| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Microbiological Research Group, National Center for Epidemiology, Pihenö u. 1, H-1529 Budapest, Hungary
2 Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany
3 Department of Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany
Correspondence
György Fejer
fejer{at}immunbio.mpg.de
Janos Minarovits
mini{at}microbi.hu
or
minimicrobi{at}hotmail.com
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
These authors contributed equally to this work. 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
for review). Transcripts for the EBV-encoded nuclear antigens (EBNAs) are initiated at alternative promoters (called Wp, Cp and Qp) that are regulated in a cell type-specific manner by epigenetic mechanisms (reviewed by Li & Minarovits, 2003). There is an inverse correlation between DNA methylation and Cp activity in lymphoid cells (Robertson et al., 1995; Salamon et al., 2001). In contrast, the regulatory region of Qp is unmethylated in all EBV latency types (I and II: Qp on; III: Qp off) (Tao et al., 1998; Salamon et al., 2001). Because the episomal copies of EBV DNA are in a chromatin structure (Dyson & Farrell, 1985), and the structure of histones affects the activity of chromatin, we used chromatin immunoprecipitation (ChIP; Weinmann & Farnham, 2002; Fejer et al., 2003) to detect covalent histone modifications (acetylation and methylation; reviewed by Verdone et al., 2005; Jenuwein & Allis, 2001) at Cp and Qp, in a panel of well characterized cell lines representing the major EBV latency types (Table 1). Two clones of the Burkitt's lymphoma (BL) line Mutu carry the same EBV strain but represent EBV latency type I and III, respectively (Gregory et al., 1990). Mutu-BL-I-Cl-216 maintains the phenotype of BL biopsy cells and expresses EBNA 1 and the small EBV-encoded RNAs, EBER 1 and 2, but EBNA 2–5 and latent membrane proteins (LMPs) cannot be detected in this clone (type I latency, Cp off, Qp on). In contrast, Mutu-BL-III-Cl-99 has a lymphoblastoid phenotype and expresses all six EBNAs, EBER 1 and 2, and LMP 1 and 2 (type III latency, Cp on, Qp off) (Gregory et al., 1990; Altiok et al., 1992; Bakos et al., 2007). The BL line Rael (latency I) and the lymphoblastoid cell line (LCL) CB-M1-Ral-STO (latency III) also carry the same EBV strain (Ernberg et al., 1989). Among the Qp-user non-BL cells, the nasopharyngeal carcinoma cell line C666-1 (Cheung et al., 1999) carries an EBV strain different from that carried by BC1, a well characterized primary effusion lymphoma cell line (Szekely et al., 1998).
|
; Fejer et al., 2003), using antibodies directed to diacetylated histone H3 and tetraacetylated histone H4 (AcH3 and AcH4). DNA from precipitated chromatin was amplified by real-time PCR. A pair-wise comparison of cell lines or clones carrying the same EBV strain but belonging to latency type III (Cp on) or latency type I (Cp off), respectively, showed that the actively used Cp in CB-M1-Ral-STO was 17-fold enriched in AcH3 compared with the silent Cp in Rael (Fig. 1a), whereas there was a 14-fold enrichment in AcH3 at the active Cp in Mutu-BL-III-Cl-99 compared with the silent Cp in Mutu-BL-I-Cl-216 (Fig. 1a). The silent C promoters were poor in AcH3 in C666-1 and BC1 cells as well (Fig. 1a). AcH4 was ninefold and almost 13-fold more abundant at the active Cp in CB-M1-Ral-STO and Mutu-BL-III-Cl-99 compared with the silent Cp in Rael and Mutu-BL-I-Cl-216, respectively, whereas the silent C promoters in C666-1 and BC1 cells were also associated with hypoacetylated histone H4 (Fig. 1b). In independent experiments, using the methods of semi-quantitative PCR and competitive PCR (Igaz et al., 1998), we obtained similar results (data not shown). A coordinated acetylation of histones H3 and H4 was observed at active cellular promoters as well (Zhang et al., 2005; Brinkman et al., 2007). Because densely methylated DNA associates with underacetylated histones in transcriptionally inactive chromatin regions (Jones et al., 1998; Nan et al., 1998; Fuks et al., 2000; Ng et al., 2000), we found that our results are consistent with the high level of CpG methylation found at inactive, but not active C promoters (Altiok et al., 1992; Robertson et al., 1995; Salamon et al., 2001). However, there is some variation between our data and the results of another laboratory (Chau & Lieberman, 2004). Chau and Lieberman observed only a marginal (1.4-fold) enrichment of AcH3 at the corresponding region of Cp in an LCL (Cp on; carrying unrelated EBV genomes than the BL cell line they studied) than in a