Interferon Regulatory Factor 8 (IRF8) Impairs Induction of Interferon Induced with Tetratricopeptide Repeat Motif (IFIT) Gene Family Members

Christine L White,Patricia M Kessler,Benjamin K Dickerman,Keiko Ozato,Ganes C Sen

doi:10.1074/jbc.m115.705467

Abstract

The chromosomally clustered interferon-induced with tetratricopeptide repeat motif (IFIT) gene family members share structural features at the gene and protein levels. Despite these similarities, different IFIT genes have distinct inducer- and cell type-specific induction patterns. Here, we investigated the mechanism for the observed differential induction of the mouse Ifit1, Ifit2, and Ifit3 genes in B cells and demonstrated that the repressive effect of the transcription factor interferon regulatory factor 8 (IRF8), which is highly expressed in B cells, played an essential role in this regulation. Although IRF8 could impair induction of all three IFIT genes following stimulation of retinoic acid-inducible gene I (RIG-I), it could selectively impair the induction of the Ifit1 gene following IFN stimulation. The above properties could be imparted to IRF8-non-expressing cells by ectopic expression of the protein. Induction of reporter genes, driven by truncated Ifit1 promoters, identified the regions that mediate the repression, and a chromatin immunoprecipitation assay revealed that more IRF8 bound to the IFN-stimulated response element of the Ifit1 gene than to those of the Ifit2 and the Ifit3 genes. Mutational analyses of IRF8 showed that its ability to bind DNA, interact with other proteins, and undergo sumoylation were all necessary to selectively repress Ifit1 gene induction in response to IFN. Our study revealed a new role for IRFs in differentially regulating the induction patterns of closely related IFN-stimulated genes that are located adjacent to one another in the mouse genome.

Highlights

The chromosomally clustered interferon-induced with tetratricopeptide repeat motif (IFIT) gene family members share structural features at the gene and protein levels
To confirm a cell-intrinsic basis for this observation and to start analyzing the mechanistic basis for impaired Ifit1 gene induction, IFIT family member induction was analyzed at the protein and RNA levels in cultures of splenocytes and purified B and T cells stimulated with IFN in vitro
Ectopic Expression of interferon regulatory factor 8 (IRF8) Impairs IFIT Family Gene Induction—In addition to the impaired Ifit1 gene induction observed in B cells stimulated in vivo [12] and in vitro (Fig. 1), impaired Ifit1 gene induction has been observed in plasmacytoid dendritic cells (pDCs) but not conventional dendritic cells [13]

Summary

Experimental Procedures

Mice—All experiments were conducted on C57Bl/6J mice between 8 and 12 weeks of age in accordance with protocols approved by the Cleveland Clinic Institutional Animal Care and Use Committee. Luciferase Promoter Reporter Assays—To analyze promoter activity, regions up to 1 kb upstream of the transcription start sites of the Ifit and Ifit genes (GenBankTM accession numbers: Ifit, NM_008331.3; Ifit, NM_008332.3) or the truncations/mutants indicated were cloned into the firefly luciferaseencoding pGL3 Basic vector (Promega), as described below These firefly luciferase constructs were cotransfected in a 10:1 excess with the pRL-TK vector containing Renilla luciferase into L929 cells stably expressing IRF8 (production described above) or vector controls. Microarray Analysis—For microarray experiments, RNA was prepared and quality-assessed as for real-time PCR from 5 ϫ 106 lentivirus-infected L929 cells stably expressing low level ectopic IRF8 (or vector control), generated as described above and treated with 1000 units/ml IFN␤ for 4 h. Statistics—p values from two-tail Student’s t tests and twoway analysis of variance with Tukey’s multiple-comparison test were calculated using GraphPad Prism statistical software (GraphPad)

Results

Primers for ChIP PCR cycling

Gene symbol

Discussion