Evidence for an Intronic Cis-Regulatory Element Within CD69 Gene

SHORT COMMUNICATION Evidence for an intronic cis-regulatory element within CD69 gene

BN Vazquez1,2, T Laguna1,2, L Notario1 and P Lauzurica1,2

CD69 is one of the earliest proteins expressed after leukocyte activation and its engagement is essential in the control of innate and adaptive immune responses. Inducible CD69 expression is strongly controlled at the transcriptional level. The molecular basis for developmental- and stage-specific regulation in T cells is beginning to be elucidated while it remains largely unknown in the rest of immune cells. DNase I hypersensitivity experiments in lymphocytes identified a novel hypersensitive region within mouse and human intron I, which was inducible upon stimulation. In silico analysis of CD69 gene revealed that this open chromatin region was present in different cell types and was associated with positioned nucleosomes. Analysis of histone post-translational modifications of intron I indicated that acetylation and lysine 4 dimethylation of histone H3 were dynamically regulated during thymocyte development and were constitutively high in resting and stimulated mature T lymphocytes. Thus, we provide evidence for the existence of a cis-acting element in intron I that is more accessible to DNase I digestion and that it is developmentally regulated at the chromatin level.

Genes and Immunity (2012) 13, 356--362; doi:10.1038/gene.2012.4; published online 29 March 2012 Keywords: CD69; intron; transcription regulation; chromatin

INTRODUCTION (TF) binding and it was able to modulate to some degree the CD69 is a transmembrane C-type lectin broadly expressed in bone expression of a reporter gene. Thus, our results point to Intron I as marrow-derived cells. Analysis of CD69 null mice indicates that it an important regulatory element of CD69 expression. has a key role in early inflammation and modulation of adaptative immune responses through the production of a variety of cytokines and lymphocyte migration.1--7 CD69 expression has RESULTS been described in human disease8,9 and, importantly, several Chromatin accessibility in mouse CD69 intron I reports support the therapeutic effect of anti-CD69 monoclonal DNase I hypersensitivity analysis is a useful tool to identify antibodies in the treatment of different animal models of cancer 18,19 2,10,11 potentially important cis-regulatory regions of genes. Our and autoimmune disorders. previous work identified multiple HSs mapping to the murine Activation-induced CD69 expression is transient and it is 12 -- 14 CD69 promoter and distal regulatory regions CNS1, CNS2, CNS3 strongly controlled at the level of transcription. The gene is and CNS4.16 These HSs presented similar pattern in CD69À and located in the NK complex in mouse chromosome 6 and human þ 13,15 CD69 cells and were important for CD69 expression in chromosome 12. CD69 gene is poised for activation. In T lymphocytes. As an attempt to identify other regulatory regions lymphocytes, constitutive high levels of permissive histone marks that may be also important for CD69 expression, we extended our are found in the transcription start site and, in the basal state, RNA 14,16 DNase I hypersensitivity mapping to the murine and human CD69- polymerase II (Pol II) is preloaded at the CD69 promoter. coding regions (Figures 1 and 2). For the murine CD69 locus, Moreover, nucleosomes surrounding the transcription start site T lymphocytes obtained from spleens of RagÀ/ÀxOT-I mice were contain predominantly H2AZ and H3 histones, which are thought 14,17 used. Purified cells were left unstimulated or were stimulated with to have a role in maintaining accessible chromatin. CD69 antiCD3/CD28 antibodies to induce CD69 expression. After 6 h, expression in a genomic context also requires the action of DNA 16 cells were treated or not with DNase I and genomic DNA was distal elements. Despite CD69 promoter confers developmen- purified. Chromatin accessibility was then measured by real-time tally regulated expression during positive selection of thymocytes, PCR (Figures 1a and b). In the basal state, the proximal region of cis-acting distal elements, named (conserved noncoding intron I was accessible to nuclease digestion (primer set 1 and 2) sequence) CNS1, CNS2, CNS3 and CNS4, are also necessary for while the distal one (primer set 3), closest to exon II, exhibited low developmental-stage and lineage-specific regulation in T cells. level of nuclease cutting (Figure 1b). CD69 gene displays rapid However, identification of other cis-acting elements is required to transcriptional induction upon activation and, consistent with this, gain further insights into the transcriptional control of CD69 in stimulation of cells resulted in a substantial increase in the T cells as well as other lymphocyte subtypes. chromatin accessibility across the entire intron I. Thus, our In this study we have examined the chromatin structure of murine experimental data identifies a HS zone that involves at least the and human CD69 intron I. We have found that intron I contains first 2 kb of CD69 intron I. several hypersensitive (HS) sites that are exclusively present in immune cells. These HS sites, and especially the one called HSa, were inducible upon stimulation. In addition, the epigenetic profile Chromatin structure of mouse CD69 Intron I of this intronic region was dynamic at different stages of lymphocyte In order to gain further insights about the regulatory capacity of maturation. Interestingly, HSa was enriched in transcription factor this intronic region, we analyzed its chromatin structure during

