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Regulation of Gene Expression by HTLV-I Tax Protein
METHODS: A Companion to Methods in Enzymology 16, 83–94 (1998) Article No. ME980646
Regulation of Gene Expression by HTLV-I Tax Protein Franc¸oise Bex*,† and Richard B. Gaynor* *Division of Hematology–Oncology, Departments of Internal Medicine and Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8594; and †Department of Molecular Biology, University of Brussels, Belgium
The human T-cell leukemia virus type I or HTLV-I is the causative agent of adult T-cell leukemia. A protein encoded by HTLV-I, Tax, activates viral gene expression and is essential for transforming T-lymphocytes. Tax activates HTLV-I gene expression via interactions with the ATF/CREB proteins and the coactivators CBP/ p300 which assemble as a multiprotein complex on regulatory elements known as 21-bp repeats in the HTLV-I LTR. Tax can also activate expression from cellular genes including the interleukin-2 (IL-2) and the IL-2 receptor genes via increases in nuclear levels of NF-kB. Tax modulation of gene expression via the ATF/CREB and NF-kB pathways is linked to its transforming properties. This review discusses the mechanisms by which Tax regulates viral and cellular gene expression. © 1998 Academic Press
The human T-cell leukemia virus types I and II (HTLV-I and HTLV-II, respectively), the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively), and the human foamy viruses (HFV) comprise the known human retroviruses. Although these viruses share a number of common structural and biological properties, each virus has unique pathological sequelae. For example, HTLV-I is responsible for adult T-cell leukemia/lymphoma (ATL) (1, 2) and tropical spastic paraparesis (TSP) or HTLV-I associated myelopathy (HAM) (3, 4) while HTLV-II is associated with a rare form of human hairy-cell leukemia. HIV-1 and HIV-2 cause the acquired immunodeficiency syndrome or AIDS (5–7). No specific diseases have been directly associated with human foamy virus infection (8). Replication of each of these retroviruses is dependent on viral gene products known as transactivators which are critical for increasing viral gene expression. HTLV-I and II encode a transactivator known as Tax, HIV-1 and HIV-2 encode a transactiva1046-2023/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
tor known as Tat, and HFV encodes a transactivator known as Bel-1. The Tax protein of HTLV-I has been the subject of intense investigation. In addition to its ability to activate HTLV-I gene expression, the Tax protein is able to transform T-lymphocytes likely by increasing the expression of a unique set of cellular genes which are involved in T-cell proliferation. Thus, activation of the expression of specific cellular genes by Tax could be critical for the induction of adult T-cell leukemia by HTLV-I. An understanding of the mechanisms involved in Tax activation of viral and cellular gene expression should increase our knowledge of how alterations in cellular gene expression correlate with cellular transformation. This review will discuss recent advances in our understanding of the mechanisms regulating Tax function.
A. TAX ACTIVATION OF GENE EXPRESSION VIA CELLULAR ACTIVATION PATHWAYS Tax is a 40-kDa nuclear phosphoprotein which is translated from a spliced HTLV-I mRNA transcribed from the 39 portion of the HTLV-I genome (9, 10). Tax does not bind DNA directly (11–13) but stimulates transcription from the HTLV-I LTR (9, 14, 15) and from the promoters of specific cellular genes (16 –21) by recruiting cellular transcription factors. Activation of gene expression by Tax involves members of the activating transcription factor/cyclic AMP response element binding protein (ATF/CREB), the nuclear factor-kB (NF-kB), and the serum response factor (SRF) as well as the two related transcriptional coactivators CREB binding protein (CBP) and p300. 83
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1. Tax Activation of Gene Expression via ATF/CREB Pathway Tax is a potent activator of gene expression from the HTLV-I LTR (9, 10, 14, 15, 22). Three imperfect 21-bp repeats present in the U3 region of the HTLV-I LTR are the cis-acting enhancer elements required for Tax activation (23–28) (Fig. 1). Each 21-bp repeat contains a central domain sharing homology with cyclic AMP response elements (CRE) flanked by short GC-rich sequences. At least two of the 21-bp repeats (23, 28, 29) are critical for Tax activation of HTLV-I gene expression in vivo (30 –35). Tax will not activate gene expression from cellular promoters such as the somatostatin gene which contain CRE sites that are not flanked by GC-rich sequences (36). Recently, the critical role of these GC-rich sequences flanking the CRE site in the 21-bp repeats was determined by the fact that Tax contacts the minor groove of the GC-rich sequences in the context of the nucleoprotein complex that assembles on the 21-bp repeats (37). Interaction of Tax with cellular factors on the 21-bp repeats is critical for the formation of complexes that activate viral gene expression. A number of studies have identified cellular factors that interact with the HTLV-I 21-bp repeats (31, 38 – 41). Tax associates with the 21-bp repeats through interaction with these cellular factors (42– 46). All the factors that are able to mediate indirect interactions of Tax with the 21-bp repeats share in common a cluster of basic amino acids involved in DNA binding and a dimerization domain known as a leucine zipper (bZIP) (40, 47–50). Among these factors, yeast two-hybrid analysis of Tax with human cDNA libraries identified interactions with CREB and the two homologous factors, ATF-1 and CREM (51–55). These proteins are also able to specifically bind to Tax in mammalian cells (47, 49, 50, 53, 54, 56 –58). These three factors form a subgroup of ATF/CREB proteins in that they are phosphorylated by protein kinase A on a conserved serine residue and have highly homologous bZIP domains. Quantitative
FIG. 1. Schematic representation of the HTLV-I promoter. The position of the three 21-bp repeats relative to the transcription start site in the HTLV-I promoter, the sequence of each of the 21-bp repeats with the three domains A, B, and C, and a comparison of domain B with a consensus CRE site are indicated.
