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Molecular Endocrinology, doi:10.1210/me.2004-0107
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Molecular Endocrinology 19 (1): 12-24
Copyright © 2005 by The Endocrine Society

CR6-Interacting Factor 1 Interacts with Orphan Nuclear Receptor Nur77 and Inhibits Its Transactivation

Ki Cheol Park, Kwang-Hoon Song, Hyo Kyun Chung, Ho Kim, Dong Wook Kim, Jung Hun Song, Eun Suk Hwang, Hye Sook Jung, Su-Hyeon Park, Insoo Bae, In Kyu Lee, Hueng-Sik Choi and Minho Shong

Laboratory of Endocrine Cell Biology (K.C.P., H.K.C., H.K., D.W.K., J.H.S., E.S.H., H.S.J., S.-H.P., M.S.), Department of Internal Medicine, Chungnam National University College of Medicine, Deajeon 301-721 Korea; Keimyung University School of Medicine (I.K.L.), Jung-Gu, Daegu 700-712, Korea; Hormone Research Center (K.-H.S., H.-S.C.), School of Biological Sciences and Technology, Chonnam National University, Kwangju 500-757, Korea; and Department of Oncology (I.B.), Lombardi Cancer Center, Georgetown University Medical Center, Northwest, Washington, D.C. 20057

Address all correspondence and requests for reprints to: Minho Shong, Laboratory of Endocrine Cell Biology, Department of Internal Medicine, Chungnam National University College of Medicine, 640 Daesadong Chungku Daejeon 301-721, Korea. E-mail: minhos{at}cnu.ac.kr; or Hueng-Sik Choi, Hormone Research Center, Chonnam National University, Kwangju, 500-757 Korea. E-mail: hsc{at}chonnam.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CR6-interacting factor 1 (CRIF1) was recently identified as a nuclear protein that interacts with the Gadd45 (growth arrest and DNA damage inducible 45) family of proteins and participates in the regulation of the G1/S phase of the cell cycle. However, the nuclear action of CRIF1 is largely unknown. In this study, we demonstrate that CRIF1 acts as a novel coregulator of transactivation of the orphan nuclear receptor Nur77. Both in vitro and in vivo studies show that CRIF1 interacts with Nur77 via the Nur77 AB domain and that it dramatically inhibits the AB domain-mediated transactivation of Nur77. Transient transfection assays demonstrate that CRIF1 inhibits steroid receptor coactivator-2-mediated Nur77 transactivation, and silencing of endogenous CRIF1 by small interfering RNA relieves this repression. CRIF1 possesses intrinsic repressor activities that are not affected by the histone deacetylase inhibitor Trichostatin A. In addition, overexpression of CRIF1 inhibits TSH/protein kinase A-induced Nur-responsive element promoter activity. CRIF1 inhibited Nur77-dependent induction of E2F1 promoter activity, mRNA expression, and Nur77-mediated G1/S progression in cell cycle. These results suggest that CRIF1 acts as a repressor of the orphan nuclear receptor Nur77 by inhibiting AB domain-mediated transcriptional activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CR6-INTERACTING FACTOR 1 (CRIF1) has been identified as a protein that interacts with three members of the Gadd45 (growth arrest and DNA damage inducible) family of proteins, Gadd45{alpha}, Gadd45ß, Gadd45{gamma} (1), and it also interacts with the papilloma virus type 16 L2 protein (2). CRIF1 is expressed ubiquitously, and at notably high levels in the endocrine organs, adrenal glands, thyroid, testis, and ovary (1). Studies of the intracellular localizations of CRIF1 revealed that it is mainly expressed in the nucleus (1, 2), although its intranuclear activities have not been fully elucidated. Chung et al. (1) reported that CRIF1 inhibits the cyclin-dependent kinases Cdc2/cyclin B1 and Cdk2/cyclin E, thus inhibiting cell cycle progression in the G1/S phase and suppressing the growth of serum-stimulated NIH3T3 cells.

The orphan nuclear receptor Nur77 (NGFI-B) subfamily consists of three members, Nur77 (NGFI-B{alpha}), Nurr1 (NGFI-Bß), and Nor1 (NGFI-B{gamma}) (3, 4, 5, 6). Nur77 is widely expressed in tissues including thymus, muscle, liver, thyroid, lung, testis, ovary, ventral prostate, and in the adrenal and pituitary glands (5, 6). Nur77 subfamily members have been shown to bind DNA as monomers or homodimers, or as heterodimers with retinoid X receptor (RXR) (7, 8, 9, 10). Recent advances have shown that Nur77 is regulated transcriptionally and posttranslationally. Nur77 has been shown to play an important role in regulating the expression of various genes in the hypothalamic-pituitary-adrenal axis that are implicated in inflammation and steroidogenesis (11). In particular, Nur77 expression is rapidly up-regulated in endocrine cells by stimulation with CRH, TSH, FSH, LH, and ACTH (12, 13, 14, 15).

Nur77 may recruit interacting partners to mediate gene activation or repression. Although the transcriptional activity of many nuclear receptor dimers depends on the binding of a ligand to the C-terminal ligand binding domain (LBD), the molecular mechanism of ligand-induced transcriptional activity requires recruitment of a coactivator protein to the activation domain (16, 17, 18). However, Nur77 is an orphan nuclear receptor for which no ligands have been identified, although recent studies have begun to elucidate coactivators and corepressors that regulate its activity. For instance, the activation function (AF) 2-dependent coactivator ASC-2 and the corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor have been found to enhance and repress Nur77 transactivation, respectively (19). Wansa et al. (20) showed that the Nur77 N-terminal AB region, which contains the AF-1 domain, is necessary for optimal Nur77-dependent transactivation in cultured C2C12 cells. In addition, they observed that the steroid receptor coactivator (SRC)-2/TIF2/GRIP1 [glucocorticoid receptor (GR)-interacting protein 1 (GRIP1)/nuclear receptor coactivator 2 complex modulates the activity of the AF-1 domain. Maira et al. (21) also showed that p160/SRC coactivators are recruited to Nur77 dimers and that coactivator recruitment to Nur-responsive elements (NurREs) is enhanced in response to CRH, which activates protein kinase A (PKA). Moreover, PKA and the coactivator-induced potentiation of Nur77 activity are primarily exerted through the Nur77 AF-1 domain. These observations suggest that the N-terminal AB region plays a major role in Nur77-mediated transactivation and coactivator recruitment and that it is an endpoint effector of hormone-induced PKA signaling pathways.

