Iranian Biomedical Journal 2 (2): 49-57 (April 1998) Transcriptional Coactivator CBP Facilitates Transcription
Initiation and Reinitiation of HTLV-I and Cyclin D2 Promoter
LRBGE, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 41, Room B403, ABSTRACT
HTLV-I is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-I-
associated myelopathy/tropical spastic paraparesis (HAM/TSP). Taxi, the major activator of this
virus, is a 40- kDa (353 amino acid) phosphoprotein, predominantly localized in the nucleus of
the host cell, which functions to trans-activate both viral and cellular promoters. Recently it has
been shown that HTLV-I and /or Taxl expressing cells have altered gene expression of some of
the cell cycle associated genes. Tax activates HTLV-I as well as number of other cellular
promoters through CREB, NF-kB and SRE elements. In this study we analyzed the effect of a
general coactivator, namely CBP, which may
be involved in activation of many cellular genes. To
analyze CBP transcription activation, we have utilized an
in vitro transcription assay that allows
the analysis of transcription initiation and reinitiation in the absence of chromatin effects. In
this assay, which utilizes a G-free cassette downstream of the Tax-responsive 21 by repeats,
polymerase II molecules responsible for the first round of transcription remain at the end of the
G-free region, effectively blocking the complete elongation of reinitiated transcripts. Addition of
Tax and a 682 amino acid fragment of CBP to the
in vitro transcription reactions increased both
full-length and shorter transcripts resulting from reinitiation. A CBP deletion mutant lacking
the N-terminal activation domain was inactive. Preliminary data is also presented to show that,
transactivation of HTLV-I and a cellular promoter, namely cyclin D2, takes place in early
GO/G1 before the restriction point, "R", where Rb function has been implicated.
Iran. Biomed. 1
2: 4957, 1998

Keywords: HTLV-I, Tax, cyclin, transcription INTRODUCTION
HTLV-I activates and immortalizes human T- lymphocytes in vitro, resulting in polyclonal prolif HTLV-I is the etiologic agent for adult T-cell eration of the infected cells, followed by oligo- clonal or monoclonal growth. The mechanism of associated myelopathy/tropical spastic para- HTLV-I transformation appears to be distinct paresis (HAM/ TSP) [1, 2]. Due to the limited
from that of chronic or acute leukemia viruses.
coding capacity of the viral genome, viral Interestingly, when the pX coding sequences were cloned into a transformation-defective herpes virus or transgenic mice containing only regulatory protein function. In HTLV-I, the pX the Tax gene [4], it was able to transform T- region contains four ORFs, X-I, X-II, X-III and lymphocytes of the same phenotype as found X-IV. Tax is encoded primarily by the X-IV with HTLV-I. Of the three proteins encoded by reading frame, while Rexi is encoded by the X-III the pX region, Tax is critical for transformation, reading frame. The AUG initiation codons for both since specific mutations of the tax gene AUG Tax and Rex are located in the second exon of the initiation codon eliminated the transformation doubly spliced mRNA. Taxi transcriptionally acti- vates viral mRNA synthesis, leading to an initial increase in the levels of Tax1 and Rex [3].
Tax is a 40-kDa (353 amino acid) phosphopro- tein, predominantly localized in the nucleus of the host cell, which functions to transactivate required for passage through START, the G1 both viral and cellular promoters. Taxi has not restriction point and transition at G1/S. Three been shown to bind directly to Taxi-responsive novel types of putative mammalian G1 cyclins were isolated by using human cDNA libraries to transactivation occurs through indirect effects of complement CLN-deficient yeast and designated Taxi on transcription factors which bind to the as cyclins C, D, and E [19]. PRAD 1 was cloned TREs [6]. Likely mechanisms for Taxi trans- uy a gene rearranged in an parathyroid tumor and activation include: (1) transcriptional induction is identical to human cyclin DI [20]. A murine of TRE-binding transcription factors; (2) post- homologue of cyclin D1 was independently isolated factors; or (3) complex formation with trans- macrophages synchronously progressing through G1 cription factors allowing indirect binding of Taxi in response to colony-stimulating factor 1. The Tax 1 has been shown to trans-activate several identify two related genes, murine cyclin D2 and D3. Unlike other types of cyclin, cyclins DE D2, transfection), including IL-2 [7], IL-2R [8], and D3 have unique cell- and tissue-specific GM-CSF [9], proliferating cell nuclear antigen patterns of expression, suggesting that each D- type cyclin may have a distinct mechanism for protein (PTHrP) [11], c-fos, csis [12] and others transcriptional regulation. Over expression of any and to negatively regulate Bpolymerase [13], a of D-type cyclins can accelerate the timing of host DNA-repair enzyme. It is likely that Tax START and shorten the G1 intervals [21]. With induction of cellular genes is critical for virus respect to general structure of cyclin D2 replication and cell transformation.
