Akirin specifies NF-κB selectivity of Drosophila innate immune response via chromatin remodeling

Abstract

The network of NF-κB-dependent transcription that activates both pro- and anti-inflammatory genes in mammals is still unclear. As NF-κB factors are evolutionarily conserved, we used Drosophila to understand this network. The NF-κB transcription factor Relish activates effector gene expression following Gram-negative bacterial immune challenge. Here, we show, using a genome-wide approach, that the conserved nuclear protein Akirin is a NF-κB co-factor required for the activation of a subset of Relish-dependent genes correlating with the presence of H3K4ac epigenetic marks. A large-scale unbiased proteomic analysis revealed that Akirin orchestrates NF-κB transcriptional selectivity through the recruitment of the Osa-containing-SWI/SNF-like Brahma complex (BAP). Immune challenge in Drosophila shows that Akirin is required for the transcription of a subset of effector genes, but dispensable for the transcription of genes that are negative regulators of the innate immune response. Therefore, Akirins act as molecular selectors specifying the choice between subsets of NF-κB target genes. The discovery of this mechanism, conserved in mammals, paves the way for the establishment of more specific and less toxic anti-inflammatory drugs targeting pro-inflammatory genes.

Keywords: Chromatin remodeling; Drosophila; Innate immune response; NF‐κB.

Figure 1. Akirin influences the expression of only a subset of Relish target genes

A, B Venn diagram (A) and table representation of microarray analysis (B). Genes in PGRP-LC-stimulated S2 cells showing a twofold reduction of their expression upon knockdown of relish or akirin compared to control (dsRNA against GFP). Numbers in brackets correspond to genes with GO terms matching immune function. Red corresponds to anti-microbial peptides, blue to negative regulators of NF-κB pathways, and beige to other immune-related functions. C Quantitative RT–PCR of Pirk, Attacin-D, Attacin-C, and Diptericin-A mRNA from sorted Drosophila S2 cells co-transfected with dsRNA against GFP, relish or akirin, and a PGRP-LCa overexpressing vector to stimulate the IMD pathway. Data information: Data are represented as mean ± standard deviation of three independent experiments performed with 1–5 × 10⁵ S2 cells. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.

Figure 2. Akirin interacts with Bap60 upon immune challenge and activates selected Relish target gene promoters

A Schematic representation of two-hybrid results and subsequent functional assay. Proteins interacting with full-length (AK) or N-terminally truncated (AK^Δ1–139) Akirin in two-hybrid assay. Proteins interacting with bait constructs encompassing full-length, or AK^Δ1–139 were tested for their ability to modulate the IMD pathway. Genes leading to increased IMD pathway activation when knocked down were identified as IMD negative regulators. Genes leading to decreased IMD pathway activation when knocked down were identified as positive IMD regulators. B, C Dual luciferase assay from S2 cell co-transfected with attacin-A- (B) or attacin-D-luciferase (C) reporter plasmids and dsRNAs targeting GFP, kenny (key), akirin, and Akirin’s putative partners extracts following 48 h of heat-killed E. coli stimulation. Data, normalized to dsRNA GFP controls, were from three independent experiments performed with 5 × 10⁵ S2 cells. D Whole-cell lysates from S2 cells stimulated with heat-killed E. coli at indicated time points were immunoprecipitated with anti-Bap60 or anti-Akirin antibodies. Whole-cell lysate (input, left panel) and immunoprecipitated samples (right panel) were immunoblotted and probed with antibodies against Bap60 and Akirin. Data information: Data are represented as mean ± standard deviation from three independent experiments. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.

Figure 3. Akirin, Relish, and Bap60 overlap non-condensed DNA regions in fat body cells

(A-D’) Fat body cells from adult Drosophila were visualized by DIC (A, F, K, P, U, Z). Immunolocalization of Akirin (C and H), Relish (M and R), Bap60 (W and B′), H3K9me2 (D, N, X), the active chromatin marker H3K9ac (I, S, C′), and DAPI staining (B, G, I, Q, V, A′) in whole fat body, 6 h after an immune challenge with E. coli. Akirin, Relish, and Bap60 sub-nuclear localizations were mostly excluded from DAPI-rich regions but partially overlapped H3K9ac regions (arrowheads) (E, J, O, T, Y, D′). In addition, Akirin systematically overlapped a small region in H3K9me2 distribution (arrows). Data information: Images are representative of at least 3 fat body samples. Scale bars (all panels): 5 μm.

