Repeat of Human Immunodeficiency Virus Type 1 - Journal of Virology

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of max and c-myc implicated in controlling cell proliferation. (6), and selected E-box ..... A. Carroll, W. Crist, B. Ozanne, M. J. Siciliano, and R. Baer. The tal gene ...
JOURNAL OF VIROLOGY, Sept. 1992, p. 5631-5634 0022-538X/92/095631-04$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 66, No. 9

Interactions of HTF4 with E-Box Motifs in the Long Terminal Repeat of Human Immunodeficiency Virus Type 1 YI ZHANG, KENNETH DOYLE, AND MINOU BINA* Department of Chemistry, Purdue University, West Lafayette, Indiana

47907-1393

Received 24 March 1992/Accepted 28 May 1992

We have identified three consensus E-box motifs in the long terminal repeat of human immunodeficiency virus type 1. One of these E boxes interacts selectively with representative members of the class A group of basic helix-loop-helix proteins, including HTF4, E47, and their heterodimers. Our analyses implicate the helix-loophelix proteins in regulation of human immunodeficiency virus type 1 gene expression.

The binding sites for a number of regulatory proteins share motif (CANNTG) known as an E box (reviewed in reference 26). For example, an E box (CACGTG) defines the core binding motif for heterodimers of max and c-myc implicated in controlling cell proliferation (6), and selected E-box motifs in the promoters and enhancers of cellular genes have been shown to interact with basic helix-loop-helix (bHLH) proteins which function in activation of gene expression (reviewed in references 9 and 26). There are three classes of bHLH proteins: A, B, and C (30). The class A proteins appear to be ubiquitous (9, 10, 17, 19, 26, 30, 41). They include several transcription factors expressed from distinct but evolutionarily related genes (41). The human E2A gene encodes several related proteins (E47/HE47, E12, and ITF1), produced from differentially spliced mRNAs (17, 22, 29); a second human gene encodes proteins related to ITF2 (10, 17); a third gene encodes proteins related to HTF4 (19, 39, 41). The class B proteins are cell type and lineage specific (see, for example, references 5, 9, 18, and 30). The members of this class interact relatively weakly with DNA, but their affinity for their cognate sites is dramatically increased when they form heterodimers with members of class A (see, for example, references 5 and 30). Gene activation mediated by bHLH proteins appears to play key roles in differentiation of lymphocytes, muscle cells, pancreatic 0i cells, and many other cell types which acquire their functional capacities through oligomeric complexes of bHLH proteins (reviewed in reference 26). In addition, the biological activity of bHLH proteins is highly regulated by Id proteins (4, 24, 36, 37). By forming heterodimers with bHLH proteins, Id proteins abolish their capacity to interact with DNA and thus negatively regulate their transcriptional activities (4, 36, 37). To investigate whether bHLH proteins could contribute to the regulation of human immunodeficiency virus type 1 (HIV-1) gene expression, we have applied a pattern recognition program (1) and identified three E-box motifs in the long terminal repeat (LTR) of an HIV-1 strain in the data base. These motifs are schematically shown as boxes I, II, and III in Fig. 1. Also shown in Fig. 1 are a predicted negative regulatory element (NRE) in the LTR, the binding sites for two of the known activators of HIV-1 gene expression (Spl and NF-KB), and the TATA element which mediates the LTR promoter function (reviewed in reference 15). One of the E-box motifs in the LTR (E box I) is located a common core

*

Corresponding author.

between the TATA element and the transcription initiation site (Fig. 1). This box appears to be conserved among the various HIV strains reported in data bases. Its sequence (CAGCTG) is identical to that of the ,uE2 core E box in the enhancer of the immunoglobulin heavy-chain gene (26) and to that in the AP4 site implicated in controlling the expression of the simian virus 40 late genes (20, 39). E box II is within the NRE (Fig. 1). Its sequence is strain dependent but conforms to a consensus (CACRTG, where R is a purine). One element (CACGTG) within the consensus is identical to the E-box recognition sites of max and c-myc heterodimers; the other (CACATG) is identical to the core KE3 and ,uE3 elements in the enhancers of the immunoglobulin genes (reviewed in reference 26). E box III is upstream of the NRE, and comparative analysis indicates that its sequence (CAGTTG) is conserved among the various known strains of HIV-1. In our initial analysis, we investigated the interactions of E box I with HTF4 (Fig. 2), which is a member of the class A group of bHLH proteins (19, 40, 41). Box I contains a ,uE2 motif found also in the simian virus 40 AP4 site which interacts specifically with HTF4 (40). This protein includes the DNA binding domain and a major portion of a longer protein (HTF4a) whose sequence has been deduced from cloned cDNA (41). A transcription factor whose predicted sequence is nearly identical to that of HTF4a has been recently described and named HEB (19). For DNA binding studies, HTF4 fused to glutathione S-transferase was expressed in Escherichia coli, isolated by affinity chromatography, and cleaved with thrombin (16, 35) in order to obtain a relatively pure HTF4. Figure 1 shows the map locations of three DNA fragments prepared for band shift analysis from recombinant constructs. Fragment Fl includes the upstream and the NRE E boxes. F2 contains the ,uE2 core sequence, the TATA element, and the binding sites for Spl and NF-KB. F3 spans the ,uE2 core and the TATA element (Fig. 1). In addition, since HTF4 interacts strongly with oligomers of the ,uE2 core motif in the simian virus 40 AP4 site (39, 40), we constructed and cloned multimers of the AP4 element and multimers of an unrelated element (AP3) for competition experiments. Incubation of radiolabelled F3 with purified HTF4 produced a prominent complex in band shift assays (Fig. 2A). The formation of this complex can be inhibited with unlabelled multimer of AP4 but not with unlabelled multimer of AP3 (Fig. 2A), suggesting that HTF4 interacts specifically with the ,uE2 core motif in the LTR. HTF4 does not show a high affinity for E box II or E box III, since a relatively high 5631

