lacZ expression as a monitoring tool for IL-2 expression in primary T-cells of transgenic mice Int. Immunol., 6, 189–197 .
Reed, W.A., Elzer, P.H., Enright, F.M., Jaynes, J.M., Morrey, J.D., White, K.L. (1997) Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropin-class lytic peptide in transgenic mice and subsequent resistance to Brucella abortus Transgenic Res., 6, 337–347 .
Minasi, L.E., Kamogawa, Y., Carding, S., Bottomly, K., Flavell, R.A. (1993) The selective ablation of interleukin 2-producing cells isolated from transgenic mice J. Exp. Med., 177, 1451–1459 .
Yui, M.A., Hernandez-Hoyos, G., Rothenberg, E.V. (2001) A new regulatory region of the IL-2 locus that confers position-independent transgene expression J. Immunol., 166, 1730–1739 .
Saparov, A., Wagner, F.H., Zheng, R., Oliver, J.R., Maeda, H., Hockett, R.D., Weaver, C.T. (1999) Interleukin-2 expression by a subpopulation of primary T-cells is linked to enhanced memory/effector function Immunity, 11, 271–280 .
Novak, T.J., White, P.M., Rothenberg, E.V. (1990) Regulatory anatomy of the murine interleukin-2 gene Nucleic Acids Res., 18, 4523–4533 .
Novak, T.J., Chen, D., Rothenberg, E.V. (1990) Interleukin 1 synergy with phosphoinositide pathway agonists for induction of interleukin-2 gene expression: molecular basis of costimulation Mol. Cell. Biol., 10, 6325–6334 .
Carleton, M., Ruetsch, N.R., Berger, M.A., Rhodes, M., Kaptik, S., Wiest, D.L. (1999) Signals transduced by CD3, but not by surface pre-TCR complexes, are able to induce maturation of an early thymic lymphoma in vitro J. Immunol., 163, 2576–2585 .
Rothenberg, E.V., Diamond, R.A., Pepper, K.A., Yang, J.A. (1990) Interleukin-2 gene inducibility in T-cells prior to T-cell receptor expression: changes in signaling pathways and gene expression requirements during intrathymic maturation J. Immunol., 144, 1614–1624 .
Rothenberg, E.V., Chen, D., Diamond, R.A. (1993) Functional and phenotypic analysis of thymocytes in SCID mice: evidence for functional response transitions before and after the SCID arrest point J. Immunol., 151, 3530–3546 .
Diamond, R.A., Ward, S.B., Owada-Makabe, K., Wang, H., Rothenberg, E.V. (1997) Different developmental arrest points in RAG2–/– and SCID thymocytes on two genetic backgrounds: developmental choices and cell death mechanisms before TCR gene rearrangement J. Immunol., 158, 4052–4064 .
Rothenberg, E.V. and Diamond, R.A. (1994) Costimulation by interleukin-1 of multiple activation responses in a developmentally restricted subset of immature thymocytes Eur. J. Immunol., 24, 24–33 .
Rozen, S. and Skaletsky, H.J. Primer3 on the WWW for general users and for biologist programmers In Krawetz, S. and Misener, S. (Eds.). Bioinformatics Methods and Protocols: Methods in Molecular Biology, Totowa, NJ Humana Press pp. 365–386 .
Garrity, P.A., Chen, D., Rothenberg, E.V., Wold, B.J. (1994) IL-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors Mol. Cell. Biol., 14, 2159–2169 .
Brunvand, M.W., Krumm, A., Groudine, M. (1993) In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T-cells Nucleic Acids Res., 21, 4824–4829 .
Chen, D. and Rothenberg, E.V. (1994) Interleukin-2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles J. Exp. Med., 179, 931–942 .
Rao, S., Procko, E., Shannon, M.F. (2001) Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene J. Immunol., 167, 4494–4503 .
Attema, J.L., Reeves, R., Murray, V., Levichkin, I., Temple, M.D., Tremethick, D.J., Shannon, M.F. (2002) The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation J. Immunol., 169, 2466–2476 .
Kim, J., Jia, L., Tilley, W.D., Coetzee, G.A. (2003) Dynamic methylation of histone H3 at lysine 4 in transcriptional regulation by the androgen receptor Nucleic Acids Res., 31, 6741–6747 .
Chen, X., Wang, J., Woltring, D., Gerondakis, S., Shannon, M.F. (2005) Histone dynamics on the interleukin-2 gene in response to T-cell activation Mol. Cell. Biol., 25, 3209–3219 .
Takahashi, I., Miyagi, H., Yoshida, T., Sato, S., Mizukami, T. (1996) Selective inhibition of IL-2 gene expression by trichostatin A, a potent inhibitor of mammalian histone deacetylase J. Antibiot., 49, 453–457 .
Litt, M.D., Simpson, M., Gaszner, M., Allis, C.D., Felsenfeld, G. (2001) Correlation between histone lysine methylation and developmental changes at the chicken ?-globin locus Science, 293, 2453–2455 .
Martens, J.H., Verlaan, M., Kalkhoven, E., Zantema, A. (2003) Cascade of distinct histone modifications during collagenase gene activation Mol. Cell. Biol., 23, 1808–1816 .
Perkins, E.J., Kee, B.L., Ramsden, D.A. (2004) Histone 3 lysine 4 methylation during the pre-B to immature B-cell transition Nucleic Acids Res., 32, 1942–1947 .
Kim, A. and Dean, A. (2004) Developmental stage differences in chromatin subdomains of the ?-globin locus Proc. Natl Acad. Sci. USA, 101, 7028–7033 .
Yamashita, M., Shinnakasu, R., Nigo, Y., Kimura, M.Y., Hasegawa, A., Taniguchi, M., Nakayama, T. (2004) IL-4-independent maintenance of histone modification of the IL-4 gene loci in memory Th2 cells J. Biol. Chem., 279, 39454–39464 .
Masternak, K., Peyraud, N., Krawczyk, M., Barras, E., Reith, W. (2003) Chromatin remodeling and extragenic transcription at the MHC class II locus control region Nature Immunol., 4, 132–137 .
Rothenberg, E.V., Diamond, R.A., Chen, D. (1994) Programming for recognition and programming for response: separate developmental subroutines in the murine thymus Thymus, 22, 215–244 .
Yui, M.A., Sharp, L.L., Havran, W.L., Rothenberg, E.V. (2004) Preferential activation of an IL-2 regulatory sequence transgenein TCR and NKT cells: subset-specific differences in IL-2 regulation J. Immunol., 172, 4691–4699 .