Figure 4. Heat-killed E. coli challenge stabilizes the interaction between Akirin and Relish

A, B Reciprocal co-immunoprecipitation assays between ectopic Akirin and Relish in S2 cells. Wild-type S2 cells were transiently transfected with V5-tagged Akirin and Flag-tagged RelishΔS29-S45. Cell lysates were immunoprecipitated with (A) anti-FLAG coupled or (B) anti-V5 coupled agarose beads. Immunoprecipitates were analyzed by Western blotting with anti-V5 or anti-Flag antibodies. C Heat-killed E. coli (HKE) promote the interaction of Akirin with Rel-68. S2 cells stably expressing V5-tagged Akirin were treated with heat-killed E. coli at the indicated time points. Cell lysates were immunoprecipitated using anti-V5 coupled agarose beads. Endogenous Relish was detected in Akirin immunoprecipitates using anti-Relish antibody. Data information: Data are representative of 3 independent experiments.

Figure 5. Akirin, Bap60, and Relish bind on Akirin-dependent immune gene promoters

A–F Chromatin IP with anti-Akirin, anti-Bap60, anti-Relish, and anti-H3K4ac antibodies on sheared chromatin from S2 cells following heat-killed E. coli stimulation at indicated time points. The graphs show recruitment of Akirin, Bap60, and Relish, relative to the values obtained with rabbit control IgG, or of H3K4ac relative to the values obtained with anti-H3 antibody on Akirin-dependent (A, p-attacin-A; C, p-drosocin; D, p-cecropin-A1), Akirin-independent (E, p-attacin-D; F, p-metchnikowin) genes proximal promoter, or on attacin-A coding sequence (B) as an internal control. G–J Chromatin IP with anti-Relish and anti-H3K4ac antibodies on sheared chromatin from S2 cells knocked down for GFP, relish, akirin, or bap60 following heat-killed E. coli stimulation at indicated time points. The graphs show recruitment of Relish (I, J) relative to the values obtained with rabbit control IgG, or of H3K4ac (G, H) relative to the values obtained with anti-H3 antibody on two Akirin-dependent (G, I, p-attacin-A; H, J, p-drosocin) proximal promoters. K Bioinformatical CpG-rich region analysis of Akirin-dependent and Akirin-independent promoters. Predicted CpG-rich regions were counted on the genomic regions −1 kb to +1 kb relative to the transcription start site for Akirin-dependent and Akirin-independent genes with Cpgplot (EMBOSS). Red squares annotate genes containing at least one CpG-rich region within its promoter. L Pie chart representation of CpG-rich region analysis of Akirin-dependent and Akirin-independent promoters. Red areas annotate genes containing at least one CpG-rich region within its promoter. Data information: Data are represented as mean ± standard deviation of three independent experiments performed on 1.5 × 10⁶ (A–F) or 5 × 10⁵ cells (G–J) per IP. Hk E. coli: heat-killed E. coli. TSS: transcriptional start site. Statistical significance was established by comparing values from stimulated (15 min, 1 h, 2 h of hk E. coli) with unstimulated conditions (NS) (A-F) or comparing Relish, Akirin, and Bap60 knockdown with GFP dsRNA control in stimulated (2 h hk E. coli) conditions (G-J). *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.

Figure 6. The Brahma BAP complex is required for Akirin-dependent immune response against Gram-negative bacteria

A, B Dual luciferase assay from S2 cell extracts co-transfected with attacin-A- (A) or attacin-D-luciferase (B) reporter plasmids and dsRNAs against GFP, kenny (key), akirin, or Brahma complex members following 48 h of heat-killed E. coli stimulation. C Dual luciferase assay from S2 cell extracts co-transfected with drosomycin-luciferase, Toll^ΔLRR pActin5C expressing vector, and dsRNAs against GFP, kenny (key), akirin, or Brahma complex members. Results were normalized to the dsRNA GFP controls. D–I Quantitative RT–PCR of Attacin-A, Attacin-C, Diptericin-A, Cecropin-A2, Cecropin-B, and Pirk mRNA on C564-gal4/UAS-RNAi flies following an E. coli 6-h challenge. J, K Survival assays following E. cloacae (J) or E. carotovora Ecc15 (K) septic infection of C564-gal4/UAS-RNAi flies. L Survival assay from E. carotovora Ecc15 septic infection of relish^E20, brahma², and moira¹ heterozygous or trans-heterozygous mutant flies. M, N Lethality calculations following E. cloacae (M) or E. carotovora Ecc15 (N) septic infection of C564-gal4/UAS-RNAi flies. O Lethality calculation from E. carotovora Ecc15 septic infection of relish^E20, brahma², and moira¹ heterozygous or trans-heterozygous mutant flies. Data information: Data are represented as mean ± standard deviation of three independent experiments performed with three batches of 15–20 flies. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.