Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1 - PubMed (original) (raw)

Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1

N Wara-aswapati et al. Mol Cell Biol. 1999 Oct.

Abstract

Potent induction of the gene coding for human prointerleukin 1beta (il1b) normally requires a far-upstream inducible enhancer in addition to a minimal promoter located between positions -131 and +12. The transcription factor Spi-1 (also called PU.1) is necessary for expression and binds to the minimal promoter, thus providing an essential transcription activation domain (TAD). In contrast, infection by human cytomegalovirus (HCMV) can strongly activate il1b via the expression of immediate early (IE) viral proteins and eliminates the requirement for the upstream enhancer. Spi-1 has been circumstantially implicated as a host factor in this process. We report here the molecular basis for the direct involvement of Spi-1 in HCMV activation of il1b. Transfection of Spi-1-deficient HeLa cells demonstrated both the requirement of Spi-1 for IE activity and the need for a shorter promoter (-59 to +12) than that required in the absence of IE proteins. Furthermore, in contrast to normal, enhancer-dependent il1b expression, which absolutely requires both the Spi-1 winged helix-turn-helix (wHTH) DNA-binding domain and the majority of the Spi-1 TAD, il1b expression in the presence of IE proteins does not require the Spi-1 TAD, which plays a synergistic role. In addition, we demonstrate that a single IE protein, IE2, is critical for the induction of il1b. Protein-protein interaction experiments revealed that the wing motif within the Spi-1 wHTH domain directly recruits IE2. In turn, IE2 physically associates with the Spi-1 wing and requires the integrity of at least one region of IE2. Functional analysis demonstrates that both this region and a carboxy-terminal acidic TAD are required for IE2 function. Therefore, we propose a protein-tethered transactivation mechanism in which the il1b promoter-bound Spi-1 wHTH tethers IE2, which provides a TAD, resulting in the transactivation of il1b.

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Figures

FIG. 1

Schematic representation of the il1b promoter showing two Spi-1-binding sites, one located adjacent to the transcription start site and the other located further upstream. The TATA sequence (TATAAAA) and the TBP are also illustrated. Two fragments of the il1b promoter, HT (−131 to +12) and DT (−59 to +12), used in the transfection experiments in this study are shown as bars (see Kominato et al. [30] for a detailed description of these sequences).

FIG. 2

The il1b promoter is strongly transactivated by IE proteins in human THP-1 monocytes. Shown are the CAT activities of the il1b promoter CAT reporters (HT and DT) containing point mutations at the distal (B) and proximal (A) Spi-1-binding sites, respectively, and cotransfected with a genomic IE1-IE2 expression vector [pEQ276(IE1+2)] which expresses both IE1 and IE2. The results are expressed as average percentages of the relative CAT activity observed for the wild-type HT promoter when it was cotransfected with the IE1-IE2 expression vector. Open bars correspond to unstimulated cells, whereas filled bars correspond to cells treated with 10 ng of LPS per ml. Standard deviations are indicated for a minimum of three repetitions. mA, mutation at site A; mB, mutation at site B; mA,B, mutations at both sites.

FIG. 3

Transactivation of il1b by HCMV IE protein is differentially mediated by discrete domains of the transcription factor Spi-1. (a) Schematic representation of Spi-1 illustrating the ETS homology DNA-binding domain (wHTH) and TAD. The previously identified functional regions are also shown, including the TBP/Rb-binding region, Q domain, and PEST sequence containing the NF-EM5/PIP-binding region. Various Spi-1 expression vectors containing different regions of Spi-1 are shown by horizontal bars. Numbers indicate the amino acid sequences. (b) The Spi-1 wHTH DNA-binding domain alone can support IE transactivation of il1b. The Q domain is important for maximal transactivation. CAT activities observed for the il1b promoter from positions −131 to +12 (HT) are shown. The antisense Spi-1 (As), wild-type Spi-1 (WT), and various mutated Spi-1 expression vectors (as shown in panel a) were each cotransfected with the il1b promoter (HT) CAT reporter along with the HCMV IE1-IE2 expression vector into HeLa cells. Twenty micrograms of the CAT vector and 10 μg of each expression vector were used for each analysis. The results are expressed as average percentages of the activity observed for the wild-type promoter and the Spi-1 expression vector after stimulation with 50 ng of PMA per ml. Open bars correspond to transfections in the absence of the IE1-IE2 expression vector. Error bars indicate the deviations in results from a minimum of three repetitions.

