Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells.

The inheritance of one defective BRCA1 or BRCA2 allele predisposes an individual to developing breast and ovarian cancers. BRCA1 is a multifunctional tumor suppressor protein, which through interaction with a vast array of proteins has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. Conversely, the oncogene, cyclin D1 is overexpressed in about 35% of all breast cancer cases. In this study, we provide detailed analyses on the phosphorylation state of BRCA1 by cyclin D1/cdk4 complexes. In particular, we have identified Ser 632 of BRCA1 as a cyclin D1/cdk4 phosphorylation site in vitro. Using chromatin immunoprecipitation assays, we observed that the inhibition of cyclin D1/cdk4 activity resulted in increased BRCA1 DNA binding at particular promoters in vivo. In addition, we identified multiple novel genes that are bound by BRCA1 in vivo. Collectively, these results indicate that cyclin D1/cdk4-mediated phosphorylation of BRCA1 inhibits the ability of BRCA1 to be recruited to particular promoters in vivo. Therefore, cyclin D1/Cdk4 phosphorylation of BRCA1 could provide a mechanism to interfere with the DNA-dependent activities of BRCA1.

The tax protein from the primate T-cell lymphotropic virus type 3 is expressed in vivo and is functionally related to HTLV-1 Tax rather than HTLV-2 Tax.

Human T-cell leukemia virus and simian T-cell leukemia virus (STLV) form the primate T-cell lymphotropic viruses group. Human T-cell leukemia virus type 1 and type 2 (HTLV-1 and HTLV-2) encode the Tax viral transactivator (Tax1 and Tax2, respectively). Tax1 possesses an oncogenic potential and is responsible for cell transformation both in vivo and in vitro. We and others have recently discovered the existence of human T-cell lymphotropic virus type 3. However, there is currently no evidence for the presence of a Tax protein in HTLV-3-infected individuals. We show that the serum of an HTLV-3 asymptomatic carrier and the sera of two STLV-3-infected monkeys contain specific anti-Tax3 antibodies. We also show that tax3 mRNA is present in the PBMCs obtained from an STLV-3-infected monkey, demonstrating that Tax3 is expressed in vivo. We further demonstrate that Tax3 intracellular localization is very similar to that of Tax1 and that Tax3 binds to both CBP and p300 coactivators. Using purified Tax3, we show that the protein increases transcription from a 4TxRE G-free cassette plasmid in an in vitro transcription assay. In all cell types tested, including transiently transfected lymphocytes, Tax3 activates its own promoter STLV-3 long terminal repeat (LTR), which contains only two Tax Responsive Elements (TREs), and activates also HTLV-1 and HTLV-2 LTRs. In addition, Tax3 also activates the NF-kappaB pathway. We also show that Tax3 possesses a PDZ-binding sequence at its C-terminal end. Our results demonstrate that Tax3 is a transactivator, and that its properties are more similar to that of Tax1, rather than of Tax2. This suggests the possible occurrence of lymphoproliferative disorders among HTLV-3-infected populations.

Enhancement of the p300 HAT activity by HIV-1 Tat on chromatin DNA.

HIV-1 Tat is able to form a ternary complex with P/CAF and p300 and increase the affinity for CDK9/P-TEFb CTD kinase complex. Our previous study demonstrated that Tat binds to p300/CBP in the minimal HAT domain (aa 1253-1790) and that the interaction results in a change of conformation on p300/CBP. Here, we show that the Tat-p300 interaction increases the HAT activity of p300 on histone H4 that is associated with nucleosomal DNA and not with free histones. Nucleosomal histone H4 was acetylated on lysines 8, 12, and 16. Acetylation of H4 was inhibited by Lys-coenzyme A (CoA), a selective inhibitor of p300 acetyltransferase activity. Unexpectedly, we also found that Tat could autoacetylate itself, which was specific to lysine residues 41 and 71. Peptides lacking these two lysines could not enhance the HAT activity of p300. Comparison of the sequences of Tat with other HIV-1 clades and HAT containing transcription factors indicated sequence identity in the acetyl-CoA binding motif A, KGXG. Furthermore, when utilizing an in vitro transcription assay, as well as a Tat mutant virus, we found that ectopic expression of only wild-type Tat in the presence of p300, and not a lysine 41 Tat mutant, could activate HIV-1 chromatin DNA, as evidenced by the absence of HIV-1 virion antigen. Therefore, transcription of integrated viral DNA in vivo requires the HAT activity of coactivators that are modulated by Tat to derepress the HIV-1 chromatin structure and aid in activated transcription.

