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• W2041639013 abstract "The human T-cell leukemia virus type 1 (HTLV-1) is integrated into the host cell DNA and assembled into nucleosomes. Within the repressive chromatin environment, the virally encoded Tax protein mediates the recruitment of the coactivators CREB-binding protein/p300 to the HTLV-1 promoter, located within the long terminal repeats (LTRs) of the provirus. These proteins carry acetyltransferase activity that is essential for strong transcriptional activation of the virus in the context of chromatin. Consistent with this, the amino-terminal tails of nucleosomal histones at the viral promoter are acetylated in Tax-expressing cells. We have developed a system in which we transfect Tax into cells carrying integrated copies of the HTLV-1 LTR driving the luciferase gene to analyze changes in “activating” histone modifications at the LTR. Unexpectedly, Tax transactivation led to an apparent reduction of these modifications at the HTLV-1 promoter and downstream region that correlates with a similar reduction in histone H3 and linker histone H1. Micrococcal nuclease protection analysis showed that less LTR-luciferase DNA is nucleosomal in Tax-expressing cells. Furthermore, nucleosome depletion correlated with RNA polymerase II recruitment and loss of SWI/SNF. The M47 Tax mutant, deficient in HTLV-1 transcriptional activation, was also defective for nucleosome depletion. Although this mutant formed complexes with CREB and p300 at the HTLV-1 promoter in vivo, it was unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results support a model in which nucleosomes are depleted from the LTR and transcribed region during Tax-mediated transcriptional activation and correlate RNA polymerase II recruitment with nucleosome depletion. The human T-cell leukemia virus type 1 (HTLV-1) is integrated into the host cell DNA and assembled into nucleosomes. Within the repressive chromatin environment, the virally encoded Tax protein mediates the recruitment of the coactivators CREB-binding protein/p300 to the HTLV-1 promoter, located within the long terminal repeats (LTRs) of the provirus. These proteins carry acetyltransferase activity that is essential for strong transcriptional activation of the virus in the context of chromatin. Consistent with this, the amino-terminal tails of nucleosomal histones at the viral promoter are acetylated in Tax-expressing cells. We have developed a system in which we transfect Tax into cells carrying integrated copies of the HTLV-1 LTR driving the luciferase gene to analyze changes in “activating” histone modifications at the LTR. Unexpectedly, Tax transactivation led to an apparent reduction of these modifications at the HTLV-1 promoter and downstream region that correlates with a similar reduction in histone H3 and linker histone H1. Micrococcal nuclease protection analysis showed that less LTR-luciferase DNA is nucleosomal in Tax-expressing cells. Furthermore, nucleosome depletion correlated with RNA polymerase II recruitment and loss of SWI/SNF. The M47 Tax mutant, deficient in HTLV-1 transcriptional activation, was also defective for nucleosome depletion. Although this mutant formed complexes with CREB and p300 at the HTLV-1 promoter in vivo, it was unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results support a model in which nucleosomes are depleted from the LTR and transcribed region during Tax-mediated transcriptional activation and correlate RNA polymerase II recruitment with nucleosome depletion. Human T-cell leukemia virus type 1 (HTLV-1) 4The abbreviations used are: HTLV-1, human T-cell leukemia virus type 1; CRE, cyclic AMP-response element; CREB, cyclic AMP-response element-binding protein; CBP, CREB-binding protein; RNAP II, RNA polymerase II; CHO, Chinese hamster ovary; Luc, luciferase; ChIP, chromatin immunoprecipitation; PCAF, p300/CBP-associated factor; LTR, long terminal repeat. 4The abbreviations used are: HTLV-1, human T-cell leukemia virus type 1; CRE, cyclic AMP-response element; CREB, cyclic AMP-response element-binding protein; CBP, CREB-binding protein; RNAP II, RNA polymerase II; CHO, Chinese hamster ovary; Luc, luciferase; ChIP, chromatin immunoprecipitation; PCAF, p300/CBP-associated factor; LTR, long terminal repeat. is the etiological agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (1Uchiyama T. Yodoi J. Sagawa K. Takatsuki K. Uchino H. Blood. 