Brown, C.T., Rust, A.G., Clarke, P.J.C., Pan, Z., Schilstra, M.J., De Buysscher, T., Griffin, G., Wold, B.J., Cameron, R.A., Davidson, E.H., et al. (2002) New computational approaches for analysis of cis-regulatory networks Dev. Biol., 246, 86–102 .
West, A.G., Huang, S., Gaszner, M., Litt, M.D., Felsenfeld, G. (2004) Recruitment of histone modifications by USF proteins at a vertebrate barrier element Mol. Cell, 16, 453–463 .
Ohlsson, R., Renkawitz, R., Lobanenkov, V. (2001) CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease Trends Genet., 17, 520–527 .
Hatzis, P. and Talianidis, I. (2002) Dynamics of enhancer-promoter communication during differentiation-induced gene activation Mol. Cell, 10, 1467–1477 .
Kim, A. and Dean, A. (2003) A human globin enhancer causes both discrete and widespread alterations in chromatin structure Mol. Cell. Biol., 23, 8099–8109 .
Morshead, K.B., Ciccone, D.N., Taverna, S.D., Allis, C.D., Oettinger, M.A. (2003) Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4 Proc. Natl Acad. Sci. USA, 100, 11577–11582 .
Kimura, H., Tada, M., Nakatsuji, N., Tada, T. (2004) Histone code modifications on pluripotential nuclei of reprogrammed somatic cells Mol. Cell. Biol., 24, 5710–5720 .(Satoko Adachi and Ellen V. Rothenberg*)
*To whom correspondence should be addressed. Tel: +1 626 395 4992; Fax: +1 626 449 0756; Email: evroth@its.caltech.edu
ABSTRACT
T-cells retain cell-type-specific programming for IL-2 inducibility through many rounds of division without being stimulated to transcribe the locus. To understand the layering of controls needed to poise this gene heritably for activation, we have used chromatin immunoprecipitation to map histone modifications across the murine IL2 locus, from –10.2 through +0.25 kb, in induction-competent and incompetent cells. In highly inducible EL4 T-lineage cells, stimulation with PMA/A23187 induced strong acetylation of histone H3 and H4, in parallel with transcriptional activation, from –4.6 through +0.25 kb. However, dimethylation of histone H3/K4 was already fully elevated across the same restricted domain before stimulation, with little change after stimulation. RNA polymerase II binding, in contrast, was only found at the known promoter region after stimulation. Similar patterns of histone modifications were seen also in normal IL-2-inducible T-lineage cells. However, neither acetylated histone H3, H4 nor dimethylated histone H3/K4 marking was detected, with or without stimulation, in expression-incompetent cells (NIH/3T3 or Scid.adh). These results identify a discrete new domain of IL2 regulatory sequence marked by dimethylated histone H3/K4 in expression-permissive T-cells even when they are not transcribing IL2, setting boundaries for histone H3 and H4 acetylation when the IL2 gene is transcriptionally activated.
INTRODUCTION
Mammalian development makes extensive use of stem-like cells for different tissues, in which a specific range of developmental potentials is programmed for future use without being overtly expressed, a condition that can persist for long periods of time (1). Epigenetic chromatin marking could be involved in establishment and long-term maintenance of the latent gene expression capabilities that must exist in these cells. However, for most mammalian systems of developmental gene regulation in which chromatin configurations have been studied closely, changes in potential for expression of a gene have been tightly linked to changes in actual transcriptional activation of that gene, making the mechanisms difficult to disentangle. Chromatin modifications, such as acetylation of histones H3 and H4, and methylation of H3 on Lys-4 (H3/K4), are known to be correlated with ‘open’ chromatin, but in most cases studied this is also transcriptionally active chromatin . Epigenetic marking strictly correlated with latent activity has been analyzed only rarely (4–6). Here, we propose that the murine IL2 gene can shed light on this more general problem in developmental molecular biology.
IL-2 is one of the important cytokines produced by mature T-cells in response to antigenic stimuli. IL2 gene regulation is subject to dual control, since it acutely depends on combinatorial signaling through particular combinations of pathways, and can only be activated in a very limited spectrum of cell types . Although strongly induced by combinations of Ca2+, Ras/MAP kinase and costimulatory signals, IL-2 transcription is transient and returns to an ‘off’ state in expression-competent cells between bouts of stimulation. For the most part, competence to express IL2 is restricted to T-lymphoid cells and a few other hematopoietic cell types, and its expression is observed in certain immature stages (10,11) as well as in the antigen responses of mature T-cells. The regulatory basis for the activation dependence of IL2 gene expression in T-cells is well-understood, with key roles for NF-AT, NF-B/c-Rel and AP-1 factors interacting with constitutive regulators such as Oct-1 in a 300 bp promoter-proximal region . Additional regulators such as HMG box factors and the Ets factor GABP may also play a role, through highly conserved sites just 100–200 bp further upstream (14,15). The binding of these factors in response to activation is correlated with changes in DNA methylation at a series of sites extending from the promoter to about –800 bp (16). However, the basis for IL2 cell-type specificity is still obscure, since all of the known transcription factors used for its induction are expressed broadly in many cell types that cannot express the gene. Meanwhile, the great majority of na?ve T-cells retain the potential to activate IL2 expression even after long periods when the gene is transcriptionally silent. The cell-type-specific regulation of IL-2 presents one of the clearest known cases of dissociation between developmentally programmed competence to express a gene, and actual expression of that gene for the great majority of a cell's lifetime.
Transgenic studies have shown that the highly conserved 600 bp region that contains the proximal promoter-enhancer is not enough for IL2 regulatory sequence activity in transgenic mice (17,18), raising the possibility that additional positive regulatory functions may be encoded elsewhere. Addition of less-characterized sequences to –2.0 to –2.7 kb (with respect to the IL2 transcriptional start site) permit correct expression in a fraction of transgenic founder lines (19,20). While ectopic expression of these constructs is rare, the locus appears to require additional sequences to counteract silencing. Experiments with hybrid regulatory sequence constructs show that one of the functions needed can be supplied by the locus control region of the human CD2 gene (21). We have therefore hypothesized that the basis of cell-type specificity might reside in a discrete regulatory function that controls IL2 chromatin accessibility (7,14). If so, then the sequences through which this effect is mediated could be expected to improve the frequency and efficiency of expression of IL2 regulatory sequence transgenes if these sequences were included.
Previously, we reported that a GFP transgene driven with 8.4 kb of IL2 5'-flanking sequence (8.4 kb IL2-GFP) displayed efficient, lineage-specific expression in transgenic mice, much improved in frequency and fidelity as compared to a 2.0 kb IL2-GFP transgene (20). Thus, the additional 6.4 kb upstream sequence contains important regulatory regions for conferring accessibility to induction on the IL2 gene. Here, we use analyses of the chromatin structure in these IL2 upstream regions to investigate the nature of the regulatory function that they confer.