Human T-lymphotropic virus type 1 p30(II) regulates gene transcription by binding CREB binding protein/p300.

The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30(II) of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30(II) directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30(II)-mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30(II)-mediated transactivation. In addition, Gal4(BD)-p30(II)-mediated transactivation was competitively inhibited by the cotransfection of CMV-p30(II)-HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30(II) colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30(II) to a highly conserved KIX region. DNA binding assays confirmed the interference of p30(II) with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30(II) and mediates its transcriptional effects in vivo.

Cell cycle effects of IL-10 on malignant B-1 cells.

IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.

Inhibition of human immunodeficiency virus type 1 transcription by chemical cyclin-dependent kinase inhibitors.

Cyclin-dependent kinases (cdk's) have recently been suggested to regulate human immunodeficiency virus type 1 (HIV-1) transcription. Previously, we have shown that expression of one cdk inhibitor, p21/Waf1, is abrogated in HIV-1 latently infected cells. Based on this result, we investigated the transcription of HIV-1 in the presence of chemical drugs that specifically inhibited cdk activity and functionally mimicked p21/Waf1 activity. HIV-1 production in virally integrated lymphocytic and monocytic cell lines, such as ACH(2), 8E5, and U1, as well as activated peripheral blood mononuclear cells infected with syncytium-inducing (SI) or non-syncytium-inducing (NSI) HIV-1 strains, were all inhibited by Roscovitine, a purine derivative that reversibly competes for the ATP binding site present in cdk's. The decrease in viral progeny in the HIV-1-infected cells was correlated with a decrease in the transcription of HIV-1 RNAs in cells treated with Roscovitine and not with the non-cdk general cell cycle inhibitors, such as hydroxyurea (G(1)/S blocker) or nocodazole (M-phase blocker). Cyclin A- and E-associated histone H1 kinases, as well as cdk 7 and 9 activities, were all inhibited in the presence of Roscovitine. The 50% inhibitory concentration of Roscovitine on cdk's 9 and 7 was determined to be approximately 0.6 microM. Roscovitine could selectively sensitize HIV-1-infected cells to apoptosis at concentrations that did not impede the growth and proliferation of uninfected cells. Apoptosis induced by Roscovitine was found in both latent and activated infected cells, as evident by Annexin V staining and the cleavage of the PARP protein by caspase-3. More importantly, contrary to many apoptosis-inducing agents, where the apoptosis of HIV-1-infected cells accompanies production and release of infectious HIV-1 viral particles, Roscovitine treatment selectively killed HIV-1-infected cells without virion release. Collectively, our data suggest that cdk's are required for efficient HIV-1 transcription and, therefore, we propose specific cdk inhibitors as potential antiviral agents in the treatment of AIDS.

Cell cycle regulation of human interleukin-8 gene expression by the human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus type 1 (HIV-1) Tat protein has been reported to transactivate several cellular genes, including the potent chemotactic factor interleukin-8 (IL-8). Consistent with these in vitro assays, elevated levels of IL-8 protein are found in the serum of HIV-infected individuals. We now extend these observations by demonstrating that Tat induction of IL-8 is linked to the cell cycle. Cells that constitutively express the Tat(1-86) protein (eTat) and control cells (pCEP) were reversibly blocked at the G(1)/S border with hydroxyurea or thymidine. The cells were subsequently released, and IL-8 expression was monitored by RNase protection assays and enzyme-linked immunosorbent assay (ELISA). RNase protection assays demonstrated that IL-8 mRNA expression is transiently induced, approximately fourfold, as the Tat-expressing cells enter S phase. Consistent with the RNase protection assay, an increase in IL-8 protein was observed in the cell supernatant using an IL-8 ELISA. Similar experiments were performed following a reversible block at the G(2)/M border with nocodazole and release into G(1). Using the RNase protection assay and ELISA, little or no increase in IL-8 expression was observed during G(1). Using gel shift as well as an immobilized DNA binding assay, we demonstrate that the increase in IL-8 gene expression correlates with a specific increase in p65 NF-kappa B binding activity only in the nucleus of the Tat-expressing cells. Moreover, the CREB-binding protein coactivator is present in the complex in the Tat cell line. Finally, we demonstrate that the presence of the proteasome inhibitor MG-132 inhibits the induction of NF-kappa B binding, as well as IL-8 expression, supporting the role of NF-kappa B.