Recently it has been shown that HTLV-I and/or vicinity of the transcription start sites, however, putative DNA binding sites for Spl, CREB, expression of some of the cell cycle associated C/EBP, PEA3, NFkB, SIF, E2F, GCF, and AP1 genes. Among these changes high levels of p53, were identified in the cyclin D2 upstream cdk inhibitor p21, Cyclin D2 and lower levels of sequence. The CAP site in the promoter was cyclin D3, cdk inhibitor p16 have been observed shown to be a loosely conserved sequence where [14]. In vitro binding assays has also indicated that number of transcription sites have been observed Tax binds pi 6INK4a (cdk/cyclin D inhibitor), but not p21c'Pl or p27kwl and it forms complexes with Here we show that Taxl associated activity p16INK4a in vivo [15, 16]. However to date, no with viral and, cellular cyclin D2 promoter, is careful analysis of Tax 1 or HTLV-I infected cells at early stages of post mitosis. Preliminary data have been performed to address the functional is presented to show that, i) Taxi efficiently consequence of these seemingly dramatic changes recruits CREB Binding Protein (CBP) to both at the cell cycle level. Of particular interest and focus of this study is the notion of very early events consequence of this event is reinitiation of post mitosis that Tax 1 and/or HTLV-I pose on the RNA Pol II, ii) The cyclin D2 over expression, host cell cycle machinery. One such early event post as observed in HTLV-I infected cells, is related mitosis is the activation of cyclin D family member, to AP2 DNA binding elements on the 5' end of Cyclins are the regulatory subunits of cdc2- HTLV-I and cyclin D2 promoter takes place at early GO/G1 before the restriction point, R, eukaryotic cell cycle. Eight different types of where Retinoblastoma (Rb) function has been cyclins have been identified in mammalian cells, and designated as cyclins A through H. CyclinsC, D (DI, D2, and D3), E, and G are believed tobe G1 cyclins [17, 18]. Cyclin A is a S phase MATERIALS AND METHODS
cyclin, and cyclin B (B1 and B2) is a mitoticcyclin. The initial studies of G1 cyclins were In vitro transcription assays. The G-free DNA performed in budding yeast, which has three templates used in the in vitro transcription assays Iranian Biomedical Journal 2 (2). 49-57 (April 1998) were pLovTATA and pTRE-lid. E. coli Tax pro- released using complete media. Whole cell tein was purified by ammonium sulfate precipita- extracts were made from 5 x 107 cells/time point.
tion as described. Prior to use in the in vitro tran- Samples were collected every three hours and the scription assay, Tax (100 ng/IA1) was incubated cells were used to make whole cell extracts for in for 30 min at 30°C with equal volume of Tax vitro transcription, isolated total cellular RNA for Northern analysis, or processed for cell mercaptoethanol, 1 mM EDTA). For the G-free sorting. Single color flow cytometric analysis of in vitro transcription reactions, preincubation DNA content was performed on all cell tested.