To analyze features of the distal sequence, we performed chromatin immunoprecipitation (ChIP) assays to define the nature and extent of any associated histone modifications. Here, we report significant levels of histone acetylation (AcH3, AcH4) and histone H3 lys-4 dimethylation (diMeH3/K4) in a discrete region from –4.6 kb through the promoter and first exon of the IL2 gene in IL-2 producing T-cells. Whereas acetylation was seen only in cells transcribing the gene in response to stimulation, diMeH3/K4 is maintained at a high level across the same region in cells competent to express IL2, even before stimulation. These results indicate the presence of transcriptionally permissive state which is marked by diMeH3/K4 without histone acetylation over a discrete 5'-flanking domain of the IL2 gene, and which is restricted to competent IL-2-inducible cells.
MATERIALS AND METHODS
Cell culture and stimulations
EL4.F4 cells (22,23), Scid.adh cells (24), NIH/3T3 cells and splenocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone, South Logan, UT), 50 μM 2-mercaptoethanol, 0.1 mM MEM non-essential amino acids (Invitrogen, Carlsbad, CA), 10 mM HEPES, 1 mM sodium pyruvate (Sigma–Aldrich, St Louis, MO), 2 mM L-glutamine and penicillin/streptomycin.
Cells were stimulated for 3 h with 10 nM PMA and 180 nM A23187 (Sigma–Aldrich). Cell concentrations were 106 cells/ml of non-adherent cells and were semi-confluent of adherent cells. For stimulations of TCR-negative SCID thymocytes, IL-1 (10 ng/ml; Biosource, Camarillo, CA) was also required in the stimulation cocktail (25–27), but IL-1 has no effect on responses of CD4+ CD8+ cortical thymocytes, as previously described (28).
Purification of B220– splenocytes and thymocytes from SCID and MHC-deficient mice
Red-blood cell depleted splenocytes from C57BL/6 mice were stained with biotin-conjugated anti-B220 Ab (Immunotech, Marseille, France), and then incubated with streptavidin-conjugated MACS microbeads (Miltenyi Biotech, Auburn, CA). Cells were passed through MACS columns to remove B220+ cells. Purity of separated cells was determined by staining with FITC-conjugated anti-B220 Ab (BD PharMingen, San Diego, CA). Thymocytes were prepared by mincing the tissue followed by filtration through a wire mesh.
Chromatin immunoprecipitation assay
Antibodies for ChIP assays were purchased from the following companies. Anti-acetyl-histone H3 (06-599), anti-acetyl-histone H4 (06-866) and anti-dimethyl-histone H3 (Lys-4) (07-030) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Pol II (N-20) (sc-899) and normal rabbit IgG (sc-2027) were from Santa Cruz Biotechnology (Santa Cruz, CA).
ChIP assays were performed by the protocol of Upstate Biotechnology with modifications. Briefly, samples of 2 x 107 cells were subjected to protein–DNA crosslinking with formaldehyde at a final concentration of 0.4% for 10 min at room temperature. Cells were lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris–HCl, pH 8.0) and DNA was sheared by sonication (Labsonic 1510, B. Braun, Melsungen, Germany) at 100 W for 1 min, to lengths between 200 and 1000 bp.
One hundred microliters of sonicated sample was kept as an input sample, and the remainder was diluted with 8.1 ml of dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.0 and 167 mM NaCl). Diluted samples were precleared with 100 μl of salmon sperm DNA/protein A agarose (Upstate Biotechnology) for 1 h, then divided into three tubes (3 ml each).
Ten micrograms of antibodies were added to each tube and incubated for 3–4 h. After that, 60 μl of salmon sperm DNA/protein A agarose was added and incubated overnight at 4°C. Agarose was washed once with 1 ml of low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0 and 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0 and 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate and 1 mM EDTA), and twice with TE buffer (pH 8.0). Immune complexes were eluted twice with 250 μl of elution buffer (1% SDS and 0.1 M NaHCO3). Protein–DNA crosslinking was reversed by incubation at 65°C for 4 h with 0.2 M NaCl, and then protein was digested at 45°C for 1 h in the presence of 10 mM EDTA (pH 8.0), 40 mM Tris–HCl (pH 6.5) and 40 μM proteinase K. The DNA was purified by phenol–chloroform extraction followed by ethanol precipitation.
Quantitative PCR analysis
Quantitations of chromatin immune precipitates were performed by real-time PCR using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Primers were designed by the Primer3 website (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (29) to amplify 100–250 bp amplicons. PCR analyses were performed in 25 or 30 μl reactions with SYBR Green (PE Applied Biosystems). All primer sets used gave no signal (CT > 40) in control reactions without template. Dissociation curve analyses showed that single products with expected Tm values were generated by each primer set. A second peak of lower Tm was detected only in products of one primer set (–8.9 to –8.7), or as a shoulder in products of one other set (–3.2 to –3.0).
Standard curves were determined for each primer set by dilution of input DNA in a 0.1–100 ng range, and only primer sets yielding slopes close to the theoretical values for 1.9- to 2.0-fold amplification per cycle were used. The amounts of each sequence in the input and precipitated DNA were calculated from the cycle thresholds (CT) for each primer set by using the standard curves. Relative units recovered for each primer set were determined by dividing the calculated amounts of precipitated sequence by the amount of that sequence in the input DNA. Due to lower cell recovery of primary thymocytes, results in Figure 7 were further normalized by the results of the S16 ribosomal protein gene.
Figure 7 Dimethylated histone H3/K4 association with IL2 5'-flanking region is correlated with IL-2 inducibility in immature thymocytes: ChIP analysis of the IL2 locus in thymocytes from SCID and MHC-deficient mice. Cells from MHC-deficient mice, >95% CD4+ CD8+ TCR-low, were cultured with or without PMA/A23187 for 3 h. Cells from SCID mice, >95% CD4– CD8– TCR-negative, were cultured with or without stimulation for 2 h, and IL-1 was also added as indicated. To correct for differences in efficiency of immune precipitation between these samples due to the extensive differences in cell physiology, ChIP analysis results for diMeH3/K4 at the IL2 locus for each sample were normalized by results at the S16 ribosomal protein locus. Two independent experiments were done with similar results.
RNA purification and real-time quantitative RT–PCR
Total RNA was extracted from the cells by RNAzol (Leedo Medical, Houston, TX) following the manufacturer's instructions. One microgram of RNA was treated with RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37°C. After that, DNase was inactivated by the incubation for 5 min at 75°C. Then, first-strand synthesis reactions were performed with Super-script II reverse transcriptase (Invitrogen, Carlsbad, CA) and 500 ng oligo dT primers (Pharmacia, Uppsala, Sweden) following standard protocols.