Gene expression array of HTLV type 1-infected T cells: Up-regulation of transcription factors and cell cycle genes.

By utilizing a human cDNA expression array blot (588 genes), we have observed overexpression of various transcription factors, cell cycle regulated kinases, and DNA repair genes in HTLV-1-infected T cells. One of the genes of interest, and focus in this study, is the cyclin-dependent kinase inhibitor, p21/waf1. The p21/waf1 transcription and protein is overexpressed in all HTLV-1-infected cell lines tested as well as ATL and HAM/TSP patient samples. While p21/waf1 has been shown to display a selectivity for G(1)/S cyclin/cdk complexes, we have observed p21/waf1 to be complexed with cyclin A/cdk2. Functionally, the association of p21/cyclin A/cdk2 decreased the histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, as well as affecting other substrates such as the C-terminus of Rb protein involved in c-Abl and HDAC1 regulation. Wild-type, but not a mutant form (M47) of Tax, was found to be able to transactivate the p21/waf1 promoter in a p53-independent manner. We found that the minimal p21/waf1 promoter (-49 to +49 sequence) was activated by Tax and the minimal promoter contained two E2A transcription factor binding sites located between the TATA box and the initiation site. E2A proteins, E12 and E47, as well as a related helix-loop-helix protein, HEB, are all up-regulated in HTLV-1-infected T cells. When using band shift analysis, we found that only the E1 site (overlapping the transcription start site) was a functional DNA binding site. By using a chromatin immunoprecipitation (ChIP) assay, we observed that histone H4, and not histone H3, was acetylated from the endogenous p21/waf1 promoter in vivo, implying that CBP/p300, and not the SAGA complex, was critical in complexing with E2A in up-regulation of p21/waf1 in HTLV-1-infected cells.

Acetylation of HIV-1 Tat by CBP/P300 increases transcription of integrated HIV-1 genome and enhances binding to core histones.

The HIV-1 Tat protein is required for viral replication and is a potent stimulator of viral transcription. Although Tat has been extensively studied in various reductive paradigms, to date there is little information as to how this activator mediates transcription from natural nucleosomally packaged long terminal repeats. Here we show that CREB-binding protein (CBP)/p300 interacts with the HIV-1 Tat protein and serves as a coactivator of Tat-dependent HIV-1 gene expression on an integrated HIV-1 provirus. The site of acetylation of Tat was mapped to the double-lysine motif in a highly conserved region, (49)RKKRRQ(54), of the basic RNA-binding motif of Tat. Using HLM1 cells (HIV-1(+)/Tat(-)), which contain a single copy of full-length HIV-1 provirus with a triple termination codon at the first AUG of the Tat gene, we find that only wild type, and not K50A, K51A, or K50A/K51A alone or in combination of ectopic CBP/p300, is able to produce full-length infectious virions, as measured by p24 gag ELISAs. Tat binds CBP/p300 in the minimal histone acetyltransferase domain (1253-1710) and the binding is stable up to 0.85 M salt wash conditions. Interestingly, wild-type peptide 41-54, and not other Tat peptides, changes the conformation of the CBP/p300 such that it can acquire and bind better to basal factors such as TBP and TFIIB, indicating that Tat may influence the transcription machinery by helping CBP/p300 to recruit new partners into the transcription machinery. Finally, using biotinylated wild-type or acetylated peptides, we find that acetylation decreases Tat's ability to bind the TAR RNA element, as well as to bind basal factors such as TBP, CBP, Core-Pol II, or cyclin T. However, the acetylated Tat peptide is able to bind to core histones on a nucleosome assembled HIV-1 proviral DNA.

