Promoter Binding and Nuclear Retention Features of Zebrafish IRF Family Members in IFN Response

Abstract

Interferon regulatory factors (IRFs) constitute a family of transcription factors that synchronize interferon (IFN) antiviral response through translocating to nucleus and binding to the promoters of IFN and IFN-stimulated genes (ISGs). Fish contain 11 IRF members; however, whether or how fish IRF family genes function in IFN response remains limited. Herein, we determine the regulatory roles of 11 zebrafish IRF family members in IFN response relevant to their subcellular localization and promoter binding. Zebrafish IRF family members display three patterns of constitutive localization, only in nucleus (IRF1/2/9/11), only in cytoplasm (IRF3/5/7), and largely in nucleus with small amounts in cytoplasm (IRF4b/6/8/10). DNA pull-down assays confirm that all zebrafish IRF proteins are capable to bind fish IFN promoters, albeit to various degrees, thus regulating IFN gene transcription as activators (IRF1/3/5/6/7/8/9/11) or repressors (IRF2/4b/10). Further characterization of distinct IFN gene activation reveals that IRF1/3/5/6/7/8/9/11 efficiently stimulate zebrafish IFNφ1 expression, and IRF1/7/11 are responsible for zebrafish IFNφ3 expression. Two conserved basic residues within the helix α3 of DNA binding domains (DBDs) contribute to constitutive or inducible nuclear import for all zebrafish IRF family members and DNA binding for most members, thereby enabling them to function as transcription factors. Our results reveal a conserved and general mechanism that specifies zebrafish IRF family proteins to nuclear import and DNA binding, thereby regulating fish IFN response.

Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

“…To strengthen the findings above, nuclear-cytoplasmic separation experiments were performed to detect nuclear translocation of IRF3, a landmark event of RLR-triggered IFN response ( 23 , 42 ). Successful nuclear-cytoplasmic separation was verified by detection of Lamin A/C only in nucleus, and α-tubulin mostly in cytoplasm ( Figure 2E ).…”

“…Successful nuclear-cytoplasmic separation was verified by detection of Lamin A/C only in nucleus, and α-tubulin mostly in cytoplasm ( Figure 2E ). Fish IRF3 is a typical IFN-inducible protein ( 23 , 42 ). Consistently, overexpression of DrLGP2 alone in EPC cells increased the protein level of IRF3 in cytoplasm (lanes 2 and 3 versus lane 1 in Figure 2E ) and also in nucleus (lanes 5 and 6 versus lane 4 in Figure 2E ), indicating that DrLGP2 alone indeed activates IFN response in fish cells.…”

“…Transfection assays were performed with polyethylenimine, linear (PEI, MW25000; Aldrich, 1μg/μl of storage concentration) according to our studies previously ( 36 , 41 , 42 ). Typically, fish cells were seeded overnight in 24-wells plates, transfected with various plasmids at a ratio of 10:10:1 (promoter-driven luciferase plasmid/expression plasmid/Renilla luciferase plasmid pRL-TK).…”

Siniperca chuatsi Rhabdovirus (SCRV)-Induced Key Pathways and Major Antiviral Genes in Fish Cells

“…In the present study, the up-regulation of five IRFs (IRF1/3/7/9/10) was observed in SCRV-infected SCSC cells. It has been reported that fish contain 11 IRFs [30]. IRF3 and IRF7 are the two main regulators of IFN expression, which are triggered by PRRs [31].…”