Caenorhabditis elegans NHR-14/HNF4α regulates DNA damage-induced apoptosis through cooperating with cep-1/p53

Nuclear hormone receptors are involved in transcriptional regulation and many important cellular processes including development and metabolism. However, its role in DNA damage-induced apoptosis remains elusive. Synchronized young adult animals were irradiated with different doses of gamma-Ray, and then put back to culture at 20 °C. Germline cell apoptosis was scored at different time point. Deletion of nhr-14 led to decreased DNA damage-induced germline apoptosis, but not the physiological programmed cell death. We also demonstrate that nhr-14 functions downstream of the DNA damage checkpoint pathway. Moreover, we show that nhr-14 regulates egl-1 and ced-13 transcription upon DNA damage. Mechanistically, NHR-14 forms a complex with CEP-1/p53 and binds directly to the egl-1 promoter to promote egl-1 transcription.. Our results indicate that NHR-14/HNF4α cooperates with CEP-1/p53 to regulate DNA damage-induced apoptosis. 3SXEF2qkT2QHPu1N_hpJxU Video abstract Video abstract

NHR-86 controls anti-pathogen responses [8], and NHR-49 controls fat consumption and fatty acid composition in C. elegans [9]. NHR-14, an orphan receptor, has been reported to regulate innate immunity and iron uptake [10]. However, the role of NHR-14 in programmed cell death has not been documented.
Programmed cell death (i.e., apoptosis) is one of the most important processes in the metazoans development. It plays key roles in animal development and DNA damage repair. DNA damage-induced apoptosis is cell death after severe DNA damage, which is associated with a number of human diseases including cancer. Caenorhabditis elegans has been used extensively to study programmed cell death induced by DNA damage responses. We previously demonstrated that prmt-5, the C. elegans homolog of mammalian type II protein arginine methyltransferase PRMT5, negatively regulates DNA damageinduced apoptosis [11]. 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 C. elegans, the p53 homolog CEP-1 acts as a key effector to mediate germ cell apoptosis triggered by ionizing irradiation [12]. Although many factors have been reported to be involved in p53/cep-1-dependent apoptotic pathway, the details of this pathway are yet to be completely understood.
In the present study, we show that RNAi knockdown of nhr-14 suppresses DNA damage-induced apoptosis in prmt-5(gk357) deletion mutants. Further, we show that nhr-14 is a new factor involved in 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 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 damageinduced apoptosis, which reveals a novel role and mechanism for NHR-14/HNF4α in apoptosis. Dysregulation of DNA damage induced apoptosis has been reported to closely correlated tumorigenesis. Our study might provide new strategy and targets for prevention and therapy of tumor.

Germ cell apoptosis assay
Synchronized young adult animals were irradiated with gamma-Ray (120 Gy), 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 a DIC Zeiss microscope. The apoptotic cells showed button-like morphology under the DIC microscope and the number of apoptotic cells were scored.

Western blot assay
Cells were scraped and lysed in lysis buffers on ice for 15 min, 15 μg total proteins were loaded on SDS-PAGE gels as co-immunoprecipitation experiment input. The SDS-PAGE gel was first run on 60 V for 30 min and then 120 V until the dye ran out of the gel, then the protein was transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk in Tris-buffered saline, 0.05% Tween for 30 min at room temperature, and then incubated with primary antibodies for 2-4 h at 4 °C, followed by incubation with secondary antibodies for 60 min at room temperature. The results were detected by an ECL-plus Western blotting detection system (Tanon-5200Multi). The primary antibodies used in this study were as follows: anti-Flag (Sigma, Cat#:F3165); anti-Myc (Sigma, Cat#:HPA055893); GAPDH (Santa CruZ, Cat#: sc-32233).

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] methionine-labeled 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.

