Functional and Placental Expression Analysis of the Human NRF3 Transcription Factor

Molecular Endocrinology, Jan 2005

Members of the Maf protooncogene and cap’n’ collar families of basic-leucine zipper transcription factors play important roles in development, differentiation, oncogenesis, and stress signaling. In this study, we performed an in vivo protein-protein interaction screen to search for novel partners of the small Maf proteins. Using full-length human MAFG protein as bait, we identified the human basic-leucine zipper protein NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3] as an interaction partner. Transfection studies confirmed that NRF3 is able to dimerize with MAFG. The resulting NRF3/MAFG heterodimer recognizes nuclear factor-erythroid 2/Maf recognition element-type DNA-binding motifs. Functional analysis revealed the presence of a strong transcriptional activation domain in the center region of the NRF3 protein. We found that NRF3 transcripts are present in placental chorionic villi from at least week 12 of gestation on through term. In particular, NRF3 is highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. The human choriocarcinoma cell lines BeWo and JAR, derived from trophoblastic tumors of the placenta, also strongly express NRF3 transcripts. We generated a NRF3-specific antiserum and identified NRF3 protein in placental choriocarcinoma cells. Furthermore, we showed that NRF3 transcript and protein levels are induced by TNF-α in JAR cells. Our functional studies suggest that human NRF3 is a potent transcriptional activator. Finally, our expression and induction analyses hint at a possible role of Nrf3 in placental gene expression and development.

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Functional and Placental Expression Analysis of the Human NRF3 Transcription Factor

Molecular Endocrinology Functional and Placental Expression Analysis of the Human NRF3 Transcription Factor 0 Lady Davis Institute for Medical Research (B.C., A.D., W.M., V.Bl.) and Department of Medicine (V.Bl.), McGill University , Montr e ́al, Que ́ bec , Canada , H3T 1E2; and Division of Stomatology (K.R.-H. , V.Be., S.J.F.), Obstetrics (S.J.F.), Gynecology and Reproductive Sciences (S.J.F.), Pharmaceutical Chemistry (S.J.F.) and Anatomy (S.J.F.) , University of California , San Francisco, California 94143-0512 Members of the Maf protooncogene and cap'n' collar families of basic-leucine zipper transcription factors play important roles in development, differentiation, oncogenesis, and stress signaling. In this study, we performed an in vivo protein-protein interaction screen to search for novel partners of the small Maf proteins. Using full-length human MAFG protein as bait, we identified the human basicleucine zipper protein NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3] as an interaction partner. Transfection studies confirmed that NRF3 is able to dimerize with MAFG. The resulting NRF3/ MAFG heterodimer recognizes nuclear factorerythroid 2/Maf recognition element-type DNAbinding motifs. Functional analysis revealed the presence of a strong transcriptional activation domain in the center region of the NRF3 protein. We - Bfactors of the Maf protooncogene and cap’n’ col ASIC-LEUCINE ZIPPER TRANSCRIPTION (bZIP) lar (CNC) families are crucial regulators of gene expression in mammals and in a variety of other organisms ( 1–4 ). The Maf and CNC protein families are defined by unique conserved regions located aminoterminal to their basic DNA-binding domain, called the extended homology region and the CNC domain, respectively ( 1, 5 ). Maf and CNC protein family members belong to the activator protein 1 (AP-1) superfamily of transcription factors, and a subset of them have been shown to form homo- and/or heterodimers with each other or with other members of AP-1 family such as the Jun, Fos, and activating transcription factor-4 proteins. The resulting complexes bind to NF-E2 (nuclear First Published Online September 23, 2004 * B.C., A.D., and W.M. contributed equally to this work and should all be considered first authors. Abbreviations: AP-1, Activator protein 1; bZIP, basicleucine zipper; CNC, cap’n’collar; GAPDH, glyceraldehyde3-phosphate dehydrogenase; GST, glutathione-S-transferase; HEK, human embryonic kidney; MARE, Maf recognition element; NF-E2, nuclear factor-erythroid 2. Molecular Endocrinology is published monthly by The Endocrine Society (, the foremost professional society serving the endocrine community. found that NRF3 transcripts are present in placental chorionic villi from at least week 12 of gestation on through term. In particular, NRF3 is highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. The human choriocarcinoma cell lines BeWo and JAR, derived from trophoblastic tumors of the placenta, also strongly express NRF3 transcripts. We generated a NRF3specific antiserum and identified NRF3 protein in placental choriocarcinoma cells. Furthermore, we showed that NRF3 transcript and protein levels are induced by TNF- in JAR cells. Our functional studies suggest that human NRF3 is a