Introduction
HIV-1, which is the primary etiologic agent of AIDS, has a characteristic gene expression system controlled by the viral Tat protein and host cell-encoded regulatory proteins that bind a number of target sequences within the viral long terminal repeat (LTR) [1-4].
Within LTR, the U3 transcriptional regulatory domain contains binding sites for a number of different transcription factors that exert direct transcriptional effects on this transcription unit [5,6]. Both constitutively expressed host cell transcription factors such as Sp1 [7], and inducible transcription factors such as nuclear factor (NF)-κB, appear to bind U3 domain and to play important roles in HIV-1 activation [8,9]. In addition, NF-κB binds the enhancer region of the LTR and activates viral gene expression in response to the processes, such as T-cell activation or macrophage differentiation [10-12] and to the stimulation by cytokines [11,13] such as tumor necrosis factor (TNF)-α and interleukin (IL)-1 through activation of NF-κB [11]. Moreover, hematopoietic growth factors including granulocyte-macrophage colony-stimulating factor and IL-3 may regulate the activity of host cell (hematopoietic cell-restricted) transcription factors, and result in the proliferation of HIV-1-infected hematopoietic cells in vivo [14-17].
In contrast to NF-κB, only a few tissue-specific transcription factors have been shown to co-ordinately regulate HIV-1 gene expression. Amongst these tissue factors, GATA-2 appears to promote the proliferation of early hematopoietic progenitors. Knock-out experiments by Tsai et al. [18] have strongly suggested that GATA-2 is indeed one of the critical transcription factors for multipotential progenitors. Although major host cells of HIV are T cells and macrophages, evidence has been accumulated that HIV-1 infects CD34+ hematopoietic stem cells [19,20], and that their infection causes decreased proliferation and differentiation of bone-marrow progenitors [21,22]. Moreover, HIV-1 can infect a certain subset of progenitors expressing CD4 and the acute leukemia cell lines with monocytic character, U937 and HL60, that express GATA-2 [23]. In addition, careful inspection of the U3 HIV-1 LTR reveals a high affinity GATA-2 binding sequence (AGATATC) [24]. Although it is not encompassed within the traditional WGATAR consensus sequence (in which W indicates A/T and R indicates A/G) [25], another candidate GATA-2 site, GATCT, which has recently been recognized as the consensus of high affinity with GATA-2 (M. Yamamoto, personal communication, 1997), exists in the HIV LTR sequence. Despite its importance in the hematopoietic system, the role of GATA-2 in HIV gene expression remains unclear. It is therefore of particular interest to examine the involvement of GATA-2 in HIV-1 transactivation.
In this report, we describe HIV-1 transactivation regulation by GATA-2, and also describe the major GATA sites on the LTR. Synergistic transactivation between GATA-2 and Tat is also presented.
Materials and methods
Plasmids
All DNA manipulations were carried out by using standard techniques. The plasmid structures were verified by sequencing. HIV-1 LTR gene 5′-flanking sequences from nucleotides -437 to +79 (relative to the transcription initiation site) were isolated from pH1CAT by partial digestion with AvaII and HindIII restriction enzymes. pH1CAT was derived from the infectious HIV-1 proviral clone pNL4-3 [26], which contains DNA from the HIV-1 isolate NY5 [27] (kindly provided by Dr M. Hatanaka, Shionogi Institute, Osaka, Japan). The excised DNA was subcloned into the promoterless pGL3-Basic vector (Promega, Madison, Wisconsin, USA) immediately adjacent to the coding sequence of the luciferase reporter gene, using the same cloning sites (the construct is referred to as LTR-compl). The ScaI-HindIII fragment of the LTR (5′LTR) was excised from pH1CAT, ligated into the SmaI/HindIII-digested pGL3-Basic vector at the HindIII site, treated with DNA polymerase I to make a blunt end, and then ligated (pGL3-LTR-del). The possible GATA-2 site encompassing nucleotides -342 to -337 (GATATC) was abrogated by changing it to GAATTC (referred to as LTR-mut1), and another two possible consensus sites ranging from nucleotides -404 to -400 (GATCT) and from nucleotides -412 to -408 (GATCT) were abrogated by changing them to GAGCA simultaneously (referred to as LTR-mut2) using standard polymerase chain reaction-directed in vitro mutagenesis (Fig. 1). LTR-mut3, which contained multiple mutations, was generated by inserting mutations at nucleotides -342 to -337, as described above, into LTR-mut2 plasmid (Fig. 1). The mutant constructs were confirmed by sequencing. The Tat expression vector pCVSV-tat was a kind gift from Dr A. Adachi (Tokushima University, Tokushima, Japan) [28].