was at 30°C for 30 mM, followed by the addition Cells were washed with PBS and approximately of 2 ul -32P-UTP (Amersham, 400 Ci/mMol), 2 x 106 cells were fixed by addition of 500 pl of 0.03% sarkosyl and incubation at 30°C for 60 70% ethanol. Cell pellets were washed with PBS (3x, 10 ml each time), incu bated in 1 ml PBS extract (25 pl), 1.5 µ g supercoiled DNA, 75 ng with 150 pg/m1RNaseA (Sigma) and 20 pg/ml Tax protein, .75 to 2.25 pg CBP(1-682) in a total volume of 50 to 65 pl. Transcription buffer minutes. The stained cells were analyzed for red (35.5 pl/reaction) contained 3 pi 20% PEG (6000), 3 41 50 mM MgC12, 3 pl 1 mM DTT, 1 Dickenson) and the distribution of cells in the G1, S and G2/M phases of the cell cycle was calculated from resulting DNA histogram using Cell FIT software, based on a rectangular S- 5'Triphosphate (Pharmacia), 20 units RNase T1 phase model (Fast Systems, Inc., Gaithersburg, (100 units/pl, Boehringer Mannheim) and 18 pl of Buffer D containing a final concentration 20mM HEPES (pH 7.9), 100 mM KCI, 12.5 mM Transfections and CAT Assays. The wild type
and various mutant cyclin D2 constructs were mM DTT. For pulse-chase assays Tax and CBP electroporated along with Taxi, into Jurkat cells were incubated in the presence of 32P-UTP for using 230 volts, and 800 uF (Cell porator, Life 30 minutes. A 10-fold molar excess of cold UTP Technologies). Extracts were prepared 18 hours was added to the reaction and the polymerase later for CAT assay. Cells were harvested, complexes were allowed to elongate for 15-60 washed once with PBS without Ca++ and Mg++, minutes. 3'-0-Methyl GTP was omitted in these pelleted, and resuspended in 150 41 of 0.25 M assays to allow pol II elongation. Sarkosyl (0.03 Tris (pH 7.8). Cells were freeze/thawed 3X, with %) was added to inhibit reinitiation complexes vortexing after each thawing. Tubes were then in duplicates of the 15 and 60 minute chase centrifugation. The supernatants were transferredto 1.5 ml Eppendorf tubes. After one final spin, Northern blot.
the supernatant was again transferred to 1.5 ml extracted using the Trizol reagent (Gibco/BRL).
Eppendorf tubes and the protein concentration Total RNA (5 pg) was spotted onto 0.2 micron was determined. CAT assays were performed nitrocellulose (Millipore Inc.), UV cross-linked with 10 pg protein according to the method of and hybridized overnight at 42°C with various 40 mer end-labelled, 32P-labelled, Cyclin D2,D3, E, HTLV-I LTR (R region) and Actinprobes [19, 22]. Next day, they were washed 2 times (10 mls), 15 minutes each, with 0.2%SDS, 2x SSC at 37°C, exposed and counted on a To analyze transcriptional activation by Tax Phosphorlmager cassette (Molecular Dynamics).
and CBP, purified proteins were added to in vitro Cell cycle analysis. Various infected or control transcription reactions with a G-free template cells were either blocked with hydroxyurea for containing the Tax-responsive 21 by repeats 18 hours, or blocked with hydroxyurea, washed upstream of the TATA box. Our initial studies and released for 1 hour followed by addition of utilized a fragment of CBP containing amino nocodazole for 14 hours (50 ng/ml). Following acids 1-682, CBP (1-682), which can activate the block, cells were washed with PBS (2x) and transcription when fused to a Gal-4 DNA binding domain and has protein interaction domains forTBP, CREB and Tax (Figure 1A). This region ofCBP is sufficient for transcription activation andformation of the Tax-CREB-CBP complex. Wesubsequently tested two deletion mutants of thisCBP domain, CBP (451-682) and CBP (509-682). In the absence of exogenous CBP andlimiting Tax protein, Tax only modestly activatedthe HTLV-I CRE promoter (Figure 1B, lanes 1and 2). The addition of Tax and CBP (1-682)stimulated the appearance of the 360 base (360 b)transcript ten-fold (Figure 1B, lane 3). Thedifficulty in obtaining an efficient in vitro Taxtransactivation system in the past may be due tothe fact that CBP is a limiting component within theextracts. CBP (451-682) or (509-682) failed to ac-tivate transcription (Figure 1B, lanes 4 and 5), sug-gesting that the CBP amino terminus is essential forTax activity. Swope et al. have recently reported thatCBP(1-400) interacts with the basal transcriptionfactor TBP [24[. Thus, our results suggest that theinteraction of CBP with TBP (TFIID) may beimportant for Tax-dependent CBP activation. Ascontrols for the in vitro transcription assays, additionof either CBP- or Tax-, but not control-, antibodiesinhibited the in vitro transcription (data not shown).
