A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation
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The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription.
A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARβ) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner.
These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.
KeywordsRetinoid Nuclear Receptor Reporter Plasmid Retinoic Acid Receptor Retinoid Receptor
retinoic acid receptor
all-trans retinoic acid
9-cis retinoic acid
hormone response element
RAR beta response element
direct repeat of the hormone response element consensus sequence separated by "n" basepairs
neural proliferation differentiation and control protein one
open reading frame
plasmid expressing rat brain cDNA
agarose bound glutathione
gel mobility shift assay
estrogen receptor alpha
estrogen receptor response element
E2F response element
cellular retinoic acid binding protein, type II
multiple tissue Northern.
Retinoids are a class of compounds that demonstrate a profound effect on cellular differentiation and proliferation [1, 2, 3, 4]. The physiologically active retinoid metabolite, all-trans retinoic acid (atRA) regulates gene expression by complexing with members of the steroid/hormone family of nuclear receptors. Two groups of nuclear receptors have been shown to mediate retinoid signaling. The three isotypes (α, β and γ) of retinoic acid receptor (RAR) bind atRA in acquiring a transcriptionally active state [3, 5, 6]. A second group of receptors, called the retinoid-X receptors (RXRs; isotypes α, β and γ), bind to an atRA isomer, 9-cis retinoic acid (9cRA), and can activate transcription as a homodimer complex [6, 7, 8]. However, the predominant physiological function of RXRs may be as an obligate heterodimer partner for a number of other nuclear receptors including the thyroid hormone receptors, the peroxisome proliferator activated receptors, the vitamin D receptor as well as the RARs [9, 10, 11].
Steroid/hormone nuclear receptors regulate the expression of specific target genes by binding to cis-acting DNA sequences called hormone response elements (HREs). RAR/RXR heterodimers preferentially bind HREs categorized as direct repeats of the half-site consensus sequence AGGTCA or AGTTCA separated by five or two nucleotides, referred to as a DR5 and DR2 respectively. One of the first and most potent RAR HRE identified was the DR5 motif in the promoter of the RARβ gene itself .
Retinoid induced transcriptional activation in mammalian cells is influenced by the cell cycle regulator E2F-1 . The E2F-1 transcription factor is part of a multi-protein family that regulates the expression of gene products needed for the initiation and completion of DNA synthesis [14, 15]. E2F-1 and other members of this family share the ability to induce the transition from G1 to S phase of the cell cycle. E2F dependent transcription is thought to be dominantly regulated by interactions with the retinoblastoma (Rb) gene product and other Rb-related proteins (reviewed in ). It has been demonstrated that co-expression of E2F-1 inhibits atRA induced transcription from a RARβ response element driven reporter plasmid . However, the E2F-1 protein could not be demonstrated to directly bind to the RARs or the βRE, suggesting an interaction with a common accessory factor or factors.
The mechanism of steroid/hormone nuclear receptor function includes interaction with a number of recently identified co-regulators [17, 18, 19, 20, 21]. The yeast two-hybrid system and far-western blotting have been used to identify several proteins that associate with nuclear receptors in a ligand-dependent manner. These include the co-repressors SMRT and N-CoR, the coactivators SRC-1 and RIP-140 and the integrator proteins p300 and CBP. Recently, a short conserved amino acid sequence motif (LXXLL) has been reported to be necessary and sufficient to mediate the binding of RIP-140, SRC-1 and CBP to liganded nuclear receptors [22, 23]. For the most part, these co-regulators have not been shown to be specifically expressed with respect to receptor function and usually demonstrate a significant degree of interaction overlap between various nuclear receptors. RXRs are promiscuous in association with multiple nuclear receptors and therefore would be an excellent target by which co-regulator function could effect sweeping changes in transcriptional regulation associated with complex cellular processes such as differentiation.