Real-time PCR was performed by using ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). Taqman probes and primers for murine IL-2 (FAM labeled) and GAPDH (VIC labeled) were purchased from PE Applied Biosystems. To normalize the samples, control GAPDH CT values were subtracted from IL-2 CT values of each sample (CT). Then, CT of the unstimulated NIH/3T3 sample (reference sample) was subtracted from CT of each sample (CT). Relative levels of IL-2 mRNA were calculated as 2–CT.
RESULTS
Stimulation with PMA/A23187 induces histone acetylation at the IL2 locus in IL-2 producing cells only
Several lines of evidence suggested that regulatory elements important for cell-type-specific accessibility of the IL2 gene might reside on the 5' side of the well-characterized promoter-enhancer region. First, a series of T-cell-specific DNase I hypersensitive sites were found between –10 and –2 kb from the transcriptional start site of the IL2 gene, including a cluster of cell-type-specific sites from about –2.8 to –4.5 kb which were hypersensitive even in resting cells (20). Second, a GFP reporter transgene controlled by an extended 8.4 kb upstream region of the IL2 gene, which includes most of the new hypersensitive sites, showed more efficient, lineage-specific expression in transgenic mice than a GFP transgene controlled by a 2.0 kb IL2 regulatory region, which does not include any of the new hypersensitive sites (20). Most recently, comparative genomics have made it possible to identify three islands of mouse–human sequence similarity upstream of the IL2 gene, as indicated in Figure 1 (for full three-way alignment of murine, human and rat sequences, see Supplementary Figure 1). Two of these islands lie within the region included in the 8.4 kb IL2 promoter transgene and one lies just beyond it.
Figure 1 Diagrams of DNase hypersensitive sites and primer positions on the IL2 locus. DNase hypersensitive sites in stimulated and unstimulated purified splenic T-cells are shown by arrows (). Regions of high homology with an upstream sequence in the human IL2 gene were determined by Blast search (H). Putative matrix attachment region (MAR) and a region of high homology with an intronic sequence in the Bruton's thymidine kinase gene (Btk) are also indicated. For full alignment of these murine IL2 sequences with human and rat orthologues using FamilyRelations and SeqComp software (46), see Supplementary Figure 1. Bottom: the functional importance of these sequences is demonstrated by the distinct behavior of IL2 regulatory sequence transgene constructs previously reported by this group (20). The sequences to –8.4 kb, but not the sequences to –2.0 kb, are sufficient to direct IL2-like expression of the reporter gene in a high frequency of independent integration sites (20).
Although indicating some functional significance for the region between –2.0 and –8.4 kb, the transgenesis results did not determine whether the sequences in this region act as auxiliary stimulation-dependent enhancers, amplifying the response upon induction, or whether they mark the locus stably as accessible to induction by a separate mechanism uncoupled from transcription. To determine which regions of the extended IL2 upstream region might participate in control of IL-2 expression, we used ChIP with quantitative real-time PCR quantitation. Although regions enriched for simple-sequence repeats needed to be excluded from analysis, we designed 17 primer sets to amplify segments from the first transcribed exon up to –10 kb of the IL2 gene (Figure 1 and Table 1), including amplicons bridging all three of the conserved regions. To correlate chromatin structure with competence to express IL-2, the murine thymoma cell line EL4, the immature pro T-cell line Scid.adh and the non-hematopoietic cell line NIH/3T3 were subjected to ChIP assay with or without PMA/A23187 stimulation. Of these, EL4 is the only one which expresses IL-2 in response to stimulation (Figure 2A). However, in unstimulated EL4 cells, the IL2 gene is virtually silent, with levels of RNA >105-fold lower than after 3 h of stimulation (note log scales in Figure 2).
Table 1 Sequences of the primers
Figure 2 Quantitative RT–PCR of IL-2 mRNA induction in different cell types. RNA was extracted from the indicated cell types, either unstimulated or stimulated with PMA/A23187 for 3 h. IL-2 mRNA levels were determined in the samples by real-time PCR following reverse transcription, and normalized to equal GAPDH levels. Units of IL-2 mRNA expression were then calculated as 2–CT relative to an unstimulated NIH/3T3 sample (20 = 1). (A) Cell lines NIH/3T3 (fibroblasts, nonhematopoietic), Scid.adh (pro-T-like thymic lymphoma), and EL4 (mature T-cell thymic lymphoma), compared with primary B220– splenic lymphocytes (mature T-cells). Levels of IL-2 in stimulated EL-4 and B220– splenic T cell samples are within 2- to 3-fold of the levels of GAPDH (data not shown). (B) Primary immature pro-T cells from SCID thymus, compared with primary CD4+ CD8+ TCR-low cortical thymocytes from MHC-deficient thymus. For SCID thymocytes, stimulation was only for 2 h, in the presence or absence of IL-1 as indicated. Levels of IL-2 in maximally stimulated SCID thymocytes are 1% of the level of GAPDH. Note that all results are displayed on a log scale. ND = not determined.
In resting EL4 cells, acetylation of histones H3 and H4 was not substantially elevated across the IL2 locus, and the recovery of all IL2 flanking sequences was low compared to the positive control. However, acetylation was greatly increased after 3 h of stimulation with PMA/A23187, in a defined interval across the sequences from –4.6 through +0.25 kb. By this time point, specific transcription factor occupancy of target sites in the enhancer is already maximal, as we have reported previously (30). The acetylated region included two islands of mouse/rat/human conserved sequence, at about –2.2 and at about –3.9 kb, but excluded the third, more distal conserved region at about –8.6 kb and all other regions tested out to –10.2 kb.
Unexpectedly, within the region from –4.6 through +0.25 kb, the sequence that was least enriched in acetylated H3 and H4 immune precipitates after stimulation was the amplicon from the IL2 proximal promoter region itself (–0.24 to –0.05 kb) (Figure 3A; top). Since the proximal promoter is known to be a core region for IL-2 transcription factor binding which undergoes dramatic changes in nuclease accessibility upon stimulation (14,30–34), the regulatory protein complexes that are assembled there upon induction might exclude nucleosomes or mask epitopes of acetylated histone. A similar result has been reported for active promoters in the ?-globin and prostate-specific antigen loci (6,35). In accordance with our results, a recent report has demonstrated directly that stimulation causes specific loss of histone protein from the proximal promoter region of the IL2 gene (36).