This study explores functional relationships between CRIF1 and the orphan nuclear receptor Nur77. Both in vitro and in vivo studies show that CRIF1 interacts with Nur77. The silencing of endogenous CRIF1 by small interfering RNA (siRNA) results in the activation of Nur77-mediated transactivation. In addition, CRIF1 inhibits hormone TSH/PKA-induced NurRE-containing promoter activities. We propose that CRIF1 is a novel repressor of Nur77 and that it plays a key role in the regulation of TSH-mediated Nur77 transactivation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Analysis of Interactions between CRIF1 and Nur77
The primary structure of CRIF1 indicates that it has a coiled-coil region and nuclear localizing signals in the C-terminal half, and it has been reported to exclusively localize within the nucleus (1). To identify the nuclear receptors that interact with CRIF1, we performed yeast two-hybrid assays. cDNA corresponding to CRIF1 amino acid residues 1–222 (223 amino acids) was fused to sequences encoding the LexA-CRIF1 [LexA DNA binding domain (DBD)], and expressed proteins were used as bait in the assays. Constructs encoding fusions of LexA with full-length CRIF1 and fusions of the B42 activation domain with CRIF1, RXR, constitutive androstane receptor (CAR), thyroid hormone receptor (TR), SMRT, or Nur77 were transformed into the Saccharomyces cerevisiae strain EGY48 containing the ß-galactosidase (ß-gal) reporter plasmid 8H18–34 (Table 1Go). In addition, constructs encoding fusions of LexA with estrogen-related receptor (ERR){gamma}, GR, short heterodimer protein (SHP), steroidogenic factor (SF)-1, and murine androgen receptor (AR) were also transformed into S. cerevisiae with B42-fused CRIF1. The LexA-CRIF1 and B42-CRIF1 fusions alone did not promote transcriptional activation of the reporter. The LexA-fused ERR{gamma}, GR, SHP, and SF-1 did not show any significant interactions with B42-fused CRIF1. In addition, no significant reporter activity was observed for cells containing LexA-fused CRIF1 and B42-fused RXR, CAR, TR, or SMRT. In contrast, the LexA-CRIF1 fusion protein strongly interacted with the B42-Nur77 fusion protein. Murine AR also interacted with CRIF1 in the presence or absence of ligand, but this interaction was relatively weak compared with that between CRIF1 and Nur77.


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  		Table 1. Characterization of the Novel CRIF1 and Nur77 Interaction

 
Nur77 has conserved functional domains (22), as shown in Fig. 1AGo. Fusions of the B42 activation domain to full-length and truncated derivatives of Nur77 were created to identify which Nur77 domains interact with CRIF1. Interestingly, the B42-Nur77 AB domain interacted with CRIF1 (Fig. 1BGo), but the B42-Nur77 CDE did not. Moreover, deletion of the AF2 region did not affect interaction between CRIF1 and Nur77. To further characterize interactions between these two proteins, we prepared in vitro-translated CRIF1 and performed pull-down assays with the glutathione-S-transferase (GST) pull-down assay-Nur77 fusion protein. In agreement with the yeast two hybrid results, CRIF1 interacted with the AB domain of Nur77. As a positive control, Gadd45{gamma} was found to interact with CRIF1, as also shown in a previous study (1) (Fig. 1CGo). Taken together, these results suggest that CRIF1 interacts with a specific region of Nur77, the AB domain.



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  		Fig. 1. CRIF1 Interacts with the AB Domain of Nur77

A, Schematic representation of the Nur77 wild-type protein and deletion mutants. B, Plasmids encoding LexA-CRIF1 fusions were cotransformed with plasmids encoding fusions of B42-AD with the full-length Nur77 or with the AB, CDE, or {Delta} AF2 deletion derivatives of Nur77 into the yeast strain EGY28. ß-gal Activity was measured as described in Materials and Methods. C, GST, GST-Nur77, GST-Nur77AB, and GST-Gadd45{alpha} were incubated independently with 35S-radiolabeled full-length CRIF1 in vitro. The input lane represents 10% of the total in vitro-transcribed and translated protein.

 
To monitor interactions between CRIF1 and Nur77 in vivo, human foreskin fibroblast cells were infected with the adenovirus constructs AdCRIF1-GFP and AdGFP. As shown in Fig. 2AGo, uninfected cells showed basal levels of Nur77 expression (lower panel, Fig. 2AGo), and in the presence of AdGFP and AdCRIF1-GFP they expressed significant levels of the GFP and CRIF1 proteins (middle panel, Fig. 2AGo). Immunoprecipitates prepared with an anti-GFP antibody contained Nur77 (upper panel, Fig. 2AGo), showing that CRIF1 interacts with Nur77 in vivo.



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  		Fig. 2. Association of CRIF1 and Nur77 in Vivo

A, Human foreskin skin fibroblast cells were infected with AdCRIF1-GFP or AdGFP at the MOI 20. Endogenous Nur77 was immunoprecipitated with an anti-Nur77 antibody. The immunoprecipitated proteins were analyzed by Western blotting using an anti-GFP antibody. Total cell lysates were prepared and subjected to Western blot analysis with the GFP and Nur77 antibodies. B, Subcellular colocalization of CRIF1 and Nur77. NIH3T3 cells were transiently transfected with pEGFP-CRIF1 and HA-Nur77. At 24 h after transfection, the cells were fixed in 4% formaldehyde for 30 min. The fixed cells were mounted onto glass slides with PBS and observed with a laser-scanning confocal microscope. GFP-fused wild-type CRIF1 was detected by autofluorescence, and Nur77 was detected by staining with a primary monoclonal anti-HA antibody and a Rhodamine-conjugated secondary antibody. The colocalization of CRIF1 and Nur77 is indicated by yellow in the merged image. Representative cells from one of three independent experiments are shown.

 
To confirm the intracellular colocalization of CRIF1 with Nur77, we performed immunofluorescence confocal microscopy on NIH/3T3 cells cotransfected with pEGFP-CRIF1 and pCDNA3-HA-Nur77 and found that CRIF1 was mainly expressed in the nucleus (Fig. 2BGo). Rhodamine fluorescence, which indicates the presence of HA-Nur77, appeared only in the nuclear compartment. CRIF1 colocalized with Nur77, as depicted in the merged image (Fig. 2BGo). Taken together, these results indicate that CRIF1 interacts with Nur77 both in vitro and in vivo.

