Abstract:
Human T-cell lymphotropic virus type-1 (HTLV-1) infects and transforms CD4 +  lymphocytes and causes Adult T-cell Leukemia/Lymphoma (ATLL), an aggressive, often fatal, lymphoproliferative disease. A conserved HTLV-1 3+ regulatory domain, pX, encodes at least five non-structural proteins, including the alternative splice-variant p30 II . HTLV-1 p30 II  may enhance the transforming activity of Myc and transcriptionally activate the human cyclin D2 promoter, dependent upon its conserved Myc-responsive enhancer elements, associated with markedly increased S-phase entry and multi-nucleation. Enhancement of Myc transforming activity by HTLV-1 p30 II  may be dependent upon the transcriptional coactivators, TRRAP/p434 6-8  and TIP60, require TIP60 histone acetyltransferase activity, and strongly correlate with interactions between HTLV-1 p30 II  and Myc-TIP60 complexes in HTLV-1-infected ATLL patient-derived lymphocytes. Thus, p30 II  may function as a novel retroviral modulator of Myc-transforming interactions that may prominently contribute to adult T-cell leukemogenesis. Thus, the present invention provides methods and compositions for screening and identifying agents that interfere with transformation.

Description:
RELATED APPLICATION  
       [0001]     This application claims priority to U.S. Provisional Patent Application Ser. No. 60/539,860, filed Jan. 27, 2004 and entitled “IDENTIFICATION OF ABERRANT MYC/TIP60 INTERACTIONS AS AN ESSENTIAL MEDIATOR OF NEOPLASTIC TRANSFORMATION: A NOVEL TARGET FOR ANTI-CANCER THERAPEUTICS.” 
     
    
     TECHNICAL FIELD  
       [0002]     The invention relates generally to methods and compositions for the treatment of cancer. The invention also relates to methods and compositions of screening candidate molecules for anticancer activity.  
       BACKGROUND OF THE INVENTION  
       [0003]     The Myc transcription factor promotes S-phase cell-cycle entry, induces apoptosis or programmed cell-death, and causes neoplastic cellular transformation. Expression of the c-Myc oncogene is deregulated in many solid tumors and hematological malignancies, including Adult T-cell Leukemia/Lymphoma, Diffuse Large-Cell Lymphomas (DLCL), Anaplastic CD30+ Large-Cell Lymphomas, and Burkitt&#39;s B-cell Lymphomas (a prominent AIDS-related malignancy). Myc is also deregulated in certain solid tumors containing the Myc gene locus mutations. The transforming viruses, human T-cell lymphotropic virus type-1 (HTLV-1) and Epstein Barr virus (EBV), deregulate Myc functions associated with development of ATLL and Burkitt&#39;s lymphomas, respectively.  
         [0004]     HTLV-1 infects CD4 +  T-cells and causes Adult T-cell Leukemia/Lymphoma (ATLL), an aggressive lymphoproliferative disease that is often fatal. HTLV-1-infected leukemic lymphocytes exhibit deregulated cell-cycle progression and characteristic multi-nucleation or polyploidy (evidenced by the appearance of ‘flower-shaped’ or lobulated nuclei). A conserved sequence, known as pX, located within the 3′ terminus of the HTLV-1 genome encodes at least five non-structural regulatory factors, including the viral trans-activator, Tax, and an alternative splice-variant, p30 II  (or Tax-ORF II, Tof), which possesses a functional trans-activation domain. The pX sequence is generally retained in the majority of ATLL patient isolates -even those containing partially-deleted proviruses, indicative of its importance for pathogenesis.  
         [0005]     The viral Tax protein transcriptionally activates numerous lymphoproliferative pathways (NF-κB, CREB/ATF, and p67 SRF ) and has been shown to inhibit transcription functions associated with the tumor suppressor p53 which likely contributes to a loss of G1/S-phase checkpoint control in HTLV-1-infected T-cells. Many of the pleiotropic effects of Tax upon cellular-signaling may derive from its aberrant recruitment of the transcriptional coactivators, p300/CREB-binding protein (p300/CBP) and p300/CBP-associated factor (P/CAF). Further, Tax interacts with cell-cycle modulators, including D-type cylin-cdk4/6 complexes, retinoblastoma (Rb) protein, and the human mitotic arrest-deficiency-1 (hMAD-1) protein. Although HTLV-1 Tax expression markedly promotes G1/S transition, Tax inhibits Myc-dependent trans-activation and prevents Myc-associated anchorage-independent cell-growth. Since ATLL patient-derived lymphocytes and tumors from HTLV-1 pX transgenic mice are known to possess deregulated Myc functions, other pX-encoded factors may influence Myc to promote cellular transformation by HTLV-1.  
         [0006]     Preliminary studies indicate that the HTLV-1 accessory protein p30 II  markedly increases S-phase cell-cycle progression and induces significant polyploidy. As relatively little is known with respect to the roles of pX-encoded accessory factors (e.g., p30 II , p13 II , p12 I , Rex p27 ) in HTLV-1-associated pathogenesis, the molecular mechanism by which p30 II  promotes Myc-dependent S-phase progression and multi-nucleation was sought. While others have proposed that HTLV-1 p30 II &#39;s functions are targeted against the viral LTR to repress HTLV-1 gene expression, it remains unclear whether these observations reflect the physiological role of p30 II . Nicot et al. (2004,  Nat. Med.  10:197-201) and Younis et al. (2004,  J. Virol.  78:11077-11083) have shown that p30 II , binds and inhibits nuclear export of the doubly-spliced Tax/Rex HTLV-1 mRNA, and it is intriguing that p30 II , might perform diverse functions to regulate viral gene expression and promote altered cellular growth—as noted for Tax which drives LTR trans-activation and deregulates host lymphoproliferative-signaling pathways. Robek et al. (1998,  J. Virol.  72:4458-4462) have previously demonstrated that p30 II  is dispensable for immortalization and transformation of human PBMCs by an infectious HTLV-1 molecular clone, ACH.p30 II , defective for p30 II  production. However, the ACH.p30 II  mutant exhibited an approx 20-50% reduction in transformation-efficiency compared to the wild-type ACH.wt suggesting that p30 II  is required for the full transforming-potential of HTLV-1.  
       SUMMARY OF THE INVENTION  
       [0007]     The present invention demonstrates that aberrant interactions between Myc (Accession No. 0907235A) and the histone acetyl transferase Tat interactive protein 60 kD (TIP60) (Accession No. U74667.1) drastically enhance the transforming potential of Myc. In some embodiments of the invention, interaction of Myc and TIP60 may be stabilized through interactions with factors associated with oncogenic viruses such as the HTLV-1 p30 II  protein (Accession No. AAB23361.1). For example, without being limited to any particular mechanism of action, HTLV-1 p30 II  may (a) recruit the transcriptional coactivator TIP60 to Myc-containing chromatin remodeling complexes assembled on conserved E-box (CACGAG) enhancer elements within promoters of Myc-responsive genes, (b) transcriptionally activate the human cyclin D2 promoter, (c) increase S-phase cell-cycle progression and polyploidy (multi-nucleation), and (d) markedly induce colony formation in transformation assays using immortalized human fibroblasts.  
         [0008]     A trans-dominant negative TIP60MAT mutant which contains an inactivating-deletion within the histone acetyltransferase (HAT) domain, abrogated foci-formation by HTLV-1 p30 II /Myc and significantly inhibited trans-activation from the human cyclin D2 promoter. These data indicate that aberrant Myc-TIP60 protein interactions prominently contribute to Myc-dependent neoplastic transformation and transcriptional activity, and further suggest that disruption of Myc-TIP60 complexes and inhibition of Myc-TIP60 complex formation are plausible approaches to impede malignancy in anti-cancer therapies.  
         [0009]     Thus, the present invention provides aberrant Myc-TIP60 interactions as a molecular target for the development of anti-cancer therapies. These therapies may impede malignancy or slow tumor progression. The present invention also provides screening methods for the identification of anti-cancer therapeutics that block, inhibit, weaken, or otherwise interfere with interactions between Myc and TIP60.  
         [0010]     Without being limited to any particular mode of action, factors encoded by transforming viruses (e.g. EBV, HPV, KSHV) or mutations of Myc may facilitate stabilization of Myc-TIP60 interactions to promote neoplastic cellular transformation.  
         [0011]     The present invention also relates to cells that may be used to detect Myc-TIP60 interaction. In some embodiments, these cells comprise: 
        a first nucleic acid including an expression control sequence having at least one E-box enhancer element and a reporter gene, wherein the expression control sequence is operatively linked to the reporter gene;     a second nucleic acid including an expression control sequence and a nucleotide sequence encoding human T-cell lymphotropic virus type-1 (HTLV-1) p30 II , wherein the expression control sequence is operatively linked to the nucleotide sequence encoding HTLV-1 p30 II ; and     a third nucleic acid including an expression control sequence and a nucleotide sequence encoding human TIP60, wherein the expression control sequence is operatively linked to the nucleotide sequence encoding human TIP60.        
 
         [0015]     In some embodiments, the expression control sequence of the first nucleic acid is selected from the group consisting of a human cyclin D2 promoter and a minimal thymidine kinase promoter. In some embodiments of the invention, the expression control sequence of the second nucleic acid and/or third nucleic acid may be a cytomegalovirus promoter. In some embodiments of the invention, the reporter gene may encode a protein selected from the group consisting of β-galactosidase, β-glucuronidase, autofluorescent proteins, including blue fluorescent protein (BFP) and green fluorescent protein (GFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).  
         [0016]     The present invention also provides methods of blocking, impeding, or otherwise interfering with the interaction of Myc and TIP60. For example, the invention provides a method of interfering with Myc and TIP60 interaction in a cell, comprising: contacting the cell with a nucleic acid, polypeptide, or organic molecule, wherein the nucleic acid, polypeptide, or organic molecule inhibits Myc-TIP60 interaction. In some embodiments of the invention, the organic molecule is a small molecule drug. The polypeptide may be a protein that comprises a TIP60 ΔHAT  protein. Similarly, the nucleic acid may be a nucleic acid that encodes a polypeptide comprising a TIP60 ΔHAT  protein.  
         [0017]     The invention further provides screening assays to identify one or more molecules that block, impede, or otherwise interfere with neoplastic transformation. For example, the invention provides a method of identifying a molecule that inhibits neoplastic transformation of a cell, comprising: 
        contacting a test cell with a test molecule;     measuring the cellular foci formed in the presence of the test molecule; and     comparing the number of foci formed in the presence of the test molecule with the number of foci formed by test cell in the absence of the test molecule, 
 
 wherein formation of fewer foci in the presence of the test molecule than in the absence of the test molecule indicates inhibition of neoplastic transformation, and wherein the test cell comprises: 
    a first nucleic acid comprising an expression control sequence and a nucleotide sequence encoding the Myc transcription factor, wherein the expression control sequence is operatively linked to the reporter gene;     a second nucleic acid comprising an expression control sequence and a nucleotide sequence encoding human T-cell lymphotropic virus type-1 (HTLV-1) p30 II , wherein the expression control sequence is operatively linked to the nucleotide sequence encoding HTLV-1 p30 II ; and     a third nucleic acid comprising an expression control sequence and a nucleotide sequence encoding human TIP60, wherein the expression control sequence is operatively linked to the nucleotide sequence encoding human TIP60. 
 
 According to the invention, the expression control sequence of the second nucleic acid and/or third nucleic acid may be a cytomegalovirus promoter. 
       
 
         [0024]     The invention also provides methods for identifying one or more molecules that block, impede, or otherwise interfere with Myc-TIP60 interactions. The invention provides, for example, a method of identifying a molecule that interferes with Myc-TIP60 interaction, comprising: 
        contacting a test cell with a test molecule wherein the test cell comprises: 
            a first nucleic acid comprising an expression control sequence comprising at least one E-box enhancer element and a reporter gene, wherein the expression control sequence is operatively linked to the reporter gene;     a second nucleic acid comprising an expression control sequence and a nucleotide sequence encoding human T-cell lymphotropic virus type-1 (HTLV-1) p30 II  wherein the expression control sequence is operatively linked to the nucleotide sequence encoding HTLV-1 p30 II ; and     a third nucleic acid comprising an expression control sequence and a nucleotide sequence encoding human TIP60, wherein the expression control sequence is operatively linked to the nucleotide sequence encoding human TIP60;     detecting the reporter gene expression in the presence of the test molecule; and     comparing the reporter gene expression in the presence of the test molecule with reporter gene expression in the absence of the test molecule, 
 
 wherein reduced reporter gene expression in the presence of the test molecule relative to reporter gene expression in the absence of the test molecule indicates inhibition of Myc-TIP60 interaction. 
   