In addition, a template which lacked the 21 byrepeats was not transactivated by the addition of Taxand CBP (Figure 1C).
To determine whether Tax and CBP affect both the initiation and reinitiation of transcription, weused 3'-0-Methyl GTP in the in vitro transcriptionassay. As described by Szentirmay and Sawadogo[25], in the presence of 3'-0-Methyl GTP, the firstround of poi II elongation complexes remain at theend of a G-free region, blocking the elongation ofpol II complexes that result from reinitiation(Figure 2C). As a result, successive polymerasesstack up from the end of the cassette, producingshorter transcripts representing rounds of reinitia-tion. Figure 2A shows that addition of both Tax andCBP stimulate the appearance of the full length 360b transcript as well as discrete shorter transcripts ofapproximately 280, 240, 210, 180, 165, and 144bases. Hybridization analysis of the full length and Fig. 1. In vitro transactivation of CRE response element by Tax
shorter RNAs confirmed that they originated from and CBP. (A) Schematic of CBP protein. Binding sites for TATA the G-free cassette (data not shown). These data binding protein (TBP), CREB, Tax, well as acetyltransferase-(HAT), zinc finger-, and glutamine rich-domains are indicated. (B) and (C) In vitro transcription assay. Tax and CBP were added at 0 transcription initiation and reinitation.
time, followed by the addition of 0.03% sarkosyl and 32P-UTP at 30 Previous results from Szentirmay and Sawadogo minutes. Samples were then incuiy,ted for 1 hour at 30°C and further have demonstrated that reinitiated pol II complexes processed for RNA product analysis [38].
are more sensitive to low concentrations of sarkosyl Iranian Biomedical Journal 2 (2). 49-57 (April 1998) nocodazole, washed and released with completemedia in the presence of sodium butyrate. Wehave previously shown that sodium butyrate canrapidly induce viral gene expression in ACH2,U I [26], and in MT-2 and MT-4 cells (data notshown). FACS analysis of blocked and releasedcells are shown in Figure 3, Panel B. Most ofthe MT-2 and CEM cells had traversed into earlyG 1 following nocodazole release. Cells at timezero and processed for RNA analysis. Results of such anexperiment are shown in Figure 4, Panel C,where both HTLV-I and cyclin D2 promotershowed an increase in gene expression in MT-2cells 2 hours post-mitosis. Cyclin D3 and E,however, were not activated under theseconditions.
Fig. 2. CBP facilitates transcription initiation and reinitiation
in vitro. (A) In vitro transcription products in the presence of
Tax and CBP using the CoPRA assays (25). (B) Reinitiation
complexes are sensitive to addition of sarkosyl. Tax and CBP
were added at 0 time, followed by the addition of sarkosyl and
32P-UTP at 30 minutes. Samples were then incubated for 1 hour
at 30°C. (C) Schematic for colliding polymerase reinitiation
[25]. To confirm that the shorter RNAs representedreinitiation transcription complexes, template DNA,extract, CBP and Tax were preincubated for 30minutes, followed by the addition of sarkosyl and32P-UTP.Consistent Sawadogo's results, our studies demonstrate that thereinitiated transcription complexes were prefer-entially sensitive to the addition of sarkosyl after thepreincubation period (Figure 2B). Following theassembly of the transcription complexes during apreincubation period, addition of sarkosyl to 0.03% Fig. 3. Endogenous promoter activities of HTLV-I and some
cyclin genes. Both MT-2 and CEM cells were blocked with inhibited the reinitiated pol II complexes, but failed Nocodazole (see Fig. 5 legend), washed next day and released to inhibit primary initiation complexes.
in presence of 5 inM sodium Butyrate. Samples were collected Promoter effect of number of genes post-mitosis at zero, or 2 hrs post release for RNA analysis. Panel A, including HTLV-I, cyclin D2, D3 and E, were represents the diagram for this experiment. Panel B, indicates the FACS analysis of both cell types using propidium IodideDNA staining (FAST systems, Gaithersburg, MD), and panel hybridization analysis. HTI_V-I-infected cells C, represents hybridization of 10 1.1g of total RNA using HTLV-I (Nick translated sequence of HTLV-1 LTR R region, +1 to +260, and cyclins D2, D3, E and actin probes [15].
