Cytomegalovirus IE2 Protein Stimulates Interleukin 1β Gene Transcription via Tethering to Spi-1/PU.1

Wara-aswapati, Nawarat; Yang, Zhiyong; Waterman, Wayne R.; Koyama, Yoshinobu; Tetradis, Sotirios; Choy, Bob K.; Webb, Andrew C.; Auron, Philip E.

doi:10.1128/mcb.19.10.6803

Cited by 37 publications

(46 citation statements)

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“…The Spi-1 pRC/CMV expression vector contains the murine Spi-1 cDNA as reported previously (25). Spi mutants were constructed using PCR mutagenesis as described previously (23) or by QuikChange (Stratagene, La Jolla, CA) site-directed mutagenesis according to the manufacturer's instructions. Spi-1/ETS DBD constructs were either mutated within parent wild type (WT) plasmids or cloned by PCR amplification into either pRC/CMV for use in transfection assays, or pEX 2T (Amersham Biosciences) for use in GST pull-down assays as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

“…C-Myb, another HTH protein, was shown to interact with the bZIP domain of C/EBP␤ while each was bound to a remote DNA site, demonstrating a model of tethering between an enhancer and promoter (21). In contrast, Spi-1 interactions with several proteins including C/EBP␤ and human cytomegalovirus (HCMV) IE2 reveal clear cooperative function but no obvious DNA binding cooperativity (18,22,23). This has led to a hypothesis of protein-tethered transactivation, whereby a direct DNA-binding protein tethers another protein, resulting in either activation or inhibition of function (18,23).…”

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confidence: 99%

“…In contrast, Spi-1 interactions with several proteins including C/EBP␤ and human cytomegalovirus (HCMV) IE2 reveal clear cooperative function but no obvious DNA binding cooperativity (18,22,23). This has led to a hypothesis of protein-tethered transactivation, whereby a direct DNA-binding protein tethers another protein, resulting in either activation or inhibition of function (18,23). Therefore, understanding the molecular basis for these kinds of interactions continues to be intensely studied.…”

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confidence: 99%

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Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

Listman

Wara-aswapati

Race

et al. 2005

Journal of Biological Chemistry

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The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1␤. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBP␤ carboxylterminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.The ETS family of transcription factors serves a variety of roles in development and differentiation (1). A highly conserved DNA-binding domain (DBD) 3 defines the family. Spi-1/PU.1 (Spi-1), one of the most diverse members, is critical for myeloid and B cell differentiation and function (2-6). It also regulates the expression of many effector genes, including IL1B, coding for the IL-1␤ protein (7). The Spi-1 molecule possesses a mutipartite transactivation domain (TAD) consisting of the amino half of the molecule that binds TBP, RB (8), and CBP/p300 (9) (Fig. 1a). The carboxyl end of the TAD contains a phosphorylatable PEST sequence that aids in the formation of a protein-protein interaction with transcription factors 11). Located at the COOH-terminal end of the protein is the ETS DBD. Besides DNA binding, this domain is also a tethering site for multiple transactivators, some of which occur at composite DNA-binding sites (reviewed in Ref. 12), and contains the previously coarsely mapped nuclear localization signal (NLS) (13). The structure of the Spi-1 DBD, like other ETS factors, consists of 85 conserved residues folded into a winged-helix-turn-helix (wHTH) conformation (14) (Fig. 1b). Two arginines, which are solventexposed in helix ␣3, are conserved in all ETS family members and form direct and indirect hydrogen bonds to a core DNA sequence of A/GGAA in Spi-1 (1, 7, 15). The Spi-1 DBD is 98% conserved at the amino acid level between mouse and human, suggesting stringent structural and functional requirements.Spi-1 plays a necessary role in the regulation of the IL1B promoter by cooperating functionally with C/EBP␤, a basic leucine zipper (bZIP) family member (16,17). This cooperativity may be derived, in part, by a physical interaction between the DBD of both molecules and the two composite DNA-binding sites in the IL1B promoter (Fig. 2a) (18). The downstream TATA-proximal site binds relatively well to Spi-1 but weakly to C/EBP␤ (7). C/EBP␤ DNA binding at this site appears to be supported by a protein interaction with the adjace...