electrophoretic mobility shift analysis indicates that Tax interacts primarily with the bZIP region of these proteins to increase their binding affinity to the 21-bp pairs repeats (47, 49, 50, 56 –58). Tax may also induce dimerization of bZIP containing proteins in the absence of DNA to stabilize ternary complexes composed of Tax-CREB bound to the 21-bp repeat (47). Crosslinking and mutational analysis suggest that Tax itself may dimerize to facilitate the assembly of the ternary complexes on the 21-bp repeats (59, 60). Substitutions in the amino-terminus of Tax can selectively affect its ability to activate gene expression from the HTLV-I promoter (61) and bind to CREB (62, 63). These studies suggest that Tax, specific members of the ATF/CREB family, and the 21-bp repeats are each critical for the assembly of a stable ternary complex. Formation of the Tax–CREB–21-bp complex leads to the recruitment of a third factor, the transcriptional coactivator CREB binding protein (CBP). CBP is a protein of 265 kDa which is part of the RNA polymerase II holoenzyme complex. It was first identified as a factor that interacts with a form of CREB that is phosphorylated on serine residue 133 (64 – 66). This factor interacts with components of the general transcription complex (65) as well as with a variety of transcription factors including the phosphorylated form of CREB (64 – 66) (Fig. 2). When recruited by transcription factors, CBP promotes strong transcriptional activation by integrating the transcriptional enhancer complex with the general transcription machinery and by inducing chromosome remodeling as a result of its intrinsic and extrinsic histone acetylase activities (67– 69). Tax interacts with a central domain of CBP termed the KIX domain (amino acids 462 to 661) (58, 70 –72) which is the domain involved in the interaction between CPB and the phosphorylated form of CREB (64, 65). Electrophoretic mobility shift assays indicated that the ternary complex formed by Tax, CREB, and the 21-bp repeat can recruit the KIX domain of CBP to assemble a quaternary complex (71). CBP enhances the footprint of Tax on the GC-rich stretches flanking the CRE sites in the 21-bp repeats, indicating that CBP stabilizes the quaternary complex (37). In the cell, the specific proteins that interact with Tax may be regulated by their intracellular distribution and/or their ability to be redistributed by Tax. Intracellular localization using immunofluorescence staining and confocal microscopy indicated that CREB, CREM, and ATF-1 display differential distribution in lymphocytes and in fibroblasts that do not express Tax. De novo expression of Tax in fibroblasts leads to the formation of nuclear bodies in which both Tax and ATF-1 colocalize (Fig. 3A), whereas CREB and CREM are not detected in these nuclear structures (72). CBP colocalizes with wild-type Tax and ATF-1 (Figs. 3A and 3B). The Tax mutant M47 (L319S, L320A) (61) which
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selectively affects activation of gene expression from the HTLV-I promoter is unable to interact in vivo and in vitro with CBP. This Tax mutant forms nuclear bodies which include ATF-1 but not CBP (72). These observations suggest that a carboxy-terminal domain of Tax delineated by mutation M47 is critical for the interaction of Tax with CBP. This domain of Tax may correspond to a previously characterized activation domain of Tax. This activation domain was delineated by mutation analysis and comprises amino acids 289 to 322 (73, 74). Tax also associates with discrete nuclear foci in HTLV-I transformed lymphocytes (Fig. 4) and these foci contain both Tax and ATF-1. A model for the assembly of the complexes between Tax and the two cellular factors ATF/CREB and CBP on the 21-bp repeats is depicted in Fig. 5. Interaction of the amino-terminal domain of Tax with the bZIP domain of ATF/CREB leads to the assembly of a ternary complex in which the ATF/CREB proteins contact the CRE site in the 21-bp repeats and Tax serves as an anchor bound to the minor groove of the GC-rich sequences flanking the CRE site. Interaction of the carboxy terminus of Tax with CBP through its KIX domain increases the stability of this quaternary complex. Contacts between CBP and the general transcription factors included in the RNA polymerase II holoenzyme and CBP-mediated effects on histone acetylation result in chromosome remodeling and activation of transcription. In vitro formation of quaternary complexes does not require phosphorylation of CREB or even the presence of the amino-terminal domain of CREB which includes phosphoserine residue 133. It is
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not yet clear whether complexes found on the HTLV-I 21-bp repeats in vivo contain phosphorylated CREB and whether CREB contacts the KIX domain of CBP in the complex. In the context of the HTLV-I promoter, Tax may serve as a binding protein leading to the recruitment of CBP to enhancer complexes containing unphosphorylated CREB. Alternatively, Tax may alter the conformation of the normal phosphorylatedCREB–CBP complex to stimulate transcriptional activation from the unusually weak CRE binding sites present in the 21-bp repeats. 2. Tax Activation of Cellular Gene Expression via NF-kB Pathway Besides activating HTLV-I gene expression, Tax is also able to regulate a unique set of cellular genes which are involved in the control of T-cell proliferation and differentiation (75). These include the genes coding for granulocyte-macrophage colony stimulating factor (GM-CSF) (76 –78), interleukin-2 (IL-2) (79 – 82), and the high affinity subunit of the IL-2 receptor, IL2Ra (83– 86). Activation of T-cells during the immune and inflammatory responses results in the induction of a family of transcription factors known as NF-kB that bind to kB enhancer elements present in the transcriptional regulatory regions of genes involved in these responses. Normal T-cell growth is in part regulated by transient expression of these genes. Tax induces the nuclear expression of NF-kB proteins but differs from the process of NF-kB activation seen with immune stimulation. For example, HTLV-I infection of T-cells leads to constitutive expression of nuclear levels of
FIG. 2. Domain organization of CREB binding protein (CBP). The map of the 2441-amino-acid CREB binding protein with domains for interaction with the general transcription factor TFIIB, the histone acetyl transferase P/CAF, and a variety of transcription factors is represented. These include the KIX domain of CBP (amino acids 462 to 661), which can interact with Tax, CREB phosphorylated on serine residue 133 (S133 [32P]CREB), and the phosphorylated form of the RelA subunit of NF-kB.
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NF-kB and immortalization of T-cells. Various aspects of the NF-kB/c-Rel signaling pathway are involved in oncogenesis, regulating apoptosis, and the inflammatory and immune responses (87– 89). NF-kB is composed of homodimers and heterodimers of the Rel family of transcription factors (Fig. 6). These include p65 or RelA (90, 91), c-Rel (92), and RelB (93), as well as p50 (94, 95) and p52 (96 –98) which are processed amino-terminal fragments of the NF-kB precursors p105 and p100, respectively. These proteins are characterized by a conserved domain, the Rel homology domain, which is involved in DNA binding,
dimerization, and nuclear localization of these proteins. The presence of NF-kB dimers in the nucleus stimulates gene transcription via the potent transactivation domain located in the carboxy-terminal half of RelA as well as in c-Rel and RelB (99). The transcriptional activity of NF-kB is controlled by its intracellular localization. In the absence of specific stimuli, the nuclear import of NF-kB is prevented by its cytoplasmic sequestration through high-affinity binding with labile inhibitors known as IkB. Members of the IkB family include IkBa or MAD-3 (100, 101), IkBb (102), and IkBe (103) in addition to the precursors
FIG. 3. Tax colocalizes with activating transcription factor -1 (ATF-1) and CREB binding protein (CBP) in discrete nuclear foci. Baby hamster kidney cells (BHK21) were infected for 18 h with a recombinant Semliki Forest virus (SFV) expressing Tax. The cells were fixed with methanol for 6 min at 220°C and stained by dual immunofluorescence staining using a monoclonal antibody directed against Tax and either a polyclonal antibody directed against ATF-1 (A) or a rabbit polyclonal antibody directed against CBP (B). After incubation with the primary antibodies, the preparations were washed and incubated with a goat anti-mouse antibody conjugated to lisamine rhodamine sulfochloride (LRSC) and a goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC). The preparations were analyzed by confocal microscopy. (Reproduced here in black and white. See special color plate section for reproduction in color.) FIG. 4. Tax displays a diffuse cytoplasmic and a punctate nuclear distribution in the HTLV-I transformed lymphocytes. The HTLV-I transformed MT2 cells were fixed on coverslips with methanol for 6 min at 220°C and stained by immunofluorescence staining using a rabbit polyclonal serum directed against Tax and a goat anti-rabbit antibody conjugated to fluorescein isothiocyanate as secondary antibody. The preparation was analyzed by confocal microscopy using a MRC1024 microscope. (Left) Immunofluorescence staining; (right) phase contrast. (Reproduced here in black and white. See special color plate section for reproduction in color.)
REGULATION OF GENE EXPRESSION BY HTLV-I TAX PROTEIN
FIG. 5. Model for Tax-mediated assembly of enhancer complexes on the HTLV-I 21-bp repeats. Interaction of an amino-terminal domain of Tax with the bZIP domain of ATF-1 leads to the assembly of ternary complexes in which ATF-1 contacts the CRE site (B domain) in the 21-bp repeat and Tax is anchored to the minor groove of the GC-rich regions (A and C domains) through a domain yet to be identified. Stabilization of this complex results in gathering CBP to the complex. Interaction of a domain in the carboxy terminus of Tax with CBP through the KIX domain increases the stability of the quaternary complex. Contacts between CBP and general transcription factors in conjunction with histone acetylation by CBP results in chromosome remodeling and activation of transcription.