1Instituto de Salud Carlos III, Centro Nacional de Microbiologıá, Majadahonda, Madrid, Spain and 2Departament de Fisiologia, Universitat de Barcelona, Barcelona, Spain. Correspondence: Professor P Lauzurica, Instituto de Salud Carlos III, Centro Nacional de Microbiologıá, Laboratorio A-20, Carretera Majadahonda a Pozuelo Km 2,2, Madrid 28220, Spain. E-mail: [email protected] Received 5 December 2011; revised 13 February 2012; accepted 22 February 2012; published online 29 March 2012 Regulatory element in the first intron of CD69 gene BN Vazquez et al 357

Figure 1. Chromatin accessibility and structure of murine CD69 intron I. (a) Schematic diagram showing murine intron I and PCR-amplified regions in DNase I and Chip experiments. (b) Chromatin accessibility in murine CD69 intron I. T lymphocytes were either stimulated with antiCD3/CD28 mAb or not and were incubated with or without 15 U mlÀ1 of DNase I enzyme. Twenty nanograms of purified genomic DNA from each treatment were subjected to quantitative PCR. Results are plotted as the percentage of digested DNA in relation to the undigested sample. The data are representative of two experiments and are expressed as the mean±s.e.m. of triplicate PCRs. (c, d) Chromatin immunoprecipitation (ChIP) performed on purified mononucleosomes obtained from total thymocytes of Rxb and HA transgenic mice using anti acetylated H3 (c) and dimethylated H3 K4 (d). (e, f) ChIP performed on sonicated chromatin from purified T cells either À1 unstimulated or stimulated with 10 ng ml PMA plus 0.5 mM Ionomycin for 6 h using anti-acetylated H3 (e) and anti-dimethylated H3 K4 antibodies (f).

Figure 2. Chromatin accessibility of human CD69 intron I. (a) Map of the human CD69 locus showing locations of signiﬁcant features (In1: Intron 1; CNS1) and the genomic fragment expected from NdeI digestion. Probe used for Southern blot (S1.H) is also shown. (b) DNase I analysis using S1.H probe. Human peripheral blood lymphocytes were activated with 10 ng mlÀ1 PMA or were left untreated, and were subsequently treated with 0, 4, 8, 12, 16 and 20 U mlÀ1 of DNase I. NdeI digested DNA was examined by Southern blot. Size markers and relevant bands (HS sites) are denoted.