We and others have shown that it is possible to reconstitute RXR transcriptional function in yeast [24, 25, 26]. In this study, we attempted to identify, from randomly expressed rat cDNA sequences, proteins that positively influence RXR mediated transcription in yeast. A cDNA sequence was isolated containing an open reading frame (ORF) which coded for a protein that has a very high amino acid identity (94%) with a previously described mouse neural proliferation, differentiation and control protein (NPDC-1) . More recently, a human isoform has been identified sharing similar structural feature but differing in its tissue distribution . Mouse NPDC-1 expression has been demonstrated to down-regulate cell proliferation in a reversible fashion and suppress transformation properties when stably transfected into cell lines . A protein-protein interaction has also been shown between mouse NPDC-1 and the cell cycle regulator E2F-1 [29, 30]. Alignment of the mouse, rat and human NPDC-1 proteins reveals a conserved LXXLL amino acid sequence proximal to their amino terminus. In this paper we demonstrate that NPDC-1 can physically interact with RXR and RAR, and can repress retinoid signaling in a mammalian cell transcription assay. We also show that NPDC-1 can repress transcription events mediated by retinoid receptors and several other steroid/nuclear receptor family members, can modulate binding of retinoid receptors to their response elements and can inhibit the endogenous activity of an E2F transcriptional reporter plasmid in mammalian cells. Together with previously published information, these data support a hypothetical role for NPDC-1 in neuronal proliferation and differentiation as a corepressor of retinoid receptor and E2F-1 mediated gene transcription events.
Functional Yeast Screen Identifies a RXR Transcription Co-regulator
Several positive colonies were expanded in solution and secondarily tested for βGal activity using the βGal substrate ONPG. The βGal enzymatic activity present in yeast extracts was determined and those cDNA expressing transformants which induced βGal transcription at least 2 fold over RXR/βRE-βGal yeast were expanded in solution and extracts were prepared for plasmid rescue by bacterial transformation . Random bacterial transformants were used to generate plasmid preps. EcoRI, Hind III restriction enzyme digests generated a DNA fragment pattern which easily identified pcRB insert containing plasmids separate from RXR expression and βRE-βGal reporter plasmids also present in every yeast extract.
A pcRB plasmid containing an insert of approximately 1.4 kb in length was isolated (pcRB1.4). To demonstrate the specific enhancement of RXR mediated transcription versus an enhancement of transcription in general, the pcRB1.4 plasmid was co-transformed with and without RXR expression plasmids. In these experiments a reporter plasmid with two copies of the βRE incorporated into the βGal reporter plasmid was used to increase the sensitivity of the assay. RXR transcription was enhanced by pcRB1.4 two fold over RXR expression alone, but pcRB1.4 did not enhance transcription when co-transformed with the reporter plasmid alone (Fig. 1C). These results demonstrated that the pcRB1.4 plasmid was autonomously capable of specifically enhancing RXR mediated transcription.
DNA Sequencing Reveals the RXR Transcription Coregulator to be NPDC-1
The cDNA for the rNPDC-1 was subsequently used to screen a human embryonic brain λgt11 cDNA library. This screen identified a 2.5 kb cDNA that contained an open reading frame of 325 amino acids and greater than 99% identity with a recently published human homolog for mNPDC-1 .
Figure 2 contains an alignment of rNPDC-1 and hNPDC with the published sequence for mNPDC-1. The overall amino acid homology of the NPDC-1 protein between species suggests that these proteins may have a conserved function. Some conserved amino acid motifs may be related to specific function. All three NPDC-1 homologues have a perfectly conserved LXXLL motif (amino acids 15 – 20, Fig. 2), a sequence that has been shown to mediate the interaction of coregulatory proteins with nuclear receptors [22, 23].
Northern Analysis of Rat and Human NPDC-1 Tissue Distribution
The human NPDC-1 probe identified two mRNA transcripts of approximately 1.5 and 6.3 kb in size in all tissues examined. These data are similar to those reported by Qu et. al.  and unlike the rat, the relative abundance of the two transcripts was more consistent in each specific human tissue. As with NPDC-1 mRNA expression in mice and rats, human NPDC-1 mRNA was most abundant in the brain. A considerable amount of NPDC-1 mRNA is present in the heart and pancreas and to a lesser extent in placenta, lung, liver, skeletal muscle and kidney. Since the cloned human cDNA fragment identified here is approximately 2.3 kb in size, has a poly-A tail representation and a TGA codon immediately upstream of the NPDC-1 ORF, it is possible that it represents a portion of the 6.3 kb transcript minus an unusually extended 5' untranslated region. The 1.5 kb transcript likely represents a splice variant isoform similar in size to the transcript that represents NPDC-1 expression in mice and rats, although the presence of an alternative, very similar gene transcript that is detectable with the human NPDC-1 probe cannot be ruled out.