potent transcriptional activator. Finally, our expression and induction analyses hint at a possible role of Nrf3 in placental gene expression and development. (Molecular Endocrinology 19: 125–137, 2005) factor-erythroid 2)/MARE (Maf recognition element)type of DNA binding motifs that typically contain an AP-1-like core recognition sequence ( 3–5 ). The founding member of the Maf protooncogene family, the v-Maf oncogene, was isolated as the transforming agent of an avian retrovirus ( 6 ). Its cellular counterpart, c-Maf, has also been implicated in neoplasia in humans ( 7 ). Related Maf proteins are divided into two subgroups: the small Mafs, including MafF, MafG, and MafK and large Mafs, including c-Maf, MafA/L-Maf, MafB/Kreisler, and Nrl. Large Mafs play important roles in the differentiation and development of a variety of hematopoietic and neural tissues ( 3, 4 ). The specific functions of the small Maf proteins are less clear. Small Mafs are widely expressed, but their transcript levels vary considerably (3). Small Maf homodimers have been reported to function as transcriptional repressors ( 8–10 ). In contrast, it was shown that small Mafs can also function as positive regulators by forming heterodimers with CNC proteins ( 10–14 ). The CNC transcription factor family includes the Drosophila cnc, Caenorhabditis elegans skn-1, and vertebrate p45, NRF1/LCR-F1/TCF11, NRF2/ECH, BACH1, and BACH2 proteins ( 15–25 ). The function of the mammalian CNC factors has been extensively studied, but only a small amount of information is Ch e´nais et al. • Analysis of NRF3 in Placental Cells available on NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3], the most recently identified member ( 14 ). We have performed gene targeting of the mouse Nrf3 locus showing that Nrf3 null mice develop normally and exhibit no obvious phenotypic differences when compared with wild-type animals ( 26 ). Complexity of CNC transcription factors was revealed by our finding that the absence of Nrf3 does not appear to cause additional lethality in compound Nrf3/ Nrf2- and Nrf3/p45-deficient mice ( 26 ). Importantly, it was shown that in humans, Nrf3 transcripts are primarily expressed in the placenta ( 14 ). The human placenta is a unique, transient organ and possesses, through its capacity to proliferate and to invade maternal tissues, qualities that are usually found in malignant tumors. However, growth and invasion at the fetal-maternal interface is under tight control, and malignant placental tumors are rare ( 27 ). Normal trophoblast cells as well as malignant choriocarcinoma cells including the JAR, BeWo, and JEG-3 cell lines, derived from trophoblastic tumors of the human placenta, have served as models to study placental cells ( 28 ). However, many aspects of the transcriptional regulation of placental gene expression and development remain to be elucidated. To better understand the in vivo roles of the small Maf proteins, we searched for novel molecules that serve as dimerization partners. We performed a twohybrid screen of a placental cDNA library using a Gal4 DNA-binding domain-MAFG fusion protein as bait and identified the human NRF3 protein as a MAFG partner. NRF3 is the most recently isolated member of the CNC protein family ( 14, 26 ), and the full-length human protein has not yet been characterized in detail. Therefore, we performed a functional analysis of the transactivation capabilities of NRF3 and examined its expression pattern in human placental cells. We showed that human NRF3 is able to bind to NF-E2/ MARE DNA binding sites by dimerization with MAFG. We also identified a strong transactivation domain in NRF3. Expression analysis showed that NRF3 transcripts are particularly expressed in placental cytotrophoblast cell populations. Furthermore, we found that in placental choriocarcinoma cells, NRF3 transcript and protein levels are inducible by TNF- . Our results suggest that human NRF3 is a strong transactivating factor that might be involved in the regulation of gene expression and development in the human placenta. RESULTS Cloning of the Full-Length Human NRF3 as a MAFG Dimerization Partner We searched for novel partners of the MAFG transcription factor using a yeast-based in vivo proteinprotein interaction screen. We fused the full-length MAFG protein (162 amino acids) to the DNA-binding domain of the GAL4 transcription factor and screened a placental library containing cDNAs fused to the GAL4 activation domain. After elimination of false positives, 11 candidates were sequenced and three of them coded for human NRF3. The isolated NRF3 cDNA was 120 bp shorter than a partial sequence reported previously ( 14 ) and included a consensus polyadenylation signal, but did not contain the 5 -end of the cDNA (Fig. 1, A and B). We recovered the missing 5 -end by performing RT-PCR. The DNA sequence of the recovered cDNA product (clone RTPlac5pr, Fig. 1A) was identical to that predicted from the bacterial artificial chromosome clone CTB-119C02 containing the human NRF3 gene. We concluded that a composite cDNA clone, containing the RT-PCR