Transcriptional and Post-Transcriptional Gene Regulation of HTLV-1

Home , CREB, KIX domain, PCAF, PDZ domain, Response element

Transcriptional and post-transcriptional gene regulation of HTLV-1

Fatah Kashanchi*,1 and John N Brady2

1Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine, 2300 Eye St, NW, Ross Hall, Washington, DC 202-994-1781, USA; 2Laboratory of Cellular Oncology, National Cancer Institute, Building 41, Bethesda, MD 20892 301-496-0986, USA

Adult T-cell leukemia (ATL) is an aggressive hematologic family. HTLV-1 is the etiologic agent of adult T-cell malignancy caused by human T-cell leukemia virus type I leukemia (ATL) and the neurological disorder tropical (HTLV-1).Tax, encoded by the HTLV-1 pX region, spastic paraparesis/HTLV-1-associated myelopathy has been recognized by its pleiotropic actions to play a (Poiesz et al., 1980; Takatsuki et al., 1984; Yamaguchi critical role in leukemogenesis.Three highly conserved et al., 1984; Yoshida et al., 1984, 1987; Gessain et al., 21-bp repeat elements located within the long terminal 1985; Osame, 1990; Kira, 1994; Yoshida, 1994b). repeat, commonly referred to as Tax-responsive element 1 Studies have shown that the transactivator protein (TRE-1), are critical to Tax-mediated viral transcrip- Tax, encoded by the pX region of HTLV-1, is a potent tional activation through complex interaction with cyclic activator of the HTLV-1 long terminal repeat (LTR) AMP-responsive element binding protein (CREB), CBP/ (Brady et al., 1987; Yoshida et al., 1989; Yoshida, 1994a, p300 and PCAF.Tax has also been shown to activate 1995; Bex and Gaynor, 1998). Tax has also been shown transcription from a number of critical cellular genes to activate transcription of a number of cellular genes, through the NF-jB and serum-responsive factor path- including interleukin-2 (IL-2) and IL-2Ra (Greene et al., ways.Tax transactivation has been attributed to the 1986a, 1986b; Ballard et al., 1988, 1989; Marriott et al., protein’s interaction with transcription factors, chromatin 1992; Franchini, 1995; Curtiss et al., 1996; Good et al., remodeling complexes, cell cycle and repair genes.In this 1996). Tax does not bind to DNA directly, but activates review, we will discuss some of the latest ﬁndings on this transcription by recruiting or modifying the activity of fascinating viral activator and highlight its regulation of cellular transcription factors, including cyclic AMP- cellular factors including CREB, p300/CBP and their responsive element binding protein (CREB), serum- effect on RNA polymerase II and chromatin remodeling, responsive factor (SRF) and NF-kB (Franklin et al., as well as its role in cytoplasmic and nuclear function.We 1993; Suzuki et al., 1993b; Adya et al., 1994; Baranger will highlight the possible contribution of each factor, et al., 1995; Yin et al., 1995, 1996; Bex et al., 1998; discuss Tax’s critical peptide domains and highlight its Kashanchi et al., 1998; Li et al., 1999; Sun and Ballard, post-transcriptional modiﬁcations.It is quite obvious that, 1999; Kuo et al., 2000; Sun et al., 2000; Jeang, 2001; collectively, Tax’s effects on a wide variety of cellular Kibler and Jeang, 2001; Portis et al., 2001). As Tax plays targets cooperate in promoting cell proliferation and such an important role in gene expression and patho- leukemogenesis.In addition, the post-transcriptional genesis of HTLV-1, numerous studies have been effects of Rex play an important role in virus replication. directed toward the mechanism of Tax transactivation. Understanding these interactions at a molecular level will Several excellent reviews (Fujisawa et al., 1993; Franklin facilitate the targeted development of drugs to effectively and Nyborg, 1995; Yoshida et al., 1995; Suzuki and inhibit or treat ATL. Yoshida, 1996; Van Orden and Nyborg, 2000; Jeang, Oncogene (2005) 24, 5938–5951. doi:10.1038/sj.onc.1208973 2001; Neuveut and Jeang, 2002; Franchini et al., 2003; Azran et al., 2004; De La Fuente and Kashanchi, 2004; Keywords: HTLV-1; Tax; transcription; p53; chromatin; Jeang et al., 2004; Kehn et al., 2004; Pise-Masison and Rex Brady, 2005) have been written concerning the classical pathways of Tax transactivation. This section will highlight recent observations that are particularly important as far as understanding Tax transactivation.

Introduction

Human T-cell leukemia virus type-I (HTLV-1) is a Tax activation of the LTR complex retrovirus belonging to the Deltaretrovirus It has been shown that Tax activates expression of viral genes via the LTR. Three highly conserved 21-bp repeat *Correspondence: F Kashanchi, Department of Biochemistry and Molecular Biology, The George Washington University School of elements located within the LTR, commonly referred to Medicine, 2300 Eye St, NW, Ross Hall, Room 551 Washington, DC as Tax-responsive element 1 (TRE-1), are critical to 202-994-1781, USA; E-mail: [email protected] Tax-mediated transcriptional activation (Felber et al., Gene regulation of HTLV-1 F Kashanchi and JN Brady 5939 1985; Rosen et al., 1985, 1987; Brady et al., 1987; high-affinity binding site for the recruitment of the Yoshida, 2001). A region denoted as TRE-2 resides multifunctional cellular coactivators CBP, p300 and between the central and proximal TRE-1 (Brady et al., PCAF (Kwok et al., 1996; Giebler et al., 1997; 1987; Marriott et al., 1989; Marriott et al., 1990) and Lenzmeier et al., 1998; Jiang et al., 1999; Harrod contains binding sites for multiple transcription factors et al., 2000) (Figure 1). The direct interaction of Tax including Ets and c-Myb, or closely related proteins with CBP allows the binding of the coactivator in the (Bosselut et al., 1990, 1992; Clark et al., 1993). Tax absence of CREB phosphorylation, permitting specific associates with the LTR primarily at TRE-1 through activation of the viral LTR (Uchiumi et al., 1996; interaction with CREB (Giam and Xu, 1989; Zhao and Laurance et al., 1997). A major focus of recent studies Giam, 1992; Adya et al., 1994; Adya and Giam, 1995; has been the function of the coactivators in chromatin Goren et al., 1995; Tie et al., 1996), although the binding modification during HTLV-1 transcription. of other transcription factors including AP-1, AP-2 and In one of the first reports of p300/CBP transcriptional Sp1 have been reported (Jeang et al., 1991; Muchardt activity on a chromatin template in vitro,Luet al. (2002) et al., 1992; Barnhart et al., 1997; Wessner and Wigdahl, demonstrated that full-length p300 and CBP facilitate 1997; Wessner et al., 1997). The importance of the CRE- transcription of a reconstituted chromatin template in binding site was first noted by Jeang et al. (1988) who the presence of Tax and CREB. The ability of p300 and identified 21-bp repeat binding proteins and showed the CBP to activate transcription from the chromatin importance of the TGACGTCT CRE-response element template was dependent on the histone acetyltransferase by functional analysis of point mutations. The forma- (HAT) activity. Moreover, coactivator HAT activity tion of the Tax-CREB promoter complex serves as a must be tethered to the template by Tax and CREB.

b Tax

Transcription Initiation Elongation Terminationermination Release

Capping

RNA processing Splicing 3’ Poly A

mRNA export Export

Figure 1 Effect of Tax on viral transcription. (a) Schematic representation of the proximal promoter of HTLV-1 LTR. Tax facilitates binding of CREB and CBP/p300 to the TRE element in a multiprotein complex, which includes Tax. Tax further facilitates the binding of basal transcription factors and formation of the preinitiation complex. The interaction of Tax with CBP/p300 and SWI/SNF leads to acetylation of histones and remodeling of the restrictive chromatin structure. (b) Potential effect of Tax on gene expression network. The sequential steps of transcription initiation, elongation and termination are intricately linked together and to mRNA processing and export. Thus, the effect of Tax on transcription initiation and possible elongation would contribute indirectly to RNA processing and export (reprinted from De La Fuente and Kashanchi, 2004)