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Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

Listman

Wara-aswapati

Race

et al. 2005

Journal of Biological Chemistry

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“…Binding of one transcription factor serves as a tether for another transcription factor. Wara-aswapati et al, 1999;Yang et al, 2000;Zhao and Sample, 2000). (f) Ets interacting partner may bridge or block the cooperation between an Ets factor and the basal transcriptional machinery (Jayaraman et al, 1999;Yang et al, 1998) signals (Yates et al, 1999).…”

Section: And Article By Mavrothalassitis and Ghysdael In This Issue)mentioning

confidence: 99%

Regulation of Ets function by protein–protein interactions

2000

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“…Therefore, IE2p86 seems to act as a transcription factor that exerts a broad activation pattern. Since IE2p86 interacts with the basal transcription factors TBP (Hagemeier et al, 1992a;Sommer et al, 1994) and TFIIB (Caswell et al, 1993) and with distinct cellular transcription factors such as CREB, Ap-1, Egr-1, Spi-1/PU.1 or Sp1, protein interactions are believed to be essential for transactivation (Lukac et al, 1994;Lang et al, 1995;Scully et al, 1995;Yoo et al, 1996;Yurochko et al, 1997;Wara-Aswapati et al, 1999).In this report we provide evidence for the mechanism by which HCMV upregulates ICAM-1 on the cell surface of endothelial cells as well as fibroblasts. We show that the regulatory proteins IE2p86 and pp71 act as strong synergistic inducers of the ICAM-1 promoter.…”

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Synergistic induction of intercellular adhesion molecule-1 by the human cytomegalovirus transactivators IE2p86 and pp71 is mediated via an Sp1-binding site

Kronschnabl¹,

Stamminger²

2003

Journal of General Virology

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Human cytomegalovirus (HCMV) infection of transplant recipients is frequently associated with allograft vasculopathy and rejection. One potential mechanism is vascular injury from HCMVtriggered, immunologically mediated processes. HCMV infection has been shown to increase the expression of intercellular adhesion molecule-1 (ICAM-1). The objective of this study was to determine the molecular basis of HCMV-enhanced ICAM-1 gene expression. Transient transfection experiments identified the IE2p86 protein as a potent activator of the ICAM-1 promoter. The tegument protein pp71 showed a strong synergistic effect on IE2p86-mediated ICAM-1 promoter activation. Mutagenesis experiments defined a DNA element from 2110 to 242 relative to the transcription start site as responsive for IE2p86. Further point mutations within this DNA element identified an Sp1-binding site that was essential for strong synergistic activation by IE2p86 and pp71. To confirm the activation of ICAM-1 gene expression, human fibroblasts (HFF) as well as endothelial cells (HUVEC) were infected with recombinant IE2p86-and pp71-expressing baculoviruses, respectively. In FACS analysis, a synergistic induction of ICAM-1 was detectable when cells were co-infected with the two recombinant baculoviruses. These findings clearly demonstrate that IE2p86 and pp71 are crucial regulatory factors for HCMV-induced ICAM-1 upregulation. INTRODUCTIONHuman cytomegalovirus (HCMV), a b-herpesvirus, is a ubiquitous human pathogen with a prevalence of infection in the adult population of 50-90 %. Although HCMV rarely causes severe disease on primary infection in immunocompetent people, it represents a highly pathogenic agent for immunosuppressed patients such as transplant recipients, as well as newborns, and establishes lifelong persistence in the infected host (Alford & Britt, 1990). So far, the exact site of latency is still uncertain; monocytes, haematopoietic precursor cells and endothelial cells have been discussed as potential reservoirs for latent HCMV (Fish et al., 1995;Sindre et al., 1996).Recent evidence suggests that HCMV not only causes acute infection in transplanted patients but may also be associated with extensive transplant vasculopathy, ultimately leading to chronic allograft rejection (Grattan et al., 1989;Skowronski et al., 1993;Koskinen et al., 1993;Lautenschlager et al., 1997a;Schnitzler et al., 1997;Borchers et al., 1999). One of the molecular mechanisms underlying the acceleration of vascular disease processes by HCMV infection may be enhancement of transplant immunogenicity. Several studies indicate that this involves the HCMV-induced upregulation of cellular adhesion molecules, which augments the adherance and infiltration of inflammatory cells that are capable of promoting vascular disease (Einsele et al., 1994;Lemstrom et al., 1995;Steinhoff et al., 1995;Koskinen et al., 1996;Yilmaz et al., 1996; Martelius et al., 1998;Waldman et al., 1998;Lautenschlager et al., 1999;The et al., 2001). For instance, cell culture experiments have demonstrated that...

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Cytomegalovirus IE2 Protein Stimulates Interleukin 1β Gene Transcription via Tethering to Spi-1/PU.1

Cited by 37 publications

References 65 publications

Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

Regulation of Ets function by protein–protein interactions

Synergistic induction of intercellular adhesion molecule-1 by the human cytomegalovirus transactivators IE2p86 and pp71 is mediated via an Sp1-binding site

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