Biochemical and Biophysical Research Communications
The mechanism of action of Spi-B in the transcriptional activation of the interferon-α4 gene
Introduction
Dendritic cells (DCs) play essential roles in the connection between innate and adaptive immunity by being activated through pathogen sensors such as Toll-like receptors (TLRs) and by producing various cytokines [[1], [2], [3]]. DCs are divided into two subsets: plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) [4]. As a unique subset within the DC lineage, pDCs are capable of producing high levels of type I interferon (IFN) through TLR7 or TLR9 signaling [5]. Type I IFN contributes to antivirus defense and to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE), myositis, Sjögren’s syndrome (SS) and systemic sclerosis (SSc) [6].
pDCs express constitutively high levels of Spi-B, which is a member of the Ets family transcription factors [7,8]. Our earlier findings demonstrated that Spi-B plays a critical role in TLR7/9 signal-induced type I IFN production in pDCs [9]. Spi-B transactivates the type I IFN promoters in synergy with transcription factor IFN regulatory factor 7 (IRF-7). IRF-7 also plays a key role in the production of type I IFN in pDCs. IRF-7 is localized in the cytoplasm and activated by phosphorylation. The activated IRF-7 then translocates into the nucleus and binds to the promoter region of type I IFN genes [10]. The Ets family transcription factors consists of approx. 30 proteins that share a DNA-binding Ets domain which binds to the consensus sequence, 5′-GGAA/T-3’ [11,12]. Spi-B associates with IRF-7 through its Ets domain and synergistically augments the IRF-7-induced transactivation of the IFN-α promoter [9]. However, little is known about the mechanism of action of Spi-B. Herein, we analyzed the detailed molecular mechanisms of the Spi-B-mediated enhancement of the Ifna4 promoter activity.
Section snippets
Plasmids
The Ifna4 promoter-driven luciferase reporter plasmids were prepared as described [13]. Deletion mutants of the Ifna4 promoter were generated by polymerase chain reaction (PCR) and subcloned into the pGL3 vector (Promega, Madison, WI, USA). The Flag-tagged murine IRF-7 expression vector (pEF-BOS-FLAG-mIRF-7) and the HA-tagged murine Spi-B (HA-SpiB-IRES2-venus, HA-SpiBdTA-IRES2-venus and HA-SpiBdEts-IRES2-venus) were generated as described [9,13]. Point mutants in the Ifna4 promoter and
The synergistic enhancement of the Ifna4 promoter by Spi-B
Spi-B can synergistically enhance the IRF-7-induced transactivation of the Ifna4 promoter [9]. To investigate how Spi-B synergistically activates the Ifna4 promoter, we first searched for Spi-B binding site(s) in the Ifna4 promoter. Spi-B has a DNA-binding Ets domain. There are five potential Ets-binding motifs in the Ifna4 promoter; they consist of the purine-rich GGAA/T consensus sequences (−486 to −55) (Suppl. Fig. S1) [12]. We constructed two mutant reporter plasmids containing the Ifna4
Discussion
The initiation of transcription is regulated by two stages: chromatin remodeling, followed by the interaction of polymerase and accessory factors with the promoter [17]. Our present results identified an Spi-B binding site around position g–150 in the Ifna4 promoter. The nucleotide sequence we found is not compatible with the consensus sequence (GGAA/T). Our findings demonstrated that the binding of Spi-B to the Ifna4 promoter is necessary for the synergistic transactivation of the Ifna4
Funding
This work was supported by JSPS KAKENHI grant numbers JP26461465 (KH), JP17K15665 (HS), JP19K07482 (HS), the Suzuken Memorial Foundation, The NOVARTIS Foundation (Japan) for the Promotion of Science, and the Daiichi-Sankyo Foundation of Life Science. This work was also supported in part by a Grant for Medical Research, Faculty of Medicine, Kagawa University.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank T. Niki for technical assistance and E. Tanaka, M. Tanaka, Y. Matsuhisa and S. Haraguchi for secretarial assistance.
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