Genomic SELEX assay
SELEX assay was done according to our previous report [13]. C. elegans genomic DNA was digested by MseI, then MseI adaptor were ligated on both sides of the digested products. The DNA fragment library was amplified by PCR using MseI adaptor primers. After GST-NHR-14 (1-87 AA) and amplified DNA fragment library were incubated for 1 h, the nonspecific binding DNA fragments were washed off with washing solution, and then the specifically bound DNA fragments were amplified for the next round of SELEX screening. After 14 rounds of screening, the obtained protein specifically binds to the DNA binding domain of GST-NHR-14 DNA fragments were recovered, cloned into T vectors and sequenced. The obtained sequences were analyzed by meme software (http:// meme. sdsc. edu/ meme4_1/ cgi-bin/ meme. cgi).

Statistical analysis
All the experiments were repeated three times and each experiment was performed in 3 replicates per sample. Data were analyzed using SPSS 19.0 and GraphPad Prism 6.0. Student's t-test, Spearman correlation, Kaplan-Meier, log-rank test and Cox regression survival and Statistical significance was defined as *P < 0.05, **P < 0.01 or ***P < 0.001.

Inactivation of nhr-14/HNF4α inhibits DNA damage-induced apoptosis
To examine whether nuclear hormone receptor is directly involved in the regulation of DNA damage-induced apoptosis, we performed RNAi screen in the background of prmt-5(gk357). We found that knockdown of nhr-14/ HNF4α reduced the DNA damage-induced programmed cell death in prmt-5(gk357) (Fig. 1A) after ionizing irradiation. nhr-14 RNAi reduced about 75% of the nhr-14 mRNA level (Fig. 1B). Further analysis showed that the C. elegans nhr-14 gene is defined by the open reading frame T01B10.4 located on the linkage group X, and encodes a protein of 435 amino acids. The nhr-14(tm1473) deletion mutant contains a deletion of 409 bp in the third exon and third intron of nhr-14, and this deletion will result in an early stop of NHR-14 translation [10].
In addition, brc-1 is the BRCA1 homolog in C. elegans and functions in DNA double-strand break repair after gamma-irradiation [18,19]. Mutation of brc-1/ BRCA1 resulted in failing to repair the double-strand break and induced germ cell apoptosis. We also found that the brc-1(tm1145); nhr-14(tm1473) double mutant dramatically decreased germ cell apoptosis compared to brc-1(tm1145) alone after DNA damage (Fig. 2D).

nhr-14/HNF4α does not affect physiological programmed cell death
Since nhr-14(tm1473) showed less apoptosis upon gamma-irradiation, we next investigated the 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 embryos. Figure 3A shows that there was no difference in the number of cell apoptosis in embryos between N2 and nhr-14(tm1473). ced-1(e1735) [20] and vps-18(tm1125) [21] 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) mutants in the background of ced-1(e1735) and vps-18(tm1125) (Fig. 3B, C). In order to further prove that nhr-14 does not affect germline physiological programmed cell death, we analyzed the expression difference of ced-3, ced-4 and ced-9 in N2 and nhr-14(tm1473) by q-PCR, our results showed that nhr-14 did not affect the mRNA levels of these three genes (Fig. 3D). These results indicate that nhr-14/HNF4α only affects the DNA damage-induced apoptosis, but not the physiological programmed cell death.