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 356 -- 362 Regulatory element in the first intron of CD69 gene BN Vazquez et al 358 T-cell development and in distinct activation cell states by line). This HS cluster is present in different human immune chromatin immunoprecipitation (Figures 1a and c--f). Levels of cells and, therefore, it is not cell-type specific. However, they are acetylated and lys4 dimethylated histone H3 (AcH3: acetylation of absent in endothelial HUVEC cells, BJ fibroblasts and embryonic histone H3 and H3K4dim: histone H3 lysine 4 dimethylation), stem cells H1-hESC and indicate that this regulatory region is marks for active and poised genes, respectively, were measured in only active in cells that express CD69 or are competent to do it. intron I, the promoter, which we reported to have constitutive The marked HS corresponds to the experimentally identified permissive histone modifications16 and Exon III. In T cells, CD69 is HSa (Figure 2b) as they both map to the middle of intron I and expressed during the positive selection of CD4 þ CD8 þ double- they are the strongest in intensity. In the same database, we also positive thymocytes, in transitional CD4 þ CD8À SP (single positive) examined the nucleosome profile of CD69 gene (Figure 3b) and, and CD4ÀCD8 þ SP thymocytes and in peripheral activated notably, the strongest HS region was adjacent to a region of lymphocytes.20,21 We used RagÀ/À mice that express a Tcrb well-positioned nucleosomes in melanoma and breast cancer transgene (Rxb) to obtain CD69À thymocytes, mice expressing a cells (compare Figures 3a and b). In mammals, well-positioned transgenic TCR specific for a hemagglutinin peptide (HA) to isolate nucleosomes may be observed in regulatory regions such as CD69 þ thymocytes and purified splenic T cells stimulated or not promoters and enhancers19,22 -- 26 and, thus, supports the with PMA to obtain CD69 þ and CD69À mature T lymphocytes. In existence of a regulatory element within this intronic site. CD69À Rxb thymocytes we observed low and moderate levels of Curiously, this nucleosome phasing was observed in nonimmune AcH3 and H3K4dim, respectively, in intron I (Figures 1c and d). cells that apparently are devoid of HSs (Figure 3a). This suggests CD69 þ HA thymocytes presented a fivefold increase in the that possibly intrinsic DNA sequence properties are responsible for amounts of AcH3 and nearly a twofold increase in the amounts this phenomenon. of H3K4dim (Figures 1c and d). CD69À Rxb and CD69 þ HA Finally, we analyzed public epigenetic data from primary non- thymocytes displayed high levels of these marks in the promoter stimulated CD4 þ T cells.22,27 Accordingly to our chip assays in the but had very lows levels in exon III. In peripheral T cells, regardless murine locus, we observed an enrichment of AcH3 and H3K4dim the expression of CD69, the chromatin of intron I always in intron I (Figure 4a). Remarkably, H3K14Ac was exclusively presented high levels of AcH3 and H3K4dim exceeding the enriched in the HSa region, and H3K4Ac, although also present in amounts observed at the promoter (Figures 1e and f). Exon III the promoter, had a peak at the same site (Figure 4a, upper rows). presented PMA-induced AcH3 and H3K4dim. The presence of H3 þ acetylation and H3K4 dimethylation in CD69 HA tg cells and DNA sequence conservation and transcription-binding sites in PMA-induced T cells is consistent with active CD69 gene CD69 intron I transcription. In CD69À Rxb thymocytes, low acetylation levels in Several distal regulatory regions for CD69 gene have been intron I may be related to a close chromatin structure of CD69- identified by means of cross-species DNA sequence comparison.16 coding region and, possibly, specific MHC--TCR interactions This approach, however, indicates that Intron I is less evolutiona- occurring during thymocyte-positive selection may result in rily conserved (Supplementary Figure 1A). A small CNS is present signal-induced acetylation and methylation marks in intron