Caenorhabditis elegans nuclear hormone receptor NHR-14 , cooperates with p 53 / cep-1 to regulate DNA damage-induced apoptosis

Nuclear hormone receptor is involved in transcription regulation and many important cellular processes including development and metabolism. However, the role of nuclear hormone receptor in DNA damage-induced apoptosis remains elusive. Here we reported that RNAi of nhr-14, which was thought to be an estrogenic hormone receptor in Caenorhabditis elegans, inhibited DNA damage-induced apoptosis in prmt5(gk357), a C. elegans homolog of mammalian type II arginine methyltransferase PRMT5, after ionizing radiation. Deletion of nhr-14 led to decreased DNA damageinduced germline apoptosis, but not in the physiological programmed cell death. We also demonstrate that nhr-14 is not a checkpoint gene and functions downstream of the checkpoint pathway. Moreover, we show that nhr-14 regulates egl-1 and ced13 transcription upon DNA damage. In addition, we provided evidence that NHR14 forms a complex with CEP-1/p53 and may function as a cofactor of CEP-1/p53. These findings indicate that NHR-14 might cooperate with CEP-1/p53 to regulate DNA damage-induced apoptosis, which reveals a novel role for nuclear hormone receptor


INTRODUCTION
Programmed cell death (i.e., apoptosis) is one of the most important processes in the metazoans development.It plays the key roles in animal development and DNA damage repair.DNA damage-induced apoptosis is the cell death happening after severe DNA damage, which is associated with a number of human diseases including cancer.Caenorhabditis elegans has been used extensively to study the programmed cell death induced by DNA damage responses.In C. elegans, the p53 homolog CEP-1 acts as a key effector to mediate germ cell apoptosis triggered by ionizing irradiation [1].Although many factors have been reported to be involved in p53/cep-1 dependent apoptotic pathway, the details of this pathway is still not completely understood.
Nuclear hormone receptors (NHRs) comprise of a large family of transcription factors distinguished by a highly conserved DNA binding domain and a structurally conserved ligand-binding domain.There are 284 predicted www.impactjournals.com/oncotarget/Oncotarget, Advance Publications 2017 www.impactjournals.com/oncotargetNHR genes in C.elegans [2].Nuclear hormone receptors have been shown to regulate important developmental process [3][4][5][6].However, the role of NHR in programmed cell death has not been documented.
We previously demonstrated that prmt-5, the C. elegans homolog of mammalian type II arginine methyltransferase PRMT5, negatively regulates DNA damage-induced apoptosis [7].prmt-5(gk357) deletion mutants have increased germline programmed cell death after DNA damage.Furthermore, genetic analyses indicated that prmt-5-mediated apoptosis depends on cep-1/p53 and requires the core cell death pathway.
In the present study, we show that RNAi knockdown of nhr-14/HNF4, which is thought to be an estrogen receptor in C. elegans [8], suppresses DNA damageinduced apoptosis in prmt-5(gk357) deletion mutant.Further, we show that nhr-14/HNF4 is a new factor involved in the DNA damage-induced apoptosis and that nhr-14 is not a checkpoint gene and functions downstream of the checkpoint genes.Our study confirmed that NHR-14/HNF4, cooperates with CEP-1/p53 to regulate egl-1 (Bcl-2 homology region 3 domain containing gene) and ced-13 (Bcl-2 homology region 3 domain containing gene) expression and DNA damage-induced apoptosis, which may offer clinical target for cancer therapy.

Inactivation of nhr-14/HNF4 inhibits DNA damage-induced apoptosis
To examine whether nuclear hormone receptor is directly involved in the regulation of DNA damageinduced apoptosis, we performed RNAi screen in the background of prmt-5(gk357).We found that knockdown of nhr-14/HNF4 RNAi reduced the DNA damage-induced programmed cell death in prmt-5(gk357) (Figure 1A) after ionizing irradiation.
C. elegans nhr-14 gene is defined by the open reading frame T01B10.4located on the linkage group X, which encodes a protein of 435 amino acids.nhr-14(tm1473)contains a deletion of 409bp in the third exon and intron resulting in an early stop of NHR-14 translation [8].
In order to test the function of nhr-14/HNF4 in DNA damage-induced apoptosis, we used nhr-14(tm1473) deletion mutant to analyze the germ cell apoptosis after ionizing irradiation, we found that nhr-14(tm1473) can inhibit DNA damage-induced apoptosis in prmt-5(gk357) at different gamma-irradiation dose (Figure 1B) and different time (Figure 1C), suggesting that nhr-14 functions downstream of prmt-5 and regulates DNA damage-induced programmed cell death.
In addition, because brc-1 is the BRCA1 homolog in C.elegans and functions in DNA double strand break (DSB) repair [13,14] after ionizing gamma-irradiation, mutation of brc-1/BRCA1 resulted in failing to repair the double strand break and induce apoptosis.We also found that brc-1(tm1145);nhr-14(tm1473) double mutant exhibited dramatic decreased apoptosis compared to brc-1(tm1145) alone after DNA damage (Figure 2C).Taken together, these findings indicate that nhr-14/HNF4 is a key regulator of DNA damage-induced programmed cell death.