FIG. 4

The −59-to-+12 il1b promoter (DT) containing only one Spi-1-binding site adjacent to the transcription start site can function as an IE-activated promoter via an enhancer-independent pathway. The upstream Spi-1-binding site is not essential for il1b transactivation by HCMV IE proteins. The il1b promoter CAT reporters were transiently cotransfected with an IE1-IE2 expression vector into HeLa cells in the presence of 50 ng of PMA per ml. The il1b promoter CAT reporter (20 μg) and IE1-IE2 expression vector (IE1+2) (10 μg) were used for each analysis. Ten micrograms of the Spi-1, antisense Spi-1 (AS.Spi-1), or Spi-1 DNA-binding domain (wHTH) expression vector was cotransfected for each analysis. The results are shown as average percentages of the CAT activity of the wild type. Error bars indicate the deviations in results from a minimum of three repetitions.

FIG. 5

IE1 and IE2 synergistically transactivate il1b expression. Shown is CAT activity of the enhancerless il1b promoter (HT) CAT vector (20 μg) cotransfected with 10 μg of the Spi-1 or antisense Spi-1 (AS.Spi-1) expression vector into PMA-treated HeLa cells, in the presence of an individual IE expression vector [pEQ273(IE1) or pEQ326(IE2)] or a genomic IE1-IE2 expression vector [pEQ276(IE1+2)] expressing both IE1 and IE2. The total amount of transfected DNA was kept constant by the addition of the parental vector [pEQ336]. The CAT data were normalized to the average activity elicited by the IE1-IE2-activated HT CAT construct in the presence of the Spi-1 expression vector. Error bars represent the deviations in results from a minimum of three repetitions.

FIG. 6

Physical interaction of Spi-1 with IE2. (a) Schematic representation of various GST–Spi-1 fusion constructs. The GST–wild-type Spi-1 fusion construct is indicated as Spi-1 (WT). Other deletion constructs are shown with numbers of amino acids deleted (in parentheses) relative to the amino acid sequence of Spi-1. Quantitation of expressed GST fusion proteins was determined by Coomassie brilliant blue staining (data not shown). (b to d) For all experiments, in vitro-translated 35S-labeled proteins (IE proteins in panels b and c or Stat 3 protein in panel d) were incubated with glutathione-Sepharose beads bound to either GST or GST–Spi-1 fusion proteins (as indicated). After incubation, the glutathione-Sepharose beads were washed extensively and bound proteins were resolved by SDS-PAGE. (b) IE2(p55) and IE2(p86) physically interacted with the Spi-1 DNA-binding domain (wHTH) (lanes 3 and 7, respectively). IE proteins bound weakly to the GST control (lanes 2, 4, and 6). Lanes 8, 9, and 10 indicate the mobilities of these radiolabeled IE proteins as well as the efficiency of in vitro translation. Molecular weight markers (in thousands [K]) are indicated at the left. (c) IE2 directly bound to Spi-1 through a portion (antiparallel β3 and β4) of the wing motif (lane 6). Lane 7 indicates the mobility of the in vitro-35S-labeled IE2(p86) protein on SDS-polyacrylamide gel. In lane 1, the radiolabeled IE2 was incubated with glutathione-Sepharose bead-linked GST alone. In lanes 2 to 6, the radiolabeled IE2 was incubated with immobilized GST–wild-type Spi-1 or various GST–Spi-1 deletion mutation fusion proteins. (d) Stat 3 is unable to bind Spi-1 wHTH. Lane 5 shows the mobility of the Stat 3 protein on SDS-polyacrylamide gel.