NPDC-1-RXR and NPDC-1-RAR form Protein-Protein complexes In Vivo
As seen in Fig. 4A recombinant NPDC was able to pull down E2F-1 (Fig. 4A), RXRα and RARβ (Fig. 4B) from PC12 lysates. The interaction with NPDC appeared to be independent of ligand stimulation. In fact, ligand appeared to slightly reduce the ability of NPDC to associate with either RXRα or RARβ. As a binding control we attempted to pull down HA-E2F1 from PC12 lysates. We demonstrate that human NPDC can bind to E2F1 expressed in mammalian cells. Although a mouse NPDC-E2F interaction has been previously demonstrated using in vitro binding and mammalian transcription protocols [29, 30], these are the first studies demonstrating a potential for in vivo complex formation between hNPDC-1 and E2F proteins.
NPDC-1 can bind directly to RXR and RXR/RAR complexes
NPDC-1 Facilitates the In Vitro DNA Binding of RXR Homodimers and RAR/RXR Heterodimers
To measure the direct effect of NPDC-1 on the DNA binding properties of retinoic receptors, RXR and NPDC-1 bacterially expressed protein was tested for binding on a DR-1 RXRE probe (Fig. 6B). As we, and others, have previously observed, RXR bound to the DR-1 probe in the absence of ligand, forming, what appears to be, a homodimer complex (upper band) and a monomer complex (lower band). The addition of NPDC-1 increased both the upper and lower complexes, again without any observable increase in complex mobility. To evaluate the presence or absence of NPDC-1 in these complexes, supershifts were performed with antibodies for RXRα and NPDC-1 (Fig. 6C). Supershift experiments demonstrated that NPDC-1 is not part of the RXR/DR-1 complex and that RXRα is a major component of both lower and upper bands.
hNPDC-1 represses E2F-1 mediated transcription
NPDC-1 Functions in Mammalian Cell Transcription Assays
To examine NPDC-1 effects on nuclear receptor mediated transcriptional activation in mammalian cells, the rat NPDC-1 gene was sub-cloned into a cytomegalovirus (CMV) promoter driven mammalian expression plasmid. Transfections of mammalian expression constructs for steroid/nuclear receptor family members hRXRα, hRARβ, rat peroxisome proliferator-activated receptor alpha (rPPARα), human estrogen receptor alpha (ERα) and human vitamin D receptor (hVDR), along with their respective luciferase reporter constructs were analyzed with respect to hNPDC effects on ligand-induced transcriptional activation mediated by these receptors (Fig. 7B). As seen in Figure 7B, NPDC-1 strongly repressed transcription mediated by the retinoid receptors, moderately repressed transcription mediated by PPARα and ERα, and had very little effect on VDR.
NPDC-1 Transcriptional Repression Appears Not to be Through its LXXLL Motif
RXR Binds the Amino Terminus of NPDC-1
In this study, the gene for the rat homologue of the mouse NPDC-1 protein was identified as a positively regulator of RXR mediated transcriptional activation in yeast. The cloning approach used took advantage of RXR's inherent ability to bind DNA and activate transcription in yeast. By creating the proper conditions for the sensitive detection of minimal transcriptional enhancement, random rat brain cDNA products were screened for their ability to facilitate RXR function. Whereas the yeast two-hybrid system has most often been used to identify a positive protein-protein interaction between cDNA products and the ligand binding domain of nuclear receptors [37, 38, 39, 40, 41, 42, 43], the strategy used here screens for a functional relationship between cDNA products and the entire receptor, without the addition of hybrid domains. A rat NPDC-1 expression plasmid autonomously affected transcription of a yeast reporter plasmid when co-expressed with RXR but had no enhancing activity when transformed alone.