nhr-14/HNF4 doesn't affect physiological programmed cell death
Since nhr-14(tm1473) showed less apoptosis upon gamma-irradiation, we next investigated underlying cellular mechanism.We performed the time lapse phenotype analysis and found that there was no germline development defect and nhr-14(tm1473) showed the same apoptosis number as N2 at any time.These data indicate the decreased programmed cell death in nhr-14(tm1473) is neither due to germline development nor the delayed cell death.We further examined whether nhr-14 affects the physiological programmed cell death in embryo.Figure 3A shows that there was no difference in the number of cell apoptosis in embryo between N2 and nhr-14(tm1473).ced-1(e1735) [15] and vps-18(tm1125) [16] has been reported to affect cell corpse clearance, we also found no difference in the number of cell apoptosis in germline between wild type and nhr-14(tm1473) mutant in the background of ced-1(e1735) [16] and vps-18(tm1125) [17] (Figure 3B, 3C).These results indicate that nhr-14 /HNF4 only affects the DNA damage-induced apoptosis, but not the physiological programmed cell death.

nhr-14/HNF4 functions downstream of the checkpoint pathway
Previous studies demonstrated that the checkpoint signaling pathways are activated upon DNA damage and play the critical role in repairing the damaged DNA or inducing programmed cell death [17,18].Mutations in checkpoint genes can restrain both DNA damage-induced cell cycle arrest and apoptosis upon gamma-irradiation in C. elegans [17].To determine whether nhr-14/HNF4 is a checkpoint gene, we first assessed the sensitivity of nhr-14(tm1473) mutant to gamma-irradiation using radiation sensitivity assay.We found that the survival rate of nhr-14(tm1473) progeny was comparable to that of wild-type animals, but was much higher than that of checkpoint gene mutant hus-1(op244) and clk-2(mn159) (Table 1).In addition, nhr-14(tm1473) worms displayed similar cell cycle arrest in germline mitotic region to that in wild type following irradiation-treatment (Figure 4A).We further made hus-1(op244); nhr-14(tm1473) and clk-2(mn159);nhr-14(tm1473) double mutants, and found that these double mutants exhibited the same phenotype as the check point mutants (Figure 4B).Therefore, nhr-14/HNF4 is not a checkpoint gene and may function downstream of the checkpoint pathway.
To determine whether nhr-14/HNF4 is involved in DNA repair, we irradiated worms containing the hus-1::gfp transgene in nhr-14(tm1473) background.We found that relocalization of HUS-1::GFP was independent of nhr-14/HNF4 (Supplementary Figure 1A), and the number of foci in nhr-14(tm1473) was the same as wild type N2(Supplementary Figure 1B).nhr-14(tm1473) is defective in irradiation induced apoptosis but is wild type for irradiation induced cell cycle arrest (Figure 4) and DNA repair.These findings suggest that nhr-14/HNF4 is not involved in DNA repair and acts downstream of the checkpoint genes.

DISCUSSION
DNA damage-induced programmed cell death is associated with various human malignancies and identification of regulators in DNA damage-induced apoptosis pathway is critical for intervention of these diseases.C.elegans has been shown to be an excellent model to study DNA damage-induced programmed cell death.And thus it is very helpful for us to understanding the mechanism of carcinogenesis by studying the regulation of DNA damage-induced apoptosis in C.elegans germline.P53 is a key tumor suppressor and its mutations were detected in more than 50% of human cancers.In C.elegans, the p53 homolog CEP-1 acts as a key effector to mediate germ cell apoptosis triggered by ionizing irradiation [21].Identification of new co-factors of CEP-1/p53 in C.elegans may offer critical targets for cancer intervention.
In response to DNA damage stimuli, the checkpoint genes will sense the signals and induce cell cycle arrest or programmed cell death.Simultaneously, CEP-1/p53 is activated and subsequently induces up-regulation of BH3 genes egl-1 and ced-13.Mutation of the checkpoint genes block the transfer of DNA damage signals and reduce DNA damage-induced apoptosis.Nuclear hormone receptor family is a key to many important cellular processes, but the role of NHR family in DNA damageinduced programmed cell death remains elusive.Previous study showed that NHR-14/HNF4, which was thought to be an estrogenic hormone receptor [8], was involved in the immune response processes via regulation of vitellogenin expression [22].In present report, we identified nhr-14/ HNF4 as an important member of NHR in regulation of DNA damage-induced apoptosis.Moreover, we showed that nhr-14/HNF4 is primarily involved in regulation of the DNA damage-induced apoptosis, but not the physiological programmed cell death (Figure 3).

Germ cell apoptosis assay
Synchronized young adult animals were irradiated with gamma-Ray (120Gy), which was located in the Peking University Health Science Center.Irradiated animals were put back to culture at 20°C at different time points.Worms with normal germline morphology were scored for germline cell apoptosis with DIC Zeiss microscope.

Western blot assay
Cells were scraped and lysed in lysis buffer on ice for 15 min, 15 μg total proteins were loaded on SDS-PAGE Gel as co-immunoprecipitation experiment input.The SDS-PAGE gel first run on 60V for 30min and then 120 V until the dye run out of the gel, then the protein was transferred to PVDF membrane.The membranes were blocked in 5% nonfat dry milk in Tris-buffered saline, 0.05% Tween for 30 minutes at room temperature, and then incubated with primary antibodies for 2~4hours at 4°C, followed by incubation with secondary antibody for 60 min at RT.The primary antibodies used in this study were as follows: anti-Flag (Sigma, Cat#:F3165) and anti-Myc (Sigma, Cat#:HPA055893).

GST pull-down assay
For GST pull-down assay, purified GST or GST-CEP-1 fusion proteins were immobilized on glutathione-Sepharose beads and incubated with [35S] methioninelabeled NHR-14 at 4°C for more than 2 h.The beads were washed extensively and bound proteins were eluted and separated on 12% SDS-PAGE and exposed to phosphoimager (Amersham) for autoradiography.
All experiments were analyzed in triplicates.

Table 1 : nhr-14 doesn't affect the survival of progeny after gamma-irradiation treatment the survival of nhr-14(tm1473) mutant progeny is not sensitive to irradiation
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