Inhibition of Nur77 Transactivation by CRIF1
Previous studies have identified the NurRE and NBRE motifs as major binding sequences for Nur77 dimers and monomers, respectively (10, 20, 23). To determine whether CRIF1 can modulate Nur77 transactivation, NurRE- and NBRE-driven reporters were cotransfected with a Nur77 expression vector in the presence or absence of CRIF1 into C2C12 cells. The NurRE and NBRE reporters, luciferase constructs under the transcriptional control of NGFI-B dimers, were strongly induced by the expression of Nur77 (Fig. 3Go). Overexpression of CRIF1 repressed Nur77-mediated reporter activities in a dose-dependent manner (Fig. 3AGo, upper). However, CRIF1 expression did not affect the endogenous or exogenous level of Nur77 expression, as shown in Fig. 3AGo (lower panels). NBRE-mediated transcription was also significantly reduced by CRIF1 (Fig. 3BGo). This result suggests that CRIF1 represses monomeric and dimeric Nur77-mediated transcriptional activities. Full-length Nur77 and derivatives were fused to the Gal4-DBD, and the ability of these chimeras to regulate the expression of a Gal4-Tk-Luc reporter in C2C12 cells was examined (20). A Gal4-Nur77 chimera encoding full-length Nur77 activated transcription by approximately 14-fold (Fig. 3CGo). However, CRIF1 did not affect the reporter activity of Gal4-Tk-Luc alone. Gal4-Nur77-induced transcriptional activities were significantly inhibited by CRIF1 in a dose-dependent manner. Increasing the amount of CRIF1 expression serially reduced the Gal4-Nur77-induced reporter activities (Fig. 3CGo). These observations suggest that CRIF1 represses Nur77 transactivation properties without affecting the level of the protein. Because CRIF1 interacts with the Nur77 AB domain (Fig. 1Go), we examined the effects of CRIF1 on transcriptional activities mediated by this motif. The Gal4-Nur77-AB fusion increased reporter activities by approximately 10-fold. However, cotransfection with CRIF1 inhibited this increase in reporter activity in a dose-dependent manner (Fig. 3DGo). The Gal4-CDE construct had no effect on the Gal4-Tk-Luc reporter (data not shown). Overall, these observations indicate that CRIF1 acts as a repressor of Gal4-Nur77 and that this inhibitory effect is mediated through the Nur77 AB region, which contains a potent ligand-independent AF-1 domain.



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  		Fig. 3. Inhibition of Nur77 Transactivation by CRIF1

A, C2C12 myoblast cells were cultured in 12-well plates until they reached 80% confluence and cotransfected with 0.2 µg of the NurRE reporter construct, 0.1 µg pCDNA 3.1-Nur77, and 0.01–0.2 µg CRIF1. The lower panels of A show a Western blot analysis of Nur77 and CRIF1 expression in cells cotransfected with the indicated expression plasmids. After 24 h incubation, cells were harvested and subjected to Western blot analysis with anti-Nur77, anti-Flag, and antiactin antibodies. B, The same cells cotransfected with 0.2 µg of the NBRE reporter construct, 0.1 µg pCDNA 3.1-Nur77, and 0.01–0.2 µg CRIF1. C, The same cells cotransfected with 0.2 µg of the Gal4-Tk-Luc reporter construct, 0.1 µg Gal4-Nur77, and 0.01–0.2 µg CRIF1. D, The same cells cotransfected with 0.2 µg of the Gal4-Tk-Luc reporter construct, 0.1 µg Gal4-Nur77-AB, and 0.01–0.2 µg CRIF1. The cells were lysed 24 h later, and luciferase activity was measured by a Berthold LB9507 luminometer and normalized to that of Renilla. The fold activation is expressed relative to the luciferase activity obtained after cotransfection of pCMV alone. The histogram represents the average of three independent sets of transfection experiments with the error bars indicating one SD.

 
Little is known about the recruitment of coactivators in Nur77-mediated transcription, SRC-2 (GRIP-1) has been identified as a possible coactivator (20). As shown in Fig. 4AGo, transfection of the Nur77 expression vector efficiently induced NurRE-dependent transcription, and this activity was significantly stimulated (2-fold) by the addition of SRC-2 to C2C12 cells. However, cotransfection of CRIF1 with SRC-2 resulted in a complete loss of the SRC-2-mediated enhancement of NurRE reporter activity. Furthermore, an increase in the amount of CRIF1 not only resulted in suppression of the SRC-2 effects but also further repressed Nur-77-mediated transcriptional activities (Fig. 4AGo).



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  		Fig. 4. HDAC-Independent Inhibition of SRC-2-Mediated Nur77 Transactivation by CRIF1

A, C2C12 myoblast cells were cultured in 12-well plates until they reached 80% confluence and cotransfected with 0.2 µg of the NurRE reporter construct, 0.1 µg Nur77, 1 µg SRC-2 and 0.01–0.2 µg CRIF1. C, The Promega TNT-coupled transcription-translation system was used to produce [35S] methionine-labeled SRC-1 and SRC-2. For assays, 5 µl of in vitro-translated SRC-2 and SRC-1 was mixed with purified GST-Nur77-AB and further reacted with increasing volumes of CRIF1 (5, 10, and 20 µl). All of the reactions were adjusted with same buffer as used in the translation reaction. C, The same cells cotransfected with 0.2 µg of the Gal4-Tk-Luc reporter construct, with or without 0.1–0.2 µg Gal4-CRIF1, and treated with 100 nM TSA or dimethylsulfoxide (DMSO). D, The same cells cotransfected with 0.2 µg of the Gal4-Tk-Luc reporter construct, 0.1 µg Gal4-Nur77, and 0.05 or 0.1 µg CRIF1 and treated with 50 or 100 nM TSA, or DMSO. The cells were lysed 24 h after transfection, and luciferase activity was measured by a Berthold LB9507 luminometer and normalized to that of Renilla. The level of activation is expressed relative to the luciferase activity obtained after cotransfection of pCMV alone. The histogram represents the average of three independent sets of transfection experiments with the error bars indicating one SD.

 
To gain an insight into the underlying mechanism through which CRIF1 reduces SRC-mediated transactivation of Nur77-activated transcription, we performed GST pull-down assays using GST-Nur77-AB, [35S] SRC-1, [35S] SRC-2, and CRIF1. Both [35S] SRC-1, [35S] SRC-2, and CRIF1 were synthesized in vitro using rabbit reticulocytes, whereas GST-Nur77-AB was purified as described in previously (24). In the absence of CRIF1, SRC-1 and SRC-2 were able to interact with Nur77-AB (Fig. 4BGo). However, the addition of increasing amounts of CRIF1 to the reaction mixture progressively inhibited the interaction of SRC-2 with Nur77-AB. In contrast, the SRC-1/Nur77-AB complex was not effectively inhibited by CRIF1.

Although CRIF1 binds to the AB domain of Nur77, it could still induce long-range conformational changes that affect the DNA binding activity of Nur77. For this reason, we performed gel shift assays with a DNA oligonucleotide probe derived from the Nur77 binding element of proopiomelanocortin promoter. The recombinant CRIF1 protein was unable to inhibit GST-Nur77/DNA complex formation (data not shown).