The rat NPDC-1 gene sequence was subsequently used to screen a human brain cDNA library from which a human NPDC-1 homologue was identified. Alignment of the mouse NPDC-1 amino acid sequence with the rat and human proteins demonstrated that these proteins are evolutionarily well conserved, indicating that they likely perform a similar and essential function. An LXXLL amino acid motif found in a number of transcription coregulators has been shown to be necessary and sufficient to mediate the binding of coregulators to liganded nuclear receptors [22, 23]. X-ray crystal structure analysis suggest the mechanism for the interaction of nuclear receptors with coregulators involves specific contacts between a coregulators LXXLL motif and a highly conserved nuclear receptors motif, termed activating function-2 (AF-2), located at the C terminus of nuclear receptors [44, 45]. The three species specific NPDC-1 proteins have a conserved LXXLL amino acid sequence proximal to their amino terminus, but mutation studies presented here suggest this domain does not play an integral role in the repression of transcription mediated by NPDC-1. Furthermore the deletion studies presented here suggest that the NPDC more probably interacts with RXR through an area localized more centrally in the protein. The most obvious protein interaction domain in this region is a coil-coil domain located between amino acids 76–112 . Finally, this observation is not unprecedented in that PNRC, a recently identified nuclear receptor coregulatory protein [46, 47], has also been observed to bind nuclear receptors through a motif other than its LXXLL motif. Further studies are ongoing to establish the role of both the NPDC-1 LXXLL motif and coil-coil domain and their functional significance in the coregulation of retinoid receptor-mediated transcription events.
Northern analysis and reverse transcriptase-PCR amplification of RNA showed that the mouse NPDC-1 was preferentially expressed in the nervous system and not detectable in the other tissues examined . In contrast, Northern analysis revealed that human and rat NPDC-1 mRNA is variably, but much more ubiquitously expressed. Similar to previously published data , our results suggest that NPDC-1 mRNA in humans exists as multiple splice variant isoforms. The cloned human cDNA isolated was larger than the predominant smaller mRNA species seen in every tissue, indicating that it likely represents a sub-fragment of the larger transcript, although we cannot rule out the existence of a unique homologous mRNA species. In rats we identified a single but abundant brain mRNA species. Its size approximates that found in mouse brain tissue  and would be largely represented by our cloned cDNA. Some of the other tissues probed displayed an mRNA species of a similar size along with the variable presence of larger hybridizing transcripts. The tissue specific relative intensities of these larger hybridizing transcripts were not consistent, which confuses their interpretation. Further examination will be required to identify them as splice variant isoforms or as cross hybridizing unique gene products.
Of special note was the seemingly opportunistic cloning of this RXR coregulator in a ligand independent yeast assay. Efficient DNA binding normally requires the ligand dependent homodimerization of RXR . Although RXR mediates significant ligand inducible transcriptional response in yeast, unliganded RXR induces minimal activation from a βRE containing reporter plasmid. RXR has a higher binding affinity for a response element from the cellular retinoic acid binding protein, type II (CRBPII) promoter region . A significant amount of constitutive activity results from a CRBPII response element containing yeast reporter plasmid and unliganded RXR , indicating that unliganded RXR is capable of facilitating transcription in yeast and a limiting condition for this function may be RXR's ability to bind DNA. From this perspective, it seems likely that the procedure by which we cloned the rat NPDC-1 gene would identify gene products that influence RXR's ability to bind DNA. The gel mobility shift assay results support the contention that NPDC-1 can facilitate the binding of DNA by unliganded RAR/RXR heterodimers or RXR homodimers. Although difficult to establish, there is precedent for an enhancement of DNA binding as a mechanism for nuclear receptor coregulator function. Human homologues of the S. cerevisiaeSWI/SNF family of genes have been shown to function as coregulators of transcriptional activation by nuclear receptors, including RAR [49, 50]. These coregulators have been characterized to function as members of large multi-subunit complexes that alter chromatin structure to facilitate receptor DNA binding. Human homologues of the S. cerevisiaeSWI/SNF gene products exhibit a protein-protein interaction with the nuclear receptor AF-2 domain and potentially require the receptor's DNA binding ability to associate with specific promoter regions for function . The yeast SWI/SNF complex stimulates the binding of DNA by the yeast transcription factor GAL4 in GMSAs without maintaining a strong association with the protein/DNA complex , similar to what is observed here with NPDC-1.