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of p50 and p52, p105 or NF-kB, and p100 or NF-kB2 (104, 105). The IkB proteins are characterized by conserved domains containing ankyrin repeats which interact with NF-kB proteins to result in their cytoplasmic retention (101, 106 –109). The IkB proteins mask the nuclear localization signal of NF-kB proteins and also inhibit NF-kB binding to DNA (102, 110, 111). The domain organization of NF-kB and IkB proteins is shown on Fig. 6. Treatment of cells with agents such as tumor necrosis factor a (TNFa), IL-1, and phorbol esters results in phosphorylation of IkBa on two serine residues at positions 32 and 36 and of IkBb on serine residues 19 and 23. These IkB proteins are subsequently ubiquitinated and degraded by the 26S proteasome (112–114). IkB degradation results in the rapid nuclear translocation of NF-kB which activates transcription of specific sets of cellular genes including the gene coding for IkBa itself (115). Resynthesis of IkBa triggers an autoregulatory loop that terminates the activation process by reinitiating NF-kB retention in the cytoplasm. Following cellular activation, IkBa also transiently accumulates in the nucleus and associates with inactive NF-
FIG. 6. Domain organization of members of the NF-kB/c-Rel and NF-kB inhibitor families. A conserved domain, the Rel homology domain (RHD), present in all members of the NF-kB/c-Rel family of transcription factors is represented as well as the nuclear localization signal (N), the phosphorylation site (P), the transactivation domain (TD), and a leucine zipper (LZ) which is present in Rel-B. The sites for endoproteolytic processing of p100 and p105 is indicated by an arrowhead which follows a glycine-rich domain (G). All NF-kB inhibitors have a conserved domain containing ankyrin repeats (ANK).
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kB/cRel complexes (116, 117). Accumulation of newly synthesized IkBa in the nucleus coincides with a decrease in the amount and activity of NF-kB. This effect results at least in part from IkBa-mediated nuclear export of NF-kB from the nucleus to the cytoplasm (118 –120). NF-kB is constitutively present in the nuclei of Taxexpressing cells and in HTLV-I infected cells (121). De novo expression of Tax in lymphocytes indicates that the process leading to the targeting of NF-kB complexes to the nucleus occurs in two phases. The first phase does not depend on new synthesis of NF-kB proteins and leads to the nuclear accumulation of p50/ RelA heterodimers (122). Nuclear pools of complexes containing other members of the NF-kB family includ-
ing p52 and c-Rel then increase (122) possibly through NF-kB dependent transcriptional activation of the genes (123). Induction of nuclear NF-kB DNA-binding activity by Tax results from induced phosphorylation, ubiquitination, and proteolytic breakdown of IkBa and IkBb leading to increased turnover rate of IkBa and virtual elimination of IkBb from Tax-expressing cells (112, 122, 124 –131). However, the mechanism involved in Tax-mediated induction of IkBa phosphorylation is not yet understood. Our results support the idea that this process requires activation by Tax of a recently identified IkB kinase known as IKK2 or IKKb (132, 173). Intracellular localization of IkBa in fibroblasts indicated that IkBa is normally distributed both in the
FIG. 7. Intracellular localization of IkBa and the RelA subunit of NF-kB in fibroblasts expressing Tax. BHK21 cells infected with the SFV vector (A, C) or with the recombinant SFV-Tax (B, D) for 18 h were fixed and stained by dual immunofluorescence staining using a monoclonal antibody directed against Tax and either a polyclonal serum directed against IkBa or a polyclonal serum directed against the RelA subunit of NF-kB. The incubation with the secondary antibodies and the analysis by confocal microscopy were as described in the legend to Fig. 3. IkBa, which is present both in the cytoplasm and the nuclei of cells that do not express Tax, was present only in the nuclei of cells expressing Tax and did not colocalize with Tax in nuclear foci. The RelA subunit of NF-kB is sequestered by IkBa in the cytoplasm of cells that do not express Tax. Expression of Tax leads to the nuclear translocation of RelA and its colocalization with Tax in nuclear foci. (Reproduced here in black and white. See special color plate section for reproduction in color.)
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cytoplasm and in the nucleus of cells that do not express Tax (Fig. 7A). Transduction of cells with a vector expressing Tax leads to the disappearance of the IkBa immunofluorescence staining in the cytoplasm (Fig. 7B). However, Tax expressing cells maintain a nuclear pool of IkBa, which is distributed in nucleoplasmic speckles excluding the nuclear foci containing Tax (Fig. 7B). Tax binding to p105 (133) and p100 (134), which are known inhibitors of NF-kB, may also release pools of NF-kB complexes from cytoplasmic sequestration (135–138). Stimulation of p105 degradation by Tax involves formation of a complex between Tax, p105, and the 20S proteasome, resulting in accelerated breakdown of p105 through the proteasome pathway (134, 139). However, interaction of Tax with p100 does not always lead to NF-kB nuclear translocation but, in some cases, this interaction leads to Tax inhibition (134, 140). Thus, p105 and p100 may have distinct effects on Tax-induced transactivation events (141). Tax functions in the cytoplasm to decrease the stability of various inhibitors of NF-kB and induce NF-kB nuclear translocation. However, to maintain constitutive NF-kB activation, Tax must also prevent nuclear events which normally lead to cessation of cellular activation. Recent studies suggest that nuclear pools of IkBa are involved in such events by preventing NF-kB interaction with DNA and resulting in NF-kB translocation from the nucleus to the cytoplasm (116 –119). Since intranuclear phosphorylation of IkBa may regulate the nuclear repressive action of IkBa (119), it is tempting to speculate that the persistence of the activation signal induced by Tax involves Tax-mediated regulation of an IkBa nuclear kinase. The possibility that Tax modulates the activity of DNA-PK, a nuclear kinase which has recently been demonstrated to specifically phosphorylate IkBa on threonine residue 273 (174) is currently being investigated. Accumulating evidence suggests that the involvement of Tax in cytoplasmic processes leading to nuclear translocation of NF-kB is not sufficient for Taxmediated activation of gene expression via the NF-kB pathway. Nuclear events involving Tax and NF-kB may also be important. This conclusion is based on the following observations. Wild-type Tax colocalizes with the two subunits of NF-kB, p50 and RelA, in nuclear foci (Figs. 7C and 7D) and the kinetics of inclusion of the RelA subunit in nuclear foci with Tax correlates with increased activity of a reporter gene under the control of a promoter containing NF-kB binding sites (142). A Tax mutant (F1-S113A, P128L), which fails to activate gene expression, is not efficiently transported to the nucleus. Nevertheless, this mutant is able to induce translocation of the RelA subunit of NF-kB to the nucleus (72). Direct interaction of Tax with RelA through the Rel homology domain has been reported
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(143, 144) as well as Tax-mediated increase in NF-kB binding to DNA by increasing NF-kB dimer formation (145). In addition, Tax interacts and colocalizes in nuclear foci with p300, a transcriptional coactivator with a high degree of homology to CBP. This interaction is likely critical for Tax activation of gene expression via the NF-kB pathway. The Tax mutant M148 (V148G) (146), which is selectively altered in its ability to activate gene expression via the NF-kB pathway, is unable to interact with p300 and recruit this factor into nuclear foci (72). Interestingly, the RelA subunit of NF-kB like the phosphorylated form of CREB is among the numerous interacting partners of both CBP and p300 (147, 148). Tax may modulate the activity of specific cellular genes containing kB binding sites in their promoter in a similar way to complexes assembled on the 21-bp repeats. The tentative model in Fig. 8 depicts contacts between Tax, NF-kB, and p300 in the assembly of complexes on NF-kB binding sites. The segregation of the roles of CBP and p300 in Tax-mediated activation of gene expression via either the ATF/CREB or the NF-kB pathway is intriguing. Although these two homologous proteins have been initially characterized through their interactions with specific ligands (phosphorylated form of CREB for CBP and adenovirus E1A protein for p300) (149, 150), they are usually studied and named as a unit (CBP/p300) having contacts with a common set of cellular factors (Fig. 2). No distinctive functions have been attributed to these related proteins. However, intracellular localization studies indicated that CBP and p300 are normally distributed in distinguishable nuclear speckles (72). Our results indicated that CBP and p300 interact with independent domains of Tax and that both of these proteins colocalize with wild-type Tax in nuclear foci (72). However, Tax mutants which are only able to activate gene expression via either the NF-kB (M47) (61) or the ATF/
FIG. 8. Model for Tax-mediated assembly of enhancer complexes on NF-kB binding sites. Tax interacts with the Rel homology domain of the RelA subunit of NF-kB and this interaction stabilizes the complex containing Tax, p50, and RelA on the NF-kB binding site. Tax recruits the transcriptional coactivator p300 which contacts general transcription factors and activates transcription via histone acetyltransferase activity.