Cell culture and DNA transfection
COS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), and transfected using the diethylaminoethyl-dextran method, as previously described [29]. Plasmid DNA including 1 μg reporter gene, 1 µg MLVpLINKLacZ (transfection control; a gift from Dr R. Marais, Institute of Cancer Research, London, UK), and varying amounts of the GATA-2 expression vector pMT2-GATA-2 (a kind gift from Dr S. Orkin, Harvard Medical School, Boston, Massachusetts, USA) were used for transfection. Transfected cells were kept in complete medium for 24 h. Cell extracts were made following the manufacturers' recommendation, and equivalent amounts of protein were used for the luciferase assays (Promega) and β-galactosidase assays (Promega). Luciferase activity was measured using a luminometer (Lumat LB9501, Berthold, Germany). The data presented were corrected for β-galactosidase levels. Each set of transfection experiments was performed in triplicate at least five times independently. The data are presented as the means ± SD. Statistical significance was analysed by the Student's t test.
The IL-3-dependent pro-B cell line BA/F3 [30] was maintained in RPMI-1640 medium supplemented with 10% FCS and 10 ng/ml murine IL-3. The transfection of plasmid DNA into BAF3 cells was performed by electroporation. The BioRad (Hercules, California, USA) electroporator was used at standard conditions (960 µF and 300 V). Luciferase activity was detected after 24 h of the transfection and was standardized by the transfection efficiency, which was determined from the β-galactosidase levels from cotransfected MLVpLINKLacZ.
Electrophoretic mobility shift assays
Preparation of nuclear extracts and electrophoretic mobility shift assays were carried out as described previously [31].
Results
The effect of GATA-2 on HIV-1 transcriptional activation through the promoter region of HIV-1 LTR was investigated: LTR of the HIV-1 strain used contained high affinity GATA-2 binding sequences [20] at nucleotides -343 to -337 (AGATATC) and at nucleotides -412 to -400 (GATCTGTGGATCT) from the transcription start site. Conventional deletion mutants of LTR constructs linked to the same reporter plasmid were generated by standard molecular biology techniques (Fig. 1). It should be noted that the mutant reporter gene construct LTR-del had a minimal promoter containing NF-κB and Sp1 sites and TATA.
These wild-type and mutant LTR-containing reporter plasmids were transfected with or without the GATA-2 expression vector, pMT2-GATA-2, into COS cells, and luciferase activity was subsequently assayed. The data are expressed as the mean relative luciferase activity with the SEM normalized to the activity obtained by the promoterless luciferase plasmid (pGL3-Basic) in each experiment. Cotransfection of the GATA-2 expression vector with the complete LTR increased the luciferase activity up to 40 times the value with the LTR vector alone (Fig. 2). LTR-del showed one-third the activity of LTR-compl, suggesting that GATA-2 transactivates HIV-1 LTR through multiple sites in GATA-2-overexpressing COS cells.
Since it remained possible that overexpression of GATA-2 might cause abnormal binding to HIV-1 LTR, we examined the effect of reduced ratio of GATA-2 transfection to the reporter genes. Whereas decreased amounts of GATA-2 abrogated the GATA-2-dependent activity of LTR-del, it strongly activated that of LTR-compl at a ratio of 1: 0.02 (reporter gene: GATA-2; Fig. 3). These results indicated that the real target of GATA-2 was located within the sites ranging from nucleotides -437 to -139, outside of the minimal promoter unit. We next examined the role of GATA-2 sites in this region. Two possible GATA-2 recognition sites in this region were disrupted by mutation, and designated LTR-mut1-3 (Fig. 1). The luciferase activity obtained by the cotransfection of these reporter plasmids with GATA-2 expression vector were significantly lower than that obtained by LTR-compl and GATA-2 (P < 0.001; Fig. 3), suggesting that these two sites are the main target of GATA-2. Previous reports have suggested that GATA-2 is the predominantly expressed member of the GATA family in murine multi-potential hematopoietic cell line BAF3 [29,32]. Although BAF3 is of murine origin, the amino-acid sequence in the zinc-finger domain of GATA-2 is highly conserved between mice and humans (M. Yamamoto, personal communication, 1997), indicating that the binding preferences of GATA-2 are similar between these species. We used a murine multipotential cell line instead of a human cell line because the transfection efficiency was much higher and more constant in BAF3 cells than in the human hematopoietic cell lines we examined (data not shown). Various reporter genes containing wild-type or mutant LTR were transfected into BAF3 cells by electroporation. Luciferase activity standardized by transfection efficiency is shown in Fig. 4. The activity obtained by LTR-mut3 was decreased to 47% of wild-type LTR (P < 0.01). These results suggest that GATA-2 may be a major transcriptional regulator of HIV-1 in hematopoietic progenitor cells and that the sequence encompassing nucleotides -343 to -337 and -412 to -400 is required for the full transactivation.
