Non-proteolytic activity of 19S proteasome subunit RPT-6 regulates GATA transcription during response to infection

Abstract

GATA transcription factors play a crucial role in the regulation of immune functions across metazoans. In Caenorhabditis elegans, the GATA transcription factor ELT-2 is involved in the control of not only infections but also recovery after an infection. We identified RPT-6, part of the 19S proteasome subunit, as an ELT-2 binding partner that is required for the proper expression of genes required for both immunity against bacterial infections and recovery after infection. We found that the intact ATPase domain of RPT-6 is required for the interaction and that inhibition of rpt-6 affected the expression of ELT-2-controlled genes, preventing the appropriate immune response against Pseudomonas aeruginosa and recovery from infection by the pathogen. Further studies indicated that SKN-1, which is an Nrf transcription factor involved in the response to oxidative stress and infection, is activated by inhibition of rpt-6. Our results indicate that RPT-6 interacts with ELT-2 in vivo to control the expression of immune genes in a manner that is likely independent of the proteolytic activity of the proteasome.

Fig 1. RPT-6 is required for activation of immune genes during infection.

A. Control or RNAi treated P_F55G11.2::gfp animals were grown on E. coli OP50 for 12 hours. B. Fluorescence images were quantified using ImageJ software. Bars represent means ± SEM; n = 3 (t-test, ***P<0.0001). C. qRT-PCR analysis of immune genes in elt-2(RNAi) or rpt-6(RNAi) animals exposed to P. aeruginosa for 12 hours relative to control animals exposed to E. coli. Red bars correspond to gene expression in control animals exposed to P. aeruginosa for 12 hours relative to control animals exposed to E. coli. Error bars indicate means ± SEM; n = 3 (t-test *P<0.05, **P<0.01, ***P<0.001). D. Control, elt-2(RNAi), and rpt-6(RNAi) animals were exposed to P. aeruginosa and scored for survival, ***P<0.0001. E. qRT-PCR analysis of recovery genes elt-2(RNAi) or rpt-6(RNAi) animals exposed to P. aeruginosa and then recovered on E. coli plus streptomycin, relative to control animals on E. coli followed by E. coli plus streptomycin. Red bars correspond to gene expression in control animals exposed to P. aeruginosa and then recovered on E. coli plus streptomycin, relative to control animals on E. coli followed by E. coli plus streptomycin. Error bars indicate means ± SEM; n = 3 (t-test ***P<0.001). F. Control, elt-2(RNAi), and rpt-6(RNAi) animals were exposed to E. coli (Ec) or P. aeruginosa (Pa) for 12 hours, treated with streptomycin, and then transferred to E. coli plus Streptomycin plates and scored for survival. Scoring started 24 hours post initial exposure to E. coli or P. aeruginosa, ***P<0.0001.

Fig 2. Knockdown of rpt-6 activates a subset of SKN-1-dependent target genes and enhances survival against infection.

A. qRT-PCR analysis of SKN-1-dependent genes in rpt-6(RNAi) or elt-2(RNAi) animals exposed to E. coli relative to control animals grown on E. coli. B. qRT-PCR analysis of SKN-1-dependent genes in rpt-6(RNAi) or elt-2(RNAi) animals exposed to P. aeruginosa for 12 hours relative to control animals infected with P. aeruginosa for 12 hours. C. Control, elt-2(RNAi), rpt-6(RNAi), and rpt-6;skn-1 co-RNAi animals were exposed to P. aeruginosa and scored for survival, ***P<0.0001, NS = not significant. All bars represent means ± SEM; n = 3 (t-test *P<0.05, **P<0.01 ***P<0.001).

Fig 3. Immune activation of ELT-2-dependent genes is independent of proteasome activity.

A. qRT-PCR analysis of immune genes in bortezomib (BTZ) and DMSO-treated animals exposed to P. aeruginosa for 4 hours relative to control animals exposed to E. coli. Error bars indicate means ± SEM; n = 3 (t-test *P<0.05, **P<0.01, ***P<0.001). B. Nuclear ELT-2::GFP in control or rpt-6(RNAi) animals. C. Quantification of number of nuclear ELT-2::GFP in control and rpt-6(RNAi) animals. D. Quantification of nuclear ELT-2::GFP intensity (per animal) in control and rpt-6(RNAi) animals. Fluorescence was quantified using ImageJ software. Shown are results of three biological replicates, all bars represent means ± SEM; n = 13 (t-test, NS = not significant).

Fig 4. ELT-2 and RPT-6 physically interact in vivo.

A. Bimolecular fluorescence complementation (BiFC) signals of animals co-expressing ELT-2::VN173 and RPT-6::VC155. Animals carrying RPT-6::VC155 and VN173 without ELT-2 were used as control. RNAi inhibition of elt-2 and rpt-6 in ELT-2::VN173/RPT-6::VC155 was also used as control. B. BiFC signals of transgenic animals co-expressing ELT-2::VN173 and RPT-6::VC155 or RNAi-treated animals. C. Quantification of bimolecular fluorescence signals of transgenic animals co-expressing ELT-2::VN173 and RPT-6::VC155 and RNAi-treated animals. The results represent three independent experiments containing a total of 150 animals (one-way ANOVA Dunnett's multiple comparisons test; **** P<0.0001, *** P = 0.0002). D. BiFC signals of transgenic animals co-expressing ELT-2::VN173 with RPT-6::VC155 or ΔRPT-6::VC155. E. Quantification of bimolecular fluorescence signals of transgenic animals co-expressing ELT-2::VN173 with RPT-6::VC155 or ΔRPT-6::VC155. Shown are three independent experiments combined, with a minimum of 399 animals used in each case. Error bars indicate means ± SEM (one-way ANOVA test; **** P<0.0001). Quantification was done using the Copas Biosort instrument (Union Biometrica, Holliston, MA).