Figure 3 Histone acetylation pattern of the IL-2 gene. (A) Acetylated histone H3 and H4 of the IL-2 gene were analyzed with quantitative ChIP assay. Results of EL4, Scid.adh and NIH/3T3 cells are shown. (B) Acetylated histone H3 and H4 on the promoter of S16 ribosomal protein. The results are shown as mean ± SD of three or four independent experiments. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
Acetylation at the IL2 locus appears to be regulated normally by an equilibrium between histone acetyltransferase and histone deacetylase (HDAC) activities, because exposure of the cells to HDAC inhibitor trichostatin A increases acetylation of H3 and especially H4, even in the absence of stimulation (Supplementary Figure 2A). This increased acetylation is not associated with transcriptional induction; in fact, trichostatin A decreases the induction of IL-2 in response to stimulation under the conditions used here (37) (Supplementary Figure 2B). Even so, most increased acetylation induced by trichostatin A is seen in the region between about –5 kb and the promoter region, where the stimulation-inducible acetylation occurs (Supplementary Figure 2A).
The IL2-nonproducing cell lines, Scid.adh and NIH/3T3, maintained very low levels of acetylation of the IL2 gene flanking sequences with no increase upon stimulation (Figure 3A; middle and bottom); however, the acetylation of a sequence from the S16 ribosomal protein promoter was comparable between all these three cell lines (Figure 3B). Controls without antibody or with normal IgG precipitation did not detectably precipitate IL2 sequences in any of these cell lines (Supplementary Figure 3). Thus, there is a specific IL2 upstream region extending to –4.6 kb, i.e. >4 kb beyond the well-characterized proximal promoter-enhancer, which is subject to histone acetylation only upon stimulation and only in IL-2 induction-competent cells.
Dimethylation of histone H3/K4 at the IL2 upstream region before stimulation in EL4 cells
To assess whether there is any other kind of modification at the IL2 locus that might mark it as accessible, we monitored dimethylation of histone H3/K4, which is also associated with active chromatin and often accompanies acetylation (6,38–41). Like AcH3 and AcH4, diMeH3/K4 was highly associated with sequences from –4.6 through +0.25 kb in EL4 cells after stimulation (Figure 4A; top). However, unlike acetylation, a significant level of diMeH3/K4 was observed from –4.6 through +0.25 kb before stimulation as well, comparable to its level after stimulation. In unstimulated EL4 cells, this modification could also be seen to extend through the proximal promoter (–0.24 to –0.05 kb), although it appeared somewhat decreased after stimulation. In the Scid.adh and NIH/3T3 cells, in contrast, which cannot express IL-2, no association of diMeH3/K4 with the IL2 gene was observed, with or without stimulation (Figure 4A; middle and bottom). These differences were specific to the IL2 gene, because diMeH3/K4 modification of chromatin associated with the S16 ribosomal protein promoter was comparable in the three cell lines (Figure 4B).
Figure 4 Dimethylated histone H3 at lys-4 is observed before stimulation in EL4 cells. (A) ChIP analyses were performed for diMeH3/K4 on the IL2 locus of EL4, Scid.adh and NIH/3T3 cells. DiMeH3/K4 on the promoter of S16 ribosomal protein is shown in (B). The results are shown as mean ± SD of three independent experiments. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
RNA polymerase II is not bound to the distal region of the IL2 gene
Both AcH3, H4 and diMeH3/K4 modifications extend >4 kb from the known promoter-enhancer of the IL2 gene. We considered possible explanations for the relatively sharp 5' border and the broad extension of the modified region all the way through the promoter-enhancer. In some cases, pol II itself is recruited to a distal regulatory region (42), from which it can spread acetylated histone in a 5' to 3' polar fashion as it moves along the gene . Therefore, we analyzed pol II binding on the IL2 gene in EL4 cells by ChIP assay. As expected, pol II immune precipitation of stimulated EL4 cell samples enriched for promoter proximal sequences (amplified with primers from –0.24 to –0.05 kb), with further enrichment of sequences immediately downstream of the transcriptional start site (+0.06 to +0.25 kb). Pol II recruitment was correlated with transcriptional activation, as little or no enrichment was seen in samples from unstimulated cells. However, even in stimulated EL4 cells, only minimal binding was found from –4.6 through +0.25 kb, or in more distal 5' sequences (20% of promoter level) (Figure 5). Thus, no evidence for specific pol II recruitment was found in any of the upstream regions examined.
Figure 5 Binding of RNA polymerase II on the IL2 locus. ChIP analyses were performed for pol II binding on the IL2 locus of EL4 cells. The results are shown as mean ± SD of three independent experiments. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
Histone modifications at the IL2 locus in primary T-cells resemble those in EL4 cells
To determine whether the distinctive patterns of chromatin modifications found in EL4 cells could be relevant to normal T-cell control of IL-2 expression, we examined three developmentally distinct populations of primary T-lineage cells: mature, peripheral splenic T-cells; cortical TCRlow CD4+ CD8+ thymocytes (>95% of cells in MHC-deficient thymus); and immature TCR– SCID thymocytes. As previously reported (10,25,44), both mature peripheral T-cells and SCID thymocytes can be stimulated to express IL-2 (Figure 2A, ‘B220-’; Figure 2B, ‘scid thy’), but the cortical thymocytes, although developmentally intermediate, cannot (Figure 2B, ‘MHC–/–’).
ChIP assays for AcH3 and diMeH3/K4 were performed on samples of stimulated or unstimulated peripheral T-cells, which were highly enriched from C57BL/6 mouse spleens by magnetic bead depletion of B220+ cells, as shown in Figure 6A. In these cells, AcH3 was increased from –4.6 kb through the IL-2 promoter after stimulation, while diMeH3/K4 was high from –4.6 kb to downstream both before and after stimulation (Figure 6B, B220– cells). These patterns in stimulated and unstimulated primary T-cells closely match those in EL4 cells. The maximum increase of AcH3 upon stimulation in B220– cells was only 4- to 5-fold, as compared to >10-fold in EL4 cells (Figure 3A; top), but this may be attributed to known quantitative differences between the frequencies of IL-2 induction in unstimulated primary T-cells and in EL4 cells (Figure 2). The fraction of primary T-cells expressing IL-2 mRNA in a particular response is never as high as the fraction of EL4 cells, which can approach 80% (E. V. Rothenberg, unpublished data), whereas the background level of IL-2 mRNA expression in unstimulated B220– cells ex vivo is often higher than that of unstimulated EL4 cells, possibly due to a low frequency of cells that received some stimulation in vivo already (45). Thus, these results indicate that chromatin modification of the IL2 locus from –4.6 kb through the promoter region may be a stable characteristic of normal mature T-cells.
Figure 6 Pattern of acetylated histone H3 and dimethylated histone H3 at lys-4 at the IL2 locus in an enriched population of mature splenic T-cells. B220+ cells were depleted from C57BL/6 mouse splenocytes by magnetic columns (A), and the remaining B220– cells (T-enriched) were subjected to ChIP assay (B). Two independent experiments were done with similar results. Open bars; unstimulated, filled bars; stimulated with PMA/A23187.