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5940 A p300 mutant that fails to interact with Tax did indicate that CBP and p300 play a prominent role in not facilitate transcription or acetylate histones. p300 Tax transactivation from chromatin templates and acetylated histones H3 and H4 within nucleosomes that the acetyltransferase activity of the coactivators located in the promoter and 50 proximal regions of the provides the major functional contribution to trans- template. Nucleosome acetylation was accompanied by criptional activation in vitro. Interestingly, a strong an increased binding of transcription factor TFIID and CBP/p300-specific acetyltransferase requirement was RNA polymerase II to the promoter. Interestingly, the observed on chromatin templates prepared from tailless investigators found distinct transcriptional activities histones. Multiple nonhistone acetylation targets, a between CBP and p300. CBP, but not p300, possesses subset of which are dependent upon Tax and CREB, an N-terminal activation domain, which directly acti- were identified. These data indicate that CBP and p300 vates Tax-mediated HTLV-1 transcription from a naked participate in critical chromatin-specific, tail-indepen- DNA template. Finally, using the chromatin immuno- dent acetylation events during transcriptional activation precipitation (ChIP) assay, the authors presented the by Tax. first direct experimental evidence that p300 and CBP Understanding the molecular mechanism of Tax are associated with the HTLV-1 long-terminal repeat transactivation may provide insight into inhibiting viral in vivo. transcription and replication in vivo. In a complex with In another in vitro study, Georges et al. (2002) used a CREB, Tax contacts the minor groove of the promoter chromatin assembly system that included recombinant DNA at guanine- and cytosine-rich sequences that core histones. The addition of Tax, CREB and p300 flank three of the off-consensus cyclic-AMP response activated transcription from the chromatin template. elements (CREs). In a recent article, Livengood Chromatin templates selectively lacking amino-terminal et al. (2004) used six Tax-directed pyrrole–imidazole histone tails demonstrated enhanced transcriptional polyamides specifically designed to block Tax bind- activation by Tax and CREB, with significantly reduced ing to DNA at each GC sequence of the three viral dependence on p300 and acetyl coenzyme A (acetyl- CREs. Four of the polyamides disrupted binding of the CoA). Interestingly, Tax/CREB activation from the Tax/CREB complex in vitro. These same molecules also tailless chromatin templates retained a substantial inhibit Tax-mediated transcription in vitro on chroma- requirement for acetyl-CoA, indicating a role for tin-assembled templates. Of these four Tax/CREB- acetyl-CoA beyond histone acetylation. These data specific polyamides, only one polyamide appears to be indicated that, during Tax transcriptional activation, uniquely Tax-specific. The polyamides can enter the the amino-terminal histone tails were the major targets nuclei of HTLV-1-infected T-cells, and two of the four of p300 and that tail deletion and acetylation are polyamides downregulated virion production in these functionally equivalent. cells. Together, these data indicate that targeted disrup- The HTLV-1 Tax protein has previously been shown tion of the Tax/CREB complex, or other complexes to bind to three of the four major transcription factor which assemble on the HTLV-1 promoter, may provide interaction domains in CBP/p300: CH1, KIX and the a novel approach for inhibiting viral replication in vivo. carboxy-terminal SRC-interacting domain (Kwok et al., 1996; Giebler et al., 1997; Harrod et al., 1998; Yan et al., 1998; Lemasson and Nyborg, 2001; Scoggin et al., 2001). The interaction between Tax and KIX has been studied Tax, chromatin and HDACs in the greatest detail and was the only Tax-CBP/p300 interaction that has been reported to occur with the Tax- Biochemical studies indicate that Tax interacts with containing, promoter-bound ternary complex (Tax/ CREB on the viral cAMP-response element enhancer CREB/viral CRE DNA) (Kwok et al., 1996; Giebler elements to recruit the pleiotropic coactivators CBP and et al., 1997; Harrod et al., 1998; Yan et al., 1998). This p300. Moreover, histone acetylation by these coactiva- observation suggested that the ternary complex interac- tors has been shown to play a major role in activating tion with KIX may play a major role in CBP/p300 HTLV-1 transcription from chromatin templates in recruitment to the HTLV-1 promoter. Georges et al. vitro. The extent of histone modification and the identity (2003) explored the transcriptional relevance of the Tax– of the cellular regulatory proteins bound at the HTLV-1 KIX interaction in Tax transactivation from chromatin- promoter in vivo have only lately began to unfold. assembled templates in vitro. Using polypeptides speci- Lemasson et al. (2002) utilized ChIP analysis to fically designed to inhibit CBP/p300 association with the investigate factor binding and histone modification at Tax/CREB/viral CRE complex, the authors demon- the integrated HTLV-1 provirus in infected T-cells strated that the KIX domain of CBP/p300 was critical (SLB-1). These studies revealed the presence of Tax, a for mediating coactivator function at the HTLV-1 variety of ATF/CREB and AP-1 family members promoter. The inhibition was specific to Tax transacti- (CREB, CREB-2, ATF-1, ATF-2, c-Fos and c-Jun), vation on chromatin templates and, unexpectedly, was and both p300 and CREB-binding protein at the independent of the four core histone amino-terminal HTLV-1 promoter. Consistent with the binding of these tails. The CBP/p300-selective acetyltransferase inhibitor coactivators, the authors observed histone H3 and H4 Lys-coenzyme A (CoA) inhibited Tax transactivation acetylation at specific sites within the proviral genome. from chromatin templates to the same levels observed Histone deacetylases were also present at the viral with the polypeptide inhibitors. These observations promoter and, following their inhibition, an increase in

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5941 histone H4 acetylation on the HTLV-1 promoter and a treatment with TSA. Consistent with the above studies, concomitant increase in viral RNA were observed. a physical interaction between Tax and HDAC1 both These results suggested that a variety of transcriptional in vitro and in vivo was observed. The N-terminal region activators, coactivators and histone deacetylases parti- of HDAC1 (amino acid residues 28–97) was required cipate in the regulation of HTLV-1 transcription in for this binding. Interestingly, HDAC1 inhibited the infected T cells. synergistic transactivation of Tax observed by ectopic The deacetylase superfamily can be divided into three expression of CBP. The repression was relieved by distinct classes based on structure. The HDACs overexpression of CBP. The authors concluded from comprise the first two classes and consist of class I these studies that HDAC1 was likely to compete with (HDACs 1, 2, 3, 8 and 11) and class II (HDACs 4, 5, 6, CBP in binding with Tax and functions as a negative 7, 9 and 10) enzymes. The class II enzymes are regulator for the transcriptional activation by Tax. distinguished by a large NH2-terminal domain or a The HDAC inhibitor FR901228, FK228 or depsipep- second catalytic site (e.g., HDAC6). The class III tide has been shown to be an effective growth inhibitor enzymes, sirtuins (SIRTs) or Sir2-related proteins, of T-cell lymphomas. Mori et al. (2004) examined deacetylate histones in yeast, while in mammalian cells whether FR901228 was effective for treatment of ATL they appear to be involved in deacetylation of other by assessing its ability to induce apoptosis of HTLV-1- proteins or transcription factors, such as p53, rather infected T-cell lines and primary leukemic cells from than histones. In general, inhibition of HDAC activity ATL patients. FR901228 induced apoptosis of Tax- by agents such as trichostatin A (TSA) or sodium expressing and -nonexpressing HTLV-1-infected T-cell butyrate leads to increased histone acetylation, correlat- lines and selective apoptosis of primary ATL cells, ing with increased mRNA expression. HDACs are especially those of patients with acute ATL. FR901228 commonly found as components of multiprotein com- efficiently reduced the DNA binding of NF-kB and AP- plexes containing DNA-histone-binding proteins (e.g., 1 in HTLV-1-infected T-cell lines and primary ATL cells NCoR, SMRT, MEF, MeCP2 and mSin3A) that use and downregulated the expression of Bcl-x(L) and cyclin HDACs to repress transcription and block the function D2, regulated by NF-kB. Although the viral protein Tax of cellular proteins such as MyoD, nuclear receptors, is an activator of NF-kB and AP-1, FR901228-induced p53, NF-kB and E2F. Conversely, histone acetylation apoptosis was not associated with reduced expression of has been correlated with transcriptionally active genes. Tax. In vivo use of FR901228 partly inhibited the The specific recruitment of a transcription factor growth of tumors of HTLV-1-infected T cells trans- complex with HAT activity to a promoter plays a planted subcutaneously in SCID mice. These results critical role in overcoming the repressive effects of indicated that FR901228 could induce apoptosis of chromatin structure on transcription. Transcriptional these cells and suppress the expression of NF-kBand regulation, therefore, is a dynamic interplay between AP-1, and suggest that FR901228 could be therapeuti- HAT and HDAC activity. cally effective in ATL. Lu et al. (2004) analysed the role of histone Transcription elongation is highly dependent upon deacetylase 1 (HDAC1) on HTLV-1 gene expression remodeling of chromatin structure. Tax has been shown from an integrated template. TSA enhanced Tax to interact with BRG1 components of the ATP- expression in HTLV-1-transformed cells. Second, using dependent chromatin-remodeling complex, SWI/SNF, a cell line containing a single-copy HTLV-1 long- and increase Tax transactivation (Wu et al., 2004). terminal repeat, the authors demonstrated that over- Disruption of BRG1 expression by siRNA led to a expression of HDAC1 represses Tax transactivation. decrease in Tax transactivation. Thus, Tax may target Furthermore, a ChIP assay allowed the analysis of SWI/SNF complexes downstream of RNA Pol II in the interaction of transcription factors, coactivators order to prevent stalling of the polymerase by chromatin and HDACs with the basal and activated HTLV-1 structure. promoter. These experiments demonstrated that HDAC1 While much has been learned about the regulated was associated with the inactive, but not the Tax- expression of transiently transfected LTR reporter transactivated, HTLV-1 promoter. In vitro and in vivo plasmids, the analysis of factors required for expression glutathione S-transferase-Tax pulldown and coimmu- of chromosomally integrated HTLV-1 LTR is of noprecipitation experiments demonstrated that there importance. Recently, Okada and Jeang (2002) con- was a direct physical association between Tax and structed cell lines which contained an integrated HTLV- HDAC1. Importantly, biotinylated chromatin pulldown 1 luciferase gene, and then compared the requirements assays demonstrated that Tax inhibits and/or dissociates for activation of transiently transfected versus stably the binding of HDAC1 to the HTLV-1 promoter. These integrated HTLV-1 LTR. In agreement with Jiang results provide evidence that Tax interacts directly with et al.’s earlier findings with transfected LTR plasmids, HDAC1 and regulates binding of the repressor to the Okada and Jeang found that P/CAF DHAT had a HTLV-1 promoter. HAT-independent activity on the stably integrated Ego et al. (2002) have also reported the physical HTLV-1 LTR. In contrast to Jiang et al.’s (1999) results interaction between Tax and the HDAC family of with the transfected LTR, the investigators reported proteins. The investigators reported that HDAC1 that when P/CAF and p300 were expressed at physio- represses the transactivation function of Tax in 293 T logic levels inside cells, a multiprotein complex contain- and MT4 cells. This repression was restored by ing CREB, p300 and P/CAF is required for Tax