               
 
         [0031]     According to the invention, the expression control sequence of the first nucleic acid may be selected from the group consisting of a human cyclin D2 promoter and a minimal thymidine kinase promoter. In addition, the expression control sequence of the second nucleic acid and/or third nucleic acid may be a cytomegalovirus promoter. In some embodiments of the invention, the reporter gene may encode a protein selected from the group consisting of β-galactosidase, β-glucuronidase, autofluorescent proteins, including blue fluorescent protein (BFP) and green fluorescent protein (GFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).  
         [0032]     In some embodiments the invention provides a method of detecting cancer in a test tissue sample, comprising: 
        detecting Myc-TIP60 complexes in the test tissue sample; and     comparing the Myc-TIP60 complexes in the tissue sample with Myc-TIP60 complexes in a corresponding non-cancerous tissue, 
 
 wherein an elevated level of Myc-TIP60 complexes in the test tissue sample relative to the non-cancerous tissue indicates the presence of cancer. The method of detecting complex formation may be accomplished by any means of detection protein-protein interactions known in the art. For example, detection may be achieved by lysing cells of the test tissue sample, forming a clear extract, and immunoprecipitating Myc-interacting complexes with an anti-HA tag antibody. The test tissue sample may be derived from any source including, without limitation, tissue biopsies.
       
 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0035]     The patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee.  
         [0036]     A more complete and thorough understanding of the present embodiments and advantages thereof may be acquired by referring to the following description taken in conjunction with the accompanying drawings, in which like reference numbers indicate like features, and wherein:  
         [0037]      FIG. 1A  is a diagram of the HTLV-1 proviral genome and its translation products with the the viral transcription factors, Tax and p30 II , are in bold;  
         [0038]      FIG. 1B  illustrates a RasMol structural prediction of the HTLV-1 p30 II  protein in which sub-domains (4 alpha-helices; 19 beta-sheets) are represented by different colors and Connelly/Richards (1.2 Å) radii are indicated in white;  
         [0039]      FIG. 1C , left panel illustrates a plot of DNA content (assayed by 7-AAD) versus DNA synthesis (S-phase; assayed by BrdU content) for Molt-4 lymphocytes that were transfected with an empty CβS vector control (3.0 μg);  
         [0040]      FIG. 1C , right panel illustrates a fluorescent activated cell sorting (FACS) analysis of the percentages of apoptotic cells in cultures of CβS-transfected Molt-4 lymphocytes stained with annexin-V-(FITC)/propidium iodide;  
         [0041]      FIG. 1D  illustrates flow cytometry results using Molt-4 lymphocytes that were transfected with an empty CβS vector control (3.0 μg)  
         [0042]     Table 1 shows the relative percentages of CβS-transfected Molt-4 lymphocytes in various stages of the cell-cycle as quantified using aneuploid analysis software (ModFit LT 3.0);  
         [0043]      FIG. 1E , left panel illustrates a plot of DNA content (assayed by 7-AAD) versus DNA synthesis (S-phase; assayed by BrdU content) for Molt-4 lymphocytes that were transfected with CMV-HTLV-1 p30 II  (HA) (3.0 μg);  
         [0044]      FIG. 1E , right panel illustrates a fluorescent activated cell sorting (FACS) analysis of the percentages of apoptotic cells in cultures of CMV-HTLV-1 p30 II -transfected Molt-4 lymphocytes stained with annexin-V-(FITC)/propidium iodide;  
         [0045]      FIG. 1F  illustrates flow cytometry results using Molt-4 lymphocytes that were transfected with CMV-HTLV-1 p30 II  (HA) (3.0 μg);  
         [0046]     Table 2 shows the relative percentages of CMV-HTLV-1 p30 II -transfected Molt-4 lymphocytes in various stages of the cell-cycle as quantified using aneuploid analysis software (ModFit LT 3.0);  
         [0047]      FIG. 2A  illustrates the results of immunofluorescence-laser confocal microscopy performed on HTLV-1-infected ATLL patient-derived T-cells (ATL-1, ATL-2, ATL-3) or Jurkat E6.1 lymphocytes as a negative control, using a monoclonal anti-Myc antibody (blue, left panels) or a rabbit polyclonal anti-HTLV-1 p30 II  antibody (green, left-middle panels), merged images of the Myc and HTLV-1 p30 II  localization (right-middle panels), and merged images with viewed with phase contrast optics;  
         [0048]      FIG. 2B  illustrates a three-dimensional Z-stack composite for ATL-3 with three rotational views of merged images (demonstrating nuclear co-localization of HTLV-1 p30 II  (green)/Myc (blue) in all focal planes) and a graphical representation of relative fluorescence-intensities for HTLV-1 p30 II /Myc-specific signals and DAPI nuclear-staining for reference;  
         [0049]      FIG. 2C  illustrates a co-immunoprecipitations performed using extracts prepared from HTLV-1-infected ATLL patient-derived lymphocytes and anti-Myc or anti-HTLV-1 p30 II  antibodies;  
         [0050]      FIG. 2D , upper panel illustrates the results of co-immunoprecipitation assays of Jurkat E6.1, HuT-102, and MJ[G11] lymphocytes transfected with an empty CβS vector control or CMV-HTLV-1 p30 II  (HA) using a monoclonal anti-HA tag antibody (CA5, Roche Molecular Biochemicals);  
         [0051]      FIG. 2D , lower panel illustrates the results of co-immunoprecipitation assays of Jurkat E6.1 whole-cell extracts using antibodies against known. RNA Polymerase II and TIP48 binding partners (anti-p300; anti-Myc) with an anti-HTLV-1 Tax monoclonal antibody (20) was used as a negative control;  
         [0052]      FIG. 2E  illustrates the results of co-immunoprecipitation assays of Jurkat E6.1, HuT-102, and MJ[G11] lymphocytes transfected with an empty CβS vector control or CMV-HTLV-1 p30 II  (HA) using a monoclonal anti-HA tag antibody (CA5, Roche Molecular Biochemicals) in which HTLV-1 p30 II -interacting proteins were detected by immunoblotting;  
         [0053]      FIG. 3A  shows a bar graph with the results of luciferase assays of HeLa cells co-transfected with a human cyclin D2 promoter-luciferase reporter plasmid and increasing amounts of CMV-HTLV-1 p30 II  (HA) and, in the lower panel, the expression of HTLV-1 p30 II  (HA), Myc, and Actin in transfected cells;  
         [0054]      FIG. 3B  shows a bar graph with the results of luciferase assays of HeLa cells co-transfected as in  FIG. 3A , lacking conserved Myc-responsive E-box enhancer elements, and increasing amounts of CMV-HTLV-1 p30 II  (HA);  
         [0055]      FIG. 3C  shows a bar graph with the results of luciferase assays of 293A Fibroblasts co-transfected as in  FIG. 3A  with a human cyclin D2 promoter-luciferase reporter plasmid and increasing amounts of CMV-HTLV-1 p30 II  (HA) and, in the lower panel, the expression of HTLV-1 p30 II  (HA), Myc, and Actin in transfected cells detected by immunoblotting;  
         [0056]      FIG. 3D  shows a bar graph with the results of luciferase assays of 293A Fibroblasts co-transfected as in  FIG. 3A  with a synthetic, E-box-containing minimal tk promoter-luciferase reporter construct (M4-tk-luc) and increasing amounts of CMV-HTLV-1 p30 II  (HA) (error bars representing standard deviations are provided);  
         [0057]      FIG. 4A  shows a bar graph with the results of luciferase assays of HeLa cells were co-transfected with a human cyclin D2 promoter-luciferase reporter plasmid (0.5 kg) and CMV-HTLV-1 p30 II  (HA) (0.15 kg) in the presence of increasing amounts of CMV-wild-type TIP60, CMV-TIP60 ΔHAT , or CMV-TIP60 L497A  and, in the lower panel, the expression of HTLV-1 p30 II  (HA) and Actin detected by immunoblotting (lower panels);  
         [0058]      FIG. 4B  shows a bar graph with the results of luciferase assays of HeLa cells co-transfected as in  FIG. 4A  with a human cyclin D2 promoter-luciferase plasmid and CMV-HTLV-1 p30 II  (HA) in the presence of increasing amounts of CβS-TRRAP anti-sense  or CβF-TRRAP 1261-1579  and, in the lower panel, the expression of the trans-dominant negative TRRAP 12611-1579 -(FLAG) mutant, HTLV-1 p30 II  (HA), Myc, and Actin proteins detected by immunoblotting using an anti-FLAG M2 monoclonal antibody (SIGMA Chemical Corp.), anti-HA (CA5) or anti-Myc monoclonal antibodies, or anti-Actin goat polyclonal antibody (error bars representing standard deviations are provided).;  
         [0059]      FIG. 4C  illustrates over-expression of the (FLAG)-TIP60 (wild-type) and (FLAG)-TIP60 ΔHAT  proteins (23) relative to endogenous TIP60 as visualized by immunofluorescence-microscopy using a rabbit polyclonal anti-TIP60 antibody (top panels) and an anti-FLAG M2 monoclonal antibody (bottom panels) with the CβS empty vector transfected as a negative control;  
         [0060]      FIG. 5A  illustrates the results of chromatin-immunoprecipitation assays performed on uninfected Molt-4 lymphocytes using antibodies that recognize various Myc-interacting factors (TIP60, TRRAP, TIP48, TIP49, hGCN5; upper panel) or acetylated forms of histone H3 (Acetyl-K9, Acetyl-K14; lower panel);  
         [0061]      FIG. 5B  illustrates the results of chromatin-immunoprecipitation assays performed on HTLV-1-infected MJ[G11] lymphocytes using antibodies that recognize various Myc-interacting factors (TIP60, TRRAP, TIP48, TIP49, hGCN5; upper panel) or acetylated forms of histone H3 (Acetyl-K9, Acetyl-K14; lower panel);  
         [0062]      FIG. 5C  illustrates the results of chromatin-immunoprecipitation assays performed on HTLV-1-infected HuT-102 lymphocytes using antibodies that recognize various Myc-interacting factors (TIP60, TRRAP, TIP48, TIP49, hGCN5; upper panel) or acetylated forms of histone H3 (Acetyl-K9, Acetyl-K14; lower panel);  
         [0063]      FIG. 5D  shows a diagram of GST-HTLV-1 p30 II  fusion proteins and relative input levels of GST-HTLV-1 p30 II  and GST-p30 II  truncation mutants, Myc, and TIP60 proteins;  
         [0064]      FIG. 5E  shows the input for GST-pull-down experiments using HeLa extracts and purified recombinant GST-HTLV-1 p30 II  or GST-p30 II  (1-98), GST-p30 II  (99-154), and GST-p30 II  (155-241) truncated mutant proteins;  
         [0065]      FIG. 5F  shows the results from GST-pull-down experiments using HeLa extracts and purified recombinant GST-HTLV-1 p30 II  or GST-p30 II  (1-98), GST-p30 II  (99-154), and GST-p30 II  (155-241) truncated mutant proteins;  
         [0066]      FIG. 5G  illustrates the results of ChIP analyses of HTLV-1 p30 II -Myc/TIP60 transcription complexes recruited to Myc-responsive E-box elements within the genomic cyclin D2 promoter in cultured HTLV-1-infected ATLL patient (ATL-1) lymphocytes in which PCR analyses of ChIP products were carried-out using PRM and UTR oligonucleotide primer pairs;  
         [0067]      FIG. 6A  illustrates expression of HTLV-1 p30 II -GFP in transfected 293A fibroblasts visualized by fluorescence-microscopy;  
         [0068]      FIG. 6B  shows the results of ChIP analyses performed on 293A fibroblasts transfected with HTLV-1 p30 II -GFP using various antibodies against specific Myc-interacting proteins;  
         [0069]      FIG. 6C  illustrates expression of GFP in 293A fibroblasts transfected with a pcDNA3.1-GFP control and visualized by fluorescence-microscopy;  
         [0070]      FIG. 6D  shows the results of ChIP analyses performed on 293A fibroblasts transfected with a pcDNA3.1-GFP control using various antibodies against specific Myc-interacting proteins;  
         [0071]      FIG. 6E  shows the results of luciferase assays in which 293A Fibroblasts were co-transfected with a human cyclin D2 promoter-luciferase reporter construct, CMV-HTLV-1 HTLV-1 p30 II -GFP, and increasing amounts of CMV-TIP60 (wild-type) or CMV-TIP60 ΔHAT  (error bars represent of standard deviations from duplicate experiments);  
         [0072]      FIG. 6F  shows the results of luciferase assays in which 293A Fibroblasts were co-transfected with a tk-promoter-renilla-luciferase reporter construct, CMV-HTLV-1 HTLV-1 p30 II -GFP, and increasing amounts of CMV-TIP60 (wild-type) or CMV-TIP60 ΔHAT  (error bars represent of standard deviations from duplicate experiments);  
         [0073]      FIG. 7A  shows a graphical illustration of microarray gene expression analyses performed on 293A fibroblasts transfected with a CβS empty vector control or CMV-HTLV-1 p30 II  (HA) using Affymetrix Human U133Plus 2.0 full-genomic chips in which transcriptional activation of cellular genes by HTLV-1 p30 II  is expressed as Fold Activation relative to the empty CβS vector control;  
         [0074]      FIG. 7B  shows a graphical illustration of microarray gene expression analyses performed on 293A fibroblasts transfected with a CβS empty vector control, CMV-HTLV-1 p30 II  (HA), or CMV-HTLV-1 p30 II  (HA) +CMV-TIP60 ΔHAT  using Affymetrix Human U133Plus 2.0 full-genomic chips in which transcriptional activation of cellular genes by HTLV-1 p30 II  is expressed as Fold Activation relative to the empty CβS vector control and TIP60-dependent genes are identified based upon their transcriptional repression in the presence of the trans-dominant-negative TIP60 ΔHAT  mutant;  
         [0075]      FIG. 7C  shows a graphical illustration of microarray gene expression analyses performed on 293A fibroblasts transfected with a CβS empty vector control or CMV-HTLV-1 p30 II  (HA) using Affymetrix Human U133Plus 2.0 full-genomic chips in which transcriptional repression of cellular genes by HTLV-1 p30 II  is expressed as Fold Repression relative to the empty CβS vector control;  
         [0076]      FIG. 7D  shows a graphical illustration of microarray gene expression analyses performed on 293A fibroblasts transfected with a CβS empty vector control, CMV-HTLV-1 p30 II  (HA), or CMV-HTLV-1 p30 II  (HA) +CMV-TIP60 ΔHAT  using Affymetrix Human U133Plus 2.0 full-genomic chips in which transcriptional repression of cellular genes by HTLV-1 p30 II  is expressed as Fold Repression relative to the empty CβS vector control and TIP60-dependent or TIP60-independent genes are identified based upon their transcriptional repression in the presence of the trans-dominant-negative TIP60 ΔHAT  mutant;  
         [0077]      FIG. 8A  illustrates representative results from triplicate foci-formation assays using immortalized human WR −/−  fibroblasts transfected with CβS empty vector (3.0 μg), CMV-HTLV-1 p30 II  (HA) (3.0 μg), CβF-FLAG-Myc (3.0 μg), and combinations of CβS (1.5 μg)/CβF-FLAG-Myc (3.0 μg) or CMV-HTLV-1 p30 II  (HA) (1.5 μg)/CβF-FLAG-Myc (3.0 μg);  
         [0078]      FIG. 8B  is a bar graph of the results shown in  FIG. 8A ;  
         [0079]      FIG. 8C  shows micrographs of CβS/CβF-FLAG-Myc-transfected (upper panels) or CMV-HTLV-1 p30 II  (HA)/CβF-FLAG-Myc-transfected (lower panels) immortalized human WRN −/−  fibroblasts viewed with phase contrast optics (left panels) or after staining with DAPI (middle panels) or a monoclonal anti-HA tag antibody and a rhodamine red-conjugated anti-mouse secondary antibody (right panels)  
         [0080]      FIG. 8D  shows HTLV-1 p30 II  (HA)/Myc-transformed fibroblasts stained with DAPI (left panel) and a monoclonal anti-HA tag antibody and a rhodamine red-conjugated anti-mouse secondary antibody viewed in the blue channel (left panel), red channel (center panel), and both the blue and red channels (right panel);  
         [0081]      FIG. 8E  shows an increased number of multi-nucleated giant cells observed in isolated HTLV-1 p30 II  (HA)/Myc-transformed WRN −/−  fibroblasts expanded in culture (arrows, micrograph) and expression of HTLV-1 p30 II  (HA) detected by immunoblotting using a monoclonal anti-HA antibody (lower panel);  
         [0082]      FIG. 9A  shows the results of a first foci-formation/transformation assay using immortalized human WRN −/−  fibroblasts transfected with CβF-FLAG-Myc (3.0 μg) and either CMV-HTLV-1 p30 II  (HA) or empty CβS vector control (1.5 μg) in the presence of CMV-TIP60, CMV-TIP60 ΔHAT , or CMV-TIP60 L497A  (3.0 μg)  
         [0083]      FIG. 9B  shows the results of a second foci-formation/transformation assay using immortalized human WRN −/−  fibroblasts transfected with CβF-FLAG-Myc (3.0 μg) and either CMV-HTLV-1 p30 II  (HA) or empty CβS vector control (1.5 μg) in the presence of CMV-TIP60, CMV-TIP60 ΔHAT , or CMV-TIP60 L497A  (3.0 μg);  
         [0084]      FIG. 9C  shows that over-expression of wild-type TIP60 results in increased foci-formation in WRN −/−  fibroblasts co-transfected with CMV-HTLV-1 p30 II  (HA), CβF-FLAG-Myc, and CMV-TIP60;  
         [0085]      FIG. 9D  shows that co-expression of the trans-dominant-negative TIP60 ΔHAT  mutant in the cells of FIG.  9 C inhibits cellular transformation by HTLV-1 p30 II  (HA)/Myc;  
         [0086]      FIG. 9E  shows representative results from duplicate foci-formation/transformation assays using immortalized human WRN −/−  fibroblasts transfected as in  FIGS. 9A  and 9B in the presence of increasing amounts of CβS-TRRAP anti-sense  or CβS empty vector (0.5, 1.5, 3.0 μg) with an asterisk denoting HTLV-1 p30 II  (HA)/Myc foci-formation; and  
         [0087]      FIG. 10  illustrates a model of HTLV-1 p30 II  modulatory interactions with Myc-TIP60 transcription complexes assembled on E-box enhancer elements within promoters of Myc-responsive genes (nucleosomal acetylation associated with transcriptional activation indicated). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0088]     HTLV-1 p30 II  increases S-phase progression and promotes polyploidy. The conserved pX domain of HTLV-1 encodes at least five non-structural regulatory factors, including the viral trans-activator, Tax, and an alternative splice-variant, p30 II  ( FIG. 1A ). The HTLV-1 p30 II  protein is comprised of 241 amino acid residues and contains Arg- and Ser/Thr-rich domains. RasMol structural prediction analyses (Brookhaven protein databank) indicate that p30 II  possesses four alpha-helices and nineteen beta-sheet regions ( FIG. 1B ). The alpha-helices likely serve as interacting or docking sites for cellular factors, whereas the Ser/Thr-rich domains may provide targets for phosphorylation by kinases that modulate p30 II &#39;s functions or interactions. As relatively little is known with respect to the functions of HTLV-1 pX accessory factors, such as p30 II , the issue of whether the p30 II  protein contributes to lymphoproliferation in HTLV-1-infected T-cells by altering cell-cycle regulation was investigated.  
         [0089]     To determine whether HTLV-1 p30 II  influences cell-cycle progression and/or apoptosis, Molt-4 and Jurkat E6.1 lymphocytes were transfected with a CMV-HTLV-1 p30 II  (HA) expression construct or an empty CβS vector control. Transfected cultures were assayed for bromodeoxyuridine (BrdU)-incorporation/cell-cycle progression or programmed cell-death using flow-cytometric analyses ( FIGS. 1C  to  1 F and Tables 1-2). HTLV-1 p30 II -expressing cells exhibited markedly increased S-phase progression and significant polyploidy, as determined by BrdU-incorporation and 7-AAD-staining of total genomic DNA ( FIGS. 1C and 1E , left panels,  FIGS. 1D and 1F  and Tables 1-2). However, p30 II  did not induce apoptosis in transfected cells as determined by annexin-V-FITC/propidium iodide-staining and FACS ( FIGS. 1C and 1E , right panels). These results suggest that p30 II  may contribute to lymphoproliferation and genomic instability in HTLV-1-infected cells during ATLL by affecting S-phase regulatory factors, such as Myc and/or E2F.  
                           TABLE 1                                       Diploid:   99.68%           Dip G1:   60.50% at 58.18           Dip G2:    2.85% at 116.37           Dip S:   36.65% G2/G1: 2.00           % CV:     9.72           Aneuploid 1:    0.32%           An1 G1:   66.73% at 67.97           An1 G2:    0.00% at 147.34           An1 S:   32.27% G2/G1: 2.17           % CV:   1.32 DI: 1.17           Total Aneuploid S-Phase:   33.27%           Total S-Phase:   36.64%           Total B.A.D.:   23.45%           Debris:   29.64%           Aggregates:    7.68%           Modeled events:   23210           All cycle events:   14547           Cycle events per channel:    161           RCS:     3.365                      
 