To determine whether CRIF1 has intrinsic repressor activities, transient transfection assays were performed to examine its effects on basal transcription in C2C12 cells. When pCMX-CRIF1, which expresses an in-frame Gal4-DBD-CRIF1 fusion, was cotransfected with Gal4-Tk-Luc into C2C12 cells, expression of the reporter gene was inhibited in a dose-dependent manner (Fig. 4CGo). The repressive effect of Gal4-CRIF1 was not influenced by treatment with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) (Fig. 4CGo). Similarly, as shown in Fig. 4CGo, TSA treatment (50 nM and 100 nM) did not affect the CRIF1-mediated repression of Gal4-Nur77 transcription (Fig. 4DGo). These observations suggest that class I HDACs are not involved in the CRIF1-mediated repression of Nur77-dependent transcription.

Identification of the CRIF1 Domain Involved in the Repression of Nur77 Transactivation
To determine which regions of CRIF1 are responsible for the repression of Nur77, a series of deletion mutants was designed (Fig. 5AGo), based on an analysis of the CRIF1 primary peptide sequence with the programs: BLAST (basic local alignment and search tool) (25), PSORT II (26), COILS (27), Pfam (28, 29) and TargetP (30). The region between amino acid residues 172 and 212 was predicted to be a coiled-coil domain, and the region between residues 184 and 200 contains a canonical nuclear localizing signal (Fig. 5AGo). The repressor activities of the CRIF1 deletion mutants were evaluated by measuring their effects on Gal4-Nur77-AB-mediated transcription. The CRIF1 1–98 construct, which lacks the mid-region and C-terminal half of wild-type CRIF1, did not exhibit repressor activity (Fig. 5BGo). However, CRIF1 1–171, which contains the N-terminal half and mid-region, retained significant repressor activity. The CRIF1 deletion derivatives CRIF1 99–171 and CRIF1 99–222, which consist of the mid-region and of the mid-region and C-terminal half, respectively, still exhibited strong repressor activity (Fig. 5BGo). These results suggest that the mid-region of CRIF1 is involved in repressing Gal4-Nur77-AB-mediated transcription.



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  		Fig. 5. Effects of CRIF1 and Its Mutants on the Nur77 Transactivation Function

A, Schematic representation of CRIF1 wild-type and deletion mutants. C-C, Coiled-coil domain; NLS, nuclear localizing signal. B, C2C12 cells were transiently cotransfected with 0.1 µg expression plasmids, 0.1 µg Gal4-Nur77-AB, and 0.2 µg of the reporter plasmid Gal4-Tk-Luc. The cells were lysed 24 h after transfection, and luciferase activity was measured by a Berthold LB9507 luminometer and normalized to that of Renilla. The activation level was expressed relative to the luciferase activity obtained after cotransfection of pCMV alone. The histogram represents the average of three independent sets of transfection experiments with error bars indicating one SD.

 
Effects of CRIF1 Silencing on Nur77-Mediated Transcription
We previously reported that the siRNA-263 construct, which targets nucleotides 263~284 of the human CRIF1 cDNA, was effective in inhibiting endogenous CRIF1 expression (1). To investigate the role of endogenous CRIF1 on Nur77 transactivation, we examined the activity of Gal4-Nur77 in the presence or absence of the 21-oligonucleotide CRIF1 siRNA-263 duplex. The construct effectively suppressed both exogenous and endogenous CRIF1, as shown in Fig. 6AGo. However, control siRNA duplex, which targets the 578- to 599-nucleotide region of human CRIF1 cDNA, did not silence either endogenous or exogenous CRIF1 expression. As a result, siRNA-578–599 was used as a control for siRNA-263. The transfection of CRIF1 siRNA-263 increased Gal4-Nur77-mediated transcription about 2-fold in the absence of exogenous CRIF1, but control siRNA had no such effect. Furthermore, the transfection of siRNA-263 inhibited the exogenous CRIF1-mediated repression of Gal4-Nur77 transcription, whereas transfection of control siRNA did not. However, functional reversal of inhibition with cotransfected CRIF1 in the presence of CRIF1 siRNA-263 was partial. This may be due to incomplete silencing of overexpressed CRIF1. These observations suggest that the level of endogenous CRIF1 may determine the transcriptional activity of Nur77. Because CRIF1 inhibits Nur77-AB domain-mediated transcription (Fig. 3DGo), we examined the effects of siRNA-263 on the activity of constructs containing only this domain. Again, transfection with CRIF1 siRNA-263 increased Gal4-Nur77-AB domain-mediated transcription in the absence of exogenous CRIF1 (Fig. 6BGo). The transfection of siRNA-263 also inhibited the exogenous CRIF1-mediated repression of Gal4-Nur77-AB transcription, whereas control siRNA did not have this effect (Fig. 6BGo). Overall, the siRNA experiments show that the intracellular level of CRIF1 may determine the ability of Nur77 to regulate transcription.



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  		Fig. 6. Knockdown of the CRIF1 Protein Increases Transactivation of Nur77

A, C2C12 skeletal muscle cells were cultured in 12-well plates until 80% confluence and cotransfected with 0.2 µg of the Gal4-Tk-Luc reporter construct, 0.1 µg Gal4-Nur77, 0.1 µg pFLAG-CRIF1, and 0.5 µg CRIF1-siRNA duplexes. The upper panel of A shows a Western blot analysis of CRIF1 expression in cells. C2C12 cells were cultured in six-well plates until 80% confluence and transfected with CRIF1-siRNA duplexes (500 ng/well). Total lysates were prepared 24 h after transfection and blotted with anti-CRIF1 and anti-ß-actin antibodies. B, The same cells cotransfected with 0.2 µg of the Gal4-Tk-Luc reporter construct, 0.1 µg Gal4-Nur77-AB, 0.1 µg pFLAG-CRIF1, and 0.5 µg CRIF1-siRNA duplexes. Cells were lysed 24 h after transfection, and luciferase activity was measured by a Berthold LB9507 luminometer normalized to that of Renilla. The fold activation is expressed relative to luciferase activity obtained after cotransfection of pCMV alone. The histogram represents the average of three independent sets of transfection experiments with error bars indicating one SD.