We show that NPDC-1 can repress the induction of transcription in mammalian cells by several nuclear/steroid receptor family members. This activity places NPDC-1 into an expanding list of proteins and RNAs whose structure appear to be targeted to DNA by transcription factors and whose function appears to be integrated into regulation of transcription by the RNA polymerase complex [53, 54, 55, 56, 57]. Data demonstrating that NPDC-1 can directly interact with nuclear receptors, can influence nuclear receptor binding to DNA and can bind to and repress E2F mediated transcription events  would strongly suggest NPDC-1 is a true corepressor protein. There also appears to be some specificity in NPDC-1's ability to corepress transcription, with very strong corepression occurring with RXR and the RXR heterodimer partner RAR, only moderate transcriptional repression with the RXR heterodimer partner PPAR and the homodimerizing receptor ER, and little if any effects on transcription mediated by the RXR heterodimerizing receptor VDR. However, RXR's promiscuous nature as a heterodimer partner with a number of other receptors implies that NPDC-1 co-regulation of transcription may be dramatic and its expression involved with sweeping changes in the transcriptional regulation of genes within a cell. The up-regulation of mouse NPDC-1 expression has been intimately linked in vivo with a variety of morphological changes as precursor nerve cells begin to terminally differentiate and this expression is maintained up to the adult age . P19 cells are a pluripotent murine embryonic carcinoma cell line that can be induced by atRA to differentiate into cells that are biochemically and morphologically similar to cells of the central nervous system . It has been shown that atRA induced differentiation of P19 cells results in the expression of mNPDC-1 . Whether NPDC-1 expression is directly regulated by an atRA activated RAR or is under the control of some other differentiation induced transcriptional regulator remains to be examined. However, atRA up-regulation of a negative mediator of its own signal would be somewhat inconsistent with this ligand's signaling pathway which is also known to up-regulate the expression of the RARα and RARβ genes .
As has been shown for mNPDC-1 [29, 30] hNPDC-1 was observed to directly interact with and suppress transcription mediated by E2F-1. E2F trans-activation of transcription is critical for the regulated G1 to S phase transition of the cell cycle . The subsequent down regulation of E2F activity during S phase is also critical for viable cell propagation and is believed to be largely the result of its association with Rb or Rb related proteins . It has been shown that the Rb-E2F complex will bind DNA but function to inhibit E2F-responsive genes. NPDC-1 may inhibit overall E2F transcription by preferentially promoting the binding of the inhibitory complex to DNA over the binding of the activating E2F complex. In the absence of ligand, RAR/RXR heterodimers have been theorized to associate with transcription repressing co-regulators [42, 60]. NPDC-1 association with RXR is ligand and RAR heterodimer independent implying that NPDC-1 may also contribute to the repression of RAR responsive genes in the absence of atRA. The E2F-1 factor is thought to play a critical role in the maintenance of cellular proliferation whereas atRA-treated cells normally proliferate at reduced rates. There are some reports suggesting that some intermediate mechanism links these apparently antagonistic pathways [13, 61]. Limited availability of NPDC-1 may be involved in the competitive relationship between these transcription factors' function.