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CREB pathway (M148) (146) have altered in vivo and in vitro interactions with CBP and p300, respectively. The Tax mutant M47 is unable to interact with CBP but this mutant activates gene expression via the NF-kB pathway at a level comparable to that of wildtype Tax. In contrast, the Tax mutant M148 is unable to interact with p300 but this mutant is able to activate gene expression via the ATF/CREB pathway at a level comparable to that of wild-type Tax. Thus, interaction of Tax with CBP is dispensable for activation of gene expression via the NF-kB pathway, but is likely required for activation of gene expression via the ATF/ CREB pathway. By contrast, the interaction of Tax with p300 is dispensable for activation of gene expression via the ATF/CREB pathway but may be required for the activation of gene expression via the NF-kB pathway (72). A summary of the cytoplasmic and nuclear events involved during Tax activation of gene expression via the ATF/CREB and NF-kB pathways is depicted in Fig. 9.
B. TRANSCRIPTIONAL ACTIVATION BY TAX CORRELATES WITH FORMATION OF TAX CONTAINING NUCLEAR BODIES Tax exhibits a diffuse cytoplasmic and a punctate nuclear distribution in HTLV-I transformed lymphocytes. Discrete nuclear foci containing Tax are also observed when a variety of cell lines transduced with Tax expression vectors are analyzed by immunofluo-
FIG. 9. Cytoplasmic and nuclear involvement of Tax in activation of viral and cellular gene transcription via the ATF/CREB and NF-kB pathways. In the cytoplasm, Tax induces IkBa phosphorylation and degradation through the proteasome pathway, resulting in the translocation of NF-kB complexes to the nucleus. In the nucleus, Tax associates with ATF/CREB proteins and the transcriptional coactivator CBP on the 21bp repeats of the HTLV-I promoter. Tax also associates in the nucleus with NF-kB heterodimers containing p50 and RelA and with the transcriptional coactivator p300. These nuclear events take place in unique nuclear structures in which activation of transcription and splicing of the pre-mRNA occur.
rescence staining and confocal microscopy (72, 142, 151). These nuclear structures not only contain Tax and the ATF/CREB and NF-kB transcription factors described above, but they contain at least three components of the RNA polymerase II holoenzyme including the large subunit of RNA polymerase II, a cyclindependent kinase, CDK8 and the transcriptional coactivators CBP/p300 (72, 142). Pulse-labeling of cells with 59-bromouridine 59-triphosphate and fluorescent in situ hybridization revealed that these foci include newly synthesized RNA and the mRNA from a gene specifically activated by Tax (151, 142). These observations suggest that the nuclear structures containing Tax may be functionally important for Tax activation of gene expression. Transcription and pre-mRNA processing are structurally organized in the nucleus and occur in particular nuclear domains which are associated with the nuclear matrix (152). Transcription by RNA polymerase II occurs in a large number of focal domains with high local concentration of RNA dispersed throughout the nucleoplasm, excluding the nucleoli (153, 154) as well as in perichromatin fibrils which contain transcripts packaged as hnRNPs that extend from active genes at the surface of the chromatin (155, 156). RNA processing is predominantly a cotranscriptional process. Components of the splicing complexes including members of the SR protein family (SC-35) (157) and core components of snRNPs (Sm) (158) are present in the transcript domains and in perichromatin fibrils. In addition to their presence at the sites of transcription, these splicing factors are also concentrated in other nuclear structures including coiled bodies and interchromatin granule clusters (IGC), which appear to be sites of storage and redistribution of splicing factors to the sites where active transcription and splicing occur (159). Interestingly, the nuclear foci containing Tax also include splicing factors Sm and SC35 and Tax coimmunoprecipitates with SC35-containing complexes (142, 160). Electron microscopy and immunogold labeling of fibroblasts expressing Tax identified the nuclear structures containing Tax as unique nuclear bodies formed of granules and fibers. Tax colocalizes with the splicing factor Sm in the fibers of these structures. These bodies are often surrounded with adjacent structures resembling coiled bodies and interchromatin granule clusters which contain a higher concentration of Sm but no Tax (142). Based on these observations, we propose that de novo expression of Tax leads to the assembly in the nucleus of transcript domains which include the components of the transcription and splicing complexes as well as select transcription factors which are involved in Tax-mediated activation of viral and cellular gene expression. Further support to this model requires demonstration that nascent RNA cor-
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responding to genes activated by Tax are present in or at the surface of these bodies.
C. TRANSFORMING PROPERTIES OF TAX HTLV-I induces neoplasia as a result of the activity of the tax gene. A number of studies have demonstrated the transforming potential of Tax in various experimental systems. Among the genes encoded by the pX 39 terminal region of the HTLV-I genome, tax is the only gene which is necessary and sufficient to induce immortalization of primary human T lymphocytes (161, 162). Tax can induce colony formation of Rat-1 fibroblast in soft agar and Tax mutants defective for activation of gene expression via either the ATF/CREB or the NF-kB pathway were unable to induce transformation of Rat fibroblasts (146, 163). This suggested that Tax transforming activity may be related to the deregulation of a common process in these two transcriptional activation pathways. Full transformation of peripheral blood lymphocytes by Tax requires activation of both the ATF/CREB and the NF- k B pathways, whereas activation of the NF- k B pathway alone only promotes growth response to IL-2 (164). Tax also cooperates with Ras to induce focus formation of primary rat embryo fibroblasts and these cells were tumorigenic in nude mice (165, 166). Transgenic mice expressing the tax gene under the control of the HTLV-I promoter develop soft tissue tumors and tumors resembling to neurofibromatosis (167). When Tax expression was targeted to the mature T-cell compartment, transgenic mice developed large granular lymphocytic leukemia, demonstrating that Tax expression in the lymphocyte compartment is sufficient for the development of leukemia (168). Tumor cell lines from mice expressing the tax transgene expressed high levels of GM-CSF which correlated with levels of Tax expression (169, 170). Interaction of Tax with the cyclin-dependent kinase inhibitor p16INK4A can lead to defects in inducing cell cycle arrest by this inhibitor and also be involved in the ability of Tax to induce transformation (171, 172). These observations suggest that Tax may have multiple cellular targets that are involved in its ability to transform cells. Further studies on the mechanism by which Tax activates viral and cellular gene expression will be essential to better understand the function of this human retroviral transforming protein.