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5942 activation of the integrated LTR. It is curious why two recruitment of TATA-binding protein to the HTLV-1 HATs (i.e. p300 and P/CAF) are frequently needed at promoter in ‘bypass’ experiments to show for the promoters even though the requirement for HAT ﬁrst time that Tax has transcriptional activity subse- activity is only required from one of the two proteins. quent to the assembly of an initiation complex at the promoter.

Transcription from both the 50 and 30 LTRs in vivo Tax, IKK and p53 During proviral integration, the 50 and 30 ends of the retrovirus are duplicated, forming LTRs. The LTRs of While another chapter will deal more extensively with the integrated provirus carry two identical U3 regions NF-kB regulation and yet another with Tax/p53 containing two identical promoters. The 50 promoter inhibition, the authors would like to discuss and perhaps directs synthesis of the genomic RNA, whereas the 30 clarify two recent reports. O’Mahony et al. (2004) promoter, if active, synthesizes RNAs that extend into recently reported a new function of IKK1 required for the adjacent host cell genome. In an intriguing study, complete activation of the NF-kB transcriptional Lemasson et al. (2004) utilized the in vivo ChIP assay to program. In IKK1À/À murine embryonic fibroblasts compare the binding of transcription regulatory proteins (MEFs), Tax normally induced early NF-kB activation at both the upstream and downstream promoters in events. However, NF-kB induced by Tax in these HTLV-1-infected cell lines and ATL-lymphoma cells. IKK1À/À cells was functionally impaired in that Tax Unexpectedly, the authors detected a nearly equal failed to activate several different NF-kB reporter distribution of activator (Tax, CREB, ATF-1, ATF-2, constructs or to induce the endogenous IkBa gene. c-Fos and c-Jun) and regulatory protein (CBP, p300, Reconstitution of IKK1À/À cells with kinase-proficient, TAF(II)250 and polymerase II) binding at both the but not a kinase dead mutant, form of IKK1 restored upstream and downstream promoters. Consistent with the Tax induction of full NF-kB transactivation. The this observation, they found that the downstream defect in NF-kB action in IKK1À/À cells correlated with promoter was transcriptionally active, suggesting that a failure of Tax to induce phosphorylation of the RelA/ the two promoters are functionally equivalent. Asym- p65 subunit of NF-kB at Ser-529 and Ser-536. Such metrical binding of histone deacetylases (HDACs 1–3) phosphorylation of RelA/p65 was readily detected in at both promoters was detected. All three HDACs wild-type (WT) MEFs. Phosphorylation of Ser-536 was strongly repressed Tax transactivation, and this repres- required for a complete response to Tax expression, sion correlated with displacement of Tax from the whereas phosphorylation of Ser-529 appeared to be less HTLV-1 promoter. These effects were reciprocal, as Tax critical. These findings highlight distinct roles for the expression reversed HDAC repression and displaced IKK1 and IKK2 kinases in the activation of NF-kBin HDACs from the HTLV-1 promoter. These data response to HTLV-1 Tax. IKK2 plays a dominant role suggest that HTLV-1 transcriptional regulation at both in signaling for IkBa degradation, whereas IKK1 the 50 and 30 LTRs was mediated, in part, through the appears to play an important role in enhancing the mutually exclusive binding of Tax and HDACs at the transcriptional activity of NF-kB by promoting RelA/ proviral promoters. p65 phosphorylation (Figure 2). In contrast to the above studies, Tax activation of the NF-kB pathway to inhibit p53 function utilizes IKK2- Specific TATAA and bZIP requirements suggest that mediated phosphorylation of p65 at Ser536 (Jeong et al., HTLV-1 Tax has transcriptional activity subsequent to 2005). Previous reports from this laboratory had the assembly of an initiation complex demonstrated that Tax inhibits p53 activity through the p65/RelA subunit of NF-kB (Jeong et al., 2004). In a Ching et al. (2004) recently reported that, in addition to recent article, the authors present evidence that suggests LTR enhancer elements, the core HTLV-1 TATAA that the upstream kinase IKK2 plays an important role motif provides specific responsiveness not seen with in Tax-induced p53 inhibition through phosphorylation either the SV40 or the E1b TATAA boxes. When of p65/RelA at Ser-536 (Figure 2). First, mouse embryo enhancer elements that can mediate Tax responsiveness fibroblast (MEF) IKK2À/À cells did not support Tax- were compared, the authentic HTLV-1 21-bp repeats mediated p53 inhibition, whereas MEFs lacking IKK1 were found to be the most effective. Related bZIP allowed Tax inhibition of p53. Second, transfection of factors such as CREB, ATF4, c-Jun and LZIP are often IKK2 WT, but not a kinase-dead mutant, into IKK2À/À thought to recognize the 21-bp repeats equivalently. cells rescued p53 inhibition by Tax. Third, the IKK2- However, among bZIP factors, CREB by far was specific inhibitor SC-514 decreased the ability of Tax to preferred by Tax for activation. When LTR transcrip- inhibit p53. Fourth, the investigators show that phos- tion was reconstituted by substituting either kBor phorylation of p65/RelA at Ser-536 is important for Tax serum response elements in place of the 21-bp repeats, inhibition of p53 using MEF p65/RelAÀ/À cells trans- Tax activated these surrogate motifs using surfaces fected with p65/RelA WT or mutant plasmids. More- which are different from that utilized for CREB over, Tax induced p65/RelA Ser-536 phosphorylation interaction. Finally, the investigators employed artificial in WT or IKK1À/À cells, but failed to induce the

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5943

Figure 2 Overlapping, but distinct, pathways for Tax activation of NF-kB are utilized for transcription activation and p53 inhibition. In the activation of transcription, Tax interacts with IKKg and stimulates IKK2 to phosphorylate IkBa, which is subsequently degraded, releasing the p50/p65 herterodimer to the nucleus. IKK1 sustains the NF-kB activation by phosphorylating p65 at Ser536. In the p53 inhibition cascade, Tax functions upstream of IKK to signal the activation of IKK2 to phosphorylate IkBa and p65 at Ser536. This event allows for interaction between p53 and p65, which then bind to the inactive p53 promoter phosphorylation of p65/RelA Ser-536 in IKK2À/À cells, sufficient for NF-kB transcriptional activation. The suggesting a link between IKK2 and p65/RelA phos- work by Jeong et al. therefore describes a novel Tax- phorylation. Consistent with this observation, blocking p65/RelA pathway that functions to inhibit p53, but IKK2 kinase activity with SC-514 decreases the does not require NF-kB transcriptional activity. phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type 1- transformed cells. While these results seem to contradict those reported by O’Mahoney et al., some important Tax modification, localization and binding partners distinctions in the systems should be noted. In the regulating transformation ubiquitination of Tax studies presented by Jeong et al., endogenous p65 phosphorylation as Ser-536 was assayed using a Ser- Recently, two labs have independently reported the Tax 536 phospho-specific antibody. On the other hand, ubiquitination in either transfected or infected cells O’Mahoney et al. assayed the in vitro kinase activity of (Chiari et al., 2004; Peloponese et al., 2004). Ubiquitin NEMO/IKKg-containing complexes. In those studies, is a small, abundant, highly conserved 76-amino-acid immunoprecipitated NEMO/IKKg complexes which did polypeptide found in all eukaryotic cells. Proteins can not contain IKK1 could not phosphorylate p65. be modified by covalent attachment of ubiquitin Together, these results suggest that distinct IKK kinase molecules to either lysine residues or their N termini complexes, which vary in composition, may exist and via an ATP-dependent process. The ubiquitination that phosphorylation of p65 at Ser-536 alone is not reaction first involves an activating enzyme (E1) that