product fused to the partial cDNA identified in the two-hybrid screen, corresponds to the full-length human NRF3 cDNA encoding a protein of 694 amino acids (Fig. 1B). This human NRF3 sequence is available in the GenBank database under accession no. AF133059. The 5 -untranslated region and the first 400 nucleotides of the coding region are extremely rich in G and C residues (76%), likely contributing to a secondary structure that is resistant to reverse transcription. The CNC and bZIP domains are highly conserved between NRF3 and other CNC protein family members such as p45 (53% identity, 66% similarity), NRF1 (59% identity, 68% similarity), and NRF2 (51% identity, 64% similarity) (Fig. 2). The best overall homology is to NRF1. Interestingly, domains identified in the amino-terminal region of other CNC proteins shown to been important for interaction with Keap1, cAMP response element binding protein (CREB)-binding protein, and WW-domain containing proteins are not conserved in NRF3 ( 29–32 ). Thus, the amino terminus of NRF3 has diverged considerably from other CNC family members, suggesting that it might perform distinct functions. Finally, we noted a peptide sequence (DEPDSDSGLSLDSS) containing a series of negatively charged amino acids and serine residues (Fig. 2), which is highly conserved among human p45, NRF1, NRF2, and NRF3 proteins as well as their orthologs. NRF3 Interacts with MAFG and Binds to NF-E2/ MARE Sites Isolation of NRF3 using a two-hybrid strategy strongly suggested that NRF3 is able to interact with MAFG. We performed EMSAs to assess DNA binding activity and protein-protein interactions of NRF3. We transiently transfected human embryonic kidney (HEK)293T cells with expression constructs coding for human MAFG and a truncated form of NRF3 (525 amino acids), corresponding to the clone isolated in the two-hybrid assay. We also used a cDNA corresponding to a truncated intermediate size version (607 amino acids) as early experiments resulted only in low level expression of the full-length clone. Thus, in subsequent transfection studies we used a higher amount of the expression vector coding for full-length NRF3 (694 amino acids). Cotransfection of expression vectors coding for MAFG and for either of the different NRF3 versions resulted in complexes binding to the NF-E2/MARE-type recognition site, whereas in mock-transfected cells the complex is not present (Fig. 3). DNA binding was specific as addition of excess unlabeled oligonucleotide corresponding to the recognition site abolished the interaction. To characterize NRF3 protein complexes biochemically, we generated a specific antiserum using a glutathione-S-transferase (GST)-NRF3 fusion protein comprising solely the center portion of NRF3 to avoid cross-reaction with other CNC proteins. Indeed, the antiserum supershifted the NRF3/MAFG heterodimer (Fig. 3) but did not appear to cross-react with other CNC proteins such as p45 and NRF1 (Fig. 4). The formerly described MAFG antiserum also disrupted formation of the DNA/protein complex ( 11 ), unique to HEK293T cells expressing NRF3 and MAFG proteins, confirming that this complex contained both factors (Fig. 3). In contrast, antisera raised against the bZIP transcription factors p45, MAFF, and MafK had no effect (Fig. 3). We also confirmed that NRF3 (525 amino acids) can dimerize with MafK (data not shown), Fig. 2. Comparison of Human CNC Family Proteins Alignment of human CNC proteins NRF3, NRF1, NRF2, and p45. Identical residues among proteins are in black. Conserved amino acids are shaded. consistent with data reported previously ( 14 ). In summary, our results show that the full-length human NRF3 transcription factor forms heterodimers with small Maf proteins resulting in complexes that can recognize NFE2/MARE-type DNA-binding motifs. Human NRF3 Comprises a Potent Transactivation Domain To determine whether human NRF3 can act as a transcriptional activator we performed transfection experiments in HEK293T cells using Gal4 DNA binding domain-NRF3 fusion protein constructs jointly with a Gal4 DNA binding site-luciferase reporter vector (Fig. 5). Full-length NRF3 is able to activate approximately 6-fold when compared with HEK293T cells transfected with Gal4 DNA binding domain (Gal4DBD) vector (pSG424) alone (Fig. 5). A chimeric protein (Gal4DBD-NRF31–536) without CNC and bZIP domains showed increased transactivation potential and stimulated the reporter gene by 18-fold. A more drastic increase was observed when the first 88 amino acids of the NRF3 amino terminus were removed. Chimeric proteins, having in common at least amino acids 298– 399 of NRF3, were extremely potent activators and induced transcription approximately 200- to 400-fold. A series of deletion mutants comprising different regions of the center portion of NRF3 were used to map the transactivation domain. We observed a drastic decrease in the transactivation potential of chimeric protein Gal4DBD-NRF3395–536 when compared with Gal4DBD-NRF3298–399 fusion protein (Fig. 5). In summary, our results reveal the presence of a potent transactivation domain in the