C. elegans nuclear receptor NHR-6 functionally interacts with the jun-1 transcription factor during spermatheca development

Abstract

The NR4A nuclear receptor NHR-6 is an essential regulator of spermatheca organogenesis in C. elegans. In this study, we perform a focused, RNAi-based screen to identify modifiers of partial nhr-6 loss of function. Ninety-eight genes that encode signaling proteins expressed in the spermatheca were screened for enhancement of the nhr-6 RNAi phenotype. We identify the C. elegans gene jun-1, which encodes the homolog of the Jun transcription factor, as a strong enhancer of nhr-6 partial loss of function. We show that nhr-6 and jun-1 function together to regulate development of the spermatheca and are necessary for generating an organ with the normal number of cells. jun-1 is expressed in all cells of the developing spermatheca. We also provide evidence that NHR-6 and JUN-1 can physically interact in a yeast two-hybrid assay. Our results provide in vivo evidence that NR4A nuclear receptor and Jun transcription factor interactions are essential in regulating developmental processes in metazoans.

Keywords: NR4A; RNAi interaction screen; modifier.

Figure 1. nhr-6 and jun-1 interaction during spermatheca development

DIC (a, c, e, g, i) and AJM-1∷GFP epifluorescence (b, d, f, h) micrographs of spermathecae from control, nhr-6(RNAi), jun-1(gk551), and jun-1(gk551), nhr-6(RNAi) animals. “P” indicates the proximal and “D” indicates the distal regions of the spermatheca. Some of spermatheca (Sp) nuclei in the plane of focus are indicated with arrows. When observed, sheath cell pair 5 nuclei (S5) are also indicated. Vu, vulva; Va, spermatheca-uterine valve; G, germline. In (i), two large, undivided spermathecal cells are indicated. Scale bar = 10µm.

Figure 2. Spermatheca nuclei number in jun-1(−); nhr-6(RNAi) animals

Spermatheca nuclei were counted in young adult animals expressing ajm-1∷GFP. N=7, 31, 27, and 30 spermathecae were counted for control, nhr-6(RNAi), jun-1(gk551), and jun-1(gk551); nhr-6 (RNAi) animals, respectively. Error bars, ± standard deviation. Due to the invariant nature of the spermatheca cell lineages, all control animals have 24 spermatheca nuclei. Spermathecal nuclei number was decreased in 100% of nhr-6 RNAi animals and in 85.2% of jun-1(−) animals. Spermatheca nuclei number was significantly decreased in the double versus single loss of function animals (p<.0001; One Way ANOVA).

Figure 3. Expression of jun-1∷GFP in the developing spermatheca

Epifluorescence (a, c, e) and DIC (b, d, f) micrographs of JUN-1∷GFP expression (strain OP234) in L4 and young adult animals. Nuclear-localized expression is seen in two rows of spermatheca (Sp) cells at mid-L4 (a, b). Expression in the uterus (Ut), sheath cell pairs 4 and 5 (S4 and S5), and in an intestinal nucleus (In) are indicated. Spermatheca expression is also observed in young adults. No expression is evident in adult valve (Va) cells but jun-1∷GFP is expressed in the spermatheca-uterine junction core (sujc) cell in L4 (e, f). The binucleate sujc cell eventually forms the core of the spermatheca-uterine valve. Contrast in (e) has been digitally enhanced to better define sujc expression.

Figure 4. NHR-6 and JUN-1 interact in a yeast two-hybrid assay

(a) Serial dilutions of transformed yeast cultures on selection plates. Positive control plasmids were provided with the ProQuest™ System. The JUN-1 protein encoded by the yeast two-hybrid expression plasmid corresponds to the JUN-1A isoform and lacks the bZIP region. The NHR-6 protein encoded by the yeast two-hybrid expression plasmid lacks the first 156 amino acids of the A/B domain. Negative control: pDEST™32 (empty) + pDEST™22 (empty) plasmids; NHR-6 (bait): pDEST™32 (GAL4DB-NHR-6) + pDEST™22 (empty) plasmids; JUN-1 (prey): pDEST™32 (empty) + pDEST™22 GAL4AD-JUN-1) plasmids; NHR-6/JUN-1: pDEST™32 (GAL4DB-NHR-6) + pDEST™22 (GAL4AD-JUN-1) plasmids. Positive control and NHR-6/JUN-1 transformants express the URA3 gene as shown by robust growth on Ura- plates and growth inhibition in the presence 5-FOA. (b) Expression of the lacZ reporter in the yeast two-hybrid assay. CPRG assays for the same transformants shown in (a), except that the positive control (provided by the ProQuest™ System) is for a weak interaction. NHR-6 (bait) shows some autoactivation in this assay but the NHR-6/JUN-1 experiment shows a significantly increased signal (p < .01(*); Student’s t test). Error bars, ± standard error. N = 3 independent assays for each experiment. (c) Both JUN-1A (FL) and JUN-1AΔbZIP interacts with the NHR-6 (same library expression clone (Lib.) as shown in (a) and (b). The JUN-1AΔbZIP is expressed from the library clone shown in (a) and (b). Both forms of JUN-1 fail to interact with NHR-6 A/B/C, which contains the same A/B/C domain sequences as the library clone but lacks the D and E domains, and NHR-6 D/E, which contains D/E domain sequence but lacks A/B/C domain sequences. Two independent transformants are shown for each test.