FIG. 7

Mapping the region of IE2(p86) responsible for cooperativity with Spi-1 in il1b transactivation. (a) Schematic representation of the HCMV IE gene products, IE2(p86), IE2(p55), and IE1(p72). The exons from which each IE protein arises and their amino acids are indicated. (b) The region between amino acids 291 and 370 of IE2 is essential for the interaction with Spi-1. GST or GST–Spi-1 β3-β4 (243-254) fusion proteins linked to glutathione-Sepharose beads were incubated with equal amounts of the in vitro-translated 35S-labeled mutated IE2 proteins (as shown in Fig. 6c). After extensive washing, the bound proteins were resolved by SDS-PAGE. All the IE2 deletion mutants bound weakly to the GST control (lanes 2, 4, and 6). Molecular weight markers (in thousands [K]) are indicated at the left. (c) Schematic representation of mutated IE2 expression vectors. All vectors are as described by Sommer et al. (52). The wild-type IE2 (WT) and the deletion mutants are illustrated with numbers representing the positions of important amino acid residues. Also shown are the locations of three previously reported TBP-binding domains (52) and an acidic activation domain (40). (d) Functional data support the significance of the IE2 interaction motif (amino acids 291 to 370), the sequence between amino acids 544 and 579, and a region within amino acids 85 to 291 of IE2 protein in the transcriptional activation of il1b. The il1b promoter luciferase reporter (pGL3B-DT) was cotransfected with Spi-1 and mutated IE2 expression vectors (as shown in Fig. 6c) into HeLa cells. The total amount of transfected DNA was kept constant by the addition of the parental vector. Shown are average relative luciferase activities. A broken line indicates the activity level in the absence of IE2. Error bars indicate the deviations in results from a minimum of three repetitions. (e) Whole-cell extracts from the transfected HeLa cells were subjected to Western blot analysis with a monoclonal antibody recognizing the IE2 protein in order to quantitate mutated IE2 protein expression. (f) Summary of the results from the protein-protein binding assay and transactivation study. Two regions of IE2 required for il1b transactivation are shown as black bars. A region providing synergistic activation is shown as a gray bar. The likely Spi-1 interaction motif of IE2 is illustrated as a dark-gray bar. The numbers indicate amino acid sequences of IE2.

FIG. 8

An in vitro ternary complex involving IE2–Spi-1 and DNA requires protein-protein interaction. (a) IE2 protein does not bind to the il1b promoter. DNA-binding assays were performed as described in Materials and Methods. GST fusion proteins were incubated with either a radiolabeled Spi-1 DNA probe (il1b) or an IE2 DNA probe (CRS). Following two rounds of washing, specific DNA-binding activities were determined by measuring the radioactive counts per minute from the Sepharose beads. Reactions with GST-IE1 were performed simultaneously as negative controls. (b) The Spi-1 wHTH is required for the interaction between IE2 and DNA. The GHIA used in vitro-translated Spi-1 wHTH, which was incubated with an equal amount of either GST-IE2 or the GST control bound to glutathione-Sepharose beads. The in vitro-translated control protein was made by the same method as was used for the Spi-1 wHTH but in the absence of the DNA template. After incubation and extensive washing, the beads were incubated with either a wild-type or a mutated Spi-1 radiolabeled DNA probe (15). Following two rounds of washing, the amount of bound radioactive probe was determined from the Sepharose beads. Error bars indicate the deviations in results from a minimum of three repetitions. Cartoons explain the details of the experiment in which the natures of individual proteins and radiolabeled DNAs ( formula image ) are indicated. βgb, β-globin-binding site; mβgb, mutated β-globin-binding site.

FIG. 9

Proposed model of PTT-mediated transcriptional activation of il1b by HCMV IE2. (a) Potent il1b transcription normally requires both a far-upstream enhancer and the −131-to-+12 il1b promoter which contains two essential Spi-1-binding sites. Two Spi-1 TAD subregions are also required. One of these is a glutamine-rich (Q) domain (hatched areas), and the other directly binds TBP (filled areas). The Spi-1 wHTH DNA-binding domain is shown as an oval, with the wing indicated by “w”. (b) IE2 replaces the functions of the Spi-1 TBP-binding domain and the il1b enhancer. Furthermore, only a minimal il1b promoter located between −59 and +12 is sufficient to support IE-activated il1b expression. IE2 is tethered to the il1b promoter via a direct interaction with the Spi-1 wing and possibly interacts with TBP. Also, an acidic domain of IE2 (shown by a minus sign) provides an activation effect eliminating the absolute requirement for the Spi-1 TAD.

References

1. Bassuk A G, Leiden J M. A direct physical association between ETS and AP-1 transcription factors in normal human T cells. Immunity. 1995;3:223–237. - PubMed
1. Behre G, Whitmarsh A J, Coghlan M P, Hoang T, Carpenter C L, Zhang D E, Davis R J, Tenen D G. c-Jun is a JNK-independent coactivator of the PU.1 transcription factor. J Biol Chem. 1999;274:4939–4946. - PubMed
1. Biegalke B J, Geballe A P. Sequence requirements for activation of the HIV-1 LTR by human cytomegalovirus. Virology. 1991;183:381–385. - PubMed
1. Boldogh I, AbuBakar S, Albrecht T. Activation of protooncogenes: an immediate early event in human cytomegalovirus infection. Science. 1990;247:561–564. - PubMed
1. Caswell R, Hagemeier C, Chiou C J, Hayward G, Kouzarides T, Sinclair J. The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation. J Gen Virol. 1993;74:2691–2698. - PubMed

Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1 - PubMed (original) (raw)