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Regulation of Gene Expression by HTLV-I Tax Protein Franc¸oise Bex*,† and Richard B. Gaynor* *Division of Hematology–Oncology, Departments of Internal Medicine and Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8594; and †Department of Molecular Biology, University of Brussels, Belgium
The human T-cell leukemia virus type I or HTLV-I is the causative agent of adult T-cell leukemia. A protein encoded by HTLV-I, Tax, activates viral gene expression and is essential for transforming T-lymphocytes. Tax activates HTLV-I gene expression via interactions with the ATF/CREB proteins and the coactivators CBP/ p300 which assemble as a multiprotein complex on regulatory elements known as 21-bp repeats in the HTLV-I LTR. Tax can also activate expression from cellular genes including the interleukin-2 (IL-2) and the IL-2 receptor genes via increases in nuclear levels of NF-kB. Tax modulation of gene expression via the ATF/CREB and NF-kB pathways is linked to its transforming properties. This review discusses the mechanisms by which Tax regulates viral and cellular gene expression. © 1998 Academic Press
The human T-cell leukemia virus types I and II (HTLV-I and HTLV-II, respectively), the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively), and the human foamy viruses (HFV) comprise the known human retroviruses. Although these viruses share a number of common structural and biological properties, each virus has unique pathological sequelae. For example, HTLV-I is responsible for adult T-cell leukemia/lymphoma (ATL) (1, 2) and tropical spastic paraparesis (TSP) or HTLV-I associated myelopathy (HAM) (3, 4) while HTLV-II is associated with a rare form of human hairy-cell leukemia. HIV-1 and HIV-2 cause the acquired immunodeficiency syndrome or AIDS (5–7). No specific diseases have been directly associated with human foamy virus infection (8). Replication of each of these retroviruses is dependent on viral gene products known as transactivators which are critical for increasing viral gene expression. HTLV-I and II encode a transactivator known as Tax, HIV-1 and HIV-2 encode a transactiva1046-2023/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
tor known as Tat, and HFV encodes a transactivator known as Bel-1. The Tax protein of HTLV-I has been the subject of intense investigation. In addition to its ability to activate HTLV-I gene expression, the Tax protein is able to transform T-lymphocytes likely by increasing the expression of a unique set of cellular genes which are involved in T-cell proliferation. Thus, activation of the expression of specific cellular genes by Tax could be critical for the induction of adult T-cell leukemia by HTLV-I. An understanding of the mechanisms involved in Tax activation of viral and cellular gene expression should increase our knowledge of how alterations in cellular gene expression correlate with cellular transformation. This review will discuss recent advances in our understanding of the mechanisms regulating Tax function.
A. TAX ACTIVATION OF GENE EXPRESSION VIA CELLULAR ACTIVATION PATHWAYS Tax is a 40-kDa nuclear phosphoprotein which is translated from a spliced HTLV-I mRNA transcribed from the 39 portion of the HTLV-I genome (9, 10). Tax does not bind DNA directly (11–13) but stimulates transcription from the HTLV-I LTR (9, 14, 15) and from the promoters of specific cellular genes (16 –21) by recruiting cellular transcription factors. Activation of gene expression by Tax involves members of the activating transcription factor/cyclic AMP response element binding protein (ATF/CREB), the nuclear factor-kB (NF-kB), and the serum response factor (SRF) as well as the two related transcriptional coactivators CREB binding protein (CBP) and p300. 83
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1. Tax Activation of Gene Expression via ATF/CREB Pathway Tax is a potent activator of gene expression from the HTLV-I LTR (9, 10, 14, 15, 22). Three imperfect 21-bp repeats present in the U3 region of the HTLV-I LTR are the cis-acting enhancer elements required for Tax activation (23–28) (Fig. 1). Each 21-bp repeat contains a central domain sharing homology with cyclic AMP response elements (CRE) flanked by short GC-rich sequences. At least two of the 21-bp repeats (23, 28, 29) are critical for Tax activation of HTLV-I gene expression in vivo (30 –35). Tax will not activate gene expression from cellular promoters such as the somatostatin gene which contain CRE sites that are not flanked by GC-rich sequences (36). Recently, the critical role of these GC-rich sequences flanking the CRE site in the 21-bp repeats was determined by the fact that Tax contacts the minor groove of the GC-rich sequences in the context of the nucleoprotein complex that assembles on the 21-bp repeats (37). Interaction of Tax with cellular factors on the 21-bp repeats is critical for the formation of complexes that activate viral gene expression. A number of studies have identified cellular factors that interact with the HTLV-I 21-bp repeats (31, 38 – 41). Tax associates with the 21-bp repeats through interaction with these cellular factors (42– 46). All the factors that are able to mediate indirect interactions of Tax with the 21-bp repeats share in common a cluster of basic amino acids involved in DNA binding and a dimerization domain known as a leucine zipper (bZIP) (40, 47–50). Among these factors, yeast two-hybrid analysis of Tax with human cDNA libraries identified interactions with CREB and the two homologous factors, ATF-1 and CREM (51–55). These proteins are also able to specifically bind to Tax in mammalian cells (47, 49, 50, 53, 54, 56 –58). These three factors form a subgroup of ATF/CREB proteins in that they are phosphorylated by protein kinase A on a conserved serine residue and have highly homologous bZIP domains. Quantitative
FIG. 1. Schematic representation of the HTLV-I promoter. The position of the three 21-bp repeats relative to the transcription start site in the HTLV-I promoter, the sequence of each of the 21-bp repeats with the three domains A, B, and C, and a comparison of domain B with a consensus CRE site are indicated.