Fig. 4. Effect of Tax and CBP on cyclin D2 promoter. CEM (12D7) cells were transfected with of the reporter genes, and
either one .tg of E. coli His-tag recombinant Tax protein, or 1 tg of CMV-CBP using the electroporation method. Cells were incubatedovernight for CAT assay. Panel A represents a diagram of the human cyclin D2 promoter with its putative DNA binding sites forvarious transcription factors (22,23). Panel B represents PCR amplified fragments of cyclin D2 promoter elements inserted upstreamof a promoter less (pCAT-basic) plasmid at Hind III site. Right panel (synchronized) represents cells blocked overnight withNocodazole (M phase blocker, 50 ng/ml), washed next day with PBS (without Ca++ or Mg++), transfected with reporter DNA and Taxprotein, released with complete medium for 2 hrs, and subsequently blocked with Hydroxy-urea (Gl/S blocker, 2 mM) for 24 hrs. Cellswere collected and processed for CAT assay after 24 hrs. This procedure scores Transcription events related to GI phase of cell cycle,since majority of cells (70-80%) remain at end of G1 phase.
Fig. 5. A model of events in GI phase of cell cycle. G1 -pm represent the time post mitosis where cyclin D2 and HTLV-I gene
expression takes place. The "R" represents the time where Rb and possibly CBP/p300 (data from this manuscript) are phosphorylated bycyclin D2 associated complex, and G1 -ps represents events before S phase, including E2F transactivation of genes such as cyclin E andPCNA synthesis. Diagram modified from Zetterberg et al [39].
Iranian Biomedical Journal 2 (2). 49-57 (April 1998) Subsequent experiments have shown that cyclin Rex , provides some initial alteration in cell me- D3 is repressed in MT-2, but not CEM, cells (data tabo‘ism predisposing the development of ATL.
not shown), consistent with a previously published Subsequently, the rearrangement or altered expres- report on cyclin D3 down regulation in all HTLV-I- sion of a cellular oncogene(s) may provide the infected cells [15, 16]. Cyclin D2 and D3 levels "second hit", leading to development of ATL. In were slightly higher in CEM control cells 2 hours fact, there have been reports that Taxi triggers DNA post-release (Figure 3, right panel), however no damage. Diverse cytogenetie abnormalities have dramatic induction of these promoters were ob- been observed in ATL patient peripheral blood served as compared to HTLV-I infected cell.
To further analyze the effect of cyclin D2 pro- abnormalities have been found, including trisomies 3 moter, we constructed a series of wild type and 5' and 7, and rearrangements in the long arm of deletion promoter plasmids using PCR and inserted chromosome 6, no single chromosomal defect is them upstream of the CAT reporter gene. The re- porter CAT gene was inserted 10 bases upstream of Tax, which is critical for viral replication, trans- the authentic cyclin D2 AUG codon, where the formation and gene regulation. Tax activates only translational start site is donated from the HTLV-I gene expression through a sequence re- CAT open reading frame. Following transfection of sembling the cyclic AMP-responsive element wild type cyclin D2 promoter into unsynchronized (CRE) in the HTLV-I LTR. The transcription coac- CEM cells, we have observed a 2.5-fold activation tivator CBP was first identified as a critical compo- with Tax protein [46] and a 9.5-fold increase with a nent of the CREB activation pathway [29, 30].
full length CBP construct (Figure 4). The same Subsequent to DNA binding, CREB is phosphory- amount of Tax protein alone (1 ug) is capable of lated at serine-133, facilitating its interaction with activating HTLV-I promoter by 40-fold (data not CBP (31). Interestingly, in the presence of Tax, the shown). Wild type full length CBP plasmid was not requirement for CREB phosphorylation is bypassed capable of activating HTLV-I, or any of the cyclin for transactivation of the viral 21 by repeats [32].