To confirm binding of GATA-2 to these sites, gel mobility shift assays were performed with BAF3 nuclear extracts and the oligonucleotides encompassing two potential regions described above. A representative gel shift using a oligonucleotide probe covering from nucleotides -343 to -337 and its mutant probe abrogating GATA sites is shown in Fig. 5. The results clearly showed that BAF3 contained GATA-2 protein binding to the sequence. A similar result was obtained by the use of oligonucleotide probe encompassing nucleotides -412 to -400 (data not shown). Thus, we confirmed that multipotential progenitor cell line BAF3 contained proteins specifically bound to these two GATA consensus sites and that, by supershift assays, GATA-2 is the major effector in the extracts among GATA family. Our results strongly suggest that the region from nucleotides -412 to -400 and -343 to -337 are major GATA-2 binding sites necessary for full GATA-dependent activation of the HIV LTR. It should be noted that at least one of these two sites was conserved among several representative clones (Fig. 6).
Finally, the effect of co-expressed Tat protein on GATA-2-dependent transactivation was examined. The Tat protein, encoded by the HIV-1 provirus, plays an important role in viral gene expression [33] and functions via trans-acting response (TAR) element, present immediately downstream of the transcription start site within HIV-1 LTR sequences [34]. It is of particular importance to examine whether the major viral transactivator, Tat, affects GATA-dependent transactivation. Tat expression vector was transfected with or without GATA-2 expression vector into COS cells. Coexpressed Tat elevated the GATA-2-derived activity five times higher than GATA-2 alone (Fig. 7). This implies that Tat and GATA-2 may act synergistically on HIV-1 LTR and raises the possibility that Tat may assemble with GATA-2 in HIV-1-infected multipotential progenitor cells.
Discussion
To date, only a few cell type-specific transcription factors have been shown to participate in the activation of HIV-1. One exception is the ETS family of transcription factors, which are predominantly expressed in T cells [35,36]. In this study, we have shown that GATA-2 highly enhances LTR-dependent transactivation. This activation appears to be GATA-site dependent. In addition, we have also shown that GATA-dependent activation may play a crucial role in regulating HIV-1 expression in BAF3 cells. The stimulation of HIV-1 transcription by infected cells is thought to depend mainly on NF-κB binding sites located directly 5′ to the core promoter element [12]. However, our results showed that the hematopoietic-specific factor GATA-2 can strongly amplify HIV-1 transactivation in hematopoietic progenitors: approximately 60% of the full activity disappeared following the disruption of GATA-2 sites in the LTR.
The next interesting issue is to identify the binding sites to GATA-2. GATA-3 has been reported to bind to the LTR of HIV-1 in vitro at six binding sites [37]. The HIV-1 clone used in that study (clone HXB2) [5] was different from the isolate we used in this study. Comparison of DNA sequences between these two clones shows only one site (nucleotides -343 to -337) of six sites in clone HXB2 exists in NY5. Together with our data, GATA-2 acts on HIV-1 LTR by binding multiple sites and the binding site preference in the LTR may be different between GATA-2 and GATA-3. Previous study revealed two major protein-binding sites within the negative regulatory element of LTR [38]. One site (site B) contained a palindromic sequence and a novel T-cell protein in Jurkat T-cell nuclear extract bound the palindromic element. One GATA-2 binding site identified here (nucleotides -343 to -337) is located between the two (5′ and 3′) palindromes. They showed that the mutation of either of the palindromes resulted in the loss of DNA binding activity of the T-cell protein. Another report [37] has shown that the GATA site is one of the six GATA-3 binding site by the use of the same HIV-1 LTR clone (HXB2) and Jurkat T cells by gel retardation assay. Our gel shift data showed that one apparent binding protein, GATA-2, existed in BAF3 nuclear extract as the binding protein for the element containing 5′ palindrome and intact GATA site. These may reflect the difference of the binding preference to the sequence. Namely, GATA-2 may bind the GATA site independent of the palindrome, whereby the novel T-cell protein, of which GATA-3 could be the component, may require complete palindromic sequence and the GATA site for its binding. Further study is required to clarify the requirement of the palindromic element for GATA-2 and a novel protein-GATA-3 binding. However, it is likely that the two GATA consensus sites identified by the present study are the main sites for GATA-2-specific binding in hematopoietic progenitors. Further study for the identification of all GATA-2 binding sites in the LTR will enable us to suppress HIV-1 expression in hematopoietic progenitor cells by targeting GATA-2 binding sites.
It has been suggested that Tat may play a crucial role in abrogating hematopoietic differentiation [39]. Although Tat seems to function via binding the TAR element, recent study has indicated the presence of a TAR-independent function of Tat [40]. Tat can alter the expression of cytokine genes such as TNF-α, transforming growth factor-β, and IL-6 [39,41,42]. More recently, it has been demonstrated that ectopically-expressed Tat protein suppressed erythroid differentiation in K562 cells [43]. Consistent with these results, GATA-2 appears to be downregulated when multipotential progenitors are differentiated into erythroid cells [18]. Given these findings, together with our data, it is fascinating to speculate that GATA-2, in cooperation with Tat, may play a central role in proliferation and differentiation through not only HIV-1 LTR sequences but also regulatory sequences of hematopoietic-specific genes in hematopoietic progenitor cells infected with HIV-1 provirus.
Acknowledgement
We are grateful to C. Wakamatsu and S. Suzuki for the technical assistance.
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