         [0090]    
       
         
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
               
             
             
               
                   
                 Diploid: 
                 65.58% 
               
               
                   
                 Dip G1: 
                 98.05% at 85.10 
               
               
                   
                 Dip G2: 
                  0.17% at 170.20 
               
               
                   
                 Dip S: 
                  1.79% G2/G1: 2.00 
               
               
                   
                 % CV: 
                   8.05 
               
               
                   
                 Aneuploid 1: 
                 34.42% 
               
               
                   
                 An1 G1: 
                 11.34% at 99.15 
               
               
                   
                 An1 G2: 
                 11.09% at 174.33 
               
               
                   
                 An1 S: 
                 77.57% G2/G1: 1.76 
               
               
                   
                 % CV: 
                 4.05 DI: 1.17 
               
               
                   
                 Total Aneuploid S-Phase: 
                 77.57% 
               
               
                   
                 Total S-Phase: 
                 27.87% 
               
               
                   
                 Total B.A.D.: 
                 28.92% 
               
               
                   
                 Debris: 
                 41.67% 
               
               
                   
                 Aggregates: 
                  6.66% 
               
               
                   
                 Modeled events: 
                 22920 
               
               
                   
                 All cycle events: 
                 11843 
               
               
                   
                 Cycle events per channel: 
                  131 
               
               
                   
                 RCS: 
                   3.211 
               
               
                   
                   
               
             
          
         
       