electrophoretic mobility shift analysis indicates that Tax interacts primarily with the bZIP region of these proteins to increase their binding affinity to the 21-bp pairs repeats (47, 49, 50, 56 –58). Tax may also induce dimerization of bZIP containing proteins in the absence of DNA to stabilize ternary complexes composed of Tax-CREB bound to the 21-bp repeat (47). Crosslinking and mutational analysis suggest that Tax itself may dimerize to facilitate the assembly of the ternary complexes on the 21-bp repeats (59, 60). Substitutions in the amino-terminus of Tax can selectively affect its ability to activate gene expression from the HTLV-I promoter (61) and bind to CREB (62, 63). These studies suggest that Tax, specific members of the ATF/CREB family, and the 21-bp repeats are each critical for the assembly of a stable ternary complex. Formation of the Tax–CREB–21-bp complex leads to the recruitment of a third factor, the transcriptional coactivator CREB binding protein (CBP). CBP is a protein of 265 kDa which is part of the RNA polymerase II holoenzyme complex. It was first identified as a factor that interacts with a form of CREB that is phosphorylated on serine residue 133 (64 – 66). This factor interacts with components of the general transcription complex (65) as well as with a variety of transcription factors including the phosphorylated form of CREB (64 – 66) (Fig. 2). When recruited by transcription factors, CBP promotes strong transcriptional activation by integrating the transcriptional enhancer complex with the general transcription machinery and by inducing chromosome remodeling as a result of its intrinsic and extrinsic histone acetylase activities (67– 69). Tax interacts with a central domain of CBP termed the KIX domain (amino acids 462 to 661) (58, 70 –72) which is the domain involved in the interaction between CPB and the phosphorylated form of CREB (64, 65). Electrophoretic mobility shift assays indicated that the ternary complex formed by Tax, CREB, and the 21-bp repeat can recruit the KIX domain of CBP to assemble a quaternary complex (71). CBP enhances the footprint of Tax on the GC-rich stretches flanking the CRE sites in the 21-bp repeats, indicating that CBP stabilizes the quaternary complex (37). In the cell, the specific proteins that interact with Tax may be regulated by their intracellular distribution and/or their ability to be redistributed by Tax. Intracellular localization using immunofluorescence staining and confocal microscopy indicated that CREB, CREM, and ATF-1 display differential distribution in lymphocytes and in fibroblasts that do not express Tax. De novo expression of Tax in fibroblasts leads to the formation of nuclear bodies in which both Tax and ATF-1 colocalize (Fig. 3A), whereas CREB and CREM are not detected in these nuclear structures (72). CBP colocalizes with wild-type Tax and ATF-1 (Figs. 3A and 3B). The Tax mutant M47 (L319S, L320A) (61) which
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selectively affects activation of gene expression from the HTLV-I promoter is unable to interact in vivo and in vitro with CBP. This Tax mutant forms nuclear bodies which include ATF-1 but not CBP (72). These observations suggest that a carboxy-terminal domain of Tax delineated by mutation M47 is critical for the interaction of Tax with CBP. This domain of Tax may correspond to a previously characterized activation domain of Tax. This activation domain was delineated by mutation analysis and comprises amino acids 289 to 322 (73, 74). Tax also associates with discrete nuclear foci in HTLV-I transformed lymphocytes (Fig. 4) and these foci contain both Tax and ATF-1. A model for the assembly of the complexes between Tax and the two cellular factors ATF/CREB and CBP on the 21-bp repeats is depicted in Fig. 5. Interaction of the amino-terminal domain of Tax with the bZIP domain of ATF/CREB leads to the assembly of a ternary complex in which the ATF/CREB proteins contact the CRE site in the 21-bp repeats and Tax serves as an anchor bound to the minor groove of the GC-rich sequences flanking the CRE site. Interaction of the carboxy terminus of Tax with CBP through its KIX domain increases the stability of this quaternary complex. Contacts between CBP and the general transcription factors included in the RNA polymerase II holoenzyme and CBP-mediated effects on histone acetylation result in chromosome remodeling and activation of transcription. In vitro formation of quaternary complexes does not require phosphorylation of CREB or even the presence of the amino-terminal domain of CREB which includes phosphoserine residue 133. It is
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not yet clear whether complexes found on the HTLV-I 21-bp repeats in vivo contain phosphorylated CREB and whether CREB contacts the KIX domain of CBP in the complex. In the context of the HTLV-I promoter, Tax may serve as a binding protein leading to the recruitment of CBP to enhancer complexes containing unphosphorylated CREB. Alternatively, Tax may alter the conformation of the normal phosphorylatedCREB–CBP complex to stimulate transcriptional activation from the unusually weak CRE binding sites present in the 21-bp repeats. 2. Tax Activation of Cellular Gene Expression via NF-kB Pathway Besides activating HTLV-I gene expression, Tax is also able to regulate a unique set of cellular genes which are involved in the control of T-cell proliferation and differentiation (75). These include the genes coding for granulocyte-macrophage colony stimulating factor (GM-CSF) (76 –78), interleukin-2 (IL-2) (79 – 82), and the high affinity subunit of the IL-2 receptor, IL2Ra (83– 86). Activation of T-cells during the immune and inflammatory responses results in the induction of a family of transcription factors known as NF-kB that bind to kB enhancer elements present in the transcriptional regulatory regions of genes involved in these responses. Normal T-cell growth is in part regulated by transient expression of these genes. Tax induces the nuclear expression of NF-kB proteins but differs from the process of NF-kB activation seen with immune stimulation. For example, HTLV-I infection of T-cells leads to constitutive expression of nuclear levels of
FIG. 2. Domain organization of CREB binding protein (CBP). The map of the 2441-amino-acid CREB binding protein with domains for interaction with the general transcription factor TFIIB, the histone acetyl transferase P/CAF, and a variety of transcription factors is represented. These include the KIX domain of CBP (amino acids 462 to 661), which can interact with Tax, CREB phosphorylated on serine residue 133 (S133 [32P]CREB), and the phosphorylated form of the RelA subunit of NF-kB.
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NF-kB and immortalization of T-cells. Various aspects of the NF-kB/c-Rel signaling pathway are involved in oncogenesis, regulating apoptosis, and the inflammatory and immune responses (87– 89). NF-kB is composed of homodimers and heterodimers of the Rel family of transcription factors (Fig. 6). These include p65 or RelA (90, 91), c-Rel (92), and RelB (93), as well as p50 (94, 95) and p52 (96 –98) which are processed amino-terminal fragments of the NF-kB precursors p105 and p100, respectively. These proteins are characterized by a conserved domain, the Rel homology domain, which is involved in DNA binding,
dimerization, and nuclear localization of these proteins. The presence of NF-kB dimers in the nucleus stimulates gene transcription via the potent transactivation domain located in the carboxy-terminal half of RelA as well as in c-Rel and RelB (99). The transcriptional activity of NF-kB is controlled by its intracellular localization. In the absence of specific stimuli, the nuclear import of NF-kB is prevented by its cytoplasmic sequestration through high-affinity binding with labile inhibitors known as IkB. Members of the IkB family include IkBa or MAD-3 (100, 101), IkBb (102), and IkBe (103) in addition to the precursors
FIG. 3. Tax colocalizes with activating transcription factor -1 (ATF-1) and CREB binding protein (CBP) in discrete nuclear foci. Baby hamster kidney cells (BHK21) were infected for 18 h with a recombinant Semliki Forest virus (SFV) expressing Tax. The cells were fixed with methanol for 6 min at 220°C and stained by dual immunofluorescence staining using a monoclonal antibody directed against Tax and either a polyclonal antibody directed against ATF-1 (A) or a rabbit polyclonal antibody directed against CBP (B). After incubation with the primary antibodies, the preparations were washed and incubated with a goat anti-mouse antibody conjugated to lisamine rhodamine sulfochloride (LRSC) and a goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC). The preparations were analyzed by confocal microscopy. (Reproduced here in black and white. See special color plate section for reproduction in color.) FIG. 4. Tax displays a diffuse cytoplasmic and a punctate nuclear distribution in the HTLV-I transformed lymphocytes. The HTLV-I transformed MT2 cells were fixed on coverslips with methanol for 6 min at 220°C and stained by immunofluorescence staining using a rabbit polyclonal serum directed against Tax and a goat anti-rabbit antibody conjugated to fluorescein isothiocyanate as secondary antibody. The preparation was analyzed by confocal microscopy using a MRC1024 microscope. (Left) Immunofluorescence staining; (right) phase contrast. (Reproduced here in black and white. See special color plate section for reproduction in color.)
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FIG. 5. Model for Tax-mediated assembly of enhancer complexes on the HTLV-I 21-bp repeats. Interaction of an amino-terminal domain of Tax with the bZIP domain of ATF-1 leads to the assembly of ternary complexes in which ATF-1 contacts the CRE site (B domain) in the 21-bp repeat and Tax is anchored to the minor groove of the GC-rich regions (A and C domains) through a domain yet to be identified. Stabilization of this complex results in gathering CBP to the complex. Interaction of a domain in the carboxy terminus of Tax with CBP through the KIX domain increases the stability of the quaternary complex. Contacts between CBP and general transcription factors in conjunction with histone acetylation by CBP results in chromosome remodeling and activation of transcription.