 
Repression of Hormone- and PKA-Mediated Nur77 Transactivation by CRIF1
TSH is known to induce and activate Nur77 in target cells (13). To determine whether CRIF1 is involved in this transactivation, we examined the influence of CRIF1 on TSH-induced increases in NurRE reporter activity. FRTL-5 thyroid cells showed very low levels of Nur77/NGFI-B mRNA, but treatment with TSH resulted in a strong increase in transcript levels within 1 h (Fig. 7AGo, upper right). Treatment with TSH also induced increases in the level of Nur77/NGFI-B protein (Fig. 7AGo, upper left). However, the level of CRIF1 RNA, which was high in untreated FRTL-5 cells, was not changed by TSH treatment. As shown in Fig. 7AGo, TSH increased the activity of the NurRE reporter, which has multiple Nur77 binding elements (Fig. 7AGo). The increase in the activity of the NurRE reporter induced by TSH was completely inhibited by the PKA inhibitor, H89 (50 µM). However, CRIF1 expression decreased TSH-induced NurRE reporter activities (Fig. 7AGo) in a dose-dependent manner.



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  		Fig. 7. Induction and Activation of Nur77 by TSH

A, FRTL-5 thyroid cells were grown to near-confluence in Coon’s modified Ham’s F-12 medium containing 5% (vol/vol) calf serum. The cells were maintained for 6 d with 5H medium not containing TSH and serum. The medium was replaced with fresh medium including the following additions as indicated: the FRTL-5 thyroid cells were maintained in 4H without serum condition for 48 h and treated with TSH (1 mU/ml), NurRE confer a higher responsiveness to the TSH treatment. The cells were treated with or without TSH (4H5). FRTL-5 thyroid cells were cotransfected with 0.2 µg NurRE and 0.01–0.2 µg CRIF1 and assayed for their luciferase activity after 48 h. Upper panel of A shows a Northern blot analysis (left) of Nur77 expression induced by TSH in FRTL-5 cells. The same cells treated with TSH (1 mU/ml) at the designated time points. Total RNA (20 µg) was analyzed by Northern blot analysis using Nur77 cDNA, CRIF1 cDNA and ß-actin probes. Western blot analysis (right) of Nur77 expression induced by TSH in FRTL-5 cells. The same cells were treated with TSH (1 mU/ml) at the designated time points, and total lysates were prepared and blotted with anti-Nur77 and anti-ß-actin antibodies. B, C2C12 myoblast cells were cultured on 12-well plates and cotransfected with 0.2 µg of the NurRE reporter construct, 0.1 µg Nur77, 0.1 µg of the wild-type PKA-catalytic domain [PKA(c) WT], or 0.1 µg of the kinase-deficient PKA catalytic domain [PKA(c) KD] and 0.01–0.2 µg CRIF1. C, The same cells were cotransfected with 0.2 µg of the Gal4-Tk-Luc reporter construct, 0.1 µg Gal4-Nur77-AB, 0.1 µg of the PKA-catalytic domain, and 0.01–0.2 µg CRIF1. The reporter assay cells were lysed 24 h after transfection, and luciferase activity was measured by a Berthold LB9507 luminometer and normalized to that of Renilla. The level of activation is expressed relative to the luciferase activity obtained after cotransfection of pCMV alone. The histogram represents the average of three independent sets of transfection experiments with the error bars indicating one SD.

 
Maira et al. (21) reported that CRH signals activate Nur77 through the cAMP/PKA pathway and suggested that PKA signaling pathways activate Nur77 via the AF1-dependent recruitment of coactivators. To determine whether CRIF1 is capable of modulating PKA-mediated Nur77 transactivation, we cotransfected cells with the wild-type PKA expression vector or the kinase-deficient PKA expression vector, together with NurRE and the Gal4-Tk-Luc reporter. Expression of the wild-type PKA catalytic subunit markedly increased Nur77-mediated transactivation of the NurRE reporter and its effects were significantly blunted by coexpression of CRIF1 (Fig. 7BGo). In addition, the expression of the wild-type catalytic PKA subunit increased Gal4-Nur77-AB transactivation markedly, as shown in Fig. 7CGo. In contrast, the kinase-deficient catalytic PKA subunit had no influence on Gal4-Nur77 transactivation. The PKA-induced increase in Gal4-Nur77-AB transactivation was significantly decreased by overexpressing CRIF1 (Fig. 7CGo). Again, CRIF1 expression decreased Gal4-Nur77-AB-mediated transactivation in a dose-dependent manner. These observations suggest that CRIF1 represses the transcriptional activities of Nur77 by inhibiting the transactivating function of the AB domain (AF1) of Nur77, which is activated by TSH and PKA.

Roles of CRIF1 in Nur77-Dependent Gene Expression and Cell Cycle Regulation
It has been reported that TR3/Nur77 can bind specifically to the TR3 response element in the –316- to –324-bp region of the E2F1 promoter (31). To determine whether CRIF1 inhibits Nur77-dependent activation of the E2F1 gene promoter, we measured the promoter activity of E2F1a, a construct containing the –728/+77 region of E2F1 gene promoter linked to a luciferase reporter gene. As shown in Fig. 8AGo, Nur77 could up-regulate E2F1a promoter activity in human embryonic kidney 293 cells. Increasing amounts of CRIF1 gradually decreased Nur77-mediated transcriptional activation of the E2F1a gene promoter (Fig. 8A).



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  		Fig. 8. Effects of Nur77 and CRIF1 on E2F1 Expression

A, The E2F1 promoter construct was cotransfected with the Nur77 expression plasmid in human embryonic kidney 293 cells. Reporter activities were observed at increasing amounts of CRIF1 expression plasmid. After 24 h, cells were collected and assayed for luciferase activity. The results were normalized with an internal control (pRLSV40-Luc) and expressed as the mean ± SE of three independent experiments. B, Regulation of E2F1 mRNA level by Nur77 and CRIF1. The FRTL-5 thyroid cells were infected with AdGFP (M.O.I 40), AdCRIF1-GFP (M.O.I 20)/AdGFP(M.O.I 20), AdNur77(M.O.I 20)/ AdGFP(M.O.I 20) and AdNur77(M.O.I 20)/AdCRIF1-GFP(M.O.I 20) for 24 h. Total RNA (20 µg) was analyzed by Northern blot analysis using rat E2F1 (XM 230765), human CRIF1, and Nur77 cDNA and ß-actin probes. Results are representative of two independent experiments. C, The recombinant adenovirus containing the cDNA encoding the Nur77 and CRIF1, was isolated as described in Materials and Methods. The cells were infected with control AdGFP, AdCRIF1-GFP, AdNur77 at a multiplicity of infection of 30. Cell cycle analysis was performed using a Becton Dickinson fluorescence-activated cell analyzer and Cell Quest version 1.2 software (Becton Dickinson Immunocytometry Systems). At least 10,000 cells were analyzed per sample. Cell cycle distribution was quantified using ModFit LT version 1.01 software (Verity Software House Inc.). Results are representative of two independent experiments.