Dimethyl H3/K4 marking correlates with competence to express in immature T-lineage cells
To determine how early in T-lineage development this chromatin modification might be detectable, SCID thymocytes were analyzed. Although SCID thymocytes cannot rearrange or express T-cell receptor genes and thus remain immature, they are inducible for IL-2 expression (Figure 2B) (up to 20% of the cells capable of expressing IL-2 mRNA in immediate response to stimulation in C.B17-SCID) (26,27). In two independent experiments, even without stimulation, SCID thymocytes showed high association of diMeH3/K4 with the region from –4.6 kb through the promoter region (Figure 7, top graph). Thus, both immature and mature T-cells specifically modify chromatin in a defined region upstream of the IL2 promoter.
The shared pattern of epigenetic marking in mature and very immature populations could be strictly correlated with competence to express IL-2, or it could be a lineage marker for all normal T-lineage cells. To distinguish these possibilities, we analyzed intermediate CD4+ CD8+ TCR-low thymocytes, which constitute >95% of the cells in the thymus of major histocompatibility complex-deficient mice (MHC–/–). These cells are more advanced than SCID thymocytes developmentally, but have lost or suspended all competence to express IL-2 (10,28,44). As shown in Figure 7 (bottom graph), these cells show much lower levels of diMeH3/K4 modification over the IL2 flanking sequences, relative to S16 positive control sequences, than EL-4 cells, mature T-cells or SCID thymocytes. The pattern is also flat, with little if any increase in modification over the region from –4.6 kb to the promoter. Thus, diMeH3/K4 modification over the region from –4.6 kb to the IL2 promoter is not universal among all T-lineage cells, but is tightly linked to the ability to express IL-2.
In summary, as shown in Figure 8, both diMeH3/K4 and AcH3 types of modifications of the IL2 flanking sequences are cell-type specific. Both are restricted to T-lineage cells that are competent to express IL-2 and not found in fibroblasts or T-lineage cells that lack this competence. However, histone acetylation is tightly linked with the recruitment of active transcription complexes to the IL2 promoter. Only diMeH3/K4 marking is a stable differentiated characteristic of these competent cells that is retained at the locus as long as they retain their developmental state, in vivo and in vitro, even in the absence of activating signals and in the complete absence (<10–5 of the induced levels) of IL-2 mRNA expression.
Figure 8 Changes of histone modifications at the IL2 gene during T-cell differentiation. The correlation between IL-2 expression and histone modifications from –4.6 through +0.25 kb of the IL2 gene is summarized.
DISCUSSION
The basis for competence to induce transcription of the IL2 gene in particular cell types has long been obscure, in contrast to the detailed knowledge that has been accumulated about the mechanism that actually triggers transcription of this locus in competent cells. Previously, we showed that sequences of the IL2 gene between –2 kb and –8.4 kb (extended 5'-flanking sequence) could be involved in such a function, since they greatly enhance the likelihood of expression of an IL2 promoter-GFP transgene in the correct cells (20). The function conferred was not defined, however, and the sequences involved in the function were not mapped. Here, we report that a particular segment of the extended 5'-flanking sequence is a site for epigenetic marking by histone H3/K4 dimethylation that occurs only in IL-2 expression-competent cells. This modification is cell-type specific, tightly linked with competence of the IL2 locus to be induced by stimulation, and coincides with the region in which increased AcH3 and AcH4 modifications occur when transcription is activated (Figures 3, 4 and 6). However, this H3/K4 methylation is uncoupled from detectable IL2 transcription, from pol II binding, and from histone acetylation, and it persists independently of stimulation. Our data provide the first direct evidence for the existence of a discrete, developmentally controlled mechanism that may control cell-type-specific accessibility of the IL2 locus, working through different sequences and different trans-acting factors than those that activate transcription of the gene in response to induction.
The relationship between H3/K4 methylation and IL-2 expression supports the model that sequences between –2 and –8.4 kb can have a discrete role in conferring cell-type-specific accessibility on this gene that is separable from any enhancer activity. Indeed, the 8.4 kb IL2 regulatory region does not drive reporter gene expression any better than the 2.0 kb regulatory region, when tested in transient transfection experiments (20). Two groups of DNase HS sites, centered around different mouse/human homology blocks, suggest that the upstream region may include two types of function. The distal portion of the H3/K4 methylation-marked region, from –4.6 to about –2.8 kb, includes the group of DNase I hypersensitive sites which are most pronounced in resting, not activated, T-cells. Activation of IL-2 transcription induces the appearance of new DNase HS sites, including a cluster around the mouse/human homology block from about –2.2 to –2.3 kb as well as in the promoter-proximal region (14,20). This –2.2 kb region may indeed have cryptic enhancer-like activity. However, activation if anything reduces the prominence of the major DNase hypersensitive site around –4.5 kb (20), at the distal boundary of the MeH3/K4 marked domain. A clear inference would be that any transcription factors binding to regulatory sites in the –2.8 to –4.6 kb upstream region would be distinct from these stimulation-activated transcription factors.
Identification of the critical regulatory elements in this interval is impeded by the remarkably poor sequence conservation between human and murine sequences beyond –2.3 kb in the mouse sequence. When the human, mouse and rat IL2 flanking sequences are aligned (46) (Supplementary Figure 1), the only well-conserved sequence from –2.8 to –4.6 kb is a 64 bp stretch at about –3.9 kb in the murine gene (in the human gene, a LINE element insertion shifts this alignment further upstream). This conserved element should thus provide a useful point of departure for identification of the factors which control the establishment or maintenance of the MeH3/K4 marked domain.
Only minimal histone modifications were seen further upstream of –4.6 kb, with or without stimulation. It remains to be seen whether known boundary-forming transcription factors such as CCCTC-binding factor (CTCF) or the bHLH-ZIP ‘Upstream Factor’ (USF) play a role in setting these limits. Interestingly, there are several E-box motifs which might bind USF between –8.0 and –8.4 kb, and a predicted double binding site around –6.2 kb. A recent article shows that USF binds to the chicken ?-globin 5'HS4 insulator, where it protects the gene from chromosomal silencing by recruitment of histone modification factors (47). It may play a role to keep the IL2 gene accessible by the same mechanisms. Closer to the region where we see discontinuities in IL2 chromatin structure, there is an unusually dense clustering of seven CCCTC motifs between –4.5 and –5.3 kb from the transcription start site. Although functional target sites of CTCF can be difficult to predict based on sequence alone (48), these sites will clearly be of interest for further investigation.