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of p50 and p52, p105 or NF-kB, and p100 or NF-kB2 (104, 105). The IkB proteins are characterized by conserved domains containing ankyrin repeats which interact with NF-kB proteins to result in their cytoplasmic retention (101, 106 –109). The IkB proteins mask the nuclear localization signal of NF-kB proteins and also inhibit NF-kB binding to DNA (102, 110, 111). The domain organization of NF-kB and IkB proteins is shown on Fig. 6. Treatment of cells with agents such as tumor necrosis factor a (TNFa), IL-1, and phorbol esters results in phosphorylation of IkBa on two serine residues at positions 32 and 36 and of IkBb on serine residues 19 and 23. These IkB proteins are subsequently ubiquitinated and degraded by the 26S proteasome (112–114). IkB degradation results in the rapid nuclear translocation of NF-kB which activates transcription of specific sets of cellular genes including the gene coding for IkBa itself (115). Resynthesis of IkBa triggers an autoregulatory loop that terminates the activation process by reinitiating NF-kB retention in the cytoplasm. Following cellular activation, IkBa also transiently accumulates in the nucleus and associates with inactive NF-
FIG. 6. Domain organization of members of the NF-kB/c-Rel and NF-kB inhibitor families. A conserved domain, the Rel homology domain (RHD), present in all members of the NF-kB/c-Rel family of transcription factors is represented as well as the nuclear localization signal (N), the phosphorylation site (P), the transactivation domain (TD), and a leucine zipper (LZ) which is present in Rel-B. The sites for endoproteolytic processing of p100 and p105 is indicated by an arrowhead which follows a glycine-rich domain (G). All NF-kB inhibitors have a conserved domain containing ankyrin repeats (ANK).
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kB/cRel complexes (116, 117). Accumulation of newly synthesized IkBa in the nucleus coincides with a decrease in the amount and activity of NF-kB. This effect results at least in part from IkBa-mediated nuclear export of NF-kB from the nucleus to the cytoplasm (118 –120). NF-kB is constitutively present in the nuclei of Taxexpressing cells and in HTLV-I infected cells (121). De novo expression of Tax in lymphocytes indicates that the process leading to the targeting of NF-kB complexes to the nucleus occurs in two phases. The first phase does not depend on new synthesis of NF-kB proteins and leads to the nuclear accumulation of p50/ RelA heterodimers (122). Nuclear pools of complexes containing other members of the NF-kB family includ-
ing p52 and c-Rel then increase (122) possibly through NF-kB dependent transcriptional activation of the genes (123). Induction of nuclear NF-kB DNA-binding activity by Tax results from induced phosphorylation, ubiquitination, and proteolytic breakdown of IkBa and IkBb leading to increased turnover rate of IkBa and virtual elimination of IkBb from Tax-expressing cells (112, 122, 124 –131). However, the mechanism involved in Tax-mediated induction of IkBa phosphorylation is not yet understood. Our results support the idea that this process requires activation by Tax of a recently identified IkB kinase known as IKK2 or IKKb (132, 173). Intracellular localization of IkBa in fibroblasts indicated that IkBa is normally distributed both in the
FIG. 7. Intracellular localization of IkBa and the RelA subunit of NF-kB in fibroblasts expressing Tax. BHK21 cells infected with the SFV vector (A, C) or with the recombinant SFV-Tax (B, D) for 18 h were fixed and stained by dual immunofluorescence staining using a monoclonal antibody directed against Tax and either a polyclonal serum directed against IkBa or a polyclonal serum directed against the RelA subunit of NF-kB. The incubation with the secondary antibodies and the analysis by confocal microscopy were as described in the legend to Fig. 3. IkBa, which is present both in the cytoplasm and the nuclei of cells that do not express Tax, was present only in the nuclei of cells expressing Tax and did not colocalize with Tax in nuclear foci. The RelA subunit of NF-kB is sequestered by IkBa in the cytoplasm of cells that do not express Tax. Expression of Tax leads to the nuclear translocation of RelA and its colocalization with Tax in nuclear foci. (Reproduced here in black and white. See special color plate section for reproduction in color.)
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cytoplasm and in the nucleus of cells that do not express Tax (Fig. 7A). Transduction of cells with a vector expressing Tax leads to the disappearance of the IkBa immunofluorescence staining in the cytoplasm (Fig. 7B). However, Tax expressing cells maintain a nuclear pool of IkBa, which is distributed in nucleoplasmic speckles excluding the nuclear foci containing Tax (Fig. 7B). Tax binding to p105 (133) and p100 (134), which are known inhibitors of NF-kB, may also release pools of NF-kB complexes from cytoplasmic sequestration (135–138). Stimulation of p105 degradation by Tax involves formation of a complex between Tax, p105, and the 20S proteasome, resulting in accelerated breakdown of p105 through the proteasome pathway (134, 139). However, interaction of Tax with p100 does not always lead to NF-kB nuclear translocation but, in some cases, this interaction leads to Tax inhibition (134, 140). Thus, p105 and p100 may have distinct effects on Tax-induced transactivation events (141). Tax functions in the cytoplasm to decrease the stability of various inhibitors of NF-kB and induce NF-kB nuclear translocation. However, to maintain constitutive NF-kB activation, Tax must also prevent nuclear events which normally lead to cessation of cellular activation. Recent studies suggest that nuclear pools of IkBa are involved in such events by preventing NF-kB interaction with DNA and resulting in NF-kB translocation from the nucleus to the cytoplasm (116 –119). Since intranuclear phosphorylation of IkBa may regulate the nuclear repressive action of IkBa (119), it is tempting to speculate that the persistence of the activation signal induced by Tax involves Tax-mediated regulation of an IkBa nuclear kinase. The possibility that Tax modulates the activity of DNA-PK, a nuclear kinase which has recently been demonstrated to specifically phosphorylate IkBa on threonine residue 273 (174) is currently being investigated. Accumulating evidence suggests that the involvement of Tax in cytoplasmic processes leading to nuclear translocation of NF-kB is not sufficient for Taxmediated activation of gene expression via the NF-kB pathway. Nuclear events involving Tax and NF-kB may also be important. This conclusion is based on the following observations. Wild-type Tax colocalizes with the two subunits of NF-kB, p50 and RelA, in nuclear foci (Figs. 7C and 7D) and the kinetics of inclusion of the RelA subunit in nuclear foci with Tax correlates with increased activity of a reporter gene under the control of a promoter containing NF-kB binding sites (142). A Tax mutant (F1-S113A, P128L), which fails to activate gene expression, is not efficiently transported to the nucleus. Nevertheless, this mutant is able to induce translocation of the RelA subunit of NF-kB to the nucleus (72). Direct interaction of Tax with RelA through the Rel homology domain has been reported
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(143, 144) as well as Tax-mediated increase in NF-kB binding to DNA by increasing NF-kB dimer formation (145). In addition, Tax interacts and colocalizes in nuclear foci with p300, a transcriptional coactivator with a high degree of homology to CBP. This interaction is likely critical for Tax activation of gene expression via the NF-kB pathway. The Tax mutant M148 (V148G) (146), which is selectively altered in its ability to activate gene expression via the NF-kB pathway, is unable to interact with p300 and recruit this factor into nuclear foci (72). Interestingly, the RelA subunit of NF-kB like the phosphorylated form of CREB is among the numerous interacting partners of both CBP and p300 (147, 148). Tax may modulate the activity of specific cellular genes containing kB binding sites in their promoter in a similar way to complexes assembled on the 21-bp repeats. The tentative model in Fig. 8 depicts contacts between Tax, NF-kB, and p300 in the assembly of complexes on NF-kB binding sites. The segregation of the roles of CBP and p300 in Tax-mediated activation of gene expression via either the ATF/CREB or the NF-kB pathway is intriguing. Although these two homologous proteins have been initially characterized through their interactions with specific ligands (phosphorylated form of CREB for CBP and adenovirus E1A protein for p300) (149, 150), they are usually studied and named as a unit (CBP/p300) having contacts with a common set of cellular factors (Fig. 2). No distinctive functions have been attributed to these related proteins. However, intracellular localization studies indicated that CBP and p300 are normally distributed in distinguishable nuclear speckles (72). Our results indicated that CBP and p300 interact with independent domains of Tax and that both of these proteins colocalize with wild-type Tax in nuclear foci (72). However, Tax mutants which are only able to activate gene expression via either the NF-kB (M47) (61) or the ATF/
FIG. 8. Model for Tax-mediated assembly of enhancer complexes on NF-kB binding sites. Tax interacts with the Rel homology domain of the RelA subunit of NF-kB and this interaction stabilizes the complex containing Tax, p50, and RelA on the NF-kB binding site. Tax recruits the transcriptional coactivator p300 which contacts general transcription factors and activates transcription via histone acetyltransferase activity.