     
         [0091]     The HTLV-1 p30 II  protein interacts in Myc-TIP60 immune-complexes in ATLL patient lymphocytes. The p30 II  protein was detected in cultured HTLV-1-infected lymphocytes, derived from three different ATLL patients (ATL-1, ATL-2, ATL-3) diagnosed with clinical acute-stage leukemias, by immunofluorescence-laser confocal microscopy ( FIGS. 2A and 2B ) and immuno-blotting ( FIG. 2C ). Three-dimensional Z-stack composite images of ATL-3 demonstrate that p30 II /Myc proteins co-localize in the nucleus in all focal planes in HTLV-1-infected cells ( FIG. 2A ). Relative fluorescence-intensities for p30 II /Myc-specific signals and DAPI nuclear-staining are shown for reference ( FIG. 2B ). HTLV-1 p30 II  is present in Myc-containing immunoprecipitated complexes in ATLL patient lymphocytes ( FIG. 2C ). Intriguingly, immunoprecipitation of Myc revealed that TIP49 (RUVBL1), TIP48 (RUVBL2), and Max are present bound to Myc, but the TIP60 histone acetyltransferase (HAT) was not detected in Myc-containing co-immune complexes in uninfected Jurkat E6.1 lymphocytes ( FIG. 2C ). The NH 2 -terminus of Myc is essential for Myc-dependent transformation and apoptosis-inducing functions and contains two conserved Myc homology domains (Myc boxes I and II, MBI and MBII, respectively) that interact with cellular factors. The transcriptional coactivator, TRRAP/p434, and the ATPases/helicases, TIP 49 (RUVBL1) and TIP 48 (RUVBL2), interact with amino acids within MBII. To determine if HTLV-1 p30 II  interacts with known Myc-binding partners, Jurkat E6.1 lymphocytes or HTLV-1-infected Hut-102 and MJ[G11] lymphocytes were transfected with CMV-HTLV-1 p30 II  (HA) or an empty CβS vector control and co-immunoprecipitations using a monoclonal anti-HA antibody were performed (CA5, Roche Molecular Diagnostics). As shown in  FIGS. 2D and 2E , HTLV-1 p30 II  (HA) immunoprecipitates with Myc, TRRAP, TIP60, and TIP49 (RUVBL1). However, TIP48 (RUVBL2) and RNA Pol II were not detected in anti-HA immunoprecipitates, although both proteins were detected in control immunoprecipitations using antibodies against known interacting proteins ( FIG. 2D , lower panels). These data suggest that HTLV-1 p30 II  may modulate Myc functions through interactions with Myc-associated transcriptional coactivators on promoters of responsive genes.  
         [0092]     HTLV-1 p30 II  trans-activates Myc-responsive E-box elements within the human cyclin D2 promoter. To investigate the possibility that HTLV-1 p30 II  might affect Myc-dependent transcription, HeLa cells were co-transfected with a human cyclin D2 promoter-luciferase reporter construct, containing two conserved Myc-responsive E-box enhancer elements (CACGTG), in the presence of increasing amounts of CMV-HTLV-1 p30 II  (HA). Results shown in  FIG. 3A  demonstrate that HTLV-1 p30 II  significantly trans-activates the human cyclin D2 promoter in a dose-dependent manner. A mutant cyclin D2 promoter, lacking Myc-responsive E-box elements, was not transcriptionally activated by p30 II  indicating that p30 II -mediated trans-activation from the human cyclin D2 promoter requires the conserved Myc-responsive E-box enhancer elements ( FIGS. 3A and 3B ). The HTLV-1 p30 II  (HA)-tagged protein was detected in transfected cells by immunoblotting using a monoclonal anti-HA antibody (CA5. Roche Molecular Biochemicals) ( FIG. 3A ). Intracellular levels of Myc were not altered by HTLV-1 p30 II  expression ( FIG. 3A , lower panels). HTLV-1 p30 II  also transcriptionally activates the human cyclin D2 promoter in transfected 293A fibroblasts in a dose-dependent manner ( FIG. 3C ). To confirm that HTLV-1 p30 II  promotes Myc-dependent transcription from E-box enhancer elements, 293A fibroblasts and HeLa cells were co-transfected with a synthetic tk minimal promoter-luciferase reporter construct (M4-tk-luc) that contains four tandem E-boxes. As shown in  FIG. 3D , HTLV-1 p30 II  trans-activates E-box enhancer elements within M4-tk-luc suggesting that p30 II  promotes S-phase progression through Myc-dependent transcriptional interactions.  
         [0093]     Interestingly, p30 II , at the lowest concentration used, induced approx 13-fold trans-activation from the synthetic M4-tk-luc promoter, whereas higher concentrations induced lower (5 to 7-fold) levels of transcriptional activation ( FIG. 3D ). These observations are consistent with findings by Zhang et al. (2000,  J. Virol.  74:11270-11277) demonstrating that p30 II -dependent trans-activation from the HTLV-1 promoter (Tax-responsive elements, TREs) maximally occurs at low p30 II  concentrations and diminishes with increased p30 II  expression.  
         [0094]     Transcriptional activation by HTLV-1 p30 II  is dependent upon the TIP60 and TRRAP/p434 coactivators. Since Myc may interact with the transcriptional coactivator/HAT, TIP60, c-Myc may be a substrate for lysine-acetylation by TIP60, and Myc may interact in chromatin-remodeling complexes with the ATM-related TRRAP/p434 protein, tests were performed to determine whether HTLV-1 p30 II -mediated trans-activation requires TIP60 and TRRAP/p434 functions. HeLa cells were co-transfected with a human cyclin D2 promoter-luciferase reporter construct and CMV-HTLV-1 p30 II  (HA) in the presence of increasing amounts of CMV-TIP60, CMV-TIP60 ΔHAT  (a trans-dominant negative HAT-inactive mutant), or CMV-TIP 60   L497A —a carboxyl-terminal mutant impaired for interactions with cellular factors, including the androgen receptor. Ectopic expression of TIP60 alone did not significantly trans-activate the human cyclin D2 promoter, however, TIP60 over-expression enhanced HTLV-1 p30 II -mediated trans-activation in a dose-dependent manner ( FIG. 4A ). The trans-dominant negative TIP60 ΔHAT  mutant potently inhibited p30 II -mediated transcriptional activation ( FIG. 4A ), suggesting that HTLV-1 p30 II  trans-activation requires TIP60-associated HAT activity. The TIP60 L497A  mutant also weakly enhanced p30 II -mediated trans-activation ( FIG. 4A ). Over-expression of wild-type TIP60 or the trans-dominant-negative TIP 60   ΔHAT  mutant did not alter expression of the HTLV-1 p30 II  (HA) protein in transfected HeLa cells ( FIG. 4A , lower panels). Inhibition of TRRAP/p434, as a result of co-expressing either TRRAP anti-sense  RNA or a trans-dominant negative TRRAP mutant, TRRAP 1261-1579  (FLAG-epitope-tagged), prevented HTLV-1 p30 II -mediated transcriptional activation from the human cyclin D2 promoter ( FIG. 4B ). The trans-dominant-negative, FLAG-tagged TRRAP 1261-1579  protein did not alter the expression of HTLV-1 p30 II  (HA) ( FIG. 4B , lower panels). Immunofluorescence-microscopy was then performed, using a monoclonal anti-FLAG M2 antibody (Sigma Chemical Corp.) and a rabbit polyclonal anti-TIP60 antibody (Upstate Biotechnology), to visualize expression of the FLAG-tagged wild-type TIP60 or TIP60 ΔHAT  proteins relative to endogenous TIP60. Results in shown  FIG. 4C  demonstrate that the FLAG-tagged TIP60 proteins were drastically over-expressed relative to endogenous TIP60 in transfected cells. These data collectively indicate that HTLV-1 p30 II  synergizes with the TIP60 HAT to trans-activate Myc-responsive E-box elements within the human cyclin D2 promoter, requiring the transcriptional coactivator TRRAP/p434.  
         [0095]     HTLV-1 p30 II  stabilizes Myc/TIP60 chromatin-remodeling transcription complexes in HTLV-1-infected lymphocytes. Since HTLV-1 p30 II  transcriptionally activates the conserved Myc-responsive E-box enhancer elements within the human cyclin D2 promoter ( FIGS. 3A and 3C ), a chromatin-immunoprecipitation (ChIP) procedure (described in Vervoorts et al., 2003,  EMBO Rep.  4:484-490) was used to determine whether p30 II  is present in Myc-containing chromatin-remodeling complexes. Formaldehyde-cross-linked genomic DNA complexes in uninfected Molt-4 lymphocytes or HTLV-1-infected MJ[G11] and HuT-102 lymphocytes were fragmented by sonication and oligonucleosomal-protein complexes were precipitated using antibodies against candidate Myc-binding factors. Cross-links were reversed and specific oligonucleotide DNA primer pairs were used in PCR reactions to amplify immunoprecipitated DNA regions spanning conserved E-box elements (PRM) or an untranslated sequence (UTR) as negative control. Results shown in  FIGS. 5A  to  5 C (top panels) demonstrate that HTLV-1 p30 II  was only detected bound to E-box enhancer elements in HTLV-1-infected lymphocytes. Myc, TRRAP, TIP49 (RUVBL1), TIP48 (RUVBL2), and the acetyltransferase hGCN5 were present in chromatin-remodeling complexes in uninfected Molt-4 cells and in HTLV-1-infected MJ[G11] and HuT-102 lymphocytes ( FIGS. 5A  to  5 C, top panels). Surprisingly, TIP60 was only detected in Myc-containing transcription complexes that contained p30 II  in HTLV-1-infected T-cells ( FIGS. 5A  to  5 C, top panels), consistent with co-immunoprecipitation results and observed effects of ectopic TIP60 in trans-activation assays (see  FIGS. 2B ,  4 A). The diminished recruitment of TIP49 to Myc-containing transcription complexes on the cyclin D2 promoter in HTLV-1-infected MJ[G11] cells was not attributable to apparent differences in p30 II /Myc/TIP60 interactions ( FIG. 5A ). Histone H3-acetylation surrounding the E-box enhancer elements within the human cyclin D2 promoter, consistent with transcriptional activation, was detected in all cell-types, with the exception that H3 appeared to be differentially-acetylated on Lys-9 and Lys-14 residues in HTLV-1-infected MJ[G11] and HuT-102 cells, respectively ( FIG. 5A , lower panels). Differences in histone H3-acetylation, however, did not correlate with the stabilization of p30 II /Myc/TIP60 transcriptional interactions in HTLV-1-infected T-cell-lines.  
         [0096]     To identify residues within HTLV-1 p30 II  that interact with Myc/TIP60 complexes in vivo, a panel of pGEX 4T.1-glutathione S-transferase (GST)-HTLV-1 p30 II  constructs, expressing full-length GST-HTLV-1 p30 II  or various truncation mutants, GST-p30 II  (residues 1-98), GST-p30 II  (residues 99-154), GST-p30 II  (residues 155-241) spanning the entire coding region of HTLV-1 p30 II  were generated ( FIG. 5D , see diagram). These proteins were expressed in  E. coli,  BL21, bacteria and purified recombinant GST-HTLV-1 p30 II  fusion proteins were used in GST-pull-down experiments as described in Harrod et al. (1998, Mol. Cell Biol. 18:5052-5061). GST-proteins were incubated with HeLa nuclear extracts at 4° C. overnight and complexes were precipitated with glutathione-Sepharose 4B (Amersham-Pharmacia Biotech). The matrices were washed and bound factors were eluted using 10 mM reduced glutathione buffer. Input levels of purified recombinant GST or GST-HTLV-1 p30 II  proteins, Myc, and TIP60 are shown in  FIGS. 5E and 5F . Results in  FIG. 5F  demonstrate that full-length GST-HTLV-1 p30 II  interacts with both Myc and TIP60 in HeLa nuclear extracts. Deletion of amino acid residues from either the NH 2 -terminus or COOH-terminus of p30 II , disrupts Myc-binding, however, the TIP60-interacting region of HTLV-1 p30 II  was mapped to residues between positions 99-154 ( FIG. 5B ).  