nhr-14/HNF4α 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 [22,23]. Mutations in checkpoint genes can restrain both DNA damage-induced cell cycle arrest and apoptosis upon gamma-irradiation in C. elegans [22]. Checkpoint mutants also showed embryonic lethality following gamma-irradiation [22]. HUS-1 is a Caenorhabditis elegans DNA damage checkpoint protein required for genome stability and CEP-1/p53-dependent activation of a BH3 domain protein in C. elegans [23].To determine where nhr-14/HNF4α functions in response to DNA damage, we first assessed the sensitivity of nhr-14(tm1473) mutants to gamma-irradiation using the 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 mutants 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 (Fig. 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 (Fig. 4B). These results indicate that nhr-14 is necessary for irradiationinduced apoptosis, but not for irradiation-induced cell cycle arrest. Our findings suggest that nhr-14/HNF4α is not involved in DNA repair and acts downstream of the checkpoint genes.
To further confirm our result, we employed dual luciferase assays to see if coexpression of NHR-14 and CEP-1 can promote egl-1 promoter-driven luciferase activity. We first performed the SELEX (systematic evolution of ligands by exponential enrichment) assay [13] to explore the NHR-14 bound DNA conserved sequence. After sequencing the NHR-14 binding sequence, we found that NHR-14 could bind to the" AANTTC AAA " motif (Fig. 6A), which is located on the egl-1 promoter region between − 950 to − 942 (Fig. 6B). CEP-1 has been reported to bind to the RRR CWW GYYY motif [26,27], which locates on the egl-1 promoter between − 1651 to − 1642 (Fig. 6B). The luciferase assay indicated that overexpression of NHR-14 or CEP-1 can increase the egl-1 promoter-driven luciferase activity, and coexpression of NHR-14 and CEP-1 has much higher luciferase activity than expression of NHR-14 or CEP-1 alone (Fig. 6C). These data suggest that NHR-14/HNF4α and CEP-1/p53 might directly interact with each other to regulate egl-1 and ced-13 transcription. However, considering that the CEP-1 and NHR-14 bindings sites are relatively far from each other, we cannot exclude the possibility that CEP-1 and NHR-14 drive egl-1 transcription in a manner independent of their direct interaction.

Discussion
DNA damage-induced programmed cell death is associated with various human malignancies and identification of regulators in the 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 understand the mechanism of carcinogenesis by studying the regulation of DNA damage-induced apoptosis in C. elegans germlines.
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 [28]. 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 the NHR family in DNA damage-induced programmed cell death remains elusive. Previous studies showed that NHR-14/HNF4α, which was thought to be an estrogenic hormone receptor [10], was involved in the immune response processes via regulation of vitellogenin expression [29]. In the present report, we identified nhr-14/HNF4α as an important member of NHR in the regulation of DNA damage-induced apoptosis. Moreover, our results indicated that nhr-14/HNF4α is involved in regulation of the DNA damage-induced apoptosis, but not the physiological programmed cell death (Fig. 3).
Mechanically, our experiments revealed that nhr-14/ HNF4α regulates DNA damage-induced transcription of egl-1 and ced-13. More significantly, we showed that NHR-14/HNF4α interacts with CEP-1/p53 and might function as a cofactor of CEP-1/p53. However, considering that the CEP-1 and NHR-14 bindings sites are relatively far from each other, it is possible that there are shared or closely spaced CEP-1 and NHR-14 sites in the egl-1 promoter region that we have not identified by the SELEX method. Another possibility is that CEP-1 and NHR-14 regulate egl-1 transcription independent of their direct interaction. In addition, the nhr-14(tm1473) mutant dramatically reduces CEP-1/p53-mediated DNA damage-induced apoptosis. Thus we consider that nhr-14 is a general positive regulator of DNA damage-induced germline apoptosis. Our study first reported a nuclear hormone receptor NHR-14/HNF4α that is involved in DNA damage-induced apoptosis. We have identified that NHR-14/HNF4α might cooperate with CEP-1/p53 to control DNA damage-induced egl-1 and ced-13 and it could provide new targets for cancer intervention.
Dysregulation of DNA damage-induced apoptosis usually leads to tumorigenesis. Nuclear receptor HNF4 alpha is one of the central elements in the liver. It was closely related to fatty acid metabolism [30][31][32][33] and can induce hepatoma differentiation and block hepatocarcinogenesis [34]. Therefore, deregulation of hepatocyte nuclear factor 4 (HNF4) could be a marker of liver cancer progression. In the future, we will further confirm the relationship between the dysregulation of DNA damage-induced apoptosis by nhr-14/HNF4α deletion and tumorigenesis and will further study the mechanism of HNF4α in tumorigenesis.

Conclusions
Our study revealed a potential function of NHR-14 in DNA damage-induced apoptosis. And nhr-14/HNF4α functions together with cep-1/p53 to regulate DNA damage-induced programmed cell death.