1977; 50: 481-492Crossref PubMed Google Scholar, 2Poiesz B.J. Ruscetti F.W. Gazdar A.F. Bunn P.A. Minna J.D. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7415-7419Crossref PubMed Scopus (3911) Google Scholar, 3Gessain A. Barin F. Vernant J.C. Gout O. Maurs L. Calender A. de The G. Lancet. 1985; ii: 407-410Abstract Scopus (2393) Google Scholar, 4Osame M. Usuku K. Izumo S. Ijichi N. Amitani H. Igata A. Matsumoto M. Tara M. Lancet. 1986; 1: 1031-1032Abstract PubMed Scopus (1900) Google Scholar). Although not fully understood, the cellular events leading to the onset of both diseases appear to be initiated by the HTLV-1-encoded Tax protein (5Azran I. Schavinsky-Khrapunsky Y. Aboud M. Retrovirology. 2004; 1: 20-43Crossref PubMed Scopus (119) Google Scholar). Tax functions as a potent activator of HTLV-1 transcription in part via the formation of a complex with CREB (or other activiting transcription factor/CREB members) and the three CRE enhancer sequences located within the HTLV-1 promoter (6Franklin A.A. Kubik M.F. Uittenbogaard M.N. Brauweiler A. Utaisincharoen P. Matthews M.A. Dynan W.S. Hoeffler J.P. Nyborg J.K. J. Biol. Chem. 1993; 268: 21225-21231Abstract Full Text PDF PubMed Google Scholar, 7Goren I. Semmes O.J. Jeang K.T. Moelling K. J. Virol. 1995; 69: 5806-5811Crossref PubMed Google Scholar, 8Adya N. Giam C.Z. J. Virol. 1995; 69: 1834-1841Crossref PubMed Google Scholar). Tax contributes to the stability of the ternary complex by binding directly to the GC-rich sequences flanking the octanucleotide CREs (9Kimzey A.L. Dynan W.S. J. Biol. Chem. 1998; 273: 13768-13775Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Lenzmeier B.A. Giebler H.A. Nyborg J.K. Mol. Cell. Biol. 1998; 18: 721-731Crossref PubMed Google Scholar, 11Lenzmeier B.A. Baird E.E. Dervan P.B. Nyborg J.K. J. Mol. Biol. 1999; 291: 731-744Crossref PubMed Scopus (47) Google Scholar, 12Lundblad J.R. Kwok R.P. Laurance M.E. Huang M.S. Richards J.P. Brennan R.G. Goodman R.H. J. Biol. Chem. 1998; 273: 19251-19259Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These sequences, called viral CREs, are absolutely required for strong Tax transcriptional activation of HTLV-1 (13Rosen C.A. Sodroski J.G. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6502-6506Crossref PubMed Scopus (72) Google Scholar, 14Rosen C.A. Park R. Sodroski J.G. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4919-4923Crossref PubMed Scopus (46) Google Scholar, 15Brady J. Jeang K.-T. Duvall J. Khoury G. J. Virol. 1987; 61: 2175-2181Crossref PubMed Google Scholar). Once associated with the promoter, Tax and CREB form a complex with the cellular coactivators CBP/p300 (16Kwok R.P. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 380: 642-646Crossref PubMed Scopus (308) Google Scholar, 17Giebler H.A. Loring J.E. Van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar). These coactivators are believed to participate in pre-initiation complex formation, culminating in strong HTLV-1 transcriptional activation (18Georges S.A. Kraus W.L. Luger K. Nyborg J.K. Laybourn P.J. Mol. Cell. Biol. 2002; 22: 127-137Crossref PubMed Scopus (51) Google Scholar). The HTLV-1 provirus is integrated into the genome of the infected host cell and assembled into nucleosomes. This chromatin packaging renders promoter DNA less accessible to the binding of transcription factors and therefore represses transcription. One mechanism for overcoming this repression is acetylation of the amino-terminal tails of the nucleosomal histones. This modification is proposed to increase the accessibility of nucleosomal DNA for transcription factor binding (19Hansen J.C. Wolffe A.P. Biochemistry. 1992; 31: 7977-7988Crossref PubMed Scopus (73) Google Scholar, 20Hansen J.C. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2339-2343Crossref PubMed Scopus (76) Google Scholar, 21Hansen J.C. Tse C. Wolffe A.P. Biochemistry. 