center portion (amino acids 298–399) of NRF3, suggesting that this protein can function as a potent activator of gene transcription. Human NRF3 Is Primarily Expressed in Placental Cytotrophoblasts We next investigated the tissue distribution of NRF3 transcripts by Northern blot analysis. A previous study has shown that human NRF3 is highly expressed in the placenta and is present at very low levels in other tissues, including small intestine, thymus, and spleen ( 14 ). The abundance of NRF3 in human placenta prompted us to explore its expression pattern in this tissue in more detail. We obtained placental chorionic villi from different stages of gestation and determined NRF3 mRNA levels (Fig. 6A). There are two human NRF3 transcripts, a major and a minor mRNA species of 2.9 kb and 4.4 kb, respectively. Interestingly, we found that NRF3 is expressed from at least week 12 of gestation to term (Fig. 6A). As chorionic villi are made up of multiple cell types, we investigated the expression patterns in specific cell populations. We isolated two of the predominant cell types in placental villi, fibroblasts, and cytotrophoblasts. Fibroblasts make up the stromal compartment whereas cytotrophoblasts line the placenta and mediate all interactions between fetal and maternal tissues. To carry out this function, cytotrophoblasts must undergo a highly regulated differentiation program, allowing them to transition from progenitor cells located in placental villi to either invasive cells that move into the uterine wall or a syncytium that covers the villi. We isolated cytotrophoblast progenitor cells from second trimester placentas and cytotrophoblasts located in cell islands from early gestation placentas, which represent a population differentiating into invasive cells. NRF3 transcripts are highly expressed in cytotrophoblast progenitors and those derived from trophoblast islands (ISL), whereas expression in placental fibroblasts was either weak or undetectable (Fig. 6B). In addition, we found that the human choriocarcinoma cell lines BeWo and JAR ( 33, 34 ), derived from trophoblastic tumors of the placenta, also strongly express NRF3 transcripts (Fig. 6C). These results suggest that, in the placenta, cytotrophoblasts are the primary source of NRF3 mRNA, implying that this transcription factor may be involved in the functioning and/or control of differentiation of these cells, which is critical to formation of the fetal-maternal interface during pregnancy. Analysis of Endogenous Human NRF3 Protein Expression As our anti-GST-NRF3 fusion protein antiserum, used in EMSA experiments, only weakly recognized fulllength NRF3 protein in immunoblot experiments, we generated a new antiserum specific for a peptide in the NRF3 carboxy terminus. We further purified the serum using affinity chromatography. We used the anti-NRF3 peptide antiserum to analyze the expression of the NRF3 protein in transfected HEK293T cells as well as in choriocarcinoma cell lines. We prepared whole extracts from HEK293T cells transfected with constructs coding for the 525 amino acids, 607 amino acids, and full-length (694 amino acids) versions of NRF3. Immunoblot analysis with preimmune serum resulted only in nonspecific staining (Fig. 7A). In contrast, incubation with purified NRF3-specific antiserum revealed the presence of the corresponding protein versions in whole-cell extracts from transfected HEK293T cells, whereas in mock-transfected cells no NRF3 is detectable (Fig. 7B). Specificity of the antiserum was further confirmed by competition with the peptide antigen in an immunoblot assay (Fig. 7C). We also analyzed whole-cell extracts from BeWo as well as JAR choriocarcinoma cells and showed that the endogenous protein displayed the same apparent molecular weight as full-length NRF3 (694 amino acids) from transfected HEK293T cells (Fig. 7B). We noticed that in transfection experiments full-length NRF3 gives rise to a shorter version, migrating at a lower molecular weight. We speculate that the faster migrating species may be a degradation product of the full-length version. The shorter product observed in transfected HEK293T cells is not seen in the choriocarcinoma cell lines that express endogenous NRF3. Our specific antisera will serve as a valuable tool for further functional characterization of the human NRF3 protein. Expression of Human NRF3 Is Stimulated by TNF TNF- produced by the placenta is believed to have important functions during pregnancy ( 35 ). We thus investigated whether NRF3 expression is modulated by TNF- in choriocarcinoma cells. We found a significant induction of NRF3 transcripts in JAR cells at 8 and 16 h after induction (Fig. 8A). Importantly, the increased human NRF3 RNA levels correlated with increased protein levels (2.3-fold) in TNF- -treated JAR cells (Fig. 8B). These results suggest that NRF3 may participate in late TNF- -mediated signaling in placental cells. DISCUSSION We used a protein-protein interaction screen to search for novel partners of the small Maf proteins. We identified human NRF3, a member of the CNC family of bZIP proteins, as