Helicobacter pylori inhibits gastric cell cycle progression.

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.

Inhibition of Tat-mediated transactivation of HIV-1 LTR transcription by polyamide nucleic acid targeted to TAR hairpin element.

Tat, an essential human immunodeficiency virus type 1 protein interacts with the transactivation response element (TAR) and stimulates transcription from the viral long-terminal repeat (LTR). Blockage of Tat-TAR interaction halts viral transcription and hence replication. We have found that polyamide nucleic acid (PNA), targeted to the TAR sequences of viral RNA genome is able to prevent Tat-TAR interaction by efficient sequestration of the TAR. Anti-TAR PNA competes for TAR and prevents Tat-mediated stimulation of HIV-1 LTR transcription in vitro but has no influence on the basal level of transcription in the absence of Tat. Using a reporter gene construct pHIV LTR-CAT and pCMV-Tat in cell culture, we have further shown that anti-TAR PNA is able to block Tat-mediated transactivation of HIV-1 LTR transcription in vivo as judged by the extent of LTR driven CAT gene expression in the absence and presence of anti-TAR PNA. Supplementation of 100 nM of anti-TAR PNA into the culture medium further enhances the suppression of transactivation. Nonspecific scrambled PNA had no influence on Tat-TAR interaction and LTR-driven CAT gene expression in cell culture. These results suggest that PNA targeted to the TAR sequence of the viral genome may be a potential inhibitor of HIV-1 gene expression.

BRCA1 is associated with a human SWI/SNF-related complex: linking chromatin remodeling to breast cancer.

Germline mutations in the tumor suppressor gene, BRCA1, predispose individuals to breast and ovarian cancers. Using a combination of affinity- and conventional chromatographic techniques, we have isolated a predominant form of a multiprotein BRCA1-containing complex from human cells displaying chromatin-remodeling activity. Mass spectrometric sequencing of components of this complex indicated that BRCA1 is associated with a SWI/SNF-related complex. We show that BRCA1 can directly interact with the BRG1 subunit of the SWI/SNF complex. Moreover, p53-mediated stimulation of transcription by BRCA1 was completely abrogated by either a dominant-negative mutant of BRG1 or the cancer-causing deletion in exon 11 of BRCA1. These findings reveal a direct function for BRCA1 in transcriptional control through modulation of chromatin structure.

Changes in E2F binding after phenylbutyrate-induced differentiation of Caco-2 colon cancer cells.

Differentiation agents use existing cellular systems to induce neoplastic cells to regain a normal phenotype and/or to cause growth arrest and therefore may offer novel chemotherapeutic approaches to treating solid tumors. In this study, we demonstrate in Caco-2 colon cancer cells that the differentiation agent phenylbutyrate (PB) causes a decrease in viable cells, an increase in cell differentiation, and a G1-S-phase block. The mechanism of this last effect is related to a PB-induced increase in p27Kip1, leading to a decrease in the activity of cyclin-dependent kinase 2 (CDK2), a positive regulator of the G1-S-phase cell cycle transition. Consistent with the decreased CDK2 kinase activity, we also observed a decrease in the phosphorylation state of the retinoblastoma protein after PB treatment. This was associated with increased binding and consequent inactivation of E2F, a transactivator of genes that regulate the G1 to S phase cell cycle transition. These data suggest that the differentiation agent PB inhibits tumor growth by limiting the availability of active E2F, with a subsequent G1-S-phase block. Additional studies should show whether PB is a clinically effective therapeutic agent against colorectal cancer.

Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2.

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G(1), S, or G(2)/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G(1) phase prior to gamma-irradiation, arrested in G(1) and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G(1)/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.

Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription.

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions +14 and +36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through +79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between +36 and +79. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.

Loss of G(1)/S checkpoint in human immunodeficiency virus type 1-infected cells is associated with a lack of cyclin-dependent kinase inhibitor p21/Waf1.

Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G(1)/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G(1)/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G(1)/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G(1)/S checkpoint in HIV-1-infected cells may in part be due to Tat's ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G(1)/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur.

Phosphorylation of the RAP74 subunit of TFIIF correlates with Tat-activated transcription of the HIV-1 long terminal repeat.