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5944 activates the ubiquitin, and then the ligation of ubiquitin Tax-binding proteins to the substrate is carried out by a complex composed of a ubiquitin-conjugating enzyme (E2) and a ubiquitin Previously, Tax has been shown to a bind to number of protein ligase (E3), in charge of substrate specificity. protein complexes regulating transformation, cell cycle Conjugation to a chain containing at least four ubiquitin and apoptosis. For example, an increased number of monomers is required to target a protein to the transformed colonies induced by Tax1 relative to Tax2 proteasome for destruction. Also, a very different role was mediated by a PDZ domain-binding motif (PBM) in for ubiquitination has been observed, where polyconju- Tax1, which is absent in Tax2. Tax1 PBM mediated the gation of ubiquitin to some transcription factors interaction of Tax1 with the discs large (Dlg) tumor positively regulates their transcriptional activity. suppressor containing PDZ domains, and the inter- The effect of Tax ubiquitination can best be seen in action correlated well with the transforming activities lysis buffers containing both proteasome and isopepti- of Tax1 and the mutants (Hirata et al., 2004). dase inhibitors, which prevent rapid deubiquitination. Tax has also been shown to interact with components Using these reagents, two labs recently showed that 10 of the INK4-CDK4/6-Rb pathway, p16 and cyclin D(s). lysine residues on Tax could potentially be ubiquitined While Tax competes with CDK4 for p16 binding, thus (Chiari et al., 2004; Peloponese et al., 2004). Tax suppressing p16 inhibition of CDK4, Tax also binds to ubiquitination occurred principally on the C-terminal cyclin D(s) with concomitant increase in both CDK4 half of Tax at lysines 263, 280 and/or 284. Tax activity and the phosphorylation of cyclin D(s), imply- ubiquitination was related to proteasome binding, but ing that the Rb protein and the G1/S phase of the cell not to rapid degradation. Finally, contrary to published cycle are deregulated by Tax (Li et al., 2003). reports on HIV-1 Tat, where overexpression of ubiquitin The Jeang lab has also found a number of critically enhanced transcriptional activity, for Tax, overexpres- important Tax-associated complexes in infected cells. sion of ubiquitin decreased transcriptional activity, and They identified novel binding partners for Tax, in- neither ubiquitinated Tat nor Tax were processed for cluding human mitotic checkpoint protein MAD1 proteasomal degradation. (TXBP181), G-protein pathway suppressor GPS2 (TXBP31), and IkB kinase regulatory subunit IKKg in addition to two new Tax partners, TXBP151 and TXBP121. A closer examination of the sequences of Tax localization eight independent cellular Tax-binding proteins identified by these investigators and others revealed that all There have been a number of attempts to localize the share a single characteristic, a highly structured coiled- nuclear Tax in either transfected or infected cells. Initial coil domain. They also noted that Tax and the Tax- studies demonstrated that Tax localized within the binding coiled-coil proteins can homodimerize (Chun interchromatin granule clusters (IGCs)/RNA splicing et al., 2000). Finally, the role of Tax-binding partners in bodies (SBs) region (Semmes and Jeang, 1996). These antiapoptosis has been described. In a yeast two-hybrid results have further been confirmed by others. Ariumi system to screen for proteins that interact with A20, et al. studied the effect of Tax on IGCs/SBs, PML and a Cys2/Cys2 zinc-finger protein which is induced by a SMRT, and found that Tax was identified within IGCs/ variety of inflammatory stimuli and characterized as an RNA SBs, not PML-NBs (Ariumi et al., 2003). Unlike inhibitor of cell death, a cDNA fragment was isolated, HFV transactivator Tas, PML significantly coactivated which encoded a portion of TXBP151. Therefore, Tax-mediated activation of HTLV-1-LTR promoter TXBP151 appears to be a novel A20-binding protein activity in 293 T, Rat1, MT-2 and HTLV-1-infected T- which might mediate the antiapoptotic activity of A20, cell lines, and no disruption or relocation of PML-NBs and which can be processed by specific caspases (De in the HTLV-1-infected T-cell lines MT-2, MT-4 and Valck et al., 1999). HUT102 was observed. This is also in contrast to HIV We and others have previously shown that Tax infection, where PML accumulates in the cytoplasm to localizes to both the cytoplasm and nucleus. In an interfere with HIV’s ability to integrate into the host attempt to define the Tax partners in either compart- genome. ment, we performed a Tax proteome experiment (Wu The nuclear corepressor SMRT also significantly et al., 2004). Results indicated that Tax could bind coactivated Tax-mediated HTLV-1 LTR-dependent to few specific proteins including cytoplasmic small gene expression, but not other promoter-dependent GTPases, which included Ras p21 proactivator 2 activities, including protein kinase A (PKA)-mediated (GAP1 m), Cdc 42/Rac effector kinase PAK-3, RhoA, CREB-dependent gene expression, p53-dependent gene Rac1 and Cdc42, all of which are GTPase proteins. expression and Tax-mediated NF-kB-dependent gene These small GTPases are turned on and off by binding expression, indicating that the SMRT coactivation to GTP/GDP nucleotides in vivo, and are best known appears to be specific for Tax. Therefore, for HTLV-1, for their effects on the actin cytoskeleton, leading to IGCs/SBs, not PML-NBs, may be the center of viral transformation. replication/transcription, and the targeting of Tax to Tax bound to several small GTPase proteins, includ- Tax-associated nuclear bodies is likely to be a pre- ing ras GAP1m, Rac1, Cdc42, RhoA and gelsolin. These requisite for CREB-dependent, but not NF-kB-depen- small GTPases function by partnering with the cyto- dent, function. skeletal proteins. Small GTPases could also regulate

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5945 downstream protein complexes such as JNK. Previous Another very interesting interaction turned out to be experiments have demonstrated that constitutive activa- that with the chromatin remodeling complex. BRG1 and tion of JNK promotes interleukin-2-independent growth hBRM, components of SWI/SNF complex, are impli- in HTLV-1-infected T-cells. Therefore, Tax modulates cated in chromatin remodeling, as well as activation and the upstream effector proteins in combination with growth control. The packaging of DNA into chromatin small GTPases, which in turn control downstream prevents access of DNA-binding factors and inhibits signaling cascades, such as JNK, p38, MEKKs and elongation by RNA polymerase II. Indeed, the activa- NF-kB complexes (Figure 3a). tion of many genes is accompanied by a disruption of

a Cytoskeleton dynamics (Actin, Intermediate filaments)

Cytoplasmic effector Arp2/3 PAK LIMK, (proliferation/ POR MLCp division/invasion Arf6 (cytoskeleton)

N-WASP ROCK mDia

1m Cdc42 Rac1 RhoA GAP Modulator TATAX X (Small GTPase)

PAKs, NIK POSH

Cytoplasmic MEK-3 MEKK-1 IKKs effector (Signal pathways)

P38 JNK/SAPK

Nuclear effector ATF2 κ AP-1 C/EBPb NF B (Transcription) Max ATF2 TCF ELK CREB

b CREB2 DNA Acidic A.A. contact contact NFκB-activating LIM-binding Cluster

Hla-A2 SH3 UR Leucine Rev-like Activation binding binding motif Zip-like NES Leucine Specific Region IKKγ binding Zip-like NLS, Zn Finger P300/CBP P/CAF PDZ CREB-binding binding Dimerization binding binding

23 62 81 95 175 2877 336 353

Tax COX1 Transpeptidase ras Fe.Asc.Oxidored Hemagglutinin LIM.bind RAS DAHP.synth.1 Flu.PB1 TFS2M

Figure 3 Schematic display of structural and functional regions in the Tax protein. (a) Schematic representation of Tax roles in regulatory pathways of small GTPase. For simplification, only the main pathways of cytoskeleton, signal transduction and their transcription effectors are shown here. Specific Tax effectors are italicized (from present results) and underlined (reported in the literature). (b) The Tax (NCBI/Accession #: 6983837) protein schematic is modified from the ‘conserved domain search’ in BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) with the E-value at 10. Each domain with its score (first data in parenthesis) and E value (second data) were as follows: Tax (166, 2e-42); transpeptidase (31.2, 0.099); hemagglutinin (30.0, 0.22); ras/Ras family (29.3, 0.38); RAS/RAS small GTPases (26.2, 3.2); COX1 (25.4, 5.4); TFS2 M (25.4, 5.4); Flu_PB1 (25.0, 7.1); Fe_Asc_oxidored (25.0, 7.1); LIM- domain-binding protein (24.6, 9.3); DAHP synthetase I family (24.6, 9.3). Above the scheme is a display of the possible functional and structural regions of Tax according to various websites (http://pbil.univ-lyon1.fr/pbil.html; http://pfam.wustl.edu) and literature searches. UR: upstream region (14); NES: nuclear export signal (2); NLS: nuclear localization signal (reprinted from Wu et al., 2004)