a partner of the MAFG transcription factor. This interaction was confirmed in transfection studies showing that NRF3 heterodimerizes with MAFG, forming a complex that can bind to typical NF-E2/MARE DNA recognition sites (Fig. 3). The expression pattern of NRF3 is striking: in contrast to the widely expressed NRF1 and NRF2 genes, NRF3 transcripts are present at high levels in the placenta (Fig. 6), although they are expressed at lower levels in a series of other tissues ( 14 ). The small Maf partners of NRF3 are also highly expressed in the placenta ( 11, 36 ). This is of interest, as many aspects of the transcriptional events governing placental gene expression remain to be uncovered. To explore the possible role of the NRF3 transcription factor in the placenta, we examined its expression pattern in this tissue in more detail. The placenta is unique, in that it involves the close association of fetal and maternal tissues, and invasion of maternal structures by cells derived from the fetus (37). NRF3 mRNA is expressed in placental chorionic villi, from at least gestation week 12 to term (Fig. 6A). NRF3 mRNA is thus present during the period when fetal cells penetrate maternal tissue and develop connections with the maternal vasculature, a process termed “endovascular invasion” that takes place during the first half of pregnancy ( 37 ). As placental chorionic villi are made up of multiple cell types including cytotrophoblasts, syncytiotrophophoblasts, fibroblasts, and macrophages among others, we performed additional analyses in specific cell types. We found that high Nrf3 transcript levels are present in cytotrophoblast progenitor cells and in cytotrophoblasts from cell islands, the latter representing cells that are differentiating down the invasive pathway. In contrast, the stromal component, placental fibroblasts, do not or only minimally express NRF3 transcripts (Fig. 6B). In this context, it is interesting to note that NRF3 transcripts are also strongly expressed in the choriocarcinoma cell lines BeWo and JAR (Fig. 6C), which were derived from malignant transformation of human cytotrophoblast progenitors ( 33, 34 ). We thus speculate that NRF3 may be involved in essential cytotrophoblast functions or possibly in the molecular mechanisms that specify their important cell fate decisions that include acquisition of tumor cell-like invasiveness. Little is known about the key regulatory factors that are important for trophoblast differentiation in humans (38). Recently, we performed gene targeting of the mouse Nrf3 locus and showed that Nrf3 and compound Nrf3/Nrf2 and Nrf3/p45 null mice exhibit no obvious abnormalities ( 26 ). Previously, it has been shown by others that CNC factor Nrf2, Bach1, and Bach 2 null mice also do not show an obvious phenotype ( 39–42 ). Although Nrf3 null mice do not display any obvious placental defects, this finding does not preclude a possible role for this transcription factor in placental biology. The difference may be explained by functional redundancy by closely related CNC proteins or AP-1-like factors binding to similar DNA recognition elements that are still present in the various single or compound CNC transcription factor-deficient mice. In addition, one might also consider differences in human and mouse biology, as evidenced by known discrepancies in both species, e.g. in innate and adaptive immunity (43). In this regard, it is of interest that the expression pattern of human and mouse Nrf3 are somewhat different ( 14, 26 ). In addition to NRF3, other transcription factors also belonging to the AP-1 family are expressed in specific placental subtypes, e.g. cytotrophoblasts switch from expressing c-Jun to JunD as they differentiate and invade the uterine wall (27). As it has been shown that NRF2 can interact with jun proteins ( 44 ), it is feasible that NRF3 may interact with other members of the AP-1 transcription factor family in the placenta to regulate specific target genes. Cytokines are important signaling molecules in the regulation of placental function ( 45 ). Interestingly, we found that NRF3 mRNA and protein expression is up-regulated by the proinflammatory cytokine TNF- . The actions of TNF- in pregnancy are not well understood, but there is evidence that it may play an important role in placental biology. For example, influences on target tissues such as the myometrium could help regulate the timing of parturition ( 35, 46 ). In addition, both placental cytotrophoblasts and choriocarcinoma cell lines express members of the TNF receptor superfamily, suggesting an autocrine role for this cytokine (46). Furthermore, circulating levels of TNF- are elevated in preeclampsia and in pathological placental tissue derived from intrauterine growth restricted pregnancies ( 47–49 ). Our data hint at a possible function for NRF3 in the placental TNF- signaling cascade. In our study we also performed structure/function analyses of NRF3. The CNC and the bZIP domains of NRF3 are highly homologous to those of other CNC family members including p45 NF-E2, NRF1, and NRF2 ( 17, 19, 23, 24 ), but the amino terminus has clearly diverged, suggesting it may have acquired specific functions. The Neh2 (Nrf2-ECH homology) domain, which has been shown to be important for interaction of NRF2 with the repressor protein Keap1 (50), is not well conserved in NRF3. Additional studies should clarify whether NRF3 is able to interact with Keap1. In addition, the Neh4 and Neh5 domains, which have been shown to be important for interaction with cAMP responsive element binding protein-binding protein, thus conferring strong transactivation properties to NRF2 ( 29 ), are also not conserved in NRF3. We investigated whether the amino-terminal domain of NRF3 has functional importance and identified a major transactivation region between amino acids 298 and 399. Our finding that NRF3 has strong transactivating capabilities is in agreement with an earlier report showing that full-length mouse Nrf3 is a transcriptional activator ( 14 ). The region is highly homologous among Nrf3 orthologs in rat and mouse and contains well-conserved potential kinase phosphorylation sites. The first 300 amino acids of NRF3 do not appear to have a significant transactivation capacity. We speculate that this part of the protein may be important for protein-protein interactions. In summary, our functional studies showed that NRF3 has strong transactivating capabilities conferred by a domain located in the center portion of the protein. In conclusion, our results suggest that human NRF3 is a potent transcriptional regulator. Its expression in placental cytotrophoblasts and choriocarcinoma cells hints at a possible role of NRF3 in the control of transcriptional events in placental cells. We also generated NRF3-specific antisera that will provide a valuable tool for future analyses. It will be important to identify placental NRF3/small Maf target genes as well as regulatory factors interacting with different domains of this protein. These studies should shed light on the regulation of gene expression governing the development and/or the endocrine functions of the placenta. MATERIALS AND METHODS Cell Culture and Cytokine Induction Human embryonic kidney 293T (HEK293T) cells were cultured in MEM . The human choriocarcinoma cell lines BeWo and JAR were maintained in F12K medium and RPMI-1640 medium plus 1 mM sodium pyruvate and 10 mM HEPES, respectively. All media contained L-glutamine and were supplemented with 10% heat-inactivated fetal bovine serum and penicillin plus streptomycin. For induction studies, the cells were serum starved overnight and then incubated with 100 ng/ml TNF- (MEDICORP, Montre´ al, Que´ bec, Canada) for the time indicated. Plasmid Constructs and Transfections A full-length NRF3, comprising the entire coding region (694 amino acids), was recovered by RT-PCR with RNA from placental tissue as template using the following primer pair: hNRF3-Fnew (5 -GCGATGAAGCACCTGAAGCGGT-3 ) and NRF3-Rnew (5 -CTCACTTTCTCTTTCCCTTTTGGG-3 ). The resulting PCR fragment was cloned into the pCR-BluntIITOPO vector (Invitrogen, San Diego, CA). As the NRF3 gene comprises an internal EcoRI site, the insert was recovered from the pCR-BluntII-TOPO plasmid by a partial EcoRI digest and inserted into the EcoRI site of the pMT2 expression vector. A shorter NRF3 version (607 amino acids) was generated by PCR with pMT2NRF3 as template by amplification with the primers NRF3-F607 (5 -GCCATGGAGGGCCAGCTGCTCCGGGAG-3 ) and NRF3-Rnew (5 -CTCACTTTCTCTTTCCCTTTTGGG-3 ). The amplicon was cloned into pMT2 via the pCR-BluntII-TOPO vector as described above for the full-length version. The shorter NRF3 version corresponding to the two-hybrid clone (525 amino acids) was generated by PCR using the oligonucleotides NRF3-MT2F1 (5-CGCGAATTCGCCATGGAGAAGGCACCCGCGGAACCG-3 ) and NRF3-GSTR1 (5 -CACGAATTCTCACTTTCTCTTTCCCTTTTG3 ). The resulting fragment was partially digested with EcoRI and inserted into the EcoRI site of pMT2. The pMT2p45 NF-E2 expression plasmid has been described previously ( 51 ). The human NRF1 cDNA has been recovered by RT-PCR using the following oligonucleotides: hNRF1-Fnew (5 -GGTCCTTCAGCAATGCTTTCTCTG-3 ), hNRF1-Rnew (5 -CCCTTCTTCCCCAGGCTCACTTT-3 ), and cloned into the pCR-BluntII-TOPO vector. Subsequently, the EcoRI fragment comprising the NRF1 cDNA was inserted into the EcoRI site of the pMT2 expression vector. Transient transfections of HEK293T cells were performed using the calcium phosphate coprecipitation procedure ( 52 ). For transfections of a 100-mm dish of 30–50% confluent HEK293T cells, we used 10 g of expression vector. In the case of the full-length NRF3 (694 amino acids) expression vector we used 30 g of the plasmid. To map the NRF3 transactivation domain, we used PCRgenerated fragments using oligonucleotides with BamHI restriction sites, to clone full-length (694 amino acids) and shorter versions of human NRF3 (Fig. 5) into the BamHI site of plasmid pSG424 in frame with the GAL4 DNA-binding domain ( 53 ). For the pSG-NRF31–297 construct we used the EcoRI site present in the NRF3 cDNA to clone the fragment into the EcoRI site of pSG424. The (Gal4)5-TK/luciferase