         [0097]     Recruitment of HTLV-1 p30 II /Myc/TIP60 chromatin-remodeling complexes to conserved, Myc-responsive E-box enhancer elements within the cyclin D2 promoter in cultured HTLV-1-infected ATLL patient lymphocytes (ATL-1) was examined next. Chromatin-immunoprecipitations were performed using antibodies that recognize endogenous HTLV-1 p30 II , Myc, and known Myc-interacting factors as described. Polymerase chain-reaction amplification of ChIP products was performed using the PRM and UTR oligonucleotide DNA primer pairs. Results shown in  FIG. 5G  demonstrate that p30 II  is present in Myc/TIP60 transcription complexes assembled on E-box enhancer elements within the cyclin D2 promoter in HTLV-1 ATLL patient lymphocytes. The transcriptional coactivators, TRRAP/p434, TIP48, TIP49, and hGCN5 were also detected in p30 II /Myc/TIP60/cyclin D2 promoter complexes ( FIG. 5G ).  
         [0098]     HTLV-1 p30 II -GFP stabilizes Myc/TIP60 interactions and trans-activates the cyclin D2 promoter in a TIP60 HAT-dependent manner. An HTLV-1 p30 II -green fluorescent protein (GFP) that is functionally identical to HTLV-1 p30 II  (HA) was used to determine whether HTLV-1 p30 II  similarly interacts in Myc/TIP60 transcription complexes in 293A fibroblasts. These cells were co-transfected 293A with CMV-HTLV-1 p30 II -GFP or a pcDNA3.1-GFP vector control and ChIP analyses were performed. Nucleoprotein complexes were cross-linked by treatment with formaldehyde and oligonucleosomal fragments were generated by brief sonication of extracted genomic DNA. Chromatin-immunoprecipitations were performed as described and ChIP products were amplified by PCR using the PRM and UTR oligonucleotide DNA primer pairs. Similar expression of HTLV-1 p30 II -GFP and GFP proteins was visualized in transfected 293A fibroblasts by fluorescence-microscopy ( FIGS. 6A and 6C ). The HTLV-1 p30 II -GFP protein was immunoprecipitated, bound to Myc-containing transcription complexes on conserved E-box elements within the cyclin D2 promoter in transfected 293A fibroblasts, using an anti-GFP antibody ( FIG. 6B ). No ChIP product was detected for the anti-GFP immunoprecipitation in 293A cells transfected with the pcDNA3.1-GFP control ( FIG. 6D ). While the transcriptional coactivators TRRAP/p434, TIP48, TIP49, and hGCN5 were present in Myc-containing complexes in both HTLV-1 p30 II -GFP and GFP-expressing cells, the TIP60 HAT was predominantly detected in HTLV-1 p30 II -GFP/Myc/TIP60 complexes (compare  FIGS. 6B and 6D ). However, TIP60 was weakly present in Myc-containing ChIP complexes in GFP-expressing cells consistent with the demonstration of pre-existing Myc-TIP60 interactions by Frank et al. (2003, EMBO Rep. 4:575-580) and Patel et al. (2004, Mol. Cell Biol. 24:10826-10834) ( FIGS. 6C and 6D ).  
         [0099]     To determine whether the HTLV-1 p30 II -GFP protein also transcriptionally activates the human cyclin D2 promoter in a TIP60-dependent manner, 293A fibroblasts were co-transfected with a tk promoter-renilla-luciferase plasmid, a human dyclin D2 promoter-luciferase reporter plasmid and CMV-HTLV-1 p30 II -GFP in the presence of increasing amounts of either CMV-TIP60 (wild-type) or CMV-TIP60 ΔHAT , which expresses a trans-dominant-negative TIP60 mutant. Results shown in  FIG. 6E  demonstrate that HTLV-1 p30 II -GFP transcriptionally activates the human cyclin D2 promoter approximately 14-fold in transfected 293A fibroblasts compared to an empty pcDNA3.1-GFP control. Over-expression of wild-type TIP60, in the presence of HTLV-1 p30 II -GFP, significantly increased p30 II -GFP-dependent transcriptional activity in a dose-dependent manner ( FIG. 6E ). Co-expression of the trans-dominant-negative TIP60 ΔHAT  mutant repressed p30 II  -GFP-dependent trans-activation from the human cyclin D2 promoter ( FIG. 6E ), consistent with results in  FIG. 4A  and an essential role for the TIP60 HAT in HTLV-1 p30 II  transcriptional activation. Relative renilla-luciferase activities for each sample are shown in  FIG. 6F  for comparison of similar transfection efficiencies.  
         [0100]     HTLV-1 p30 II  transcriptionally activates numerous cellular genes in a TIP60-dependent or TIP60-independent manner. To comprehensively identify cellular gene sequences whose expression is altered by HTLV-1 p30 II -TIP60 transcriptional interactions, 293A fibroblasts were co-transfected with a COS empty vector control, CMV-HTLV-1 p30 II  (HA), or CMV-HTLV-1 p30 II  (HA) +TIP60 ΔHAT  which expresses a trans-dominant-negative mutant that interferes with endogenous TIP60 functions. Total cellular RNAs were extracted and microarray gene expression analyses were performed using Affymetrix Human U133Plus 2.0 full-genomic chips. Transcriptional activation of cellular target genes is expressed as Fold-Activation relative to the empty CβS vector control and the lower-limit for trans-activation was set at 2.5-fold.  FIG. 7A  shows a graphical representation of cellular target genes transcriptionally activated by HTLV-1 p30 II  (HA) (red lines). TIP60-dependent gene sequences were identified based upon their transcriptional repression in the presence of the TIP60 ΔHAT  mutant and are indicated by green lines ( FIG. 7A ). In general, the fold trans-activation by HTLV-1 p30 II  (HA) ranged between 2.5-fold to 393-fold for specific target genes ( FIG. 7A ). Numerous cellular genes are also transcriptionally repressed as a result of HTLV-1 p30 II  expression. Results in  FIG. 7B  are a graphical representation of cellular target genes transcriptionally repressed (with levels ranging between 2.5-fold to 125-fold trans-repression) by HTLV-1 p30 II  (HA) (red lines). Effects of the trans-dominant-negative TIP60 HT mutant upon transcriptional repression by HTLV-1 p30 II  (HA) are indicated by green lines ( FIG. 7B ) Table 3 is a representative list of the major target gene sequences that are transcriptionally activated by HTLV-1 p30 II  (HA) as determined by Affymetrix microarray gene expression analyses. TIP60-dependent gene sequences are indicated. Numerous cellular genes were transcriptionally induced by HTLV-1 p30 II  (HA) in a TIP60-dependent or TIP60-independent manner, suggesting that p30 II  may participate in multiple, distinct transcription complexes (Table 3).  
                                                   TABLE 3                           Target sequences transcriptionally-activated by       HTLV-1 p30″ (HA) in a TIP60-dependent or TIP60-independent       manner                HTLV-1       TIP60-       HTLV-1   p30 II /       Depen-       p30 II     TIP60 ΔHAT     Gene or Sequence Identity   dent                    393.8725   396.7248   TITLE = zinc finger protein                   236 /DEF =  Homo sapiens  cDNA               FLJ20840 fis, clone               ADKA02336.       69.33333   2.666667     Homo sapiens , clone   Yes               IMAGE: 4813412, mRNA       65.5   7   Hs.42369 /UG_TITLE = ESTs   Yes       56   46.4   UG = Hs.66114 /UG_TITLE = ESTs       52.75   1   CPX chromosome region,   Yes               candidate 1 /DEF =  Homo                   sapiens  cDNA FLJ25780 fis,               clone TST06618.       49.09091   0.909091   Hs.131856 /UG_TITLE = ESTs   Yes       48   43   Hs.23196 /UG_TITLE = ESTs       45.4   43.06667   Hs.116301 /UG_TITLE = ESTs       40.44444   18.22222   Hs.200286 /UG_TITLE = ESTs   Yes       40.16667   22     Homo sapiens , clone   Yes               IMAGE: 4812574, mRNA.       34.625   49.375     Homo sapiens , clone               IMAGE: 5172609, mRNA.       34.44444   3.444444   Hs.122442 /UG_TITLE = ESTs   Yes       31.85714   3.857143     Homo sapiens  cystic   Yes               fibrosis transmembrane               conductance regulator               isoform 36 (CFTR) mRNA,               partial cds.       31.1875   1     Homo sapiens  myeloid cell   Yes               nuclear differentiation               antigen (MNDA), mRNA       31   2.75   Hs.145611 /UG_TITLE = ESTs   Yes       30.88889   3.555556   Hs.120414 /UG_TITLE = ESTs   Yes       28.06667   10.66667   Hs.125291 /UG_TITLE = ESTs   Yes       27.72222   1.722222     H. sapiens  mRNA HTPCRX06 for   Yes               olfactory receptor.       27.625   28     Homo sapiens , clone               IMAGE: 5223057, mRNA.       26.65217   7.73913   TESTI2017113.   Yes       26.1875   1.5625   protocadherin 15 /DEF =  Homo     Yes                 sapiens  mRNA; cDNA               DKFZp667A1711 (from clone               DKFZp667A1711).       25.5   1.333333   Hs.279616 /UG_TITLE = ESTs,   Yes               Highly similar to KIAA1387               protein ( H. sapiens )       25.16667   11.55556     Homo sapiens  full length   Yes               insert cDNA clone YW25E05       24.83333   29.83333   Hs.208486 /UG_TITLE = ESTs       24.7619   11.90476     Homo sapiens  mRNA; cDNA               DKFZp313L0839 (from clone               DKFZp313L0839).       24.71429   14.92857     Homo sapiens  synaptonemal               complex protein 1 (SYCP1),               mRNA. /PROD = synaptonemal               complex protein 1               /FL = gb: NM_003176.1 gb: D67       24.64286   2.785714     Homo sapiens  cDNA FLJ14020               fis, clone HEMBA1002508.       24.09091   27.18182     Homo sapiens , clone               IMAGE: 5269594, mRNA.       23.73333   1.933333   Hs.99578 /UG_TITLE = ESTs,   Yes               Highly similar to               PTPD_HUMAN PROTEIN-               TYROSINE PHOSPHATASE               DELTA PRECURSOR               ( H. sapiens )       23.58065   22.03226     H. sapiens  mRNA for               gonadotropin-releasing               hormone receptor, splice               variant. /PROD = gonadotropin-               releasing               hormone receptor       23.3913   4.913043     Homo sapiens  cDNA FLJ12548   Yes               fis, clone NT2RM4000657,               weakly similar to 1-               PHOSPHATIDYLINOSITO       23   2.833333     Homo sapiens  cDNA FLJ37910   Yes               fis, clone CTONG1000040.       22.65   17.15     Homo sapiens  mRNA for pH-               sensing regulatory factor               of peptide transporter,               complete cds.       22.35714   2     Homo sapiens , clone   Yes               IMAGE: 4398590, mRNA.       22.125   0.625     Homo sapiens  cDNA: FLJ20870   Yes               fis, clone ADKA02524.       21.83333   13.58333     Homo sapiens  cDNA FLJ11096               fis, clone PLACE1005480.       21.8   7.1   Hs.60556 /UG_TITLE = ESTs   Yes       21.47059   1.058824     Homo sapiens , clone   Yes               IMAGE: 5742085, mRNA.       21.4   1.2   Hs.130544 /UG_TITLE = ESTs   Yes       21   7.208333   Hs.222222 /UG_TITLE = ESTs   Yes       20.85714   7.214286     Homo sapiens  osteoglycin   Yes               (osteoinductive factor,               mimecan) (OGN), mRNA       20.73913   17.82609   Hs.222120 /UG_TITLE = ESTs       20.71429   19.78571     Homo sapiens  cDNA FLJ25595               fis, clone JTH13269.       20.7   4.3     Homo sapiens  cDNA FLJ13003   Yes               fis, clone NT2RP3000418.       20.42857   7.035714   Human DNA sequence from   Yes               clone 733D15 on chromosome               Xp11.3. Contains a Zinc-               finger (pseudo?) gene and G       20.42857   6.714286   Hs.132649 /UG_TITLE = ESTs   Yes       20.28235   10.15294     Homo sapiens  cDNA FLJ11475   Yes               fis, clone HEMBA1001734,               moderately similar to               CADHERIN-11 PRECURSOR       20.25   4.75     Homo sapiens  epiregulin   Yes               (EREG), mRNA       20.2381   15.52381     Homo sapiens  cDNA FLJ39700               fis, clone SMINT2011588,               weakly similar to Kruppe       20.04545   9.136364     Homo sapiens  mRNA; cDNA   Yes               DKFZp434P2450 (from clone               DKFZp434P2450).       