D2 promoters, in the absence of Tax (data not Similar to transcription activators, CBP has been shown). Similar data, albeit less overall activity, reported to interact with basal transcription factors was obtained with transfection of G1 cells where such as TFIIB and TBP. The N-terminal half of the Taxi and CBP effect is more pronounced than CBP, which contains the TBP binding site, is im- Taxi alone (Figure 4, synchronized panel). G1 cells portant for CREB-mediated transcription and con- represent cells blocked with nocodazole at M (for tains a strong activation domain. It has also been 18 hrs), washed with PBS, transfected with various reported that CBP interacts with holo pol II [33], plasmids, released for 2 hours with complete media and contains a histone acetyltransferase domain.
and blocked for the second time with hydroxyurea Thus, CBP may contain several domains which are at Gl/S border. Cells were harvested for CAT assay important for transcription. Given the importance 24 hours post-hydroxyurea block. Interestingly, of CBP/p300 in CREB, NF- B, AP1, MAPK, PKA, transfection in unsynchronized and synchronized cells show the possible effect of AP2 DNA binding elucidating the mechanism of action of the sites to be most crucial in cyclin D2 promoter. High CBP/p300 co-activators has important implications levels of AP2 activity have been reported in for gene regulation [34, 35]. These observations, collectively, suggest that Tax and CBP interactionshave broad transcriptional implications andpossibly are involved in more than just activating DISCUSSION
the HTLV-I promoter. To that end, result fromFigure 3 is an indication to that effect, where cyclin The human T-lymphotropic virus type I (HTLV-I) is associated with an aggressive malignancy of Genetically, the human cyclin D2 gene (CCND2) mature CD4+ T-cells, adult T-cell leukemia (ATL) has been mapped to chromosome 12p13 and tri- and the degenerative neuromuscular disease tropical somy 12 which is the commonest chromosomal spastic paraparesis/HTLV-I-associated myelopathy change in B-CLL and immunocytomas. Cyclin D2 mRNA was found to be over expressed in 29 of 34 It seems likely that the HTLV-I virus, through ex- B-CLL cases and in all cases of LPL. The level of pression of the viral regulatory proteins Taxi and cyclin D2 expression in these disorders was, on average, 5- to 10-fold higher than in normal resting talization of primary human lymphocytes in culture.
Brady, J.N. (1996) Biology of HTLV-I: Host cell in- patients, whereas cyclin D1 was expressed only teractions. in Human T-cell lymphotropic virus typeI. (Per Hollsberg and David A. Hafler, eds). John in the 3 cases (LPL, 1; MCL, 2) associated with a t(11;14) translocation. Other interesting Leung, K. and Nabel, G.J. (1988) HTLV-I transacti- observations on the Cyclin D2 gene have also vator induces interleukin-2 receptor expression been noted where retroviral sequences found through an NF-kappa B-like factor. Nature (Lond.) adjacent to the open reading frame. The vin-1 gene first identified as the common site of Green, J. E., Begley, C.G., Wagner, D.K., Waldmann, granulocyte-macrophage colony-stimulating factor identical to the cyclin D2 gene [37]. The and the interleukin-2 receptor in transgenic mice car- rying the human T-lymphotropic virus type 1 taxgene. Mol. Cell. Biol. 9: 4731-4737.
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Executive Summary The Commission to Inquire into Child Abuse was established in 2000 with functions including theinvestigation of abuse of children in institutions in the State. It was dependent on people givingevidence which they did in large numbers. The Commission expresses its gratitude to all thosewho participated and contributed with their testimony and documents. The witnesses who cameto

Report of drugs magnesium Sulphate Misoprostol ACKNOWLEDGMENTS We are grateful to the Dr.Yasin, Hargeisa Hospital, Berbera Hospital, Burco Hospital, and Adna Aden Hospital as a whole in Somaliland region for their cooperation, time and provision of information individually and in focus group that helped the survey team get a better understanding of the medical report in We wis

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