BL line (Cp off). The 898 bp fragment they amplified (nt 10504–11402 of the prototype B95-8 EBV genome; P.M. Lieberman, personal communication) is considerably larger than (although covers) the 129 bp region amplified by our primers (covering nt 10981–11110 of the prototype B95-8 EBV genome). Identical results were achieved when analysing AcH4 levels. The differences between the results from the Lieberman laboratory and our laboratory regarding the AcH3 and AcH4 levels observed at Cp may be due to the different cell lines analysed and the different primers used. Treating Mutu-BL-I-Cl-216 and C666-1 cells (Cp off) with trichostatin A (TSA), an inhibitor of histone deacetylases, resulted in an enrichment of AcH4 at Cp (Fig. 1b). To confirm the functional significance of histone acetylation, we measured the level of Cp-initiated transcripts in cells differing in Cp usage (Fig. 1d) and in TSA-treated and control Mutu-BL-I-Cl-216 cells (Fig. 1f). Cp was active only in type III latency, as described previously (Woisetschlaeger et al., 1991; Altiok et al., 1992; reviewed by Györy & Minarovits, 2005). TSA alone or combined with cycloheximide (CHX) induced Cp-initiated transcription in Mutu-BL-I-Cl-216, although this induced transcription did not reach the level of Cp activity detected in the latency type III Mutu clone (Mutu-BL-III-Cl-99) (Fig. 1e). Because histone acetylation is associated with a chromatin structure permissive for transcription (Verdone et al., 2005), these data fit well with the enrichment of AcH4 at Cp in TSA-treated Mutu-BL-I-Cl-216 cells (Fig. 1b). Enrichment in histone H3 methylated at the lysine 4 (K4) residue (H3mK4) correlates with the activity of certain promoters (Jenuwein & Allis, 2001). Therefore, we analysed the level of H3K4me2 at active and silent C promoters. Surprisingly, we detected comparable H3K4me2 levels at Cp in both Mutu clones, although they differ in Cp usage (Fig. 1f). In contrast, H3K4me2 was approximately three times less abundant in Rael (Cp off) than in CB-M1-Ral-STO (Cp on). The lowest H3K4me2 signal was observed in C666-1 and BC1 (Cp off). These data show that even a high level of H3K4me2 is insufficient, by itself, to switch on Cp in Mutu-BL-I-Cl-216 cells, and are in contrast with the conclusion of Chau and Lieberman, who suggested that Cp activity is connected to enrichment in H3K4me2 (Chau & Lieberman, 2004). We suggest that H3K4me2 may contribute to the maintenance of a ‘poised’ chromatin state at the silent Cp in Mutu-BL-I-Cl-216, similar to inactive promoters at the β-globin locus (Schneider et al., 2004). We also report here that active Qp is enriched in AcH3, AcH4 and H3K4me2. We found elevated levels of AcH3, AcH4 and H3K4me2 at the active Q promoters (Rael, Mutu-BL-I-Cl-216 and C666-1) compared with the silent ones (CB-M1-Ral-STO and Mutu-BL-III-Cl-99) (Fig. 2). Among the cells actively using Qp the highest levels of chromatin-opening histone modifications were observed in C666-1 cells (Fig. 2). This corresponds to the high level of Qp-initiated transcripts detected in these cells (Bakos et al., 2007).
|
|
; Robertson et al., 1995; Salamon et al., 2001; Bakos et al., 2007) because methylated CpG-rich regions are known to be associated with histone deacetylases (Jones et al., 1998; Nan et al., 1998; Fuks et al., 2000; Ng et al., 2000). Our data show that the active (but not the silent) C promoters in latent EBV genomes are localized to a region rich in AcH3 and AcH4, which is similar to the acetylation islands characteristic to the active chromatin domains in human T cells, as described by Roh et al. (2005). Our data also fit well with our observation that enhanced levels of acetylated histones H3 and H4 mark the upregulated LMP2A promoter of EBV in lymphoid cells (Gerle et al., 2007). Using an LCL conditional for EBNA 2, Alazard et al. (2003) observed that in the presence of β-oestradiol binding of an EBNA 2/oestrogen receptor fusion protein to Cp results in a significant local increase in histone H4 acetylation, and a slight increase of histone H3 acetylation. In contrast, binding to another EBNA 2 activated regulatory region, the promoter of the LMP1 gene, correlated with an increase in AcH3, but not AcH4 (Alazard et al., 2003). These data fit well with our observation that active Cp is located on an acetylated island, although a fusion protein may induce somewhat different local histone modifications than native EBNA 2. The data of Alazard et al. (2003) are also in agreement with our observation that in Mutu-BL-I-Cl-216 cells inhibition of histone deacetylase by TSA upregulates Cp activity (Fig. 1e