19.95238   1   paraneoplastic   Yes               encephalomyelitis antigen               {5 region, alternatively               spliced} (human, lung               cancer cell line, mRNA               Partial, 10       19.78261   15.65217     Homo sapiens  aldehyde               oxidase 1 (AOX1), mRNA       19.77778   13.77778   Hs.293582 /UG_TITLE = ESTs       19.75   6.833333     Homo sapiens  cDNA: FLJ21221   Yes               fis, clone COL00570.       19.71795   15.89744     Homo sapiens  cDNA FLJ40624               fis, clone THYMU2013981.       19.69565   23.95652   colony stimulating factor 2               receptor, beta, low-               affinity (granulocyte-               macrophage)       19.6   12.46667   Human deleted in               azoospermia protein (DAZ)               mRNA, complete cds       19.57143   19.85714   glutamate receptor,               ionotropic, kainate 2               /DEF =  Homo sapiens  mRNA for               GluR6 kainate receptor               (GRIK2 gene), isoform-b       19.55556   2.111111   hypothetical protein   Yes               LOC285419 /DEF =  Homo                   sapiens , clone               IMAGE: 4839001, mRNA       19.52941   1.235294     Homo sapiens  sperm   Yes               associated antigen 11               (SPAG11), transcript               variant B, mRNA.       19.25926   9.185185     Homo sapiens  mRNA; cDNA   Yes               DKFZp434L1717 (from clone               DKFZp434L1717); complete               cds       19.24138   20.31034     Homo sapiens  cDNA FLJ35054               fis, clone OCBBF2018380.       19.16667   14.11111   Hs.36683 /UG_TITLE = ESTs       19.07143   8   Hs.106645 /UG_TITLE = ESTs   Yes       18.93333   37.53333     Homo sapiens  cDNA FLJ11602               fis, clone HEMBA1003908       18.90476   15.38095     Homo sapiens , clone               IMAGE: 5164933, mRNA       18.71429   40.71429   Hs.176420 /UG_TITLE = ESTs       18.7037   2.222222     Homo sapiens , Similar to   Yes               BCL2-associated athanogene,               clone IMAGE: 4310445, mRNA       18.66667   20.06667   DNA segment on chromosome X               (unique) 9928 expressed               sequence       18.5   5.958333     Homo sapiens  PIAS-NY   Yes               protein mRNA, complete cds       18.47368   4.368421     Homo sapiens  full length   Yes               insert cDNA clone YI41H11       18.46154   4.615385     Homo sapiens  pre-TNK cell   Yes               associated protein (1D12A),               mRNA       18.45455   17.24242     Homo sapiens  mRNA               differentially expressed in               malignant melanoma, clone       F       MM K2       18.23529   3.529412     Homo sapiens  cDNA FLJ32062   Yes               fis, clone OCBBF1000042.       18.14286   6   Hs.204562 /UG_TITLE = ESTs   Yes       18.03226   7.451613   Hs.269931 /UG_TITLE = ESTs   Yes       18   13.58333     Homo sapiens , clone               IMAGE: 4393885, rnRNA,               partial cds       17.76471   24.88235   Hs.23187 /UG_TITLE = ESTs       17.71429   23.07143   Hs.42993 /UG_TITLE = ESTs       17.625   1.5     Homo sapiens  glypican 5   Yes               (GPC5), mRNA       17.6129   12.12903     Homo sapiens  mRNA; cDNA               DKFZp686C1636 (from clone               DKFZp686C1636)       17.5   21.53125   Hs.213386 /UG_TITLE = ESTs       17.48276   2   Hs.99200 /UG_TITLE = ESTs   Yes       17.47826   2.913043   Hs.17388 /UG_TITLE = ESTs   Yes       17.47368   6.710526   MCF.2 cell line derived   Yes               transforming sequence-like               /DEF =  Homo sapiens , clone               IMAGE: 5185971, mRNA       17.40541   3.486486   Hs.6656 /UG_TITLE = ESTs   Yes       17.36842   15.73684   Hs.22249 /UG_TITLE = ESTs       17.31169   16.42857   Hs.20103 /UG_TITLE = ESTs       17.09091   17.09091   Human (clone CTG-A4) mRNA               sequence       17.09091   22.36364     Homo sapiens  cDNA FLJ36285               fis, clone THYMU2003470.       17   16.44444     Homo sapiens  SAM domain,               SH3 domain and nuclear               localisation signals, 1               (SAMSN1), mRNA. /PROD = SAM               domain, SH3 domain and               nuclear       16.83333   17.69444     Homo sapiens , clone               MGC: 34025 IMAGE: 4828588,               mRNA, complete cds.               /PROD = Unknown (protein for               MGC: 34025)       16.83333   15.58333     Homo sapiens , clone               IMAGE: 4838843, mRNA       16.81818   0.727273   Hs.97977 /UG_TITLE = ESTs   Yes       16.75556   4.377778     Homo sapiens  mRNA; cDNA   Yes               DKFZp586O2023 (from clone               DKFZp586O2023)       16.66667   17.75   Hs.36409.0 /TIER = ConsEnd               /STK = 4 /UG = Hs.36409               /UG_TITLE = ESTs       16.65789   11.65789   Human hepatocyte nuclear               factor-6 alpha (HNF6) mRNA,               complete cds       16.625   3.5   Hs.188950 /UG_TITLE = ESTs   Yes       16.625   11.0625   Hs.277419 /UG_TITLE = ESTs       16.61111   12.77778   pancreatic ribonuclease               (human, mRNA Recombinant               Partial, 491 nt)       16.54545   15.09091     Homo sapiens , clone               IMAGE: 5277680, mRNA,               partial cds.       16.53488   3.860465     Homo sapiens  glutamate   Yes               receptor, ionotrophic, AMPA               4 (GRIA4), mRNA.               /PROD = glutamate receptor,               ionotrophic /FL = gb: U16129.1               gb: NM_0       16.46575   11.61644   endothelin receptor type A               /FL = gb: NM_001957.1               gb: L06622.1       16.42857   4.761905   Hs.99472 /UG_TITLE = ESTs   Yes       16.42105   5.631579     Homo sapiens  mRNA for type   Yes               I keratin. /PROD = HHa5 hair               keratin type I intermediate               filament       16.40909   3.227273     Homo sapiens  protein   Yes               tyrosine phosphatase,               receptor-type, Z               polypeptide 1 (PTPRZ1),               mRNA       16.36   22.4     Homo sapiens  neuropeptide Y               receptor Y5 (NPY5R), mRNA       16.2   48.8     Homo sapiens  cDNA: FLJ20890               fis, clone ADKA03323.       16.05882   3.882353   syntaphilin   Yes       16   13.5   Human DNA sequence from               clone RP4-545L17 on               chromosome 20p12.2-13.               Contains the 5 end of the               gene for a novel protein               similar to RAD21 (S. pom       15.95522   8.552239     Homo sapiens  cDNA FLJ36177   Yes               fis, clone TESTI2026515.       15.91667   0.5   Hs.40840 /UG_TITLE = ESTs   Yes       15.89474   25.12281     Homo sapiens  cDNA FLJ13602               fis, clone PLACE1010089,               highly similar to  Homo                   sapiens  mRNA for       15.88235   1.156863   Human CB-4 transcript of   Yes               unrearranged immunoglobulin               V(H)5 gene /DEF = Human CLL-               12 transcript of               unrearranged immuno       15.875   18.5     Homo sapiens , clone               IMAGE: 4823434, mRNA       15.85185   1.888889   Hs.173596 /UG_TITLE = ESTs   Yes       15.83333   3.944444     Homo sapiens  GPBP-   Yes               interacting protein 90               mRNA, complete cds       15.81481   0.888889     Homo sapiens , Similar to   Yes               recombination activating               gene 1, clone MGC: 43321               IMAGE: 5265661, mRNA,               complete cds       15.72414   2.241379     Homo sapiens  cDNA FLJ37001   Yes               fis, clone BRACE2008172.       15.71429   11.42857     Homo sapiens , Similar to               RIKEN cDNA 4833427G06 gene,               clone IMAGE: 5561932, mRNA       15.6875   9.0625     Homo sapiens , clone               IMAGE: 4831108, mRNA       15.6875   0.78125     Homo sapiens , clone   Yes               IMAGE: 5295305, mRNA       15.63636   5.681818   Hs.98388 /UG_TITLE = ESTs   Yes       15.61538   11.11538   methyl-CpG binding domain               protein 2       15.59677   267.7419   Hs.154993 /UG_TITLE = ESTs       15.54545   9.181818     Homo sapiens  transmembrane               phosphatase with tensin               homology (TPTE), mRNA.       15.52632   10.31579   Hs.105620 /UG_TITLE = ESTs       15.42857   23.28571     Homo sapiens  acetyl LDL               receptor; SREC = scavenger               receptor expressed by               endothelial cells (SREC),               mRNA. /PROD = acetyl LDL               receptor; SR       15.36842   0.684211     Homo sapiens  cDNA: FLJ21351   Yes               fis, clone COL02762       15.36364   1.454545     Homo sapiens  cDNA FLJ30168   Yes               fis, clone BRACE2000750.       15.3   12.83333     Homo sapiens  clone 148022               iduronate-2-sulfatase               (IDS2) pseudogene, mRNA               sequence       15.27027   3.324324     Homo sapiens  microtubule-   Yes               associated protein tau               (MAPT), transcript variant               1, mRNA.       15.23077   12.15385     Homo sapiens , clone               IMAGE: 4830182, mRNA.       15.21429   2.642857     Homo sapiens  mRNA; cDNA   Yes               DKFZp434H0872 (from clone               DKFZp434H0872).       15.21053   1.684211     Homo sapiens  cDNA: FLJ22630   Yes               fis, clone HSI06250.       15.19231   11.76923     Homo sapiens  H2B histone               family, member N (H2BFN),               mRNA       15.19048   1.47619     Homo sapiens  cDNA FLJ11921   Yes               fis, clone HEMBB1000318.       15.16667   1.233333   Hs.168268 /UG_TITLE = ESTs,   Yes               Moderately similar to               A35969 heparin-binding               growth factor receptor K-               sam precursor ( H. sapiens )       15.125   1.1875   Human EST clone 53125   Yes               mariner transposon Hsmar1               sequence       15.09375   4     Homo sapiens , clone   Yes               MGC: 47837 IMAGE: 6046539,               mRNA, complete cds.               /PROD = Unknown (protein for               MGC: 47837)       15.07407   4.277778     Homo sapiens  cDNA: FLJ21710   Yes               fis, clone COL10087.       15.06667   15.96667   Hs.143834 /UG_TITLE = ESTs       15.05128   5.512821   hypothetical protein   Yes               FLJ20271 /FL = gb: NM_017734.1       15.02564   5.974359     Homo sapiens  full length   Yes               insert cDNA clone YP60H04       15.02326   11.44186     Homo sapiens  calsyntenin-2               (CS2), mRNA.               /PROD = calsyntenin-2       15   1.5   Hs.104572 /UG_TITLE = ESTs   Yes       14.9403   12.67164     Homo sapiens  inhibin, beta               C (INHBC), mRNA.               /PROD = inhibin beta C               subunit precursor       14.88462   11.82692     Homo sapiens  cDNA FLJ10146               fis, clone HEMBA1003327       14.80851   15.65957   gb: AW451826               /DB_XREF = gi: 6992602               /DB_XREF = UI-H-BI3-alk-e-07-               0-UI.s1               /CLONE = IMAGE: 2737236               /FEA = EST /CNT = 8               /TID = Hs.258791.0               /TIER = ConsEnd /STK = 4               /UG = Hs.258791               /UG_TITLE = ESTs       14.78788   5.348485   gb: BF590323   Yes               /DB_XREF = gi: 11682647               /DB_XREF = nab22h10.x1               /CLONE = IMAGE: 3266922               /FEA = EST /CNT = 33               /TID = Hs.55256.0 /TIER = Stack               /STK = 30 /UG = Hs.55256               /UG_TITLE = ESTs       14.71154   0.442308     Homo sapiens , clone   Yes               IMAGE: 4815474, mRNA       14.69565   12.67391     Homo sapiens  RAGE mRNA for               advanced glycation               endproducts receptor,               complete cds.       14.65217   22.82609     Homo sapiens , clone               MGC: 10724, mRNA, complete               cds. /PROD = Unknown (protein               for MGC: 10724)       14.64286   2.571429     Homo sapiens  mRNA; cDNA   Yes               DKFZp761D191 (from clone               DKFZp761D191)       14.61538   1.307692   Hs.313876 /UG_TITLE = ESTs   Yes       14.53488   8.348837   CGI-67 protein       14.5   9.772727   Hs.132650 /UG_TITLE = ESTs       14.42857   1.619048   Hs.293685 /UG_TITLE = ESTs   Yes       14.41667   4.708333   Hs.143789 /UG_TITLE = ESTs   Yes       14.40909   5.181818     Homo sapiens  cDNA FLJ13755   Yes               fis, clone PLACE3000363.       14.33333   2.888889   Hs.327117 /UG_TITLE = ESTs   Yes       14.29032   1.903226   Hs.161566 /UG_TITLE = ESTs   Yes       14.27778   1.055556     Homo sapiens , clone   Yes               IMAGE: 4778480, mRNA.       14.23077   15.84615     Homo sapiens , Similar to               hypothetical protein               FLJ22792, clone MGC: 22933               IMAGE: 4905554, mRNA,               complete cds       14.20513   1   Hs.162565 /UG_TITLE = ESTs   Yes       14.14815   21.37037     Homo sapiens , Similar to               sex comb on midleg-like 3               ( Drosophila ), clone               MGC: 25118 IMAGE: 4509724,               mRNA, complete cds.       14.14286   11.57143     Homo sapiens  olfactory-like               receptor JCG8 (JCG8) mRNA,               complete cds.               /PROD = olfactory-like               receptor JCG8       14.1 1   5.3     Homo sapiens , clone               IMAGE: 5267701, mRNA       14.06667   5.15     Homo sapiens  cDNA FLJ34667   Yes               fis, clone LIVER2000769.               /DEF =  Homo sapiens  cDNA               FLJ34667 fis, clone               LIVER2000769.       14.05882   7.588235   major histocompatibility   Yes               complex, class II, DR beta 3       14.05882   3.176471   Hs.125962 /UG_TITLE = ESTs   Yes       14.05833   6.35   Hs.293118 /UG_TITLE = ESTs   Yes       14.03896   13.66234   Hs.20726 /UG_TITLE = ESTs       14.02778   9.555556     Homo sapiens , clone               MGC: 14510, mRNA, complete               cds. /PROD = Unknown (protein               for MGC: 14510)       14.02632   17.39474     Homo sapiens  CD84 antigen               (leukocyte antigen) (CD84),               mRNA. /PROD = CD84 antigen               (leukocyte antigen)       14   11.90909   Hs.296235 /UG_TITLE = ESTs       14   38.29412   prostate specific G-protein               coupled receptor /DEF =  Homo                   sapiens  prostate specific               G-protein coupled receptor               gene, comple       14   22.2     Homo sapiens  cystic               fibrosis transmembrane               conductance regulator               isoform 36 (CFTR) mRNA,               partial cds       13.97826   12.54348     Homo sapiens  testis               transcript Y 9 (TTY9) mRNA,               complete cds       13.90625   0.9375   Hs.88450 /UG_TITLE = ESTs   Yes       13.9   1.1   Hs.20468 /UG_TITLE = ESTs   Yes       13.89474   3.421053     Homo sapiens  cDNA: FLJ21618   Yes               fis, clone COL07487.       13.85294   2.352941     Homo sapiens  fibroblast   Yes               growth factor 20 (FGF20),               mRNA       13.81818   3.136364     Homo sapiens  mRNA; cDNA   Yes               DKFZp761J1323 (from clone               DKFZp761J1323).       13.80769   2.307692   Hs.407438   Yes               /UG_TITLE = neurogenic               differentiation 1       13.78788   12.45455     Homo sapiens  hypothetical               protein FLJ12983               (FLJ12983), mRNA       13.7549   9.77451   Human DNA sequence from               clone RP5-1184F4 on               chromosome 20q11.1-11.23.               Contains the 3 end of gene               KIAA0978, two genes for               novel proteins similar       13.73333   8.8   Hs.201420 /UG_TITLE = ESTs       13.71429   12.17857     Homo sapiens  cDNA FLJ12573               fis, clone NT2RM4000979       13.7   23.3   Hs.244710 /UG_TITLE = ESTs       13.68293   5.585366     Homo sapiens  tenascin R   Yes               (restrictin, janusin)               (TNR), mRNA. /PROD = tenascin               R (restrictin, janusin)       13.66667   1.606061   Hs.99336 /UG_TITLE = ESTs   Yes       13.64045   9.280899     Homo sapiens  testis-               specific ankyrin motif               containing protein               (LOC56311), mRNA.       13.61538   1.846154   Hs.130922 /UG_TITLE =  Homo     Yes                 sapiens , Similar to likely               ortholog of yeast ARV1,               clone IMAGE: 5265646, mRNA       13.59259   0.814815   olfactory receptor, family   Yes               2, subfamily M, member 4               /DEF =  H. sapiens  mRNA for               TPCR100 protein.       13.57143   3.142857     Homo sapiens , clone   Yes               IMAGE: 4694422, mRNA.       13.55882   3.661765     Homo sapiens  small   Yes               intestine aquaporin mRNA,               complete cds       13.55172   3.689655     Homo sapiens  mRNA; cDNA   Yes               DKFZp564I083 (from clone               DKFZp564I083)       13.55172   1.448276   gb: H47594   Yes               /DB_XREF = gi: 923646               /DB_XREF = yp75c01.s1               /CLONE = IMAGE: 193248               /TID = Hs2.407314.1 /CNT = 3               /FEA = mRNA /TER = ConsEnd               /STK = 1 /UG = Hs.407314               /UG_TITLE =  Homo sapiens  full               length insert cDNA clone               YP75C01       13.53846   7.807692     Homo sapiens  cDNA FLJ39005               fis, clone NT2RI2024496       13.52941   11.2549   gb: H46217               /DB_XREF = gi: 922269               /DB_XREF = yo14h12.s1               /CLONE = IMAGE: 177959               /FEA = EST /CNT = 4               /TID = Hs.268805.0               /TIER = ConsEnd /STK = 4               /UG = Hs.268805               /UG_TITLE = ESTs       13.5   8.535714   Hs.250113 /UG_TITLE = ESTs,               Moderately similar to               thyroid hormone receptor-               associated protein complex               component TRAP150 (H. sap       13.40909   9.863636     Homo sapiens , clone               IMAGE: 3933453, mRNA       13.36842   0.842105   Hs.28714 /UG_TITLE = ESTs   Yes       13.35714   1.357143     Homo sapiens , clone   Yes               IMAGE: 5266862, mRNA.       13.35135   12.81081   Hs.158937 /UG_TITLE = ESTs       13.35   1.65     Homo sapiens  cDNA FLJ13136   Yes               fis, clone NT2RP3003139       13.33333   6.6     Homo sapiens  non-coding RNA   Yes               HANC       13.30769   7.410256   Hs.25046 /UG_TITLE = ESTs       13.29384   8.21327     Homo sapiens  protein kinase               C, alpha binding protein               (PRKCABP), mRNA       13.2807   4.017544     Homo sapiens  hypothetical   Yes               protein FLJ10979               (FLJ10979), mRNA.               /PROD = hypothetical protein               FLJ10979       13.25   7.15     Homo sapiens  full length               insert cDNA clone YI41B09       13.24138   0.896552     Homo sapiens , clone   Yes               IMAGE: 4818264, mRNA       13.2381   10.2619     Homo sapiens , clone               IMAGE: 4824978, mRNA       13.23333   15.1   gb: AA776626               /DB_XREF = gi: 2835960               /DB_XREF = ae86f02.s1               /CLONE = IMAGE: 971067               /FEA = EST /CNT = 12               /TID = Hs.62183.0               /TIER = ConsEnd /STK = 1               /UG = Hs.62183 /UG_TITLE =               ESTs       13.2   8   myelin oligodendrocyte               glycoprotein /DEF = Human DNA               sequence from clone RP11-               145L22 on chromosome               6p21.32-22.2       13.18182   0.818182     Homo sapiens  regulator of   Yes               G-protein signaling 1               (RGS1), mRNA.               /PROD = regulator of G-               protein signaling 1       13.11111   12.11111     Homo sapiens  clone HQ0202               PRO0202 mRNA, partial cds       13.09091   17.30303   cytoplasmic linker               associated protein 2       13.09091   17.63636     H. sapiens  AA1 mRNA       13.04762   1.380952     Homo sapiens , clone   Yes               IMAGE: 4825614, mRNA.       13   1.75   Human clone 23909 mRNA,   Yes               partial cds. /PROD = unknown       13   1.657143     Homo sapiens  cDNA FLJ12289   Yes               fis, clone MAMMA1001788       12.93333   11.66667     Homo sapiens  mRNA for               keratin associated protein               4.7 (KRTAP4.7 gene)       12.93103   0.413793   Hs.43052 /UG_TITLE = ESTs   Yes       12.92308   4.846154     Homo sapiens  cDNA FLJ14152   Yes               fis, clone MAMMA1003089.       12.88   1.52   Hs.118342 /UG_TITLE = ESTs   Yes       12.86207   6.965517     Homo sapiens , clone               IMAGE: 4042783, mRNA.       12.84   11.68     Homo sapiens  POU domain,               class 4, transcription               factor 2 (POU4F2), mRNA.               /PROD = POU domain, class 4,               transcription factor 2       12.83333   8.111111   Hs.190319 /UG_TITLE = ESTs       12.8   3.72   Hs.231951 /UG_TITLE = ESTs   Yes       12.8   9.766667     Homo sapiens  olfactory               receptor-like protein JCG3               (JCG3), mRNA       12.78947   1.263158     Homo sapiens , clone   Yes               IMAGE: 4413783, mRNA.       12.78788   1.181818     Homo sapiens , clone   Yes               IMAGE: 4800001, mRNA.       12.76923   2     Homo sapiens , clone   Yes               IMAGE: 4828930, mRNA.       12.76471   1.705882     Homo sapiens  mRNA expressed   Yes               only in placental villi,               clone SMAP41       12.75   7.08333   Hs.259168 /UG_TITLE = ESTs       12.69565   17.47826     Homo sapiens  hypothetical               protein FLJ21272               (FLJ21272), mRNA       12.6875   7.333333   Hs.92955 /UG_TITLE = ESTs       12.67857   4.071429   hypothetical protein   Yes               FLJ10024 /DEF =  Homo sapiens                 cDNA FLJ13978 fis, clone               Y79AA1001665.       12.66102   9.050847     Homo sapiens  RNA binding               motif protein, Y               chromosome, family 2,               member B (RBMY2B) mRNA.               /PROD = RNA binding motif               protein, Y chromos       12.61538   1.384615   Hs.127556 /UG_TITLE = ESTs   Yes       12.57576   9.636364   Hs.44736 /UG_TITLE = ESTs       12.55172   0.724138   hypothetical protein   Yes               LOC285965 /DEF =  Homo sapiens                 mRNA; cDNA DKFZp686O0656               (from clone DKFZp686O0656).       12.54839   7.709677   Hs.276363               /UG_TITLE = hypothetical               protein LOC283112       12.53333   14.86667   REPL1S /UG_TITLE = ret finger               protein-like 1 antisense       12.50847   1.559322   Hs.98945 /UG_TITLE = ESTs   Yes       12.5   6.136364   Hs.213371 /UG_TITLE = ESTs   Yes       12.45946   7.297297     Homo sapiens , similar to               hypothetical protein, clone               MGC: 27103 IMAGE: 4831323,               mRNA, complete cds.       12.42424   16.54545     Homo sapiens  GLB2 gene,               upstream regulatory region                  
 