 
To observe the effect of CRIF1 on Nur77-mediated gene expression, we infected FRTL-5 thyroid cells with AdNur77 adenovirus in the absence or presence of AdCRIF1-GFP infection. As shown in Fig. 8BGo, uninfected cells showed a basal level of E2F1 RNA that was increased in cells infected with AdNur77. Interestingly, cells infected with AdCRIF1-GFP did not show a basal level of E2F1 RNA and cells coinfected with Adnur77 and AdCRIF1 showed much less E2F1 RNA level compared with cells infected with AdNur77 alone.

A recent study showed that ectopic expression of Nur77 in both H460 and Calu-6 lung cancer cell lines promoted cell cycle progression, whereas the inhibition of TR3/Nur77 expression by the small interfering RNA (siRNA) approach suppressed the mitogenic effect of epidermal growth factor and serum (32). We performed flow cytometric analysis with FRTL-5 thyroid cells infected with control AdGFP, AdNur77, AdCRIF1-GFP, and AdNur77/AdCRIF1-GFP (Fig. 8CGo). AdNur77 enhanced the S and G2/M-phase population. Twenty-four percent of AdGFP-infected cells were in S and G2/M phase, and this level increased to 57% upon AdNur77 infection. AdCRIF1 slightly increased cell populations in G1 phase compared with cells infected with AdGFP. Cells coinfected with AdNur77/AdCRIF1-GFP showed significantly fewer cell populations in S and G2/M phase compared with cells infected with AdNur77 alone. In summary, these observations suggest that CRIF1 inhibits Nur77-dependent E2F1 gene expression, which in turn inhibits cell cycle progression mediated by Nur77.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study provides compelling evidence that CRIF1 interacts with the orphan nuclear receptor Nur77 and inhibits its transactivation potential. In particular, CRIF1 interacts with the AB domain of Nur77, which contains the activation function AF-1, and inhibits AB domain-mediated transactivation. Members of the Nur77 subfamily, which includes Nur77, Nor-1, and Nurr1, share common structural features, as well as a similar transactivation domain in the N-terminal portion, a central DBD and a C-terminal LBD. Although the DBDs and LBDs are highly conserved, the N-terminal transactivation (AB) domains are not well conserved, with a shared homology of 27% for Nur77 and Nurr1 and 21% for Nur77 and Nor1 (5, 6). Recent advances have shown that the AB domain is involved in the coactivator- and kinase-induced transcriptional activation of Nur77 (20, 21). Moreover, the AB domain facilitates the recruitment of coactivators such as SRC-2/GRIP-1 (20, 21). These results suggest that it plays a major role in Nur77-mediated transcriptional activation and cofactor recruitment (20, 21). Interestingly, CRIF1 inhibits Nur77 AB domain-mediated transcriptional activities, suggesting that it competes with coactivators that act at this site. However, it is unknown whether CRIF interacts with SRCs, p300, or pCAF. We have tested several nuclear receptors, such as CAR, ERR{gamma}, and SF-1. These receptors do not interact with CRIF1 in yeast two-hybrid assays. Cotransfection with CRIF1 did not inhibit CAR-, ERR{gamma}-, or SF-1-mediated transactivation using corresponding reporter constructs for these factors. These observations suggest that the primary binding of CRIF1 to the nuclear receptor is a prerequisite for the inhibition of transactivation of nuclear receptors.

It was shown that Gal4-CRIF1 has intrinsic repressor activities. Furthermore, CRIF1 repression of Gal4-Nur77 and Gal4-Nur77-AB domain-mediated transcription was not inhibited by the HDAC inhibitor TSA. Although these observations suggest that CRIF1 has an intrinsic repressor function, the domain that possesses repressor activity has not been characterized. The mid-region of CRIF1 can inhibit Gal4-Nur77-AB domain-mediated transcription. This region may have intrinsic repressor activities in CRIF1.

The AB domain of Nur77 was also shown to be hyperphosphorylated in response to variety of signals, and such effects would be consistent with the involvement of the AF-1 domain in PKA action (21). Serine/threonine-rich domains in the N terminus have been implicated in the regulation of Nur77-dependent transcription (33, 34). The role of phosphorylation in the Nur77 AB domain potentiates AF-1-mediated transactivation by the recruitment of coactivators. As shown in Fig. 7Go, the catalytic subunit of PKA increases Nur77 and Gal4-Nur77-AB-mediated transcription, but this effect is inhibited by CRIF1. Several hypothalamic and pituitary hormones, such as CRH, LH, and TSH, activate the PKA system and are involved in the induction and activation of Nur77 (13, 14, 21, 35). The finding that CRIF1 expression modulates the influence of TSH on NurRE-dependent activity suggests that CRIF1 may be involved in the hormone-mediated regulation of Nur77 transcriptional activity.

Chung et al. (1) reported that CRIF1 is involved in regulating the cell cycle, and that it acts as a negative regulator of G1 to S phase progression by inhibiting cyclin-dependent kinases. The relationship between the regulation of the cell cycle and the repression of the Nur77 activities remains to be elucidated. Recently, Nur77 was demonstrated to be involved in the apoptotic process (36). Nur77 translocates from the nucleus to the cytoplasm, where it targets mitochondria to induce the release of cytochrome c and to promote apoptosis. However, CRIF1 may not modify apoptotic responses caused by Nur77 because the apoptotic role of Nur77 does not require its transcriptional or DNA binding activities. Nur77, which is induced by growth factors, epidermal growth factor, and serum, is involved in cell cycle progression and proliferation (32). The mitogenic effect of Nur77 requires its DNA binding and transactivation functions (32). We make a few observations about Nur77 target genes involved in growth and proliferation of endocrine thyroid cells. We observed the effect of Nur77 on E2F1 regulation in thyroid cells. The up-regulation of E2F1 by Nur77 overexpression was inhibited by CRIF1. We have presented a representative FACS analysis that shows that a low dose of CRIF1 adenovirus inhibits Nur77-mediated cell cycle progression. From these observations, we speculate that one of the cell cycle mechanisms regulated by CRIF1 is mediated by CRIF1 inhibition of Nur77 transactivation.