Transcription from the HIV-1 long terminal repeat (LTR) is regulated by the viral transactivator Tat, which increases RNA polymerase II (RNAP II) processivity. Previous reports have demonstrated that phosphorylation of the RNAP II carboxy-terminal domain by TFIIH and P-TEFb is important for Tat transactivation. Our present results demonstrate that phosphorylation of the RAP74 subunit of TFIIF is also an important step in Tat transactivation. Interestingly, while the general transcription factor TFIIF is required for both basal and Tat-activated transcription, phosphorylation of the RAP74 subunit occurs in the presence of Tat and correlates with a high level of transcription activity. Using a biotinylated DNA template transcription assay, we provide evidence that RAP74 is phosphorylated by TAF(II)250 during Tat-activated transcription. Depletion of RAP74 from the HeLa nuclear extract inhibited HIV-1 LTR-driven basal transcription and Tat transactivation. The addition of TFIIF, reconstituted from recombinant RAP30 and RAP74, to the depleted HeLa nuclear extract resulted in restoration of Tat transactivation. Of importance, the exogenous RAP74 was rapidly phosphorylated in the presence of Tat. These results suggest that RAP74 phosphorylation is one important step, of several, in the Tat transactivation cascade.

Cell cycle-regulated transcription by the human immunodeficiency virus type 1 Tat transactivator.

Cyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G(2) phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G(1). This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G(2) and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1, vpr, which arrests infected cells at the G(2)/M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions.

Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia virus type 1-infected cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax(1) is a 40-kDa phosphoprotein, predominantly localized in the nucleus of the host cell, which functions to transactivate both viral and cellular promoters. It seems likely that HTLV-1, through expression of the viral regulatory protein Tax(1), provides some initial alteration in cell metabolism predisposing the development of ATL. Here, we demonstrate that HTLV-1 infection in T-cell lines and patient samples causes overexpression of an early G(1) cyclin, cyclin D2. The transcriptional up-regulation of the cyclin D2 gene is due to activation of Tax on the cyclin D2 gene. More important, we find that overexpression of cyclin D2 is accompanied by acquisition of new partners such as cyclin-dependent kinase 2 (cdk2), cdk4, and cdk6 in infected cells. This is in contrast to uninfected T cells, where cyclin D2 associates only with cdk6. Functional effects of these cyclin-cdk complexes in infected cells are shown by hyperphosphorylation of Rb and histone H1, indicators of active progression into S phase as well as changes in cellular chromatin and transcription machinery. These studies link HTLV-1 infection with changes of cellular cyclin gene expression, hence providing clues to development of T-cell leukemia.

Pax-6 interactions with TATA-box-binding protein and retinoblastoma protein.

PURPOSE: To identify proteins that physically interact with Pax-6, a paired domain- and homeodomain (HD)-containing transcription factor that is a key regulator of eye development. METHODS: Protein-protein interactions involving Pax-6, TATA-box-binding protein (TPB), and retinoblastoma protein were studied using affinity chromatography with Pax-6 as ligand, glutathione-S-transferase (GST) pull-down assays, and immunoprecipitations. RESULTS: The authors have shown that Pax-6 is a sequence-specific activator of many crystallin genes, all containing a TATA box, in the lens. Others have shown that lens fiber cell differentiation, characterized by temporally and spatially regulated crystallin gene expression, depends on retinoblastoma protein. In the present study it was shown that Pax-6 interacted with the TBP, the DNA-binding subunit of general transcription complex TFIID. GST pull-down assays indicated that this interaction was mediated by the Pax-6 HD, with a substantial role for its N-terminal arm and first two alpha-helices. The experiments also indicated a binding role for the C-terminal-activation domain of the protein. In addition, the present study showed that the HD of Pax-6 interacted with retinoblastoma protein. Immunoprecipitation experiments confirmed retinoblastoma protein/Pax-6 complexes in lens nuclear extracts. CONCLUSIONS: Blending the present results with those in the literature suggests that Pax-6 and retinoblastoma protein participate in overlapping regulatory pathways controlling epithelial cell division, fiber cell elongation, and crystallin gene expression during lens development.