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5946 the pattern of nucleosomes over promoters and tran- element modulation and CREB and stimulate transcrip- scribed regions. Consistent with the current view of tional activity in yeast and mammalian cells in the SWI/SNF recruitment of site-specific activators, we also absence of the classical CBP/p300 pathway (Fimia et al., found that the Tax/SWI/SNF binding may be recruited 1999, 2000). to an active promoter. In addition, two different classes Finally, Tax can also interact through a coiled-coil of chromatin remodelers may function at separate and structure (Chun et al., 2000), ERM and myosin tail-like successive steps in gene activation. Therefore, according domains, a TRAF-interacting protein, T6BP (almost to our previous results and those of others with Tax/ identical to TXBP151), myotubularin-related protein, CBP interactions, we suspect that, much like the HO and Armadillo/beta-catenin-like repeats which mediate promoter in yeast, there may be a cell cycle-regulated interaction of beta-catenin with its ligands. Collectively, wave of SWI/SNF-dependent histone modification that the biochemical and computational data suggest that is restricted to B1 kb of the Tax responsive viral and LIM, SH3, PDZ, coiled-coil and myotubularin-related cellular promoters (i.e., the HTLV-1 promoter elements structures may be partly sufficient for Tax binding and upstream of the TATA box and the cyclin D2 possibly induce transformation by Tax. promoter). This would explain, in part, Tax’s pleiotropic effect on both viral and many cellular genes regulated by CREB and NF-kB. Tax and C/EBP: repression of viral transcription

The interaction of Tax with bZIP domain-containing Tax interacting domains transcription factors such as the activating transcription factor/CRE-binding protein (ATF/CREB) family, that Using various published biochemical and computational is, CREM (Suzuki et al., 1993a), CREB (Zhao and methods, we have identified the most critical Tax-1 Giam, 1992; Franklin et al., 1993; Yin et al., 1996), and interacting domains shown in Figure 3b. The upper half CREB-2 (Reddy et al., 1997; Gachon et al., 1998; of the scheme lists the functional regions of the Tax Lemasson et al., 2002), results in potent stimulation of protein. They include binding regions to CREB, p300/ viral transcription. However, another bZIP factor, CBP, NF-kB and others from previously published C/EBPb, was recently shown to downregulate Tax- results (Harrod et al., 1998; Nicot et al., 1998; Gachon mediated transactivation from the HTLV-1 LTR and et al., 2000; Xiao et al., 2000; Jeang, 2001). There the TRE-1 (three 21-bp repeats composed of a central are two leucine zipper-like (Leucine Zip-like) regions cAMP-response element (CRE)-like motif located with- (sequences 116–145 and 213–248) in Tax, both of which in the U3 region of the LTR; Hivin et al., (2004). are missing one leucine when compared with a typical C/EBPb, also called NF-IL6, LAP or CRP2, is part of leucine zipper motif (LX(6)LX(6)LX(6)L). The total the distinct CCAAT/enhancer-binding protein (C/EBP) length of these regions is larger than a typical leucine family of transcription factors, that contains at least zipper, and is involved in protein dimer formation (Jin six different members (Bakker and Parker, 1991; and Jeang, 1997; Xiao et al., 2000). There is a PDZ- Lekstrom-Himes and Xanthopoulos, 1998). Within their binding motif at the C-terminal region of Tax with an homologous C-termini, these factors contain the bZIP XTXV consensus sequence. This region is involved in domain that is involved in their homo- or heterodimer- the interaction of Tax with six proteins containing a ization. In addition to forming dimers with other C/EBP PDZ domain (Rousset et al., 1998). PDZ domains play protein family members, C/EBPb can heterodimerize critical roles in interaction with the cytoskeleton, in the with CREB-2. In this context, the heterodimer prefer- organization of the Rho pathway (both upstream and entially binds to CRE sites with high affinity (Podust downstream) and in scaffolding (Reynaud et al., 2000; et al., 2001). Bezprozvanny and Maximov, 2001). Amino acids (aa) Results from Hivins et al. (2004) suggest that C/EBPb 106–111 of Tax encompass a conserved region with a can decrease the recruitment of Tax to the TRE-1, even predicted a-helix and may function as an interaction in the presence of CREB-2, resulting in decreased surface with IKK gamma (Xiao and Sun, 2000). Two transactivation of this response element. While these other possible active domains in Tax are an SH3- results are preliminary, they imply that C/EBPb and Tax binding region (aa 73–79) and a LIM-binding domain may have antagonistic functions since Tax was shown to (aa 207–219), both of which are critical for protein– reduce C/EBPb transcriptional activity as well. Inter- protein interactions. estingly, another cellular bZIP factor that is a strong LIM domains are cysteine-rich domains composed of transcriptional transrepressor without an activation two special zinc-fingers that are joined by a two-amino- domain, inducible cAMP early repressor (ICER; Bodor acid spacer. LIM proteins form a diverse group, which et al., 1996; Newbound et al., 2000), also downregulates includes transcription factors and cytoskeletal proteins. Tax transactivation. These results suggest that, in LIM-only proteins are also implicated in the control of addition to virally encoded proteins, HBZ and p30II cell proliferation, because several genes encoding such (Gaudray et al., 2002; Nicot et al., 2004), cellular factors proteins are associated with oncogenic chromosome like C/EBP and ICER can influence Tax activity, translocation (Dawid et al., 1995). LIM-only proteins, decreasing viral transcription. While speculative, the such as ACT, specifically associate with cAMP response overall benefits of decreasing viral transcripts may

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5947 be to limit cytotoxic T lymphocyte (CTL) response within residues 66–118 that is vital for Rex function and against viral transcripts such as Tax, which is an has been speculated to be important for interaction with immunodominant antigen for CTL response to other cellular factors (Hope et al., 1991; Weichselbraun HTLV-1 (Kannagi et al., 1993; Pique et al., 1996). In et al., 1992b). Furthermore, the activation domain is this way, the virus would be able to evade detection by necessary for targeting Rex to the nuclear pore complex the immune system. (Rehberger et al., 1997), possibly due to the nuclear export signal (NES) located within this domain. The NES is required for the shuttling of Rex between the nucleus and cytoplasm (Palmeri and Malim, 1996). Post-transcriptional regulator of HTLV-1: Rex Finally, a third less-well-defined domain is the multimerization domain (Weichselbraun et al., 1992a; Bogerd Rex is a 27 kDa RNA-binding protein that is essential and Greene, 1993). Through a series of missense for splicing and transport of viral mRNAs. While it is mutation, Weichselbraun et al. (1992a) identified aa not required for cellular immortalization in vitro,itis 54–69 as being critical for multimerization. Both HIV-1 required for viral spread and persistence in vivo (Ye Rev and HTLV-1 Rex are found in multimers that are et al., 2003). Rex acts at the post-transcriptional level to required for their function (Malim et al., 1990). In Rex, increase the cytoplasmic levels of singly spliced (env) aa 60–70 can functionally replace the N-terminal multi- and unspliced (gag/pol/pro) mRNAs at the expense of merization domain in Rev (Weichselbraun et al., 1992a), the doubly spliced messages (Hidaka et al., 1988). The indicating that this region is involved in multimeriza- exact mechanism of action is not known; however, it is tion. More recently, a C-terminal region has also been known that Rex expression results in the reduced rate of shown to be important for protein oligomerization, but splicing and increased stability of mRNAs (Grone et al., not nuclear export (Heger et al., 1998). 1996). Multiple cellular proteins can either enhance or The HTLV-1 proviral genome contains a sequence inhibit Rex, including the heterogenous nuclear ribonu- within the U3 and R regions of the 30 LTR that mediates cleoprotein A1 (hnRNP A1), the splicing factor SF2, the interaction with Rex (Seiki et al., 1988). This nucleolar protein B-23 and the exportin protein, CRM1. sequence, the Rex-responsive element (RexRE), forms hnRNP was found to bind RexRE sequences, resulting a stable and complex secondary structure, consisting of in interference of Rex binding to the RexRE (Dodon four stem loops and a long stretch of stem structure et al., 2002). In addition, hnRNP siRNA-treated (Toyoshima et al., 1990; Ballaun et al., 1991). Interest- cells exhibited increased levels of viral transcription, ingly, one stem loop (stem loop D) is sufficient to viral production and cytoplasmic expression of viral mediate Rex responsiveness in vivo (Grone et al., 1996). mRNAs (Kress et al., 2005). Expression of hnRNP A1 Inhibitory sequences within the viral RNA have also also resulted in increased exon skipping. Conversely, been identified, which are termed cis-acting repressive overexpression of another splicing factor SF2 did sequences (CRS). The first identified CRS spans the U5 not increase exon skipping, but rather resulted in regions of the LTR (Seiki et al., 1990), whereas a more differential pX splice site utilization (Princler et al., recently identified CRS overlaps the 30 RexRE (King 2003). B-23 has been implicated in the shuttling of et al., 1998). Deletion of the CRS located in the U5 ribosomal components between the nucleus and cyto- region does not result in complete release of mRNAs plasm (Borer et al., 1989). Utilizing a peptide containing from the nucleus, but rather deletion of both CRS is the NOS/NLS, B-23 was found to be the major protein needed (King et al., 1998). that bound to this region of Rex (Adachi et al., 1993). The activity of Rex can be affected through phos- Similarly, data indicate that B-23 helps import Rex from phorylation. Adachi et al. (1990) showed that treatment the cytoplasm to the nucleolus, further facilitating the of infected cells with the protein kinase C inhibitor export of unspliced mRNAs ((Adachi et al., 1993). More H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] re- recently, when utilizing a dominant-negative form of sulted in the accumulation of unspliced mRNA and Rex, which results in decreased export and multi- decreased gag protein synthesis. Furthermore, Rex is merization of Rex, CRM1 was shown to restore both phosphorylated in vivo on Ser-70, Ser-177 and Thr-174, events (Hakata et al., 1998). Further studies have with Ser-70 phosphorylation being 12-O-tetradecanoyl- indicated that the interaction between Rex and CRM1 phorbol-13-acetate (TPA) dependent (Adachi et al., is mediated through the NES of Rex (Bogerd et al., 1992). 1998). Rex has multiple domains that are important for its While many important studies have analysed Rex function, the first of which is a highly basic N-terminal function, more mechanistic details are needed. Further RNA-binding domain located within aa 1–19, which is studies addressing the cellular binding partners of Rex essential for binding to the RexRE (Bogerd et al., 1991; and post-translational modifications such as phosphor- Grassmann et al., 1991). This domain also serves as a ylation are needed in the near future. Rex is invaluable nucleolus targeting signal (NOS)/nuclear localization to the virus; therefore, knowing the precise molecular signal (NLS), and is necessary for the transport of mechanism of action is essential. This knowledge will unspliced viral mRNAs to the cytoplasm (Siomi et al., facilitate targeted drug therapies and much needed 1988; Nosaka et al., 1989; Bohnlein et al., 1991). advances in the treatment of ATL and HAM/TSP Secondly, an activation (or effector) domain is found patients.