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CREB pathway (M148) (146) have altered in vivo and in vitro interactions with CBP and p300, respectively. The Tax mutant M47 is unable to interact with CBP but this mutant activates gene expression via the NF-kB pathway at a level comparable to that of wildtype Tax. In contrast, the Tax mutant M148 is unable to interact with p300 but this mutant is able to activate gene expression via the ATF/CREB pathway at a level comparable to that of wild-type Tax. Thus, interaction of Tax with CBP is dispensable for activation of gene expression via the NF-kB pathway, but is likely required for activation of gene expression via the ATF/ CREB pathway. By contrast, the interaction of Tax with p300 is dispensable for activation of gene expression via the ATF/CREB pathway but may be required for the activation of gene expression via the NF-kB pathway (72). A summary of the cytoplasmic and nuclear events involved during Tax activation of gene expression via the ATF/CREB and NF-kB pathways is depicted in Fig. 9.
B. TRANSCRIPTIONAL ACTIVATION BY TAX CORRELATES WITH FORMATION OF TAX CONTAINING NUCLEAR BODIES Tax exhibits a diffuse cytoplasmic and a punctate nuclear distribution in HTLV-I transformed lymphocytes. Discrete nuclear foci containing Tax are also observed when a variety of cell lines transduced with Tax expression vectors are analyzed by immunofluo-
FIG. 9. Cytoplasmic and nuclear involvement of Tax in activation of viral and cellular gene transcription via the ATF/CREB and NF-kB pathways. In the cytoplasm, Tax induces IkBa phosphorylation and degradation through the proteasome pathway, resulting in the translocation of NF-kB complexes to the nucleus. In the nucleus, Tax associates with ATF/CREB proteins and the transcriptional coactivator CBP on the 21bp repeats of the HTLV-I promoter. Tax also associates in the nucleus with NF-kB heterodimers containing p50 and RelA and with the transcriptional coactivator p300. These nuclear events take place in unique nuclear structures in which activation of transcription and splicing of the pre-mRNA occur.
rescence staining and confocal microscopy (72, 142, 151). These nuclear structures not only contain Tax and the ATF/CREB and NF-kB transcription factors described above, but they contain at least three components of the RNA polymerase II holoenzyme including the large subunit of RNA polymerase II, a cyclindependent kinase, CDK8 and the transcriptional coactivators CBP/p300 (72, 142). Pulse-labeling of cells with 59-bromouridine 59-triphosphate and fluorescent in situ hybridization revealed that these foci include newly synthesized RNA and the mRNA from a gene specifically activated by Tax (151, 142). These observations suggest that the nuclear structures containing Tax may be functionally important for Tax activation of gene expression. Transcription and pre-mRNA processing are structurally organized in the nucleus and occur in particular nuclear domains which are associated with the nuclear matrix (152). Transcription by RNA polymerase II occurs in a large number of focal domains with high local concentration of RNA dispersed throughout the nucleoplasm, excluding the nucleoli (153, 154) as well as in perichromatin fibrils which contain transcripts packaged as hnRNPs that extend from active genes at the surface of the chromatin (155, 156). RNA processing is predominantly a cotranscriptional process. Components of the splicing complexes including members of the SR protein family (SC-35) (157) and core components of snRNPs (Sm) (158) are present in the transcript domains and in perichromatin fibrils. In addition to their presence at the sites of transcription, these splicing factors are also concentrated in other nuclear structures including coiled bodies and interchromatin granule clusters (IGC), which appear to be sites of storage and redistribution of splicing factors to the sites where active transcription and splicing occur (159). Interestingly, the nuclear foci containing Tax also include splicing factors Sm and SC35 and Tax coimmunoprecipitates with SC35-containing complexes (142, 160). Electron microscopy and immunogold labeling of fibroblasts expressing Tax identified the nuclear structures containing Tax as unique nuclear bodies formed of granules and fibers. Tax colocalizes with the splicing factor Sm in the fibers of these structures. These bodies are often surrounded with adjacent structures resembling coiled bodies and interchromatin granule clusters which contain a higher concentration of Sm but no Tax (142). Based on these observations, we propose that de novo expression of Tax leads to the assembly in the nucleus of transcript domains which include the components of the transcription and splicing complexes as well as select transcription factors which are involved in Tax-mediated activation of viral and cellular gene expression. Further support to this model requires demonstration that nascent RNA cor-
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responding to genes activated by Tax are present in or at the surface of these bodies.
C. TRANSFORMING PROPERTIES OF TAX HTLV-I induces neoplasia as a result of the activity of the tax gene. A number of studies have demonstrated the transforming potential of Tax in various experimental systems. Among the genes encoded by the pX 39 terminal region of the HTLV-I genome, tax is the only gene which is necessary and sufficient to induce immortalization of primary human T lymphocytes (161, 162). Tax can induce colony formation of Rat-1 fibroblast in soft agar and Tax mutants defective for activation of gene expression via either the ATF/CREB or the NF-kB pathway were unable to induce transformation of Rat fibroblasts (146, 163). This suggested that Tax transforming activity may be related to the deregulation of a common process in these two transcriptional activation pathways. Full transformation of peripheral blood lymphocytes by Tax requires activation of both the ATF/CREB and the NF- k B pathways, whereas activation of the NF- k B pathway alone only promotes growth response to IL-2 (164). Tax also cooperates with Ras to induce focus formation of primary rat embryo fibroblasts and these cells were tumorigenic in nude mice (165, 166). Transgenic mice expressing the tax gene under the control of the HTLV-I promoter develop soft tissue tumors and tumors resembling to neurofibromatosis (167). When Tax expression was targeted to the mature T-cell compartment, transgenic mice developed large granular lymphocytic leukemia, demonstrating that Tax expression in the lymphocyte compartment is sufficient for the development of leukemia (168). Tumor cell lines from mice expressing the tax transgene expressed high levels of GM-CSF which correlated with levels of Tax expression (169, 170). Interaction of Tax with the cyclin-dependent kinase inhibitor p16INK4A can lead to defects in inducing cell cycle arrest by this inhibitor and also be involved in the ability of Tax to induce transformation (171, 172). These observations suggest that Tax may have multiple cellular targets that are involved in its ability to transform cells. Further studies on the mechanism by which Tax activates viral and cellular gene expression will be essential to better understand the function of this human retroviral transforming protein.
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