reporter construct (a kind gift from Dr. Rongtuan Lin) was generated by inserting a 140-bp HindIII/XbaI fragment from the (Gal4)5-TK/chloramphenicol acetyltransferase vector ( 54 ) treated with Klenow enzyme into the SmaI site of the TK/ pGL3 vector. As a transfection control we used the pRL-TK vector (Promega Corp., Madison, WI). Transient transfections of HEK293T cells for reporter assays were performed in 24TNF- for 4, 8, or 16 h. Ten micrograms of RNA were analyzed per lane. Human NRF3 and GAPDH transcripts are indicated. Quantification of NRF3 transcript levels normalized to GAPDH mRNA levels was done by PhosphorImager analysis of three independent experiments. Significant statistical difference is indicated by an asterisk (P 0.01). B, Immunoblot analysis of JAR choriocarcinoma cell extract treated with 100 ng/ml TNF- for 16 h. Total proteins (30 g) were subjected to immunoblotting using purified antipeptide antiserum raised against NRF3. Quantification of NRF3 protein levels normalized to GAPDH protein levels was performed using NIH image software analysis of three independent experiments. Significant statistical difference is indicated by an asterisk (P 0.01). well plates using the calcium phosphate coprecipitation procedure ( 52 ). HEK293T cells (104 per well) were plated and transfected with 100 ng of (Gal4)5-TK/luciferase reporter, 5 ng of pRL-TK, and 500 ng of either of the various pSG-NRF3 constructs. A dual luciferase assay was performed 48 h post transfection according to the instructions of the manufacturer (Promega). Two-Hybrid Screen A Matchmaker (CLONTECH, Palo Alto, CA) two-hybrid screen was performed according to the instructions of the manufacturer. Human MAFG cDNA comprising the entire coding region was subcloned into the EcoRI and BamHI sites of the pGBT9 vector coding for the GAL4 DNA-binding domain. The pGBT9-MAFG vector was introduced into HF7c cells. Subsequently, a pGAD10 vector-based placental Matchmaker library (CLONTECH), containing cDNA sequences fused to the GAL4 activation domain, was transformed into the HF7c (pGBT9-MAFG) cells. The transformation mixture was then plated on Trp , Leu , His selection plates. Twenty-two of 80 colonies showed lacZ activity. The cDNA insert of 11 independent clones was recovered by PCR and sequenced. The sequence of seven clones corresponded to that of NRF2 and one to a cDNA of unknown function. The remaining three clones were identical and contained nucleotides 735-2827 (Fig. 1B) of the NRF3 gene fused to the GAL4 activation domain. One of the clones, 2Hyb-33 (Fig. 1A), was further characterized. The 5 -end of NRF3 mRNA (clone RT-Pla5pr) was recovered by standard RTPCR, in the presence of 10% dimethylsulfoxide, using human placental RNA as template. To this end, we designed specific primers, located upstream of the putative start codon using sequence data from human bacterial artificial chromosome clone (no. CTB-119C02) and in the second exon of the NRF3 gene: hNRF3–5 F1 (5 -GTGGCTCCTTCTTCGCTTCT-3 ) and hNRF3–5 R1 (5 -CAGTGGTCTTTTCTGCCTCC-3 ). Isolation of Chorionic Villi Informed consent was obtained from all patients. Placentas from elective terminations of normal pregnancies (12–22 wk) or from normal term deliveries (34–40 wk) were collected immediately after delivery, washed thoroughly in PBS with antibiotics, and placed on ice. Chorionic villi were dissected free from the rest of the placenta and lysed immediately in TRIzol (Invitrogen). RNA was extracted according to the manufacturer’s instructions. Isolation of Cytotrophoblasts and Placental Fibroblasts Cytotrophoblast progenitors were isolated from pooled second-trimester human placentas by published methods ( 55, 56 ). Briefly, placentas were subjected to a series of enzymatic digests, which detached cytotrophoblast progenitors from the stromal cores of the chorionic villi. Once detached, the cells were purified over a Percoll gradient (Collaborative Biomedical Products, Bedford, MA). Cytotrophoblast cell islands, visualized by using a dissecting microscope, were dissected from the surface of early-gestation placentas (57). Fibroblasts were isolated from first-trimester placentas and passaged as previously described ( 56 ). The lines were used after the third passage to ensure that contaminating cells were no longer present. RNA was extracted from these cell populations as described above for chorionic villi. Preparation of NRF3 Antisera To facilitate biochemical studies of NRF3 complexes, we raised a high-titer polyclonal antiserum against a GST-NRF3 fusion protein. To this end, we generated a partial human NRF3 cDNA by PCR using the following oligonucleotides: FMG-GSTF3 (5 -CTCGGATCCAGCCAGGCTATAAGTCAGGAT-3 ) and FMG-GSTR2 (5 -CGCGAATTCAGTTTCTATCTGTGTCTTCAAG-3 ). The amplicon was digested with EcoRI and BamHI and cloned into the corresponding sites of the pGEX-2TK vector (Amersham Pharmacia Biotech, Arlington Heights, IL). Female New Zealand White rabbits were immunized with the GST fusion protein containing amino acids 306–534 of human NRF3. We chose the center portion of NRF3 as antigen to avoid