The mechanism maintaining the MeH3/K4-marked IL2 5'-flanking region in unstimulated cells is clearly distinct from the highly coordinated assemblies of transcription factors, histone acetyltransferases and RNA pol II which are seen associated with ‘open chromatin’ in other sites (42,43,49,50). In the case of the IL2 gene, a stable pattern of H3/K4 methylation appeared to be maintained even in the absence of any substantial pol II binding to the region between –4.6 kb and the promoter. The minimal level of binding that was seen could be consistent with co-precipitation by interaction of distal regulatory regions with the proximal promoter via looping, rather than with direct binding. The IL2 gene thus joins a small number of cases where diMeH3/K4 is associated with accessibility to transcription before transcription itself is induced. In some cases, this modification appears to demarcate the boundaries of a region within which transcription and/or DNA recombination can occur (40,51). Recently, a two-step chromatin modification sequence, with diMeH3/K4 modification preceding histone acetylation, was shown in a variety of genes activated during hybridization between ES cells and somatic cells (52). Even if this sequence is used for regulation of many genes, the regulatory style may differ for each gene. For example, in the activation of HNF-4 gene concomitant with the enterocyte differentiation, diMeH3/K4 is restricted to the promoter region through differentiation while acetylated histones develop from the distal enhancer toward the promoter (49) And at the IgH locus in pro B-cells, peaks of diMeH3/K4 are observed at the borders of active DJ regions and inactive V and C regions (51). For immune regulatory cytokines, such as IL-2, maintaining a transcriptionally permissive stage which is marked by long-range diMeH3/K4 modification alone may be important to preserve the potential for immediate gene activation in response to precisely calibrated external stimuli.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
The authors would like to thank Dr Mary Yui and members of the Rothenberg group for helpful discussions, and the staff of the Caltech Office of Laboratory Animal Research for expert care of the different mouse strains. This work was supported by USPHS grant AG13108. Funding to pay the Open Access publication charges for this article was provided by AG13108.
REFERENCES
Reya, T., Morrison, S.J., Clarke, M.F., Weissman, I.L. (2001) Stem cells, cancer, and cancer stem cells Nature, 414, 105–111 .
Fischle, W., Wang, Y., Allis, C.D. (2003) Binary switches and modification cassettes in histone biology and beyond Nature, 425, 475–479 .
Marmorstein, R. (2001) Protein modules that manipulate histone tails for chromatin regulation Nature Rev. Mol. Cell Biol., 2, 422–432 .
Guo, L., Hu-Li, J., Zhu, J., Watson, C.J., Difilippantonio, M.J., Pannetier, C., Paul, W.E. (2002) In TH2 cells the Il4 gene has a series of accessibility states associated with distinctive probabilities of IL-4 production Proc. Natl Acad. Sci. USA, 99, 10623–10628 .
Yamashita, M., Ukai-Tadenuma, M., Miyamoto, T., Sugaya, K., Hosokawa, H., Hasegawa, A., Kimura, M., Taniguchi, M., DeGregori, J., Nakayama, T. (2004) Essential role of GATA3 for the maintenance of type 2 helper T (Th2) cytokine production and chromatin remodeling at the Th2 cytokine gene loci J. Biol. Chem., 279, 26983–26990 .
Bottardi, S., Aumont, A., Grosveld, F., Milot, E. (2003) Developmental stage-specific epigenetic control of human ?-globin gene expression is potentiated in hematopoietic progenitor cells prior to their transcriptional activation Blood, 102, 3989–3997 .
Rothenberg, E.V. and Ward, S.B. (1996) A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin-2 gene regulation Proc. Natl Acad. Sci. USA, 93, 9358–9365 .
June, C.H., Ledbetter, J.A., Lindsten, T., Thompson, C.B. (1989) Evidence for the involvement of three distinct signals in the induction of IL-2 gene expression in human T lymphocytes J. Immunol., 143, 153–161 .
Powell, J.D., Ragheb, J.A., Kitagawa-Sakakida, S., Schwartz, R.H. (1998) Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy Immunol. Rev., 165, 287–300 .
Chen, D. and Rothenberg, E.V. (1993) Molecular basis for developmental changes in interleukin-2 gene inducibility Mol. Cell. Biol., 13, 228–237 .
Wang, H., Diamond, R.A., Yang-Snyder, J.A., Rothenberg, E.V. (1998) Precocious expression of T-cell functional response genes in vivo in primitive thymocytes before T-lineage commitment Int. Immunol., 10, 1623–1635 .
Jain, J., Loh, C., Rao, A. (1995) Transcriptional regulation of the IL2 gene Curr. Opin. Immunol., 7, 333–342 .
Serfling, E., Avots, A., Neumann, M. (1995) The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation Biochim. Biophys. Acta, 1263, 181–200 .
Ward, S.B. and Rothenberg, E.V. (1998) Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions Nucleic Acids Res., 26, 2923–2934 .
Avots, A., Hoffmeyer, A., Flory, E., Cimanis, A., Rapp, U.R., Serfling, E. (1997) GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction Mol. Cell. Biol., 17, 4381–4389 .
Bruniquel, D. and Schwartz, R.H. (2003) Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process Nature Immunol., 4, 235–240 .
Brombacher, F., Sch?fer, T., Weissenstein, U., Tschopp, C., Andersen, E., Bürki, K., Baumann, G. (1994) IL-2 promoter-driven lacZ expression as a monitoring tool for IL-2 expression in primary T-cells of transgenic mice Int. Immunol., 6, 189–197 .
Reed, W.A., Elzer, P.H., Enright, F.M., Jaynes, J.M., Morrey, J.D., White, K.L. (1997) Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropin-class lytic peptide in transgenic mice and subsequent resistance to Brucella abortus Transgenic Res., 6, 337–347 .
Minasi, L.E., Kamogawa, Y., Carding, S., Bottomly, K., Flavell, R.A. (1993) The selective ablation of interleukin 2-producing cells isolated from transgenic mice J. Exp. Med., 177, 1451–1459 .
Yui, M.A., Hernandez-Hoyos, G., Rothenberg, E.V. (2001) A new regulatory region of the IL-2 locus that confers position-independent transgene expression J. Immunol., 166, 1730–1739 .
Saparov, A., Wagner, F.H., Zheng, R., Oliver, J.R., Maeda, H., Hockett, R.D., Weaver, C.T. (1999) Interleukin-2 expression by a subpopulation of primary T-cells is linked to enhanced memory/effector function Immunity, 11, 271–280 .
Novak, T.J., White, P.M., Rothenberg, E.V. (1990) Regulatory anatomy of the murine interleukin-2 gene Nucleic Acids Res., 18, 4523–4533 .