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protein concentration is required to produce a shifted band with radiolabelled Fl which contains both motifs (data not shown). Thus, the binding assays indicate that in the LTR, the ,uE2 core represents the preferred HTF4 binding site. This analysis was further extended by two other binding studies. In the first, we added unlabelled F2, which contains the ,uE2 core, and Fl, which contains the NRE and upstream E boxes, as competitor DNAs to reaction mixtures containing HTF4 and radiolabelled F2 as the probe (Fig. 2B). The results show that the NRE and upstream E boxes do not compete effectively with the probe for binding HTF4. In the second analysis, we used 1,10-phenanthroline copper ion as a footprinting reagent (25). HTF4 produced a prominent footprint on radiolabelled F2 (Fig. 2C) in a region (nucleotides -26 to -13) which includes the ,uE2 core motif (Fig. 2C) and extends to include a perfect palindrome (AAG CAGCTGCTT) surrounding the E box. This finding lends support to the suggestion that the flanking sequence of the E box contributes to the specificity of protein binding (19). Since the ,uE2 core motif in the LTR is the only E box that interacts detectably with HTF4, we examined the interactions of this motif with several other bHLH proteins of class A (Fig. 3). The proteins were synthesized in rabbit reticulocyte extracts, since this system provides a relatively efficient way for producing heterodimers of bHLH proteins for binding assays (29, 30). The selected class A proteins were translated from in vitro-transcribed RNA encoding HTF4, ITF2, E12, HE47, ITF1, and ITF1S (see Fig. 3 for the constructs used for transcription). In band shift assays, the in vitro-translated HTF4 produces a shifted band with radiolabelled F3 (Fig. 3, lane 2), as observed for the purified protein (Fig. 2). The probe also formed a detectable complex with HE47 and ITFlS (Fig. 3, lanes 3 and 7) but not with E12, ITF2, and ITF1 (Fig. 3). Cotranslation and DNA binding experiments further revealed interactions between the probe and HTF4-HE47 and HTF4-ITF1S heterodimers. Multiple E-box motifs appear to act cooperatively or

synergistically in the activation of cellular genes (26). Therefore, similar mechanisms might also contribute to the regulation of HIV-1 gene expression by the three E-box motifs in the viral LTR (Fig. 1). The results described above have revealed interactions between E box I and at least two members of bHLH proteins of class A: HTF4, HE47, and their heterodimers. E box II in the NRE has previously been shown to bind USF (14, 27). This box may also interact with TFE3 and TFEB (2, 3, 7, 14) and with heterodimers of c-myc and max. E box III conforms to the consensus binding site for the product of the cellular proto-oncogene c-myb (11). The expression of c-myb is induced in mitogen-stimulated peripheral blood lymphocytes and is constitutive in several CD4+ T-cell and myeloid cell lines, all of which represent potential targets for HIV-1 infection (11). A systematic linker-scanning mutational analysis indicates that sequences between the TATA element and transcription start site (-21 to -4) are required for optimum LTRmediated gene expression in unstimulated, stimulated, and particularly tat-expressing Jurkat cells (38). Interestingly, in the mutated sequences, E box I is replaced by an E box (CATATG) that does not appear to constitute a high-affinity binding site for HTF4. Potential significance of E box I in the wild-type sequence can also be inferred from the results of methylation protection analysis showing that in HIV-1infected H9 cells, a G residue in E box I is protected against chemical modification (12). Generally, G residues in E-box motifs represent critical contact sites for specific interactions of DNA with bHLH proteins, including E47, E12 (30), and HTF4 (data not shown). Since the results described above indicate that the class A group of proteins might contribute to the activation of HIV-1 gene expression, it would be of interest to identify their potential lymphoid-specific class B partners and to determine the physiological conditions under which their biological activities are regulated (4, 36, 37). The protein Tal-1 appears to be a likely candidate for a lymphoid-specific class B protein. Heterodimers of Tal-1 with E47 and E12 have