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5948 Conclusion and future directions compartment, as well as post-translational modification of the NF-kB subunits by either phosphorylation and/or During the past decade, many talented and dedicated acetylation. In the Tax transactivation cascade, IKK1 researcher have contributed to our understanding of specifically phosphorylates p65 and induces transcrip- Tax and its role in transcription, transformation and tion activity. In the p53 cascade, IKK2 phosphorylates viral pathogenesis. A large part of the research has p65 to facilitate inhibition of p53 activity. Obviously, focused on Tax transactivation, primarily at the initia- unweaving the complex and functionally distinct NF-kB tion level. Tax associates with the LTR primarily at activation pathways is of critical importance. TRE-1 through interaction with CREB. The formation Tax has also been shown to bind to a number of of the Tax–CREB promoter complex serves as a high- important cytoplasmic factors regulating the cell cycle affinity binding site for the recruitment of the multi- and apoptosis. They include: (i) components of the functional cellular coactivators such as CBP, p300 and INK4-CDK4/6-Rb pathway, p16 and cyclin D(s) PCAF. The direct interaction of Tax with CBP allows directly regulating the Rb protein and the G1/S phase the binding of the coactivator in the absence of CREB of the cell cycle; (ii) human mitotic checkpoint protein phosphorylation, permitting specific activation of the MAD1 (TXBP181), and G-protein pathway suppressor viral LTR. p300/CBP transcriptional activity on a GPS2 (TXBP31), TXBP151 and TXBP121, all of which chromatin template has also been shown in a recon- contain highly structured coiled-coil domains; (iii) stituted chromatin template in the presence of Tax and TXBP151-binding protein A20, a Cys2/Cys2 zinc-finger CREB. Nucleosome acetylation was accompanied by an protein which is induced by a variety of inflammatory increased binding of RNA polymerase II transcription stimuli and characterized as an inhibitor of cell death; factor TFIID and RNA polymerase II to the promoter. (iv) and cytoplasmic GTPases, which included RhoA, The extent of histone modification and the identity of Rac1 and Cdc42, all of which regulate cytoskeletal and the cellular regulatory proteins bound at the HTLV-1 JNK proteins promoting interleukin-2-independent promoter in vivo has recently been explored. ChIP assays growth in HTLV-1-infected T cells. These interactions have shown factor binding and histone modification at are largely possible due to Tax’s many interacting the integrated HTLV-1 provirus, and the presence of domains, including coiled-coil domains, two leucine ATF/CREB and AP-1 family members (CREB, CREB- zipper-like regions involved in protein dimer formation, 2, ATF-1, ATF-2, c-Fos and c-Jun), p300 and CREB PDZ-binding motif at the C-terminal region critical in have been documented. The effect of Tax on HDAC is interaction with the cytoskeleton, Rho pathway and also of considerable interest, since this is a natural in scaffolding, a-helix domain which may function as an suppressor of gene expression, and its regulation by Tax interaction surface with IKKg, an SH3-binding region may be of critical importance. More importantly, the and a LIM-binding domain, both of which are critical HDAC inhibitor FR901228 has been shown to be an for protein–protein interactions. effective growth inhibitor of T-cell lymphomas, and For the past 15 years, our analysis of Tax has largely induces apoptosis of Tax-expressing and selective been toward understanding the molecular aspects of apoptosis of primary ATL cells, especially those of transactivation, cell cycle regulation and transforma- patients with acute ATL. Transcription elongation is tion. This understanding has led to the use and highly dependent upon remodeling of chromatin struc- development of novel therapies for treatment of ATL, ture, and Tax has been shown to interact with BRG1 including anti-Tac strategies, specific NF-kB inhibitors components of the ATP-dependent chromatin-remodel- and HDAC inhibitors. With our present knowledge, we ing complex, SWI/SNF. Thus, Tax may target SWI/ are poised to take bold new steps in translational SNF complexes downstream of RNA PolII in order to research, which will improve the treatment of ATL and prevent stalling of the polymerase by chromatin other HTLV-1-associated diseases. structure. Again, how this interaction contributes to Tax’s effect on cellular gene expression needs to be clearly defined in future. Acknowledgements This work was supported by grants from the George Tax has been shown to regulate cell cycle and check Washington University REF funds to FK and Akos Vertes, point repression through NF-kB and p53. Activation of NIH grants AI44357, AI43894 and 13969 to FK and the NIH NF-kB can be viewed as occurring in separate phases, IATAP to JNB. We would also like to thank Cynthia de la including degradation of the IkBa inhibitor and the Fuente, Kylene Kehn, Soo-Jin Jeong and Cynthia Pise- translocation of the NF-kB complex into the nuclear Masison for their critical editing, writing and figures.

References

Adachi Y, Copeland TD, Hatanaka M and Oroszlan S. (1993). Adya N and Giam CZ. (1995). J. Virol., 69, 1834–1841. J. Biol. Chem., 268, 13930–13934. Adya N, Zhao LJ, Huang W, Boros I and Giam CZ. (1994). Adachi Y, Copeland TD, Takahashi C, Nosaka T, Ahmed A, Proc. Natl. Acad. Sci. USA, 91, 5642–5646. Oroszlan S and Hatanaka M. (1992). J. Biol. Chem., 267, Ariumi Y, Ego T, Kaida A, Matsumoto M, Pandolﬁ PP and 21977–21981. Shimotohno K. (2003). Oncogene, 22, 1611–1619. Adachi Y, Nosaka T and Hatanaka M. (1990). Biochem. Azran I, Schavinsky-Khrapunsky Y and Aboud M. (2004). Biophys. Res. Commun., 169, 469–475. Retrovirology, 1, 20.