Novak, T.J., Chen, D., Rothenberg, E.V. (1990) Interleukin 1 synergy with phosphoinositide pathway agonists for induction of interleukin-2 gene expression: molecular basis of costimulation Mol. Cell. Biol., 10, 6325–6334 .
Carleton, M., Ruetsch, N.R., Berger, M.A., Rhodes, M., Kaptik, S., Wiest, D.L. (1999) Signals transduced by CD3, but not by surface pre-TCR complexes, are able to induce maturation of an early thymic lymphoma in vitro J. Immunol., 163, 2576–2585 .
Rothenberg, E.V., Diamond, R.A., Pepper, K.A., Yang, J.A. (1990) Interleukin-2 gene inducibility in T-cells prior to T-cell receptor expression: changes in signaling pathways and gene expression requirements during intrathymic maturation J. Immunol., 144, 1614–1624 .
Rothenberg, E.V., Chen, D., Diamond, R.A. (1993) Functional and phenotypic analysis of thymocytes in SCID mice: evidence for functional response transitions before and after the SCID arrest point J. Immunol., 151, 3530–3546 .
Diamond, R.A., Ward, S.B., Owada-Makabe, K., Wang, H., Rothenberg, E.V. (1997) Different developmental arrest points in RAG2–/– and SCID thymocytes on two genetic backgrounds: developmental choices and cell death mechanisms before TCR gene rearrangement J. Immunol., 158, 4052–4064 .
Rothenberg, E.V. and Diamond, R.A. (1994) Costimulation by interleukin-1 of multiple activation responses in a developmentally restricted subset of immature thymocytes Eur. J. Immunol., 24, 24–33 .
Rozen, S. and Skaletsky, H.J. Primer3 on the WWW for general users and for biologist programmers In Krawetz, S. and Misener, S. (Eds.). Bioinformatics Methods and Protocols: Methods in Molecular Biology, Totowa, NJ Humana Press pp. 365–386 .
Garrity, P.A., Chen, D., Rothenberg, E.V., Wold, B.J. (1994) IL-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors Mol. Cell. Biol., 14, 2159–2169 .
Brunvand, M.W., Krumm, A., Groudine, M. (1993) In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T-cells Nucleic Acids Res., 21, 4824–4829 .
Chen, D. and Rothenberg, E.V. (1994) Interleukin-2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles J. Exp. Med., 179, 931–942 .
Rao, S., Procko, E., Shannon, M.F. (2001) Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene J. Immunol., 167, 4494–4503 .
Attema, J.L., Reeves, R., Murray, V., Levichkin, I., Temple, M.D., Tremethick, D.J., Shannon, M.F. (2002) The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation J. Immunol., 169, 2466–2476 .
Kim, J., Jia, L., Tilley, W.D., Coetzee, G.A. (2003) Dynamic methylation of histone H3 at lysine 4 in transcriptional regulation by the androgen receptor Nucleic Acids Res., 31, 6741–6747 .
Chen, X., Wang, J., Woltring, D., Gerondakis, S., Shannon, M.F. (2005) Histone dynamics on the interleukin-2 gene in response to T-cell activation Mol. Cell. Biol., 25, 3209–3219 .
Takahashi, I., Miyagi, H., Yoshida, T., Sato, S., Mizukami, T. (1996) Selective inhibition of IL-2 gene expression by trichostatin A, a potent inhibitor of mammalian histone deacetylase J. Antibiot., 49, 453–457 .
Litt, M.D., Simpson, M., Gaszner, M., Allis, C.D., Felsenfeld, G. (2001) Correlation between histone lysine methylation and developmental changes at the chicken ?-globin locus Science, 293, 2453–2455 .
Martens, J.H., Verlaan, M., Kalkhoven, E., Zantema, A. (2003) Cascade of distinct histone modifications during collagenase gene activation Mol. Cell. Biol., 23, 1808–1816 .
Perkins, E.J., Kee, B.L., Ramsden, D.A. (2004) Histone 3 lysine 4 methylation during the pre-B to immature B-cell transition Nucleic Acids Res., 32, 1942–1947 .
Kim, A. and Dean, A. (2004) Developmental stage differences in chromatin subdomains of the ?-globin locus Proc. Natl Acad. Sci. USA, 101, 7028–7033 .
Yamashita, M., Shinnakasu, R., Nigo, Y., Kimura, M.Y., Hasegawa, A., Taniguchi, M., Nakayama, T. (2004) IL-4-independent maintenance of histone modification of the IL-4 gene loci in memory Th2 cells J. Biol. Chem., 279, 39454–39464 .
Masternak, K., Peyraud, N., Krawczyk, M., Barras, E., Reith, W. (2003) Chromatin remodeling and extragenic transcription at the MHC class II locus control region Nature Immunol., 4, 132–137 .
Rothenberg, E.V., Diamond, R.A., Chen, D. (1994) Programming for recognition and programming for response: separate developmental subroutines in the murine thymus Thymus, 22, 215–244 .
Yui, M.A., Sharp, L.L., Havran, W.L., Rothenberg, E.V. (2004) Preferential activation of an IL-2 regulatory sequence transgenein TCR and NKT cells: subset-specific differences in IL-2 regulation J. Immunol., 172, 4691–4699 .
Brown, C.T., Rust, A.G., Clarke, P.J.C., Pan, Z., Schilstra, M.J., De Buysscher, T., Griffin, G., Wold, B.J., Cameron, R.A., Davidson, E.H., et al. (2002) New computational approaches for analysis of cis-regulatory networks Dev. Biol., 246, 86–102 .
West, A.G., Huang, S., Gaszner, M., Litt, M.D., Felsenfeld, G. (2004) Recruitment of histone modifications by USF proteins at a vertebrate barrier element Mol. Cell, 16, 453–463 .
Ohlsson, R., Renkawitz, R., Lobanenkov, V. (2001) CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease Trends Genet., 17, 520–527 .
Hatzis, P. and Talianidis, I. (2002) Dynamics of enhancer-promoter communication during differentiation-induced gene activation Mol. Cell, 10, 1467–1477 .
Kim, A. and Dean, A. (2003) A human globin enhancer causes both discrete and widespread alterations in chromatin structure Mol. Cell. Biol., 23, 8099–8109 .
Morshead, K.B., Ciccone, D.N., Taverna, S.D., Allis, C.D., Oettinger, M.A. (2003) Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4 Proc. Natl Acad. Sci. USA, 100, 11577–11582 .
Kimura, H., Tada, M., Nakatsuji, N., Tada, T. (2004) Histone code modifications on pluripotential nuclei of reprogrammed somatic cells Mol. Cell. Biol., 24, 5710–5720 .(Satoko Adachi and Ellen V. Rothenberg*)