1998; 37: 17637-17641Crossref PubMed Scopus (213) Google Scholar). Additionally, this and other histone modifications can serve as a platform for the binding of transcriptional regulatory proteins (22Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7468) Google Scholar). Previous studies have demonstrated that histone deacetylase inhibitors increase the level of HTLV-1 transcription and that Tax and histone deacetylase complex occupancy are mutually exclusive (23Lemasson I. Polakowski N. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 49459-49465Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 24Lu H. Pise-Masison C.A. Fletcher T.M. Schiltz R.L. Nagaich A.K. Radonovich M. Hager G. Cole P.A. Brady J.N. Mol. Cell. Biol. 2002; 22: 4450-4462Crossref PubMed Scopus (52) Google Scholar). It has also been shown that an increase in histone tail acetylation on HTLV-1 promoter-associated nucleosomes correlates with an increase in viral RNA in HTLV-1-infected T-cells. Among proteins that carry intrinsic histone acetyltransferase activity, the coactivators CBP/p300 have been shown to have an essential role in HTLV-1 transcriptional activation. Our laboratories, as well as others, detected CBP/p300 at the integrated HTLV-1 promoter in T-cells expressing high levels of Tax (23Lemasson I. Polakowski N. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 49459-49465Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 24Lu H. Pise-Masison C.A. Fletcher T.M. Schiltz R.L. Nagaich A.K. Radonovich M. Hager G. Cole P.A. Brady J.N. Mol. Cell. Biol. 2002; 22: 4450-4462Crossref PubMed Scopus (52) Google Scholar). This finding is consistent with several previous studies showing that Tax directly facilitates the recruitment of CBP/p300 to the HTLV-1 promoter (16Kwok R.P. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 380: 642-646Crossref PubMed Scopus (308) Google Scholar, 17Giebler H.A. Loring J.E. Van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar, 25Kashanchi F. Duvall J.F. Kwok R.P. Lundblad J.R. Goodman R.H. Brady J.N. J. Biol. Chem. 1998; 273: 34646-34652Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 26Harrod R. Tang Y. Nicot C. Lu H.S. Vassilev A. Nakatani Y. Giam C.Z. Mol. Cell. Biol. 1998; 18: 5052-5061Crossref PubMed Scopus (158) Google Scholar). Furthermore, we and others have shown that the histone acetyltransferase activity of p300 is essential for strong HTLV-1 transcription in a chromatin context (18Georges S.A. Kraus W.L. Luger K. Nyborg J.K. Laybourn P.J. Mol. Cell. Biol. 2002; 22: 127-137Crossref PubMed Scopus (51) Google Scholar, 27Okada M. Jeang K.T. J. Virol. 2002; 76: 12564-12573Crossref PubMed Scopus (40) Google Scholar). Using chromatin assembled with core histones lacking their amino-terminal tails and using specific inhibitors of CBP/p300, we have found that CBP/p300 participate in critical chromatin-specific, histone tail-independent acetylation events during transcriptional activation by Tax (28Georges S.A. Giebler H.A. Cole P.A. Luger K. Laybourn P.J. Nyborg J.K. Mol. Cell. Biol. 2003; 23: 3392-3404Crossref PubMed Scopus (44) Google Scholar). These data suggest that, in addition to the core histone tails, another target of acetylation by p300 functions in mediating strong Tax transactivation in a chromatin environment. In previous studies we have examined transcription factor binding and histone modifications at the viral promoter in Tax-expressing, HTLV-1-infected cells (23Lemasson I. Polakowski N. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 49459-49465Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 29Lemasson I. Polakowski N.J. Laybourn P.J. Nyborg J.K. Mol. Cell. Biol. 2004; 24: 6117-6126Crossref PubMed Scopus (48) Google Scholar). In this study, we sought to better define the epigenetic changes that occur during Tax-dependent activation of HTLV-1 LTR-directed transcription in vivo. These analyses were performed in cells carrying chromosomally integrated HTLV-1 LTR-luciferase constructs in the absence or presence of Tax. This approach enabled direct correlation of histone modifications and transcription factor occupancy with or without Tax expression. We characterized histone H3 acetylation associated with the HTLV-1 promoter and within the downstream coding region. This histone tail modification is typically associated with active genes. Unexpectedly, we found