I as a proximal to Exon I and it is adjacent to a weak HS. However, result of transcription elongation. Resting lymphocytes do neither the strong HS nor the rest of the intronic region fulfill the not transcribe CD69 gene and, therefore, the presence of permissive criteria to be considered a CNS (70% identity over 100 bp length). histone marks in intron I in these cells was unexpected. Considering Therefore, and as described for other tissue-specific enhancers,28 that we have identified a new HS region in intron I, our Chip CD69 gene contains an intronic DNA regulatory region that is not data suggest that intron I may contain a developmental- sequence conserved under canonical basis. specific regulatory region that may become active in mature When searching for conserved binding sites for TFs, however, we lymphocytes. found several candidates within HSa (Supplementary Figure 2A). An interferon regulatory factor- (IRF) binding site was conserved in Chromatin accessibility and structure of human CD69 intron I all six mammal sequences analyzed including human and mouse. This finding was in agreement with Chip-seq data from the We extended our analysis to the human CD69 locus by using ENCODE consortium in which IRF4 was actually bound to HSa site peripheral blood lymphocytes. Cells were cultured with or without in non-stimulated lymphoid cells (Supplementary Figure 2B). PMA and were subsequently treated with increasing amounts of Interferon is a well-known inducer of CD69 expression in DNase I. Genomic DNA was purified, digested with NdeI enzyme response to infection by virus and, therefore, it is possible that and analyzed by southern blot using probe S1.H (Figure 2a). This IRF4 binding increases after cell stimulation. In addition to this assay identified multiple bands in resting and activated peripheral TF, this analysis also revealed that HSa also functioned as a blood lymphocytes (Figure 2b). Remarkably, a HS was clearly binder for HEY1, POU2F2, PAX5, BATF, PU.1, c-Myc and Pbx3 located in intron I (indicated as HSa) in unstimulated and (Supplementary Figure 2B). stimulated cells, being more prominent after stimulation. Importantly, this is the first inducible HS described for the CD69 gene. It is possible that this region participates in the induction of Luciferase activity of HSa region CD69 expression through the recruitment of stimulation dependent- To examine whether HSa may regulate CD69 gene transcription, a TFs. Pre-bound TFs in CD69À cells to this intronic site may also 400-bp spanning murine HSa region (chr6:129, 223, 614--129, 223, exist, as the HS is also present in unstimulated cells. 869) was examined in a luciferase reporter vector containing the Human peripheral blood lymphocytes contain a mixture of CD69 promoter and compared with CNS2, which has an important different immune cells such as T and B-lymphocytes and NK cells. enhancer activity as previously reported (Figure 5). When this As an attempt to identify whether this intronic HS was cell-type intronic fragment was placed upstream of the CD69 promoter, we specific, we analyzed open chromatin regions using public data did not observe any luciferase activity in the conditions assayed from the ENCODE consortium in the University of California Santa (data not shown). When this intronic element was placed Cruz (UCSC) Genome Browser (Figure 3a). Two remarkable results downstream and in sense orientation to the luciferase gene were obtained from this analysis: first, a marked HS in intron I is (Figure 5), there was a mild increase in the reporter activity. observed in primary human Th1 and Th2 immune cells and in Intriguingly, when using the same construct with reverse untreated GM12878 B cells and erythroleukemic K562 cells, and, orientation, we observed a small decrease. Therefore, this intronic second, additional weaker HSs are also present in human Intron I element is able to modulate the reporter activity driven by the concentrated 2 Kb downstream of the promoter (Figure 3a, upper CD69 promoter.