) and results in an increased AcH4 level at Cp (Fig. 1b). We found that in clones of the BL line Mutu, H3K4me2 may be enriched at Cp independent of promoter activity (Fig. 1f). However, a significant enrichment of H3K4me2 at Cp in the Mutu-I cells was not observed in another laboratory (Chau & Lieberman, 2004; Day et al., 2007). This discrepancy may be due to the different passage history of the cells used. While Cp activity is regulated by both CpG methylation (Altiok et al., 1992; Minarovits et al., 1994; Robertson et al., 1995; Salamon et al., 2001; Bakos et al., 2007) and histone modifications (this study), Qp is unmethylated independently of its activity (Tao et al., 1998; Salamon et al., 2001). We observed that similar to active Cp, active Q promoters in lymphoid and epithelial cells are also located on an acetylation island enriched in AcH3 and AcH4. There is variation between our data and that of Day et al. (2007), who did not detect an enrichment in AcH3 at the active Qp in Mutu-I cells. This may be due to differences in the cells and methods used in the two laboratories. In addition to enrichment in AcH3 and AcH4, we also found that active Q promoters are selectively marked by high levels of H3K4me2. Day et al. (2007) also detected elevated H3K4me2 at Qp in the Mutu-I cells they studied, as well as in Raji cells (a BL line not known to use Qp). We suggest that Qp usage is regulated, in addition to a putative repressor protein (Salamon et al., 2001), by regional histone modifications. To our knowledge this is the first study connecting upregulation of Cp and Qp activity with a local, coordinated increase in the acetylation of histones H3 and H4.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Altiok, E., Minarovits, J., Hu, L. F., Contreras-Brodin, B., Klein, G. & Ernberg, I. (1992). Host-cell-phenotype-dependent control of the BCR2/BWR1 promoter complex regulates the expression of Epstein-Barr virus nuclear antigens 2–6. Proc Natl Acad Sci U S A 89, 905–909.
Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrel, P. J., Gibson, T. G., Hatfull, G., Hudson, G. S., Satchwell, S. C. & other authors (1984). DNA sequence and expression of the B95–8 Epstein-Barr virus genome. Nature 310, 207–211.[CrossRef][Medline]
Bakos, A., Banati, F., Koroknai, A., Takacs, M., Salamon, D., Minarovits-Kormuta, S., Schwarzmann, F., Wolf, H., Niller, H. H. & Minarovits, J. (2007). High resolution analysis of CpG methylation and in vivo protein-DNA interactions at the alternative Epstein-Barr virus latency promoters Qp and Cp in the nasopharyngeal carcinoma cell line C666–1. Virus Genes 35, 195–202.[CrossRef][Medline]
Brinkman, A. B., Pennings, S. W. C., Braliou, G. G., Rietveld, L. E. G. & Stunnenberg, H. G. (2007). DNA methylation immediately adjacent to active histone marking does not silence transcription. Nucleic Acids Res 35, 801–811.
Chau, C. M. & Lieberman, P. M. (2004). Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus. J Virol 78, 12308–12319.
Cheung, S. T., Huang, D. P., Hui, A. B. Y., Lo, K. W., Tsang, Y. S., Whitney, N. & Lee, J. C. K. (1999). Nasopharyngeal carcinoma cell line (C666–1) consistently harbouring Epstein-Barr virus. Int J Cancer 83, 121–126.[CrossRef][Medline]
Day, L., Chau, C. M., Nebozhyn, M., Rennekamp, A. J., Showe, M. & Lieberman, P. M. (2007). Chromatin profiling of Epstein-Barr virus latency control region. J Virol 81, 6389–6401.
Dyson, P. J. & Farrell, P. J. (1985). Chromatin structure of Epstein-Barr virus. J Gen Virol 66, 1931–1940.
Ernberg, I., Falk, K., Minarovits, J., Busson, P., Tursz, T., Masucci, M. G. & Klein, G. (1989). The role of methylation in the phenotype-dependent modulation of Epstein-Barr nuclear antigen 2 and latent membrane protein genes in cells latently infected with Epstein-Barr virus. J Gen Virol 70, 2989–3002.
Fejer, G., Medveczky, M. M., Horvath, E., Lane, B., Chang, Y. & Medveczky, P. G. (2003). The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts preferentially with the terminal repeats of the genome in vivo and this complex is sufficient for episomal DNA replication. J Gen Virol 84, 1451–1462.
Fuks, F., Burgers, W. A., Brehm, A., Hughes-Davies, L. & Kouzarides, T. (2000). DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat Genet 24, 88–91.[CrossRef][Medline]
Gerle, B., Koroknai, A., Fejer, G., Bakos, A., Banati, F., Szenthe, K., Wolf, H., Niller, H. H., Minarovits, J. & Salamon, D. (2007). Acetylated histone H3 and H4 mark the upregulated LMP2A-promoter of Epstein-Barr virus in lymphoid cells. J Virol 81, 13242–13247.