In summary, we have identified CRIF1 as a novel interaction factor for the AB region of the Nur77 orphan nuclear receptor. CRIF1 may repress Nur77-mediated transcription by inhibiting the binding of coactivators to the AB region of the Nur77 receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Plasmids
Highly purified bovine TSH and TSA and all other materials were obtained from Sigma-Aldrich (St. Louis, MO). The CRIF1 expression vector and pEGFP-CRIF1 construct were described previously (1). The CRIF1 mutant expression plasmids pCMV-Tag2-CRIF1 (1–98), pCMV-Tag2-CRIF1 (1–171), pCMV-Tag2-CRIF1 (99–171), and pCMV-Tag2-CRIF1 (99–222) were constructed by amplification of the respective regions of pCMV-Tag2-CRIF1 and subcloning of PCR fragments into the vector pCMV-Tag2. For the Gal4-Luciferase assay, pCMX-CRIF1 was constructed by inserting cDNA into the EcoRI/BamH1 sites of the vector pCMX. All clones were confirmed by DNA sequencing.

The mouse Nur77 cDNA, Gal4-Nur77, Gal4-Nur77-AB domain, and NBRE-Tk-Luc reporter construct were described previously (20, 21). The NurRE 3 copy-POMC-Luc reporter construct and Nurr-1 were obtained from Dr. Jacques Drouin (Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada) and Dr. Thomas Perlmann (Ludwig Institute for Cancer Research, Stockholm, Sweden), respectively.

Yeast Two-Hybrid Protein Interaction Assay
Interactions between CRIF1 and Nur77 in yeast were measured by activating the lacZ reporter constructs, as detected by (ß-Gal assays. The yeast strain EGY48 (p80p-lacZ) (CLONTECH Laboratories, Inc., Palo Alto, CA) was transformed with appropriate plasmids encoding fusions of Lex A-DBD to CRIF1, ERR{gamma}, murine AR, GR, SHP, and SF-1 and plasmids encoding fusions of B42-AD to the CRIF1, RXR, CAR, TR, SMRT, and Nur77 proteins. Colonies were selected on synthetic medium lacking uracil, histidine, and tryptophan (SC-UHW) at 30 C for 3 d, and the ß-gal activity in the extracts prepared from the liquid culture was determined. Five independent colonies from each plate were grown overnight in 2 ml SC-UHW with or without the indicated concentration of bisphenol A. The cells were harvested and assayed for ß-gal activity as described previously (24).

Cell Culture
Murine fibroblasts, NIH3T3 cells, human foreskin fibroblasts and C2C12 murine skeletal muscle cells were cultured in DMEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal calf serum (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 g/ml streptomycin in a humidified of 5% CO2 atmosphere at 37 C.

FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a fresh subclone (F1) that exhibited all previously detailed properties (37), with a doubling time of 36 ± 6 h in the presence of TSH, and they did not proliferate without TSH. After 6 d in medium lacking TSH, the addition of 1 x 10–10 M TSH stimulated thymidine incorporation into DNA at least 10-fold. The cells were diploid between their fifth and 20th passages. The cells were grown in a six-hormone medium consisting of Coon’s modified F-12 supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture of six hormones (6H): bovine TSH (1 mU/ml), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Fresh medium was added every 2 or 3 d, and cells were passaged every 7 or 10 d. In individual experiments, cells were shifted to 5H medium without TSH and 5% calf serum.

In Vitro Translation
CRIF1 was transcribed and translated in vitro with a coupled rabbit reticulocyte system (Promega, Inc., Madison, WI) in the presence or absence of [35S] methionine (Amersham Bioscience, Inc., Arlington Heights, IL) according to the manufacturer’s protocol.

GST Pull-Down Assay
GST pull-down assays were performed according to a previously described method (1). Briefly, GST fusion proteins or GST protein alone were expressed in Escherichia coli BL21 (DE3) pLys cells, which were recovered on glutathione-Sepharose-4B beads (Amersham Bioscience, Inc.). GST fusion proteins were analyzed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels to confirm their integrity and to normalize the amount of each protein. The Promega TNT-coupled transcription-translation system was used to produce [35S] methionine-labeled CRIF1, SRC-1, and SRC-2, which was visualized by SDS-PAGE. In vitro binding assays were performed with glutathione-agarose beads (Amersham Bioscience, Inc.) coated with 500 ng of GST fusion protein and 2–20 µl of [35S] methionine-labeled protein in 200 µl of a binding buffer containing 100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40, 5 µg ethidium bromide, and 100 µg BSA. The reaction was allowed to proceed for 1–2 h at 4 C with constant agitation. The beads were then collected by centrifugation and washed five times with 1 ml binding buffer without ethidium bromide and BSA, resuspended in 20 µl SDS-PAGE sample buffer and boiled for 5 min. The eluted proteins were fractionated by SDS-PAGE, and the gel was treated with Amersham Amplify Fluor, dried at –70 C, and autoradiographed.

Immunoblot Analysis
Cells were lysed by the addition of SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 6% (wt/vol) SDS, 30% glycerol, 125 mM dithiothreitol, and 0.03% (wt/vol) bromophenol blue]. Total cell lysates were denatured by boiling for 5 min, resolved on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked in TBS containing 5% (wt/vol) milk and 0.1% Tween for 1 h, and incubated for 2 h with primary antibodies. The blot was developed using horseradish peroxidase-conjugated secondary antibodies (Phototope-HRP Western Blot Detection Kit, New England Biolabs, Beverly, MA).

Immunoprecipitation
All immunoprecipitation procedures were carried out at 4 C. Cells grown on 100-mm dishes were washed twice with PBS before lysis. Radioimmunoprecipitation assay buffer containing protease inhibitors (20 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml chymostatin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) was added to induce cell lysis and incubated for 30 min. The cell lysate was collected, triturated, and centrifuged at 1000 x g for 10 min. To preclear the cell lysate, the supernatant was mixed with 20 µl protein A/G beads (Santa Cruz Biotechnology, Inc.), incubated for 30 min with agitation, and centrifuged for 15 min at 1000 x g. Precleared samples were incubated with a primary antibody for 2 h with agitation, and protein A beads were added, incubated for 1 h, and centrifuged at 1000 x g. The immunoprecipitates were collected and washed three times with radioimmunoprecipitation assay buffer.

Generation and Transduction of Recombinant Adenovirus
Recombinant adenoviral vectors were generated by a standard homologous recombination method (38). Briefly, cDNAs encoding pEGFP-CRIF1, Nur77 or pGFP were amplified by PCR and cloned into the shuttle vector pxcx2dCMV (cytomegalovirus). AdCRIF1-GFP, AdNur77, and AdGFP adenovirus constructs were generated through homologous recombination between a cotransfected pBHG10 plasmid and shuttle plasmids expressing pGFP-CRIF1 or pGFP in 293 cells as described previously (38). Recombinant adenoviruses were amplified after plaque purification and titrated using the plaque assay as described (39). All expression cassettes were confirmed by DNA sequencing and Western blot analysis. The cells were infected with AdNur77 and either control AdGFP or AdCRIF1-GFP at a multiplicity of infection of 30. One hundred percent of the cells were infected as determined by visualizing green fluorescent protein fluorescence under a fluorescence microscope. Twenty-four hours after transfection, the cells were harvested for Northern blot and flow cytometric analysis.