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FIG. 2. HTF4 binding specificity on the HIV-1 LTR. (A) HTF4 was expressed in E. coli, purified, and subsequently incubated with end-labelled F3 (0.5 ng) and 0.5 ,ug of poly(dI-dC) in binding buffer (10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.8], 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 8% glycerol) for 30 min at room temperature. The products were fractionated on a 5% native polyacrylamide gel containing 2.5% glycerol in TGE (12.5 mM Tris [pH 8.3], 95 mM glycine, 0.5 mM EDTA). Lane 1 shows the electrophoretic mobility of the probe in the absence of any protein. The binding reactions contained no competitor (lane 2), 25 and 80 ng of unlabelled AP4 multimer (lanes 3 and 4), and equivalent amounts of unlabelled AP3 multimer (lanes 3' and 4'). (B) Band shift assays were performed by using HTF4 and 0.5 ng of F2 probe as described above. Lane 1 shows the mobility of the probe in the absence of added protein. The reactions contained no competitor (lane 2), 25 and 75 ng of unlabelled F2 (lanes 3 and 4), and equivalent amounts of unlabelled Fl (lanes 3' and 4'). (C) For footprinting analysis, HTF4 was incubated with singly-end-labelled F2 probe as described above; the free and protein-bound DNAs were separated by gel electrophoresis. Chemical footprinting was performed within the gel slices (25) containing free and HTF4-bound DNA identified by autoradiography. The cleavage products were isolated and analyzed on a 10% denaturing polyacrylamide gel. Lane G represents a Maxam-Gilbert (28) G reaction with the probe; lanes F and B represent, respectively, the cleavage products of 1,10phenanthroline copper ion reactions with free and HTF4-bound DNA. The sequence of the protected area is indicated.

been shown to interact specifically with an E-box motif in vitro (18); in patients with T-cell acute lymphoblastic leukemia, abnormalities in the tal-1 gene result in accumulation of immature lymphoblasts in bone marrow and peripheral blood (8). Id protein represses the activity of several bHLH proteins. DNA binding assays have shown that Id inhibits complex formation between E47 and muscle-specific factors (4, 36, 37) and also abolishes the DNA binding activity of HTF4 and its complexes with myogenic factors (data not shown). It has been postulated that Id functions as a general inhibitor of cell differentiation (4, 24). Recent studies indicate that during myeloid differentiation, there is a correlation between a decrease in Id mRNA and a concomitant appearance of E-box binding activities in nuclear extracts (24). These findings considered in the context of the results described above indicate that the potential effects of bHLH proteins on the activation of HIV-1 gene expression may be detectable only in appropriately differentiated cells and may be of significance during development.

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FIG. 3. Interaction of E box I with bHLH proteins of class A. The proteins indicated above the lanes were translated separately or cotranslated in vitro in rabbit reticulocyte lysates (Promega). For band shift analysis, a sample (5 ,ul) of the translation reactions was incubated with radiolabelled F3 probe (0.2 ng), and the products were analyzed by electrophoresis as described in the legend to Fig. 2. Lane F shows the mobility of the probe in the absence of added protein. NS represents a nonspecific band derived from a component in reticulocyte lysates. For in vitro protein synthesis, E12 RNA was transcribed from an E12R cDNA containing plasmid obtained from D. Baltimore (22, 30); ITF1, ITFlS (a shorter version of ITF1), and ITF2 RNAs were transcribed from plasmids T7PE2-5, T7PE25S, and T7,BE2-2, respectively, obtained from T. Kadesch (17). HE47 and HTF4 RNAs were transcribed from plasmids which contained the proteins' cDNAs cloned into the pBSATG vector described in reference 30. HE47 represents the carboxy terminus (141 residues) of a HeLa protein (40) related to E47, an immunoglobulin enhancer-binding protein (29). We thank D. Baltimore and T. Kadesch for providing plasmids used in the in vitro transcription-translation experiments. This research was supported by grants awarded by NIH. REFERENCES 1. Ambrose, C., and M. Bina. 1990. Strategy for statistical-mapping of potential regulatory regions in the human genome. J. Mol. Biol. 216:485-490. 2. Beckman, H., and T. Kadesch. 1991. The leucine zipper of TFE3 dictates helix-loop-helix dimerization specificity. Genes Dev. 5:1057-1066. 3. Beckmann, H., L.-K. Su, and T. Kadesch. 1990. TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer ,uE3 motif. Genes Dev. 4:167-179. 4. Benezra, R., R. L. Davis, D. Lockshon, D. L. Turner, and H. Weintraub. 1990. The protein Id: a negative regulator of helixloop-helix DNA binding proteins. Cell 61:49-59. 5. Blackwell, T. K., and H. Weintraub. 1990. Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science 250:1104-1110. 6. Blackwood, E. M., and R. N. Eisenman. 1991. Max: a helix-loophelix zipper protein that forms a sequence-specific DNA-binding complex with myc. Science 251:1211-1217. 7. Carr, C. S., and P. A. Sharp. 1990. A helix-loop-helix protein related to the immunoglobulin E box-binding proteins. Mol. Cell. Biol. 10:4384-4388. 8. Chen, Q., J.-T. Cheng, L.-H. Tsai, N. Schneider, G. Buchanan, A. Carroll, W. Crist, B. Ozanne, M. J. Siciliano, and R. Baer. The tal gene undergoes chromosome translocation in T cell

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