The evidence presented here demonstrates that NPDC-1 has remained highly conserved through human and rodent evolution and is capable of binding retinoid receptors, influencing their associations with DNA binding and repressing their ability to regulate gene expression. These results, along with previously published data demonstrating mNPDC-1' s ability to repress proliferation and stimulate differentiation, suggest that NPDC-1 plays an important role in retinoid signaling as cells differentiate. Cumulatively these data categorize NPDC-1 as a tissue-specific corepressor of retinoid-mediated transcription directly linked to differentiation of neuronal cells. Although NPDC-1 has yet to be aligned with any specific neuronal tumorigenesis event it's intrinsic role in proliferation and differentiation suggest it might function as a tumor suppressor gene. Further studies will be necessary to establish this possibility
Materials and methods
Plasmids and Constructions
The yeast expression plasmids YEphRXRα(TRP), YEphRARγ(LEU) and the yeast transcription reporter plasmids YRpβRE(1) and YRpβRE(2) have been described elsewhere . The mammalian expression plasmid pCMVβ (Clontech) was modified to introduce a bacterial T7 promoter enabling overlapping expression from this promoter and the CMV immediate early gene promoter/enhancer. A polycloning site was also introduced downstream from these promoters, replacing the β-galactosidase (βGal) gene, and the resulting plasmid was designated pCMV/T7PC. NotIfragments containing the cloned rat and human NPDC-1 genes were sub-cloned into pCMV/T7PC creating pCMVrNPDC-1. The mammalian expression plasmids pRShRARβ•, pRShRXRα• , pRShERα , pRSrPPARα•, pRShVDR  and pRSβGal  are described elsewhere. The mammalian reporter plasmids βRE-tk-luc and E2FRE-tk-luc were constructed by sub-cloning a single copy of annealed synthetic oligonucleotides into HindIIIor SalI– BamHI, respectively, prepared pBL-tk-luc vector . The sequence of the oligonucleotides used were as follows: βRE, 5'-AGCTTAAGGGTTCACCGAAAGTTCACTCGCAT-3' and 3'-ATTCCCAAGTGGCTTTCAAGTGAGCGTATCGA-5'; E2FRE, 5'-TCGACATGCAATTTCGCGCCAAACTTGGGA-3' and 3'-GTACGTTAAAGCGCGGTTTGAACCCTCTAG-5'. The mammalian reporter plasmid ERE(5)-MTV-luc was constructed by sub-cloning five copies of annealed synthetic oligonucleotides (5'-AGCTGTCAGGTCACAGTGACCTGAT-3' and 3'-CAGTCCAGTGTCACTGGACTATCGA-5') into HindIIIprepared MTV-luc .
Single amino acid mutations in the LLRLLL motif of the pCMV-hNPDC-1 cDNA were made using the QuikChange Site Directed Mutagenesis Kit (Stratagene) and protocols as described in the users manual. Oligonucleotides used in the construction of the various mutations include M1-ALRLLL
5'-GGACGAGGCCGGAGAGCAGCAGCCGCAGCGCCCGCAGGTGCCGCG-3'), M2-LLRLAL (5'-CGCGGCACCTGCGGCTGCTGCGGCTGgcGCTCTCCGGCCTCGTCC-3' and
5'-GGACGAGGCCGGAGAGCGCCAGCCGCAGCGCCCGCAGGTGCCGCG-3') and M3-AARLLL
5'-GGACGAGGCCGGAGAGCAGCAGCCGCGCCGCCCGCAGGTGCCGCG-3'). The integrity of introduced mutations was verified by DNA sequencing.
A bacterial expression construct for full-length hNPDC-1 and a series of hNPDC-1 mutant constructs were generated in pET-32a (Novagen). Synthetic oligonucleotide primers for full-length hNPDC-1 (forward: 5'-TTGAATTCATGGCGACGCCGCTGC-3' and reverse: 5'-TTGGATCCAGTGGCAGTGCAGGCGGTG-3'), a D1 mutant containing amino acids 1–268 (forward: 5'-TTGAATTCATGGCGACGCCGCTGC-3' and reverse: 5'-TTGGATCCATGGGTGGCTCTTTATGC-3'), a D2 mutant truncating the predicted transmembrane domain of hNPDC-1 and containing amino acids 1–182 (forward: 5'-TGAATTCATGGCGACGCCGCTGC-3' and reverse: 5'-TTGGATCCATGCCGTCGCCTTGCCCTC-3'), a D3 mutant truncating NPDC-1 just before the putative coil-coil domain and containing amino acids 1–76 (forward: 5'-TTGAATTCATGGCGACGCCGCTGC-3' and reverse: 5'-TTGGATCCATCCCTTGCTGGTCCTCC-3'), a D4 mutant retaining the putative transmembrane domain but deleting the LXXLL motif and containing residues 112–325 (forward: 5'-TTGAATTCGGACACTCAACTCCGC-3') and reverse: 5'-TTGGATCCAGTGGCAGTGCAGGCGGTG-3') and a D5 mutant retaining all residues after the putative transmembrane domain and containing residues 205–325 (forward: 5'-TTGAATTCTGCTGGTGCAGGCTGCAG-3' and reverse: 5'-TTGGATCCAGTGGCAGTGCAGGCGGTG-3') were used in polymerase chain reaction technology (PCR) reactions to generate a full-length hNPDC-1 and mutant fragments containing terminal EcoRI(5') and BamHI(3') restriction sites. Subsequently the hNPDC-1 and hNPDC-1 mutant PCR fragments were subcloned into EcoRI/BamHIrestricted pET32-a vector. These constructs generated hNPDC-1 proteins tagged with thioredoxin (TRX), 6xHis, and S-protein at its amino terminus. The integrity of all plasmid constructs and the composition of unique cloned cDNAs were confirmed by dideoxy sequencing.