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5949 Bakker O and Parker MG. (1991). Nucleic Acids Res., 19, Franklin AA, Kubik MF, Uittenbogaard MN, Brauweiler A, 1213–1217. Utaisincharoen P, Matthews MA, Dynan WS, Hoefﬂer JP Ballard DW, Bohnlein E, Hoffman JA, Bogerd HP, and Nyborg JK. (1993). J. Biol. Chem., 268, 21225–21231. Dixon EP, Franza BR and Greene WC. (1989). New Biol., Franklin AA and Nyborg JK. (1995). J. Biomed. Sci., 2, 1, 83–92. 17–29. Ballard DW, Bohnlein E, Lowenthal JW, Wano Y, Franza BR Fujisawa J, Hirai H, Suzuki T and Yoshida M. (1993). Hum. and Greene WC. (1988). Science, 241, 1652–1655. Cell, 6, 266–272. Ballaun C, Farrington GK, Dobrovnik M, Rusche J, Hauber J Gachon F, Peleraux A, Thebault S, Dick J, Lemasson I, and Bohnlein E. (1991). J. Virol., 65, 4408–4413. Devaux C and Mesnard JM. (1998). J. Virol., 72, 8332–8337. Baranger AM, Palmer CR, Hamm MK, Giebler HA, Gachon F, Thebault S, Peleraux A, Devaux C and Mesnard Brauweiler A, Nyborg JK and Schepartz A. (1995). Nature, JM. (2000). Mol. Cell. Biol., 20, 3470–3481. 376, 606–608. Gaudray G, Gachon F, Basbous J, Biard-Piechaczyk M, Barnhart MK, Connor LM and Marriott SJ. (1997). J. Virol., Devaux C and Mesnard JM. (2002). J. Virol., 76, 71, 337–344. 12813–12822. Bex F and Gaynor RB. (1998). Methods, 16, 83–94. Georges SA, Giebler HA, Cole PA, Luger K, Laybourn PJ and Bex F, Yin MJ, Burny A and Gaynor RB. (1998). Mol. Cell. Nyborg JK. (2003). Mol. Cell. Biol., 23, 3392–3404. Biol., 18, 2392–2405. Georges SA, Kraus WL, Luger K, Nyborg JK and Laybourn Bezprozvanny I and Maximov A. (2001). Proc. Natl. Acad. PJ. (2002). Mol. Cell. Biol., 22, 127–137. Sci. USA, 98, 787–789. Gessain A, Barin F, Vernant JC, Gout O, Maurs L, Calender Bodor J, Spetz AL, Strominger JL and Habener JF. (1996). A and de The G. (1985). Lancet, 2, 407–410. Proc. Natl. Acad. Sci. USA, 93, 3536–3541. Giam CZ and Xu YL. (1989). J. Biol. Chem., 264, 15236– Bogerd H and Greene WC. (1993). J. Virol., 67, 2496–2502. 15241. Bogerd HP, Echarri A, Ross TM and Cullen BR. (1998). J. Giebler HA, Loring JE, van Orden K, Colgin MA, Garrus JE, Virol., 72, 8627–8635. Escudero KW, Brauweiler A and Nyborg JK. (1997). Mol. Bogerd HP, Huckaby GL, Ahmed YF, Hanly SM and Cell. Biol., 17, 5156–5164. Greene WC. (1991). Proc. Natl. Acad. Sci. USA, 88, Good L, Maggirwar SB and Sun SC. (1996). EMBO J., 15, 5704–5708. 3744–3750. Bohnlein S, Pirker FP, Hofer L, Zimmermann K, Bachmayer Goren I, Semmes OJ, Jeang KT and Moelling K. (1995). H, Bohnlein E and Hauber J. (1991). J. Virol., 65, 81–88. J. Virol., 69, 5806–5811. Borer RA, Lehner CF, Eppenberger HM and Nigg EA. (1989). Grassmann R, Berchtold S, Aepinus C, Ballaun C, Boehnlein Cell, 56, 379–390. E and Fleckenstein B. (1991). J. Virol., 65, 3721–3727. Bosselut R, Duvall JF, Gegonne A, Bailly M, Hemar A, Brady Greene WC, Leonard WJ, Depper JM, Nelson DL and J and Ghysdael J. (1990). EMBO J., 9, 3137–3144. Waldmann TA. (1986a). Ann. Intern. Med., 105, 560–572. Bosselut R, Lim F, Romond PC, Frampton J, Brady J and Greene WC, Leonard WJ, Wano Y, Svetlik PB, Peffer NJ, Ghysdael J. (1992). Virology, 186, 764–769. Sodroski JG, Rosen CA, Goh WC and Haseltine WA. Brady J, Jeang KT, Duvall J and Khoury G. (1987). J. Virol., (1986b). Science, 232, 877–880. 61, 2175–2181. Grone M, Koch C and Grassmann R. (1996). Virology, 218, Chiari E, Lamsoul I, Lodewick J, Chopin C, Bex F and Pique 316–325. C. (2004). J. Virol., 78, 11823–11832. Hakata Y, Umemoto T, Matsushita S and Shida H. (1998). J. Ching YP, Chun AC, Chin KT, Zhang ZQ, Jeang KT and Jin Virol., 72, 6602–6607. DY. (2004). Retrovirology, 1, 18. Harrod R, Kuo YL, Tang Y, Yao Y, Vassilev A, Nakatani Y Chun AC, Zhou Y, Wong CM, Kung HF, Jeang KT and Jin and Giam CZ. (2000). J. Biol. Chem., 275, 11852–11857. DY. (2000). AIDS Res. Hum. Retroviruses, 16, 1689–1694. Harrod R, Tang Y, Nicot C, Lu HS, Vassilev A, Nakatani Y Clark NM, Smith MJ, Hilﬁnger JM and Markovitz DM. and Giam CZ. (1998). Mol. Cell. Biol., 18, 5052–5061. (1993). J. Virol., 67, 5522–5528. Heger P, Rosorius O, Koch C, Casari G, Grassmann R and Curtiss VE, Smilde R and McGuire KL. (1996). Mol. Cell. Hauber J. (1998). J. Virol., 72, 8659–8668. Biol., 16, 3567–3575. Hidaka M, Inoue J, Yoshida M and Seiki M. (1988). EMBO Dawid IB, Toyama R and Taira M. (1995). C. R. Acad. Sci. J., 7, 519–523. III, 318, 295–306. Hirata A, Higuchi M, Niinuma A, Ohashi M, Fukushi M, Oie De La Fuente C and Kashanchi F. (2004). Retrovirology, 1, 19. M, Akiyama T, Tanaka Y, Gejyo F and Fujii M. (2004). De Valck D, Jin DY, Heyninck K, Van de Craen M, Contreras Virology, 318, 327–336. R, Fiers W, Jeang KT and Beyaert R. (1999). Oncogene, 18, Hivin P, Gaudray G, Devaux C and Mesnard JM. (2004). 4182–4190. Virology, 318, 556–565. Dodon MD, Hamaia S, Martin J and Gazzolo L. (2002). Hope TJ, Bond BL, McDonald D, Klein NP and Parslow TG. J. Biol. Chem., 277, 18744–18752. (1991). J. Virol., 65, 6001–6007. Ego T, Ariumi Y and Shimotohno K. (2002). Oncogene, 21, Jeang KT. (2001). Cytokine Growth Factor Rev., 12, 207–217. 7241–7246. Jeang KT, Boros I, Brady J, Radonovich M and Khoury G. Felber BK, Paskalis H, Kleinman-Ewing C, Wong-Staal F and (1988). J. Virol., 62, 4499–4509. Pavlakis GN. (1985). Science, 229, 675–679. Jeang KT, Chiu R, Santos E and Kim SJ. (1991). Virology, Fimia GM, De Cesare D and Sassone-Corsi P. (1999). Nature, 181, 218–227. 398, 165–169. Jeang KT, Giam CZ, Majone F and Aboud M. (2004). J. Biol. Fimia GM, De Cesare D and Sassone-Corsi P. (2000). Mol. Chem., 279, 31991–32004. Cell. Biol., 20, 8613–8622. Jeong SJ, Pise-Masison CA, Radonovich MF, Park HU and Franchini G. (1995). Blood, 86, 3619–3639. Brady JN. (2005). J. Biol. Chem., 280, 10326–10332. Franchini G, Nicot C and Johnson JM. (2003). Adv. Cancer Jeong SJ, Radonovich M, Brady JN and Pise-Masison CA. Res., 89, 69–132. (2004). Blood, 104, 1490–1497.