         [0101]     With respect to the potential role of HTLV-1 p30 II  in adult T-cell leukemogenesis, transcriptional activation of the following genes is of significant interest: myeloid cell nuclear differentiation 1 antigen (31.1-fold; TIP60-dependent), protocadherin 15 (26.1-fold; TIP60-dependent), human protein tyrosine-phosphatase delta precursor (23.3-fold; TIP60-dependent), cadherin 11-like precursor (20.2-fold; TIP60-dependent), colony stimulating factor 2 receptor, beta (19.6-fold; TIP60-independent), human protein tyrosine-phosphatase receptor-type Z polypeptide (16.4-fold; TIP60-dependent),  S. pombe  RAD21-like protein (16-fold; TIP60-independent), human transmembrane phosphatase with tensin homology (15.5-fold; TIP60-independent), H2B histone family member N (15.1-fold; TIP60-independent), major histocompatibility complex class II DR beta 3 (14.0-fold; TIP60-dependent), human CD84 leukocyte antigen (14.0-fold; TIP60-independent), prostate-specific G-protein coupled receptor (14.0-fold; TIP60-independent), fibroblast growth factor 20 (13.8-fold; TIP60-dependent), protein kinase C alpha-binding protein (13.2-fold; TIP60-independent), regulator of G-protein-signaling 1 (13.1-fold; TIP60-dependent), cytoplasmic linker associated protein 2 (13.0-fold; TIP60-independent), POU domain 4 transcription factor 2 (12.8-fold; TIP60-independent), and RNA-binding motif protein (RBMY2B) (12.6-fold; TIP60-independent). Infectious HTLV-1 molecular clone, ACH.p30 II , exhibits an approx 20-50% reduction in transformation-efficiency compared to the wild-type ACH.wt suggesting that p30 II  is required for the full-transforming potential of HTLV-1. Microarray analyses indicates that numerous cellular genes are transcriptionally activated by p30 II , and proteins encoded by these genes may contribute to HTLV-1 leukemic transformation and development of ATLL.  
         [0102]     HTLV-1 p30 II  enhances Myc transforming potential and requires the TIP60 HAT and TRRAP/p434. Since the c-Myc oncogene is known to cause cellular transformation, foci-formation assays using immortalized human WRN −/−  fibroblasts, which lack Werner&#39;s Syndrome helicase functions were used to determine whether HTLV-1 p30 II  might influence Myc-associated transforming activity. This cellular background was chosen because ATLL is an aging-related malignancy requiring clinical latency periods of 25-40 years prior to disease onset, which suggests that genetic mutations linked to the aging process likely contribute to leukemogenesis. Werner&#39;s Syndrome is a premature-aging disorder that mimics or recapitulates many of the clinical and cellular features of normal aging; and WRN locus (8p11-12) mutations have been found in HTLV-1-infected ATLL patient lymphocytes and in HTLV-1-infected mycosis fungoides/Sezary syndrome cells. Neither Myc nor HTLV-1 p30 II  (HA) alone significantly induces foci-formation in immortalized human WRN −/−  fibroblasts ( FIG. 8A ). Surprisingly, in combination, HTLV-1 p30 II  (HA)-Myc co-expression reproducibly induces between 35-58 foci in different assays ( FIGS. 8A and 8B ). HTLV-1 p30 II  (HA) expression was detected in transformed colonies by immunofluorescence-microscopy ( FIG. 8C ); and the p30 II  protein appeared to be distributed throughout the nucleoplasm ( FIG. 8D ). A high-incidence of multi-nucleated giant cells were also observed in isolated HTLV-1 p30 II  (HA)-Myc-transformed fibroblasts that were expanded in culture, consistent with HTLV-1 p30 II -induced polyploidy observed during BrdU-FACS analyses ( FIG. 8E ; compare to control cells in  FIG. 8C ). Expression of HTLV-1 p30 II  (HA) in transformed fibroblasts was confirmed by immunoblotting using a monoclonal anti-HA antibody ( FIG. 8E ). Indeed, these findings indicate that HTLV-1 p30 II  markedly enhances the transforming potential of Myc and may promote genomic instability resulting in polyploidy.  
         [0103]     The foregoing transcriptional activation data suggested that enhancement of Myc functions by HTLV-1 p30 II  requires the coactivators TIP60 and TRRAP/p434. Therefore, tests were performed to determine whether foci-formation induced by co-expressing HTLV-1 p30 II  (HA)-Myc might be affected by over-expressing wild-type TIP60 or TIP60 ΔHAT  and TIP60 L497A  mutant proteins. Results from two independent experiments shown in  FIG. 9A  indicate that none of the TIP60 expression constructs, either alone or in combination with Myc, significantly induces foci-formation in immortalized human WRN −/−  fibroblasts. However, ectopic TIP60 markedly increases foci-formation induced by HTLV-1 p30 II  (HA)-Myc co-expression ( FIG. 9A ). The trans-dominant negative TIP60 ΔHAT  mutant completely abrogated colony formation by HTLV-1 p30 II  (HA)-Myc, and the TIP60 L497A  mutant partially inhibited foci-formation ( FIG. 9A ). Increased colony formation by HTLV-1 p30 II  (HA)/Myc/TIP60, compared to inhibition of foci-formation by the trans-dominant-negative TIP60 ΔHAT  mutant, is shown in  FIGS. 9C and 9D . Finally, inhibition of TRRAP/p434, as a result of co-expressing increasing amounts of TRRAP anti-sense  RNA, also significantly decreased foci-formation by HTLV-1 p30 II  (HA)-Myc ( FIG. 9E ). These findings collectively agree with the transcriptional activation data, and suggest that HTLV-1 p30 II  enhances Myc transcriptional and transforming activities in a TIP60 HAT-and TRRAP-dependent manner ( FIG. 10 ).  
         [0104]     The HTLV-1 infects CD4 +  T-cells and promotes deregulated cell-growth and lymphoproliferation associated with development of ATLL. While numerous studies have demonstrated that the viral Tax protein transcriptionally-activates growth/proliferative-signaling pathways, it has become increasingly evident that other pX-encoded regulatory factors (p12 I , p13 II , p30 II , Rex) are likely to perform essential functions during adult T-cell leukemogenesis. Indeed, the majority of partially-deleted HTLV-1 proviruses in ATLL patient isolates contain intact pX sequences; and alternatively-spliced ORF I and ORF II mRNAs have been detected in HTLV-1-infected transformed T-cell-lines and ATLL patient samples. Cytotoxic T-lymphocytes (CTLs) specifically targeted against ORF I and ORF II peptides have been obtained from ATLL patients suggestive that these proteins are present during in vivo HTLV-1 infections. Zhang et al. (2001) reported that p30 II  interacts with p300/CREB-binding protein and represses Tax-mediated trans-activation from the HTLV-1 LTR (83) and differentially modulates CREB-dependent transcription (84). Nicot et al. (2004. ref. 46) and Younis et al. (2004. ref. 82) have demonstrated that p30 II  prevents nuclear export of the doubly-spliced Tax/Rex mRNA and others have shown that p30 II  is required for maintenance of high viral titers in a rabbit model of ATLL using an infectious HTLV-1 molecular clone, ACH.30 II , defective for p30 II  production (4, 68). Interestingly, Robek et al. (1998) have previously demonstrated that p30 II  is dispensable for immortalization and transformation of human PBMCs by ACH.p30 II , however, this mutant exhibited an approx 20-50% reduction in transformation-efficiency compared to the wild-type ACH.wt (60) suggesting that p30 II  is required for the full transforming-potential of HTLV-1. The physiological role of p30 II  in HTLV-1 pathogenesis remains unclear and it is intriguing that, similar to Tax, p30 II , may perform multiple functions to control viral gene expression and promote deregulation of CD4 +  T-cell growth/proliferative pathways.  
         [0105]     Therefore, the data presented herein demonstrates that HTLV-1 p30 II  drastically enhances Myc-associated transcriptional and transforming activities and markedly increases S-phase progression-and polyploidy through interactions with the coactivator/HAT, TIP60 ( FIG. 10 ). HTLV-1 p30 II  significantly trans-activates conserved E-box enhancer elements within promoters of Myc-responsive genes, requiring TIP60 HAT activity and the transcriptional coactivator TRRAP/p434. The data presented herein indicate that, in the absence of HTLV-1 p30 II -interactions, ectopic TIP60 over-expression does not significantly alter Myc transcriptional and transforming activities in functional assays (see  FIGS. 4A and 9A ). Further, TIP60 is not detectably present in Myc-containing chromatin-remodeling complexes on the human cyclin D2 promoter, in absence of HTLV-1 p30 II , in Molt-4 lymphocytes ( FIG. 5C ). Aberrant Myc-TIP60 interactions, as a result of HTLV-1 p30 II  or other stabilizing factors, may prominently contribute to neoplastic transformation in hematological malignancies and solid tumors where Myc functions are deregulated or that contain Myc locus mutations. Indeed, disruption of Myc-TIP60 complexes is a plausible approach for anti-cancer therapies designed to impede malignancy.  
         [0106]     The present invention provides the first evidence, based upon biological-functional assays, that HTLV-1 p30 II  is a novel retroviral enhancer of Myc-TIP60 transcriptional and transforming activities that likely plays an important role during adult T-cell leukemogenesis.  
       EXAMPLES  
     Example 1  
     Plasmids, Transfections, and Cell-Culture  
       [0107]     HeLa cells (ATCC, CCL-2) were grown in Dulbecco&#39;s Modified Eagle&#39;s Medium (D-MEM, ATCC) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), 100U/ml penicillin and 100 μg/ml streptomycin sulfate (Invitrogen-Life Technologies) and cultured at 37° C. under 5% CO 2 . 293A Fibroblasts (Quantum Biotechnology) were cultured in ATCC 46-X medium supplemented with sodium bicarbonate (Invitrogen-Life Technologies), 10% FBS, and 100U/ml penicillin and 100 g/ml streptomycin-sulfate. Molt-4 (ATCC, CRL-1582), Jurkat E6.1 (ATCC, TIB-152) and HTLV-1-infected MJ[G11] (ATCC, CRL-8294) and HuT-102 lymphocytes (ATCC, TIB-162) were grown in RPMI medium (ATCC) supplemented with 20% FBS, 100U/ml penicillin, 100 μg/ml streptomycin-sulfate, and 20 μg/ml gentamicin-sulfate (SIGMA Chemical Corp.) and cultured under 10% CO 2 . Primary HTLV-1-infected lymphocytes were obtained, after informed consent from three ATLL patients (ATL-1, ATL-2, ATL-3), and were cultured in RPMI medium supplemented with 20% FBS, 50U/ml hIL-2 (Invitrogen-Life Technologies), 100U/ml penicillin, 100 μg/ml streptomycin-sulfate, and 20 μg/ml gentamicin-sulfate. The CMV-HTLV-1 p30 II  (HA) expression construct was kindly provided by Dr. G. Franchini (NCI, NIH) and has been reported in Koralnik et al. (1993, J. Virol. 67:2360-2366). In order to generate the human cyclin D2 promoter-luciferase reporter construct, sequences encompassing the human cyclin D2 promoter were located in GeneBank accession number U47284 clone; according to these sequences a PCR product was generated that contains 1622 nucleotides upstream of the ATG start codon. Two closely-spaced E-boxes (5′-CACGTG) are localized within the promoter region which bind Myc/Max/Mad network components (2001, Genes Dev. 15:2042-2047). This fragment was cloned into the pGL3-luciferase vector. Both E-box sequences were mutated to 5′-CTCGAG using the quick change method. The M4-tk-luciferase reporter plasmid was reported (2001, Genes Dev. 15:2042-2047; 1998, Cell 93:81-91). The CβF-FLAG-Myc, CβF-FLAG-TRRAP 1261-1579 , CβS-TRRAP anti-sense , and CβS constructs were described in McMahon et al. (1998, Cell 94:363-374). The pOZ-wildtype-TIP60 and pOZ-TIP60sAT expression constructs were reported in Ikura et al. (2000, Cell 102:463-473); and the CMV-TIP60 L497A  expression plasmid was reported in Gaughan et al. (2001, J. Biol. Chem. 276:46841-46848). All transfections were performed using Lipofectamine (Invitrogen-Life Technologies) or Superfect (Qiagen) reagents as recommended.  
       Example 2  
     Cell-Cycle and FACS Analyses  
       [0108]     Molt4 and Jurkat E6.1 lymphocytes were seeded in 100 mm 2  tissue-culture dishes and transfected with CMV-HTLV-1 p30 II  (HA) or an empty CβS vector. Following 48 hr, cultures were split and either labeled for 4 hr by adding BrdU (BD-Pharmingen) to the medium or immediately stained using annexin-V-(FITC)/propidium iodide (BD-Pharmingen). For cell-cycle analyses, transfected BrdU-labeled cells were permeabilized and stained with a FITC-conjugated anti-BrdU antibody; and total genomic DNA was stained using 7-AAD (BD-Pharmingen). Flow cytometry was performed and data were analyzed using ModFit LT 3.0 software.  
       Example 3  
     Foci-Formation/Transformation Assays  
       [0109]     Immortalized Werner&#39;s Syndrome (WRN −/− ) fibroblasts (2000, Nucleic Acids Res. 28:648-654) were seeded at 6×10 5  cells in 60 mm 2  tissue-culture dishes in D-MEM supplemented with 10% FBS and cultured at 37° C. under 5% CO 2 . Cells were transfected with an empty CβS vector, CMV-HTLV-1 p30 II  (HA), CβF-FLAG-Myc, and combinations of CMV-HTLV-1 p30 II  (HA)/CβF-FLAG-Myc or CβS/CβF-FLAG-Myc using Superfect reagent. Foci were observed within 2 weeks and quantified by direct counting. Expression of HTLV-1 p30 II  (HA) was detected by fixing plates with 0.2% gluteraldehyde, 1% formaldehyde in PBS and immuno-staining using a monoclonal antibody against the HA-epitope tag (CA5, Roche Molecular Biochemicals), diluted 1:1000 in BLOTTO buffer (50 mM Tris-HCl, pH 8.0, 2 mM CaCl 2 , 80 mM NaCl, 0.2% v/v NP-40, 0.02% w/v sodium azide, 5% w/v non-fat dry milk). HTLV-1 p30 II  (HA) was visualized by immunofluorescence-microscopy. Six p30 II -expressing fibroblast colonies were isolated and expanded in 6-well tissue-culture plates in D-MEM supplemented with 10% FBS, 100U penicillin, and 100 mg/ml streptomycin-sulfate.  
       Example 4  
     Immunoprecipitations and ChIPs  
       [0110]     Myc-interacting complexes were immunoprecipitated from transfected Jurkat E6.1 or HTLV-1-infected MJ[G11] and HuT-102 lymphocytes expressing HTLV-1 p30 II  (HA) using a monoclonal anti-HA tag antibody. Immunoprecipitation of endogenous p30 II , from cultured HTLV-1-infected ATLL patient-derived lymphocytes was performed using a rabbit polyclonal antibody against the COOH-terminus of p30 II  (anti-HTLV-1 p30 II  antibody was generously provided by Dr. G. Franchini, NCI, NIH):(J. Virol. 67:2360-2366). Briefly, 3×10 6  cells were harvested by centrifugation and lysed in RIPA buffer (1× PBS, 1% (v/v) IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors: bestatin, pepstatin, antipain-dihydrochloride, chymostatin, leupeptin (50 ng/ml each. Roche Molecular Biochemicals) followed by passage through a 27.5-gauge tuberculin syringe. Immunoprecipitations were carried-out by incubating pre-cleared extracts with primary antibodies. Ten microliters of recombinant protein G-agarose (Invitrogen-Life Technologies) were added and reactions were incubated with agitation at 4° C. overnight. Matrices were pelleted by centrifugation at 6500 rpm for 5 min and washed twice with RIPA buffer. Samples were resuspended in 40 μl 2× SDS-PAGE loading buffer and bound proteins were resolved by electrophoresis through 4-15% gradient or 12.5% Tris-glycine SDS-polyacrylamide gels. Chromatin-immunoprecipitations were performed using a kit from Upstate Biotechnology. Nucleoprotein complexes were cross-linked in vivo by adding 270 μl formaldehyde to approximately 3×10 6  Molt-4 or HTLV-1-infected MJ[G11] and HuT-102 lymphocytes in 100 mm 2  tissue-culture dishes for 10 min. Cells were pelleted by centrifugation and resuspended in 200 μl SDS lysis buffer. Chromatin DNA was fragmented by sonication and oligonucleosomal-protein complexes were immuno-precipitated using primary antibodies and 60 μl salmon sperm DNA/protein A agarose. Precipitated oligonucleosomal-protein complexes were washed, cross-links were reversed, and bound DNA fragments were amplified by PCR using specific oligonucleotide primer pairs:  
                                   PRM,   5′ -CCCCTTCCTCCTGGAGTGAAATAC-3′;   (SEQ ID NO:1)           and                   5′ -CGTGCTCTAACGCATCCTTGAGTC-3′   (SEQ ID NO:2)          
 
 that flank conserved E-box elements within the human cyclin D2 gene promoter or anneal within an untranslated region, UTR, 5′-ATCAGACCCTATTCTCGGCTCAGG-3′ (SEQ ID NO:3) and 5′-CAGTCAGTAAGGCACTTTATTTCCCC-3′ (SEQ ID NO:4) as described in Vervoorts et al. (2003, EMBO Rep. 4:484-490). 
 
         [0111]     PCR products were electrophoresed through a 2% TAE agarose gel and visualized by ethidium bromide-staining.  
         [0000]     Documents Cited  
         [0112]     All sequences, patents, patent applications or other published documents cited anywhere in this specification are herein incorporated in their entirety by reference to the same extent as if each individual sequence, publication, patent, patent application or other published document was specifically and individually indicated to be incorporated by reference.