Yeast Expression Library Screening and Transcription Assay
Reconstitution of RXR transcriptional activation in the auxotrophic mutant S. cerevisiaecell line BJ5409 (MAT•, leu2D, his3D200, ura3–52, tr 1) has been described elsewhere . An established YEpRXRα (TRP), YRpβRE(1) transformant was further transformed with a rat brain cDNA yeast expression library , a generous gift of Dr. Robert Dickson, University of Kentucky, by the method of lithium acetate . Transformed yeast were plated onto yeast nitrogen base, 2% bacto agar plates supplemented with 2% glucose, 20 μg/ml adenine sulfate, 20 μg/ml histidine-HCl, 10 μM CuSO4, 0.5 μM all-trans retinoic acid and 0.06 mg/ml 5-bromo-4-chloro-3-indolyl-β-o-galactopyranoside (X-GAL). Activation of βGal transcription by yeast grown in solution was determined as previously described  with the following modifications. The βGal enzymatic activity present in yeast extracts after 22–24 hr incubation in minimal media plus 25 μM CuSO4 was determined by measuring the conversion of the o-nitrophenyl-β-D-galactopyranoside (ONPG) colorimetric substrate (A415) normalized to cell number approximations (A600). Yeast ONPG transcription assays were repeated at least twice with separate transformants and yielded similar results.
Cloning human NPDC-1
A full-length cDNA fragment for rNPDC-1 was labeled with 32P by nick translation and used to screen a λgt-11 human fetal brain cDNA library (Clontech) . Utilizing dideoxy DNA sequencing techniques, a 2.5 kb insert containing λgt-11 clone was established to harbor the complete coding sequence for the human homolog of mNPDC-1 .
Northern Blot Analysis
Rat and human Multiple Tissue Northern (MTN) blots were purchased from Clontech Laboratories Inc. The rat and human cDNA clones were used to prepare [α-32P]-labeled DNA probes by nick translation of double-stranded fragments . The blots were probed using ExpressHyb solution as suggested by the manufacturer (Clontech).
Bacterial Protein Expression
Bacterially expressed NPDC-1 (Trx-S-NPDC) was purified from BL21-CodonPlus-RP E. coli (Stratagene) harboring the pET32a-hNPDC-1 expression construct. An overnight culture was diluted 1:5 into 2xYT bacterial media and incubated at 37°C for 2 hours. Subsequently, cells were moved to room temperature, grown to 0.6 OD600 and induced with 1 mM IPTG for an additional 4 hours. Cells were collected at 6000 × g for 10 minutes. The pellet was resuspended in 20 ml triple detergent lysis buffer (50 mM Tris-8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate) supplemented with 1 mg/ml lysozyme, protease inhibitors (Roche) and Benzonase nuclease (Novagen). Cells were allowed to lyse for 20 minutes at room temperature. Lysate was clarified in 50 ml Oakridge tubes at 25,000 × g for 20 minutes. Clarified lysate was injected onto HiTrap chelating HP columns (Amersham Pharmacia) preloaded with nickel. Bound protein was washed with 10 column volumes of His-wash (50 mM Tris-8.0, 500 mM NaCl, and 20 mM Imidazole). Proteins were eluted in His-elution buffer (50 mM Tris-8.0, 500 mM NaCl, and 500 mM Imidazole) and subsequently desalted on a HiTrap desalting column (Amersham Pharmacia) into TBS-P (10 mM Tris-7.6, 150 mM NaCl, and 10% glycerol).