that the level of modified histones was reduced at the promoter and within the luciferase coding region in the presence of Tax. We determined that this reduction was a result of a Tax-dependent decrease in nucleosome density. To begin to investigate the mechanism of Tax-mediated nucleosome depletion, we used the activation-deficient M47 Tax mutant (30Smith M.R. Greene W.C. Genes Dev. 1990; 4: 1875-1885Crossref PubMed Scopus (346) Google Scholar). We found that this mutant is also deficient in nucleosome depletion. However, it is fully competent for the formation of a complex with CREB and p300 on the chromosomally integrated viral promoter. Prompted by the Tax-dependent loss of nucleosomes from the coding region of the construct, we compared RNA polymerase II (RNAP II) binding at the viral promoter in cells expressing wild type or M47 Tax. Significantly, we found that M47 Tax is disabled for recruitment of RNAP II. Interestingly, we also found that the SWI/SNF chromatin remodeling complexes are displaced by wild type Tax and that M47 Tax is defective for their displacement. These data correlate Tax-mediated SWI/SNF displacement and RNAP II recruitment with nucleosome depletion. We propose that Tax, SWI/SNF, and RNAP II each plays a role in nucleosome eviction and transcriptional activation of HTLV-1 transcription. Cell Culture and Transient Transfection Assays—CHOK1-Luc hamster ovary cells (27Okada M. Jeang K.T. J. Virol. 2002; 76: 12564-12573Crossref PubMed Scopus (40) Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, penicillin-streptomycin, and 500 μg of G418/ml (Geneticin; Invitrogen). For chromatin immunoprecipitation (ChIP) assays, cells were electroporated as described previously (31van den Hoff M.J. Christoffels V.M. Labruyere W.T. Moorman A.F. Lamers W.H. Methods Mol. Biol. 1995; 48: 185-197PubMed Google Scholar). Briefly, 2 × 107 cells were electroporated with a GenePulser Xcell electroporation device from Bio-Rad in the presence of 20 μg of total DNA. The pSG-Tax and pSG-Tax M47 expression plasmids have been described previously (30Smith M.R. Greene W.C. Genes Dev. 1990; 4: 1875-1885Crossref PubMed Scopus (346) Google Scholar, 32Rousset R. Desbois C. Bantignies F. Jalinot P. Nature. 1996; 381: 328-331Crossref PubMed Scopus (126) Google Scholar). The cells were then harvested at 20 h for luciferase and ChIP analysis. For each experiment, luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega) with a Turner Designs model TD 20-e luminometer. The electroporation transfection protocol used in this study produced transfection efficiencies approaching 50%. To enrich the transfected population in most experiments, cells were selected using the MACSelect System (Miltenyi Biotec). Briefly, the cells were co-transfected with pSG-Tax and pMACS 4.1 at a 2.5:1 molar ratio of pSG-Tax:pMACS 4.1. After 20 h, the cells were incubated with magnetic beads conjugated to a monoclonal antibody against the surface marker encoded by pMACS 4.1 and then passed through a magnetic field. Antibodies—Antibodies against p300 (catalog number N-15), RNA polymerase II (catalog number H-224), Brg1 (catalog number H-88) and hBrm (catalog number N-19) were purchased from Santa Cruz Biotechnology. Antibodies against acetylated lysines 9/14 of histone H3 (catalog number 06–599), acetylated lysines 5/8/12/16 of histone H4 (catalog number 06–866 or 06–598), dimethylated lysine 4 of H3 (catalog number 07–030), CREB (catalog number 06–863), and linker histone H1 (catalog number 05–457) were purchased from Upstate Biotechnology. The trimethylated lysine 4 of H3 (catalog number Ab 8580) and histone H3 (catalog number Ab 1791) antibodies were purchased from Abcam. Antibodies against acetylated lysine 9 of H3 (catalog number 9671) and acetylated lysine 8 of H4 (catalog number 2594) were purchased from Cell Signaling Technology. Tax monoclonal antibody (hybridoma 168B17–46-92) was obtained from the National Institutes of Health Aids Research and Reference Reagent Program. ChIP Assays, Real-time PCR, and Primers—ChIP assays, real-time PCR, and data analysis were performed as previously described (23Lemasson I. Polakowski N. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 49459-49465Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 29Lemasson I. Polakowski N.J. Laybourn P.J. Nyborg J.K. Mol. Cell. Biol. 2004; 24: 6117-6126Crossref PubMed Scopus (48) Google Scholar). PCR primers were as follows: HTLV-1 promoter (–349/–81), 5′-AATGACCATGAGCCCCA/GTGAGGGGTTGTCGTCA-3′; luciferase proximal (+708/+804), 5′-ATTTATCGGAGTTGCAGTTGCGCC/AACAAACACTACGGTAGGCTGCGA-3′; luciferase distal (+1527/+1717), 5′-TGAAGCGAAGGTTGTGGATCTGGA/AGAAGTGTTCGTCTTCGTCCCAGT-3′; luciferase Region 2 (+799/+1041), 5′-CGCAGTATCCGGAATGATTTGATTGCCA/ACGGATTACCAGGGATTTCAGTCG-3′; pGL3 vector 5′ (Region 1), 5′-GATCGGTGCGGGCCTCTTCGCTATTA/TCGATAGAGAAATGTTCTGGCACCTGCACT-3′; pGL3 vector 3′ (Region 3), 5′-GGG AGGTGTGGGAGGTTT/ACCGTATTACCGCCTTTGAGTGAG-3′; β-globin promoter, 5′-TCACACACTTGACCCTGTGCCATA/TTATATGCCCTGTCCTGGCTCCTT-3′. Standard curves were generated for all primer sets using 5-fold serial dilutions of CHOK1-Luc input DNA and were included on each experimental plate. PCR efficiencies for all of the primer sets ranged from 89–112% with correlation coefficients of 0.99 or greater. Quantitation was done by comparing threshold cycle values for co-immunoprecipitated DNA to the threshold cycle value for the input DNA in each ChIP experiment as described previously (33Frank S.R. Schroeder M. Fernandez P. Taubert S. Amati B. Genes Dev. 2001; 15: 2069-2082Crossref PubMed Scopus (418) Google Scholar). Western Blot Analysis—CHOK1-Luc cells transfected with pSG-Tax or pSG-M47 were resuspended in SDS sample dye. Proteins were separated on a 10% SDS-polyacrylamide gel and analyzed by Western blot analysis with the indicated antibodies. Micrococcal Nuclease/Dot Blot Assay—Segments of the LTR-luciferase construct indicated in Fig. 4A and the β-globin promoter were amplified by standard PCR from CHOK1-Luc genomic DNA. The primers used were (see Fig. 3B) (1) –319/–102, 5′-GGCTTAGAGCCTCCCAGTGAAA/CTGAGGGCGGCTTGACAAACAT-3′; (2) –67/+145, 5′-TCATGGCACGCATATGGCTGAA/CGGTCTCGACCTGAGCTTTAAACTTACC-3′; (5) +799/+1041, 5′-TTTCGCGGTTGTTACTTGACTGGC/ACTGGGACGAAGACGAACACTTCT-3′; (4) +1335/+1573, 5′-TGTCAATCAAGGCGTTGGTCGCTT/AGGGATACGACAAGGATATGGGCT-3′; (3) +1607/+1863, 5′-CGCAGTATCCGGAATGATTTGATTGCCA/ACGGATTACCAGGGATTTCAGTCG-3′. A region of the HTLV-1 env gene, used as a control, was amplified from HTLV-1-infected SLB-1 genomic DNA (5′-ACTCTAACCTAGACCACATC/CGTTACCATTTAACTGGACC-3′). PCR products were purified, spotted in triplicate onto a positively charged nylon membrane (3 μg of DNA/spot), and hybridized with 25 ng of radiolabeled total mononucleosomal DNA from CHOK1-Luc cells. Total mononucleosomal DNA was prepared by first isolating nuclei from CHOK1-Luc cells that had been treated with formaldehyde (1% final concentration at 37 °C for 10 m) as described previously (34Hager G.L. Fragoso G. Methods Enzymol. 1999; 304: 626-638Crossref PubMed Scopus (8) Google Scholar). Nuclei were exposed to micrococcal nuclease, and the nuclear DNA was extracted and purified as described previously (35Fragoso G. Hager G.L. Methods. 1997; 11: 246-252Crossref PubMed Scopus (33) Google Scholar). Purified DNA (found to be at least 80% mononucleosomal) was resolved on a 1.5% agarose gel, and the band corresponding to mononucleosome-length DNA was excised and the DNA purified from the gel slice. The mononucleosomal DNA was radiolabeled using the Random Primed Labeling Kit (Roche Diagnostics) for hybridization with the dot blot membrane. Following hybridization, membranes were exposed to a phosphorimaging screen and scanned using a Storm phosphorimaging device (Molecular Dynamics). Signal intensities for each triplicate were averaged, and the average signal for HTLV-1 env was used for background subtraction. Values for LTR-luciferase segments were normalized to the value obtained for the β-globin promoter. We confirmed that β-globin DNA was represented in the mononucleosomal DNA population by Southern blot analysis of DNA from MNase-treated CHOK1-Luc cells (data not shown).FIGURE 3Tax expression leads to nucleosome depletion from the HTLV-1 promoter and luciferase gene. A, schematic representation of the integrated HTLV-1 LTR-luciferase construct in CHOK1-Luc cells. The positions of the probes used in the dot blot analysis are indicated. B, CHOK1-Luc cells