cross-reaction of the antiserum with other CNC family members. Antiserum was tested in parallel with preimmune serum to confirm the specificity to human NRF3. In addition, we raised an antiserum specific for a peptide (KLHDLYHDIFSRLLRDDQGRPVNPN) in the carboxy terminus of human NRF3. The peptide was coupled to keyhole limpet hemocyanin and used to immunize female New Zealand White rabbits (Pocono Rabbit Farm and Laboratory, Canadensis, PA). The serum was purified using peptide coupled to Affi-Gel 10 according to the instructions of the manufacturer (Bio-Rad Laboratories, Inc., Hercules, CA). Nuclear Protein Extract and EMSA For EMSA experiments, nuclear extracts were prepared as described previously ( 58 ) and incubated at room temperature for 20 min in the binding reaction containing 20 mM HEPESKOH (pH 7.9), 60 mM KCL, 6 mM MgCL2, 0.2 mM EDTA, 1 mM dithiothreitol, 10% glycerol, poly(dI-dC), 70 g/ml, and 7,000–30,000 cpm -[32P]ATP-labeled probe ( 59 ). Reaction mixtures were analyzed by native 5% PAGE and autoradiography. The NF-E2 binding site oligonucleotide derived from the human porphobilinogen deaminase promoter, endlabeled with T4-polynucleotide kinase, was used as a probe ( 17 ). A 400 molar excess of unlabeled oligonucleotide was used for competition. For supershift experiments 1 l of either preimmune or immune serum was added to the reaction mixture as indicated. NRF1 and MafK antisera were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The p45 and MAFG antisera have been reported previously ( 11, 17 ); the generation of the MAFF antiserum will be described elsewhere (Massrieh, W., A. Derjuga, F. DovallaBell, C.-Y. Ku, B. M. Sanborn, and V. Blank, in preparation). Northern Analysis Total RNA was prepared by using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Northern blotting was performed using standard procedures loading 10 g of RNA per lane. As a probe, a 950-bp BglII fragment of human NRF3 cDNA, corresponding to amino acids 171– 487 of the full-length protein, was labeled using the random priming labeling kit (Roche Diagnostics, Indianapolis, IN) and hybridized as described ( 60 ). To provide a control for loading, membranes were stripped and hybridized with a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. Immunoblot Analysis Cell pellets were resuspended in the lysis buffer (10 mM Tris-HCl, pH 8.0; 420 mM NaCl; 250 mM sucrose; 2 mM MgCl2; 1 mM CaCl2, 1% Triton X-100) supplemented with 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, Complete protease inhibitor cocktail (Roche) and then centrifuged at 10,000 rpm for 3 min. Total protein was separated on precast NuPAGE Novex 4–12% Bis-Tris gradient gels according to the instructions of the manufacturer (Invitrogen). SeeBlue Plus 2 (Invitrogen) prestained protein standard was run with the samples. Proteins were then transferred to a polyvinylidine difluoride membrane (Immobilon, Millipore Corp., Bedford, MA). Blocking of the membrane was carried out overnight at 4 C in 1 Tris-buffered saline (25 mM Trisbase, pH 7.5; 150 mM NaCl) plus 5% milk. Subsequently, the membrane was incubated for 2 h at room temperature with NRF3-specific peptide antiserum or GAPDH-specific monoclonal antibody (Research Diagnostics, Flanders, NJ) in 1 Tris-buffered saline plus 5% milk, 0.05% Tween-20, and 350 mM NaCl. After washing, the membrane was incubated for 1 h at room temperature with goat antirabbit antibody conjugated to horseradish peroxidase (Pierce). The proteins were detected by using the Super-Signal West Pico chemiluminescent reagent from Pierce according to the manufacturer’s instructions. For competition experiments, the peptide antigen was added to the antiserum mixture at an excess concentration of 50 M before incubation of the blot. Quantification and Statistical Analysis Quantification of Northern experiments was performed using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and ImageQuant software (version 5.2). Immunoblots were quantified by densitometry using NIH image software (version 1.63). Data were analyzed for significance by Student’s t test using Prism software (version 4.0). Acknowledgments We thank Bena Teo for her participation in the early phase of this project, Lewis Rubin for providing the choriocarcinoma cell line BeWo, and Rongtuan Lin for the GAL4 constructs. Received September 29, 2003. Accepted September 13, 2004. Address all correspondence and requests for reprints to: Volker Blank, Lady Davis Institute for Medical Research, 3755 Coˆ te-Sainte-Catherine Road, Montre´ al, Que´ bec, Canada H3T 1E2. E-mail: . This work was supported by a Canadian Institutes of Health Research/Institut National de la Sant e´ et de la Recherche Me´ dicale exchange program fellowship (to B.C.); McGill Cancer Consortium and J. W. 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Chénais, Benoı̂t, Derjuga, Anna, Massrieh, Wael, Red-Horse, Kristy, Bellingard, Valerie, Fisher, Susan J., Blank, Volker. Functional and Placental Expression Analysis of the Human NRF3 Transcription Factor, Molecular Endocrinology, 2005, 125-137, DOI: 10.1210/me.2003-0379