Genes and Immunity (2012) 356 -- 362 & 2012 Macmillan Publishers Limited Regulatory element in the ﬁrst intron of CD69 gene BN Vazquez et al 359

Figure 3. UCSC Genome Browser display on human CD69 locus. (a) ENCODE DNase I hypersensitivity track from Duke and Washington University in Th1, Th2, K562, GM12878, HUVEC, BJ and H1-hESC cells. (b) Nucleosome occupancy track in melanoma and breast cancer cells. For nucleosome positioning, a positive score corresponds to of frequent nucleosome occupancy and a negative score to a random nucleosome positioning.

Conclusion and/or in a chromatin context. Interestingly, HSa was enriched in We provide evidence for the existence of several cis-acting several TFs according to Chip-seq data in different cell lines. IFR4 is elements within CD69 intron I. These cis-acting elements exist as a an important candidate for future studies as IFN is clearly involved cluster of several DNase I HSs in human and mouse loci. in the induction of CD69 expression during viral infections. Future Remarkably, the accessibility of this region is inducible after experiments will be required in order to delineate the major lymphocyte stimulation. Despite CD69 is early expressed during regulatory function of this intronic region. To this purpose, lymphocyte activation, any inducible HS had been described modified bacterial artificial chromosomes containing the CD69 previously. It is possible that this intronic region may provide the locus are under construction in our laboratory. means to ensure a fast induction of the gene. Analysis of histone modifications in both human and mouse loci indicates that this intronic region presents an active epigenetic signature in mature MATERIALS AND METHODS lymphocytes and, at least for the murine locus, this epigenetic DNase I hypersensitivity assay signature appears to be developmentally regulated. The region Human peripheral blood lymphocytes and Rag-1 deficient OT-1 (Rag1À/À containing HSa was able to mildly modulate the expression of a XOT-I) transgenic splenocytes were used for the experiments. In both reporter gene in regular luciferase assays. However, and as we cases, red blood cells were lysed in 0.15 M NH4Cl, 1 mM KHCO3,1mM have previously shown for other CD69 regulatory regions, its Na2-EDTA, pH 7.4 for 3 min at 4 1C, subsequently washed in 30 ml of cold function may be only revealed in conjunction with other elements PBS and resuspended at a concentration of 2 Â 106 per ml in complete

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 356 -- 362 Regulatory element in the ﬁrst intron of CD69 gene BN Vazquez et al 360

Figure 4. Chromatin structure of human CD69 intron I. UCSC Genome Browser display on human CD69 locus for H3 acetylation at K14, K4 and K9 residues and H3 dimethylation at K4 (indicated above panels as H3K4Ac, H3K14Ac, H3K9Ac and H3K4me2). HSa is denoted by an arrow.

(10 mM Tris-HCl pH 8, 1 mM EDTA) and for quantitative PCR analysis in modiﬁed TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA).

Measurement of chromatin accessibility by southern blot For human samples, purified DNA (10 mg) was incubated overnight at 37 1C with an excess of NdeI restriction enzyme and was separated by 0.7% (w/vol) agarose gel electrophoresis. DNA was denatured by soaking the gel in 0.5 M NaOH/1.5 M NaCl for 45 min and neutralizing it in 1 M Tris-HCl pH 8, 1.5 M NaCl for 45 min. DNA was transferred to nitrocellulose membranes by capillary action in 20 Â SSC overnight, followed by cross-linking with UV light (Stratalinker, Stratagene, La Jolla, CA, USA). Blots were prehybridized for 3 h at 42 1C with hybridization solution (50% formamide, 5Â Denhardt’s solution, 0.5% (w/vol) SDS, 50 mM Hepes pH 7) containing À1 Figure 5. Luciferase activity of reporter constructs. Unstimulated or 100 mgml denatured salmon sperm DNA and were probed with 32 stimulated Jurkat cells were transfected with the indicated vectors. denatured P-labeled DNA fragments (Invitrogen Random primer kit, Arrows above constructs indicate sense (-) and antisense (’) Invitrogen, Carlsbad, CA, USA). Primers used to generate probe S1-H were orientations of the insert. Luciferase assays were performed after S1 (F): 50-CCCTCTGTACAATGGTGAAACA-30 and S2 (R): 50-GCAACTTCTCTT 48 h of transfection and results are expressed as the mean±s.e.m. of GGTCCAGTTT-30. Blots were washed twice in 1 Â SSC, 0.5% (w/vol) SDS for two experiments performed in duplicate transfections. 15 min at room temperature, twice in 0.1 Â SSC, 0.1% (w/vol) SDS for 15 min at 601 C and once in 0.1ÂSSC, 0.1% (w/vol) SDS, 0.1 mg mlÀ1 proteinase K for 30 min at 37 1C. DNA bands were visualized by media. Before DNase I digestion, human lymphocytes were stimulated autoradiography. À1 in vitro with 10 ng ml PMA and 0.5 mM Calcium Ionophore (Sigma-Aldrich, St Louis, MO, USA), and murine lymphocytes with 5 mgmlÀ1 plate-bound Measurement of chromatin accessibility by real-time PCR anti-CD3 (clone 145-2C11, eBiosciences, San Diego, CA, USA) and anti- For mouse CD69 intron I, accessibility to DNase I digestion was analyzed by CD28 (clone 37.51, eBiosciences) monoclonal antibodies for 6 h at 37 1C. quantitative PCR as described previously with only minor alterations.29 After this time, cells (107 per ml) were permeabilized with 0.067 mg mlÀ1 Samples were quantified using an ND-1000 spectrophotometer (Thermo- lysolecithicin (Sigma-Aldrich) in buffer C (0.15 M Sucrose, 80 mM KCl, 30 mM Fisher Scientific, Waltham, MA, USA). Twenty nanograms of purified

