Stabilization of RNT-1 protein, runt-related transcription factor (RUNX) protein homolog of Caenorhabditis elegans, by oxidative stress through mitogen-activated protein kinase pathway

Abstract

RUNX proteins are evolutionarily conserved transcription factors known to be involved in various developmental processes. Here we report a new role for a RUNX protein: a role in stress response. We show that RNT-1, the Caenorhabditis elegans RUNX homolog, is constantly produced and degraded by the ubiquitination-proteasome pathway in the intestine of the nematode. RNT-1 was rapidly stabilized by oxidative stress, and the rnt-1-mutant animals were more sensitive to oxidative stress, indicating that rapid RNT-1 stabilization is a defense response against the oxidative stress. The MAP kinase pathway is required for RNT-1 stabilization, and RNT-1 was phosphorylated by SEK-1/PMK-1 in vitro. ChIP-sequencing analysis revealed a feedback loop mechanism of the MAP kinase pathway by the VHP-1 phosphatase in the RNT-1-mediated oxidative stress response. We propose that rnt-1 is regulated at the protein level for its role in the immediate response to environmental challenges in the intestine.

FIGURE 1.

Continuous degradation of RNT-1 proteins in the intestine. A, the upper panel shows the genomic structure of the rnt-1 gene, and the bottom panels show the in situ hybridization results of rnt-1 at different developmental stages. The white line and the white dotted line in the adult indicate the intestine and gonadal arm, respectively. The red arrow in the upper panel represents the location of the in situ probe of rnt-1. A sense probe was used as the negative control. B, the top two rows show GFP expression in N2 animals containing the RNT-1::GFP transgene in the absence or presence of MG132, respectively. The third row shows the results of knockdown of ubq-1 (polyubiquitin), uba-1 (E1), and math-33 (ubiquitin-specific hydrolase) at the young adult stage. L4440 was used as s negative control. Scale bars = 50 μm. C, ubiquitination of RNT-1 in 293T cells. The ubiquitinated FLAG-tagged RNT-1 was detected in lanes 4 and 5. Treatment of MG132 increased ubiquitinated RNT-1 (lane 5).WB, Western blotting; IP, immunoprecipitation.

FIGURE 2.

Oxidative and osmotic stresses stabilize RNT-1::GFP in the intestine. A, oxidative stress (paraquat, t-butyl peroxide) and osmotic stress (NaCl, sucrose) stabilize RNT-1::GFP in wild-type animals expressing a RNT-1::GFP transgene. Heat shock, cold shock, hypoxia, or UV light did not affect the stability of RNT-1. B, time course changes in the RNT-1::GFP abundance after treating paraquat. Late L4 or young adults were used. C, quantification of the animals for their levels of stabilized RNT-1::GFP under oxidative stress conditions. The expressions of GFP in int1 cells were analyzed. Scale bars = 50 μm.

FIGURE 3.

RNT-1 is required for response to oxidative stress. A, survival rates of N2 and rnt-1(tm491) animals after 5-min exposure to 0.4 m paraquat for acute oxidative stress. B, stabilization of RNT-1 in the intestine by oxidative stress is dependent on nsy-1, sek-1, and pmk-1. ***, p <0.001.

FIGURE 4.

Purified PMK-1 can directly phosphorylate the C-terminal regions of RNT-1 in vitro. A, the result of the in vitro kinase assay of RNT-1 with SEK-1/PMK-1 kinases. B, domain studies of RNT-1 using GST-fused polypeptides containing 1–200, 201–250, or 251–301 amino acids. The asterisk is a nonspecific signal or a dimerized protein signal. The arrowhead is the signal of RNT-1 full sequence. The arrow is the GST-partial RNT-1 fusion protein signal. C, the result of the in vitro kinase assay with wild-type and the mutation of serine 255 to alanine of RNT-1.

FIGURE 5.

vhp-1 is a target of RNT-1 in oxidative stress response. A, chromatin immunoprecipitation quantitative PCR analysis on the promoter of vhp-1. The arrows indicate the locations of the putative RNT-1 binding sequences. In A, two putative RNT-1 binding sequences are closely located. B, relative mRNA levels of vhp-1 in the absence or presence of 0.1 m paraquat in wild-type N2 and rnt-1 mutant animals. C, effects of vhp-1 RNAi on the survival rates of N2 and rnt-1(tm491) in M9 buffer containing 0.1 m paraquat. Additional experiments are presented in supplemental Fig. 4. **, p <0.01; ***, p <0.001.

FIGURE 6.

The effect of the vhp-1 RNAi on the stability of RNT-1 in the oxidative stress condition. The photos show time course changes in the intestinal GFP of wild-type animals containing an RNT-1::GFP transgene in the control (L4440, upper panels) and vhp-1 RNAi background (bottom panels). Scale bar = 25 μm.

FIGURE 7.

A model of RNT-1 action in oxidative stress. Oxidative stress induces the transcription of vhp-1, a phosphatase, which, in turn, negatively regulates the stabilization of RNT-1 in time under the oxidative stress condition.