were transfected with pSG-Tax or control DNA (pUC19). Nuclei from formaldehyde-treated cells were subjected to micrococcal nuclease digestion 20 h following transfection. Mononucleosome-length DNA was purified and used to probe PCR-amplified regions of the LTR-luciferase construct spotted in triplicate onto a dot blot membrane. Boundaries for each amplicon with respect to the transcription start site are indicated. The left and right membranes were probed with mononucleosome-length DNA from control-transfected or Tax-expressing cells, respectively. C, graphical representation of the experimental results shown in B. The average signal from the HTLV-1 env gene region (negative control; absent in CHOK1-Luc cells) was subtracted from all other average signals. Values for the LTR-luciferase regions were then standardized to that of the β-globin gene promoter. Values were normalized to the signal obtained in absence of Tax (set to 1). The weaker signal from the β-globin promoter (which should have been fully nucleosomal) was because of the shorter length of this amplicon (140 base pairs) relative to the LTR-luciferase amplicons (212–257 base pairs). Use of the shorter β-globin amplicon was a consequence of the limited genomic sequence information available for hamster and related species. Numbers correspond to the LTR-luciferase regions indicated in A. The graph shows results from two independent experiments performed in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tax Expression Reduces Histone Tail Acetylation Associated with the HTLV-1 Promoter and Transcribed Region—There are several lines of evidence that link Tax-mediated HTLV-1 transcriptional activation with amino-terminal tail acetylation on core histones occupying the viral promoter (18Georges S.A. Kraus W.L. Luger K. Nyborg J.K. Laybourn P.J. Mol. Cell. Biol. 2002; 22: 127-137Crossref PubMed Scopus (51) Google Scholar, 23Lemasson I. Polakowski N. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 49459-49465Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 24Lu H. Pise-Masison C.A. Fletcher T.M. Schiltz R.L. Nagaich A.K. Radonovich M. Hager G. Cole P.A. Brady J.N. Mol. Cell. Biol. 2002; 22: 4450-4462Crossref PubMed Scopus (52) Google Scholar). However, the precise role that Tax plays in mediating these changes has not been examined. Therefore, we were interested in directly comparing histone acetylation and methylation patterns on HTLV-1 promoter nucleosomes in the absence and presence of Tax. To perform these studies, we optimized a system in which we transfected a Tax expression plasmid into CHOK1-Luc cells that carried two to four integrated copies of the HTLV-1 LTR driving expression of the firefly luciferase gene (Fig. 1A) (27Okada M. Jeang K.T. J. Virol. 2002; 76: 12564-12573Crossref PubMed Scopus (40) Google Scholar). Histone modifications at the viral promoter and within the luciferase gene were analyzed using the ChIP assay. Changes in acetylation were subsequently determined using real-time PCR with a primer set that specifically amplified a region of the HTLV-1 promoter surrounding the three viral CREs and primer sets that amplified a proximal and distal segment of the transcribed luciferase coding region (Fig. 1A). Data was quantified by comparing the signal from the co-immunoprecipitated DNA to that of the input DNA in each experiment. The CHOK1-Luc cells were initially developed to compare activation from a transiently transfected versus an integrated HTLV-1 promoter, which demonstrated the importance of using chromosomally integrated promoter constructs (27Okada M. Jeang K.T. J. Virol. 2002; 76: 12564-12573Crossref PubMed Scopus (40) Google Scholar). The integrated HTLV-1 promoter is the true physiological substrate for transcription factor binding and Tax transactivation, as the provirus is packaged into nucleosomes following integration into the host cell chromosome. We have used these cells in a previous study to demonstrate mutually exclusive binding of Tax and histone deacetylase complexes at the HTLV-1 promoter (29Lemasson I. Polakowski N.J. Laybourn P.J. Nyborg J.K. Mol. Cell. Biol. 2004; 24: 6117-6126Crossref PubMed Scopus (48) Google Scholar). Using the quantitative ChIP assay, we measured a histone modification typically associated with active genes (acetylation at H3 Lys-9/14) on nucleosomes present on the LTR-luciferase construct in CHOK1-Luc cells. Many “activating” histone modifications were previously identified on nucleosomes associated with the provirus in Tax-expressing HTLV-1 infected cells (23Lemasson I. Polakowski N. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 49459-49465Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). CHOK1-Luc cells were transfected with a Tax expression plasmid or a control plasmid, and samples were analyzed 20 h following transfection. Fig. 1B shows the results of this study. Unexpectedly, we found that Tax expression led to a >50% decrease in the detection of H3 Lys-9/14 acetylation at the HTLV-1 promoter and within the luciferase gene. A similar decrease was observed in a time course experiment examining H3 acetylation levels following transfection of Tax (data not shown). These results indicate that Tax recruitment to the HTLV-1 promoter directly correlates with the loss of histone acetylation at the promoter and within the luciferase gene. We also looked at other activating modifications of H3 and H4 and obtained similar results (data not shown). Fig. 1C shows that Tax expression did not produce a global reduction in the H3 acetylation level in the transfected CHOK-Luc cells. Loss of Chromatin Modifications during Tax-mediated Activation Correlates with the Loss of Histone H3 and Linker Histone H1—The decrease in histone acetylation at the HTLV-1 promoter upon Tax transactivation was unexpected and is antithetical to widely accepted models of chromatin modifications associated with transcriptional activation. Therefore, we hypothesized that the observed decline in histone tail acetylation upon transcriptional activation by Tax was due to a loss of histones from the integrated LTR-luciferase construct. To test this hypothesis, we used the ChIP assay to analyze histone H3 binding in the absence and presence of Tax. The antibody used in these experiments recognizes the carboxyl-terminal region of histone H3, which lacks sites of post-translational modification and should provide a constitutively available epitope. Using this antibody, we showed that the level of histone H3 associated with the HTLV-1 promoter was reduced by nearly 50% during Tax transactivation (Fig. 2A). Furthermore, the level of histone H3 detected within the proximal region of the luciferase gene was reduced in the presence of Tax. We also examined the binding of linker histone H1 in parallel experiments. Similar to our observations with H3, histone H1 binding at the HTLV-1 LTR and within the luciferase gene decreased following transfection of Tax into the CHOK1-Luc cells (Fig. 2B). These data suggest that the observed Tax-dependent reduction in activating histone modifications is because of a loss of core histones and H1 and thus intact nucleosomes from the LTR-luciferase construct. Our data do not support the replacement of H3 with the transcription-associated variant histone H3.3, as the H3 antibody recognizes both of these histones. In these experiments, the data were standardized against histones H3 and H1 measured at the transcriptionally silent β-globin promoter. We found that Tax expression had no effect on the level of H3 and H1 at the β-globin promoter (Fig. 2C). This control was not feasible in our examination of histone acetylation described in Fig. 1, as activating modifications were undetectable at the β-globin promoter (data not shown). Micrococcal Nuclease and Dot Blot Analysis Shows Nucleosome Loss in the Presence of Tax—The observed reduction in histone H3 from the HTLV-1 LTR could be due to either a reduc" @default.
• W2041639013 created "2016-06-24" @default.
• W2041639013 creator A5008442693 @default.
• W2041639013 creator A5022174147 @default.
• W2041639013 creator A5062123500 @default.
• W2041639013 creator A5084169286 @default.
• W2041639013 date "2006-05-01" @default.
• W2041639013 modified "2023-09-29" @default.
• W2041639013 title "Tax-dependent Displacement of Nucleosomes during Transcriptional Activation of Human T-Cell Leukemia Virus Type 1" @default.
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