Gregory, C. D., Rowe, M. & Rickinson, A. B. (1990). Different Epstein-Barr virus-B cell interactions in phenotypically different clones of a Burkitt's lymphoma cell line. J Gen Virol 71, 1481–1495.
Györy, I. & Minarovits, J. (2005). Epigenetic regulation of lymphoid specific gene sets. Biochem Cell Biol 83, 286–295.[CrossRef][Medline]
Igaz, P., Fejer, G., Szalai, C., Toth, S. & Falus, A. (1998). Development of competitive mRNA PCR for the quantification of interleukin-6-responsive junB oncogene expression. Biotechniques 24, 854–860.[Medline]
Jenuwein, T. & Allis, C. D. (2001). Translating the histone code. Science 293, 1074–1080.
Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J. & Wolffe, A. P. (1998). Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 19, 187–191.[CrossRef][Medline]
Li, H. & Minarovits, J. (2003). Host cell-dependent expression of latent Epstein-Barr virus genomes: regulation by DNA methylation. Adv Cancer Res 89, 133–156.[Medline]
Liebowitz, D. (1998). Epstein-Barr virus pathogenesis. In Human Tumor Viruses, pp. 173–199. Edited by D. J. McCance. Washington, DC: ASM Press.
Minarovits, J. (2006). Epigenotypes of latent herpesvirus genomes. Curr Top Microbiol Immunol 310, 61–80.[Medline]
Minarovits, J., Hu, L. F., Minarovits-Kormuta, S., Klein, G. & Ernberg, I. (1994). Sequence-specific methylation inhibits the activity of Epstein-Barr virus LMP 1 and BCR2 enhancer-promoter regions. Virology 200, 661–667.[CrossRef][Medline]
Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N. & Bird, A. (1998). Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386–389.[CrossRef][Medline]
Ng, H. H., Jeppesen, P. & Bird, A. (2000). Active repression of methylated genes by the chromosomal protein MBD1. Mol Cell Biol 20, 1394–1406.
Niller, H. H., Salamon, D., Banati, F., Schwarzmann, F., Wolf, H. & Minarovits, J. (2004). The LCR of EBV makes Burkitt's lymphoma endemic. Trends Microbiol 12, 495–499.[CrossRef][Medline]
Robertson, K. D., Hayward, S. D., Ling, P. D., Samid, D. & Ambinder, R. F. (1995). Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial. Mol Cell Biol 15, 6150–6159.[Abstract]
Roh, T. Y., Cuddapah, S. & Zhao, K. (2005). Active chromatin domains are defined by acetylation islands defined by genome-wide mapping. Genes Dev 19, 542–552.
Salamon, D., Takacs, M., Ujvari, D., Uhlig, J., Wolf, H., Minarovits, J. & Niller, H. H. (2001). Protein-DNA binding and CpG methylation at nucleotide resolution of latency-associated promoters Qp, Cp and LMP1p of Epstein-Barr virus. J Virol 75, 2584–2596.
Schneider, R., Bannister, A. J., Myers, F. A., Thorne, A. W., Crane-Robinson, C. & Kozuarides, T. (2004). Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat Cell Biol 6, 73–77.[CrossRef][Medline]
Szekely, L., Chen, F., Teramoto, N., Ehlin-Henriksson, B., Pokrovskaja, K., Szeles, A., Manneborg-Sandlund, A., Lennette, E. T. & Klein, G. (1998). Restricted expression of Epstein-Barr virus (EBV)-encoded, growth transformation-associated antigens in an EBV- and human herpesvirus type 8-carrying body cavity lymphoma line. J Gen Virol 79, 1445–1452.[Abstract]
Tao, Q., Robertson, K. D., Manns, A., Hildesheim, A. & Ambinder, R. F. (1998). The Epstein-Barr virus major latency promoter Qp is constitutively active, hypomethylated and methylation sensitive. J Virol 72, 7075–7083.
Verdone, L., Caserta, M. & Di Mauro, E. (2005). Role of histone acetylation in the control of gene expression. Biochem Cell Biol 83, 344–353.[CrossRef][Medline]
Weinmann, A. S. & Farnham, P. J. (2002). Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26, 37–47.[CrossRef][Medline]
Woisetschlaeger, M., Jin, X. W., Yandava, C. N., Fumarsky, L. A., Strominger, J. L. & Speck, S. H. (1991). Role of the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection. Proc Natl Acad Sci U S A 88, 3942–3946.
Zhang, Y., Fatima, N. & Dufau, M. L. (2005). Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription. Mol Cell Biol 25, 7929–7939.
Received 16 November 2007;
accepted 12 February 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