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5950 Jiang H, Lu H, Schiltz RL, Pise-Masison CA, Ogryzko VV, Nicot C, Dundr M, Johnson JM, Fullen JR, Alonzo N, Nakatani Y and Brady JN. (1999). Mol. Cell. Biol., 19, Fukumoto R, Princler GL, Derse D, Misteli T and 8136–8145. Franchini G. (2004). Nat. Med., 10, 197–201. Jin DY and Jeang KT. (1997). Nucleic Acids Res., 25, 379–387. Nicot C, Tie F and Giam CZ. (1998). J. Virol., 72, Kannagi M, Matsushita S and Harada S. (1993). Int. J. 6777–6784. Cancer, 54, 582–588. Nosaka T, Siomi H, Adachi Y, Ishibashi M, Kubota S, Maki Kashanchi F, Duvall JF, Kwok RP, Lundblad JR, Goodman M and Hatanaka M. (1989). Proc. Natl. Acad. Sci. USA, 86, RH and Brady JN. (1998). J. Biol. Chem., 273, 34646–34652. 9798–9802. Kehn K, Berro R, de la Fuente C, Strouss K, Ghedin E, Okada M and Jeang KT. (2002). J. Virol., 76, 12564–12573. Dadgar S, Bottazzi ME, Pumfery A and Kashanchi F. O’Mahony AM, Montano M, Van Beneden K, Chen LF and (2004). Front Biosci., 9, 2347–2372. Greene WC. (2004). J. Biol. Chem., 279, 18137–18145. Kibler KV and Jeang KT. (2001). J. Virol., 75, 2161–2173. Osame M. (1990). Tanpakushitsu Kakusan Koso, 35, King JA, Bridger JM, Lochelt M, Lichter P, Schulz TF, 1320–1326. Schirrmacher V and Khazaie K. (1998). Oncogene, 16, Palmeri D and Malim MH. (1996). J. Virol., 70, 6442–6445. 3309–3316. Peloponese JM, Jr, Iha H, Yedavalli VR, Miyazato A, Li Y, Kira J. (1994). Mol. Neurobiol., 8, 139–145. Haller K, Benkirane M and Jeang KT. (2004). J. Virol., 78, Kress E, Baydoun HH, Bex F, Gazzolo L and Duc Dodon M. 11686–11695. (2005). Retrovirology, 2, 8. Pique C, Connan F, Levilain JP, Choppin J and Dokhelar Kuo YL, Tang Y, Harrod R, Cai P and Giam CZ. (2000). MC. (1996). J. Virol., 70, 4919–4926. AIDS Res. Hum. Retroviruses, 16, 1607–1612. Pise-Masison CA and Brady JN. (2005). Front Biosci., 10, Kwok RP, Laurance ME, Lundblad JR, Goldman PS, Shih H, 919–930. Connor LM, Marriott SJ and Goodman RH. (1996). Podust LM, Krezel AM and Kim Y. (2001). J. Biol. Chem., Nature, 380, 642–646. 276, 505–513. Laurance ME, Kwok RP, Huang MS, Richards JP, Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD Lundblad JR and Goodman RH. (1997). J. Biol. Chem., and Gallo RC. (1980). Proc. Natl. Acad. Sci. USA, 77, 272, 2646–2651. 7415–7419. Lekstrom-Himes J and Xanthopoulos KG. (1998). J. Biol. Portis T, Harding JC and Ratner L. (2001). Blood, 98, Chem., 273, 28545–28548. 1200–1208. Lemasson I and Nyborg JK. (2001). J. Biol. Chem., 276, Princler GL, Julias JG, Hughes SH and Derse D. (2003). 15720–15727. Virology, 317, 136–145. Lemasson I, Polakowski NJ, Laybourn PJ and Nyborg JK. Reddy TR, Tang H, Li X and Wong-Staal F. (1997). (2002). J. Biol. Chem., 277, 49459–49465. Oncogene, 14, 2785–2792. Lemasson I, Polakowski NJ, Laybourn PJ and Nyborg JK. Rehberger S, Gounari F, DucDodon M, Chlichlia K, Gazzolo (2004). Mol. Cell. Biol., 24, 6117–6126. L, Schirrmacher V and Khazaie K. (1997). Exp. Cell. Res., Lenzmeier BA, Giebler HA and Nyborg JK. (1998). Mol. Cell. 233, 363–371. Biol., 18, 721–731. Reynaud C, Fabre S and Jalinot P. (2000). J. Biol. Chem., 275, Li J, Li H and Tsai MD. (2003). Biochemistry, 42, 33962–33968. 6921–6928. Rosen CA, Park R, Sodroski JG and Haseltine WA. (1987). Li XH, Murphy KM, Palka KT, Surabhi RM and Gaynor Proc. Natl. Acad. Sci. USA, 84, 4919–4923. RB. (1999). J. Biol. Chem., 274, 34417–34424. Rosen CA, Sodroski JG and Haseltine WA. (1985). Cell, 41, Livengood JA, Fechter EJ, Dervan PB and Nyborg JK. (2004). 813–823. Front Biosci., 9, 3058–3067. Rousset R, Fabre S, Desbois C, Bantignies F and Jalinot P. Lu H, Pise-Masison CA, Fletcher TM, Schiltz RL, Nagaich (1998). Oncogene, 16, 643–654. AK, Radonovich M, Hager G, Cole PA and Brady JN. Scoggin KE, Ulloa A and Nyborg JK. (2001). Mol. Cell. Biol., (2002). Mol. Cell. Biol., 22, 4450–4462. 21, 5520–5530. Lu H, Pise-Masison CA, Linton R, Park HU, Schiltz RL, Seiki M, Hikikoshi A and Yoshida M. (1990). Virology, 176, Sartorelli V and Brady JN. (2004). J. Virol., 78, 6735–6743. 81–86. Malim MH, Tiley LS, McCarn DF, Rusche JR, Hauber J and Seiki M, Inoue J, Hidaka M and Yoshida M. (1988). Proc. Cullen BR. (1990). Cell, 60, 675–683. Natl. Acad. Sci. USA, 85, 7124–7128. Marriott SJ, Boros I, Duvall JF and Brady JN. (1989). Mol. Semmes OJ and Jeang KT. (1996). J. Virol., 70, 6347–6357. Cell. Biol., 9, 4152–4160. Siomi H, Shida H, Nam SH, Nosaka T, Maki M and Marriott SJ, Lindholm PF, Brown KM, Gitlin SD, Duvall JF, Hatanaka M. (1988). Cell, 55, 197–209. Radonovich MF and Brady JN. (1990). Mol. Cell. Biol., 10, Sun SC and Ballard DW. (1999). Oncogene, 18, 6948–6958. 4192–4201. Sun SC, HarhajEW, Xiao G and Good L. (2000). AIDS Res. Marriott SJ, Trinh D and Brady JN. (1992). Oncogene, 7, Hum. Retroviruses, 16, 1591–1596. 1749–1755. Suzuki T, Fujisawa JI, Toita M and Yoshida M. (1993a). Proc. Mori N, Matsuda T, Tadano M, Kinjo T, Yamada Y, Natl. Acad. Sci. USA, 90, 610–614. Tsukasaki K, Ikeda S, Yamasaki Y, Tanaka Y, Ohta T, Suzuki T, Hirai H, Fujisawa J, Fujita T and Yoshida M. Iwamasa T, Tomonaga M and Yamamoto N. (2004). J. (1993b). Oncogene, 8, 2391–2397. Virol., 78, 4582–4590. Suzuki T and Yoshida M. (1996). Tanpakushitsu Kakusan Muchardt C, Seeler JS, Nirula A, Gong S and Gaynor R. Koso, 41, 1249–1257. (1992). EMBO J., 11, 2573–2581. Takatsuki K, Yamaguchi K, Kawano F, Hattori T, Nishimura Neuveut C and Jeang KT. (2002). Front. Biosci., 7, d157–63. H, Tsuda H and Sanada I. (1984). Princess Takamatsu Newbound GC, O’Rourke JP, Collins ND, Andrews JM, Symp., 15, 51–57. DeWille J and Lairmore MD. (2000). J. Med. Virol., 62, Tie F, Adya N, Greene WC and Giam CZ. (1996). J. Virol., 70, 286–292. 8368–8374.

Oncogene Gene regulation of HTLV-1 F Kashanchi and JN Brady 5951 Toyoshima H, Itoh M, Inoue J, Seiki M, Takaku F and Yan JP, Garrus JE, Giebler HA, Stargell LA and Nyborg JK. Yoshida M. (1990). J. Virol., 64, 2825–2832. (1998). J. Mol. Biol., 281, 395–400. Uchiumi F, Maruta H, Inoue J, Yamamoto T and Tanuma S. Ye J, Silverman L, Lairmore MD and Green PL. (2003). (1996). Biochem. Biophys. Res. Commun., 220, 411–417. Blood, 102, 3963–3969. Van Orden K and Nyborg JK. (2000). Gene Expr., 9, Yin MJ, Paulssen E and Gaynor RB. (1996). J. Biol. Chem., 29–36. 271, 4781–4790. Weichselbraun I, Berger J, Dobrovnik M, Bogerd H, Grass- Yin MJ, Paulssen EJ, Seeler JS and Gaynor RB. (1995). mann R, Greene WC, Hauber J and Bohnlein E. (1992a). J. Virol., 69, 3420–3432. J. Virol., 66, 4540–4545. Yoshida M. (1994a). Leukemia, 8 (Suppl 1), S51–S53. Weichselbraun I, Farrington GK, Rusche JR, Bohnlein E and Yoshida M. (1994b). AIDS Res. Hum. Retroviruses, 10, Hauber J. (1992b). J. Virol., 66, 2583–2587. 1193–1197. Wessner R and Wigdahl B. (1997). Leukemia, 11 (Suppl 3), Yoshida M. (1995). J. Cancer Res. Clin. Oncol., 121, 521–528. 21–24. Yoshida M. (2001). Annu. Rev. Immunol., 19, 475–496. Wessner R, Yao J and Wigdahl B. (1997). Leukemia, 11 Yoshida M, Inoue J, Fujisawa J and Seiki M. (1989). Genome, (Suppl 3), 10–13. 31, 662–667. Wu K, Bottazzi ME, de la Fuente C, Deng L, Gitlin SD, Yoshida M, Osame M, Usuku K, Matsumoto M and Igata A. Maddukuri A, Dadgar S, Li H, Vertes A, Pumfery A and (1987). Lancet, 1, 1085–1086. Kashanchi F. (2004). J. Biol. Chem., 279, 495–508. Yoshida M, Seiki M, Yamaguchi K and Takatsuki K. (1984). Xiao G, HarhajEW and Sun SC. (2000). J. Biol. Chem., 275, Proc. Natl. Acad. Sci. USA, 81, 2534–2537. 34060–34067. Yoshida M, Suzuki T, Fujisawa J and Hirai H. (1995). Curr. Xiao G and Sun SC. (2000). Oncogene, 19, 5198–5203. Top. Microbiol. Immunol., 193, 79–89. Yamaguchi K, Seiki M, Yoshida M, Nishimura H, Kawano F Zhao LJ and Giam CZ. (1992). Proc. Natl. Acad. Sci. USA, 89, and Takatsuki K. (1984). Blood, 63, 1235–1240. 7070–7074.

Oncogene