Confocal Microscopy
NIH/3T3 cells were grown on coverslips and transfected with pEGFP-CRIF1 and pCDNA3-HA-Nur77 using the LipofectAMINE method (Invitrogen Life Technologies) (40). Quiescent cells were washed three times with cold PBS and fixed in 3.7% formaldehyde for 10 min. The fixed cells were mounted on glass slides with PBS and observed by laser-scanning confocal microscopy (Olympus, Japan). To detect pCDNA3-HA-Nur77, cells mounted on glass slides were permeabilized with 2 ml PBS containing 0.1% Triton X-100 and 0.1 M glycine at room temperature, incubated for 15 min, washed three times with 1x PBS and blocked with 3% (wt/vol) BSA in PBS for 10 min at RT. The cells were incubated with the primary anti-HA antibody for 1 h at 37 C, washed three times with 1x PBS and incubated for 1 h with the rhodamine-conjugated antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 37 C.

Transient Transfection and Luciferase Assay
The pNBRE3-Tk-Lluc promoter and Gal4-Tk-Luc promoter constructs were transfected into FRTL-5 thyroid cells or C2C12 mouse myoblast cells by lipofection with a Lipofectamine reagent (Invitrogen Life Technologies). In each well of a 12-well plate, 4 µl Lipofectamine was combined with 200 µl OptiMEM (Invitrogen Life Technologies), to which was then added 0.2 µg of the pNBRE3-Tk-Luc promoter and Gal4-Tk-Luc promoter construct. The cells were incubated overnight with the DNA/Lipofectamine mixture. 5H5% medium or DMEM applied, and the cells were incubated for an additional 24 h before determining the luciferase activity.

The cells were washed with PBS, lysed with 180 µl lysis buffer, and were cotransfected with 0.1 µg of the pRL-CMV plasmid containing the Renilla luciferase gene (Promega, Inc.) according to the manufacturer’s protocol. The extracts were assayed for luciferase activity in triplicate, and light intensity was measured using a luminometer (EG&G Berthold, Bad Wildbad, Germany). The luciferase activity was integrated over a 10-sec period. Firefly luciferase values were standardized to the Renilla values.

siRNA Experiments
A 21-nucleotide siRNA was synthesized and purified using the Silencer siRNA construction kit (Ambion, Inc., Austin, TX). The siRNA sequence targeting the human CRIF1 (GenBank accession no. AF479749) corresponds to the coding region (1). Desalted sense and antisense oligonucleotides targeting the four different regions on the human CRIF1 were synthesized, and the eight nucleotides at the 3-end of both oligonucleotides had the following sequence: 5-CCTGTCTC-3, which is complementary to the T7 promoter. To produce an efficient transcription template, the sense and antisense oligonucleotides for each siRNA were converted to double stranded DNA with a T7 promoter at 37 C. Sense and antisense siRNA transcripts were transcribed for 2 h in separate reactions with T7 RNA polymerase. The reactions were then mixed, and the combined reaction was incubated overnight at 37 C to recover double-stranded RNA. A single-strand specific ribonuclease and a deoxyribonuclease digestion procedure was used to eliminate the five-overhanging leader sequence and the DNA template, respectively. The resulting siRNA was recovered from the mixture of nucleotides, enzymes, short oligomers, and salts in the reaction by column purification.

Northern Blot Analysis
cDNA probes for Nur77, CRIF1, and E2F1 were radiolabeled in a mixture containing Klenow DNA polymerase, 32P deoxycytidine 5'-triphosphate, and random hexamers, and purified with Sephadex-G-50 spin columns. Prehybridization and hybridization were carried out in QuickHyb hybridization solution according to the manufacturer’s protocol (Stratagene, Inc., La Jolla, CA). Low-stringency washes were done at room temperature x 30 min with three changes of wash solution containing 0.3 M NaCl, 0.3 M Na citrate (2x SSC), and 0.1% SDS. High-stringency washes were done at 50 C x 40 min with two changes of wash solution containing 0.2x SSC, 0.1% SDS.

Flow Cytometry Analysis
Samples were prepared for flow cytometry essentially as described previously (1). Briefly, cells were washed with 1x PBS and then fixed with ice-cold 70% ethanol. Samples were washed with 1x PBS and stained with propidium iodide 60 µg/ml (Sigma) containing ribonuclease 100 µg/ml (Sigma) for 30 min at 37 C. Cell cycle analysis was performed using a Becton Dickinson fluorescence-activated cell analyzer and Cell Quest version 1.2 software (Becton Dickinson Immunocytometry Systems, Mansfield, MA). At least 10,000 cells were analyzed per sample. Cell cycle distribution was quantified using ModFit LT version 1.01 software (Verity Software House Inc., Topsham, ME).


   FOOTNOTES
 
First Published Online September 30, 2004

This work was supported by grants from the National Research Laboratory Program (M1-0104-00-0014), and Korea Science and Engineering Foundation grant (R02-2004-000-100030-0) (to M.S.), Korea Research Foundation Grant C00126 (to H.-S.C.), the Ministry of Science and Technology, Korea and by the Department of Defense, Breast Cancer Research Program Grant DAMD-17-02-1-0525, USA (to I.B.).

Abbreviations: AF, Activation function; AR, androgen receptor; CAR, constitutive androstane receptor; CRIF1, CR6-interacting factor 1; DBD, DNA binding domain; ERR, estrogen-related receptor; Gadd45, growth arrest and DNA damage inducible; ß-gal, ß-galactosidase; GR, glucocorticoid receptor; GRIP1, GR-interacting protein 1; GST, glutathione-S-transferase; HDAC, histone deacetylase; LBD, ligand binding domain; NGFI-B, nerve growth factor-inducible gene B; NurRE, Nur-responsive element; PKA, protein kinase A; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor 1; SHP, short heterodimer protein; siRNA, small interfering RNA; SMRT, silencing mediator of retinoid and thyroid hormone receptor; SRC, steroid receptor coactivator; TR, thyroid hormone receptor; TSA, Trichostatin A.

Received for publication March 15, 2004. Accepted for publication September 22, 2004.


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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   knrl  |  TRα  |  CAR  |  RXRα  |  ERRγ  |  GR  |  AR  |  NGFIB  |  SF-1
Coregulators:   CRIF1  |  SRC-1  |  GRIP1  |  SMRT



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