Hepes pH 7.4, 5 mM MgCl2,5mM CaCl2) for 5 min and were left untreated or genomic DNA were used to perform SYBR Green real time PCR according were treated with DNase I for 10 min on ice. DNase I was added at a final to manufacturer’s instructions (Roche, Branchburg, NJ, USA). The PCR- concentration of 0, 4, 8, 12, 16 and 20 U mlÀ1. Reactions were stopped by cycling program included a touchdown phase in which the annealing the addition of EDTA, SDS and Proteinase K to final concentrations of temperature was progressively lowered from 65 1Cto601Cby11C every À1 10 mM, 0.4% (w/vol) and 0.4 mg ml , respectively, and were incubated two cycles followed by 30 cycles at 60 1C. After amplification, a melting overnight at 37 1C. DNA was purified by phenol, phenol--chloroform curve and peak profile was obtained to verify the specificity of each primer and chloroform extractions and ethanol precipitation. For southern pair. A standard curve using serial dilutions of genomic DNA was

blot, genomic DNA was re-suspended in conventional TE buffer generated to quantify cycle threshold (Ct) values of each amplicon. Each

Genes and Immunity (2012) 356 -- 362 & 2012 Macmillan Publishers Limited Regulatory element in the first intron of CD69 gene BN Vazquez et al 361 PCR was performed in triplicates and DNase I sensitivity was expressed UCSC Genome Browser, the alignment was finally displayed in combina- as the percentage of accessibility [ ¼ 100 Â (1ÀDnase I-treated sample/ tion with the DNase I clusters from the ENCODE consortium. untreated DNA)]. Primer sequences are CONFLICT OF INTEREST 0 0 Set1F 5 -TGATAAATTGCCTGGCTTGAC-3 , The authors declare no conflict of interest. Set1R: 50-TGGATAAGGAAAGAGCAAGAAC-30, Set2F 50-TTGAGTCCAAGCAATGTGAC-30, 0 0 Set2R 5 -TAGGTCTGTTTTCCCCTGC-3 , ACKNOWLEDGEMENTS Set3F 50-TCTGAGCATTCCATCTCCC-30 This work was supported by grants (SAF2010-15649) from the Ministerio de and set3R 50-TTTAAGACTTCTAAGCACCCAC-30. Educacioń, Ciencia y Tecnolog´ıa and La Fundacio´ La Marato´ de TV3 2004. B Vazquez and T Laguna were supported by pre-doctoral fellowships from the Spanish Ministry Luciferase reporter constructs and cell transfections of Education and Science. We would like to thank Dr Mike Krangel at Duke University for providing scientific advice and Dr Elisenda Alari-Pahissa for the critical review of For the firefly luciferase vector we used the pGL3 vector (Promega, the manuscript. Fitchburg, WI, USA). Primers used to amplify the promoter were 50-GTCCAATTGAGAGAGAGGG-30 and 50-CCTTTTTAATTTTTTTCCCC-30 (primer promt F contained a BglII restriction site and the Promt R HindIII REFERENCES restriction site). Primers used to amplify the intron fragment were 0 0 0 1 Allende ML, Tuymetova G, Lee BG, Bonifacino E, Wu YP, Proia RL. S1P1 receptor 5 -GGAGGTACCGACCCTTGGAGATGGAAATAAA-3 and 5 -GGACTCGAGAGA directs the release of immature B cells from bone marrow into blood. J Exp Med 0 CATCACATGGCTAGTATCA-3 . To clone the intronic region upstream of the 2010; 207: 1113 -- 1124. 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