Bacterially expressed pGEX-hRXRα was purified using standard GST-purification techniques except as modified below. Cells were collected and clarified lysates were produced as above. Lysates were injected onto a GST-rap HP column (Amersham Pharmacia) and washed with 10 column volumes of TBS-P. Proteins were eluted with 25 mM glutathione pH 7.0 and desalted into TBS-P.
Gel Mobility Shift Assay
Gel mobility shift assays were performed essentially as described previously . Briefly, annealed synthetic oligonucleotides coding for the DR-5 RARE (5'-AGCTTCAGGTCAGAAGGTCAGAGAGCT-3') and DR-1 RARE (5'-TCGAGGGTAGGGTTCACCGAAAGTTCACTCG-3') were radioactively end-labeled with [γ-32P]-ATP using T4 polynucleotide kinase (PNK) . Labeled probes were incubated with variable combinations of recombinant protein or PC12 nuclear extract in a 20 ul reaction mixture (5 mM Tris pH 7.9, 15 mM HEPES-KOH pH 7.9, 5 mM EDTA, 3.5 mM MgCl2, 5 mM DTT, 0.1% Tween-20, 10% Glycerol, 50 mM KCl) as follows: NPDC (10 μg) and RXR (5 μg) plus PC12 (25 μg). The protein/DNA complexes were separated from free radiolabeled DNA by electrophoresis in non-denaturing polyacrylamide gels and autoradiographed. In order to identify proteins present in complexes, supershifts with antibodies to RXRα (D-20, Santa Cruz) and NPDC-1 (custom antibody and α-thioredoxin, Thio-probe, Santa Cruz) were used with the above reactions.
In Vivo Pull-downs
The ability of NPDC to interact with in vivo expressed RARβ and RXRα was assayed by using Trx-S-NPDC as a pull-down reagent. PC12 cells were transfected with either pCMV-RARβ or pCMV-RXRα using Effectene reagent (Qiagen). After 48 hours cells were lysed on the plate in 200 μL of lysis buffer-PD (50 mM Tris-7.5, 150 mM NaCl, 20 mM MgCl2, 10% glycerol, 1% NP-40) supplemented with 1X protease inhibitors (Calbiochem).
Trx-S-NPDC was prebound to S-protein covalently attached to agarose beads for 20 minutes. 30 μL of a 50% slurry of beads was added to 300 μL of buffer PD. 100 μg of cell lysate expressing HA-E2F-1, RARβ, or RXRα were added to the tubes and incubated with rotation for 1 hour at 4°C. Beads were collected and washed three times in buffer PD. Bound-proteins were eluted by adding 20 μL of 2X SDS-PAGE sample buffer. Samples were run on a 12.5% SDS-Page gel and immunoblotted onto nitrocellulose filters. Filters were probed with anti-HA (Cell Signaling), anti-RARβ (C-19, Santa Cruz), or anti-RXRα (D-20, Santa Cruz).
In Vitro Pull-Down Assays
Agarose-glutathione (Ag-G) bound Glutathione-S-Transferase (GST) and GST-RXR fusion protein were prepared from BL21 (DE3) LysS cell transformants as described above, with the exception that agarose bound recombinant protein was not eluted but rather stored bound to beads. Bacterial extracts containing pETCFlag, pETrNPDC(F) and pEThRARγ were generated as described above with the exception that cleared soluble fractions were used or stored at -70°C without further affinity purification. Protein concentrations were determined using Bio-Rad Protein Assay per the manufacturer's instructions (Bio-Rad).
The pull-down assays were performed as described previously . Briefly, 30 μl of a GST-RXR agarose bound 33% protein slurry was pre-equilibrated with NENT buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) and incubated for 60 min at room temperature with 40 μg of pETrNPDC(F) extracts without and/or with 60 μg of pEThRARγ extracts or 1 μM 9-cis retinoic acid. Successive washes with NENT removed unbound material and bound material was eluted from Ag-G by incubation in electrophoresis sample buffer. Eluted proteins were Western blotted onto nitrocellulose filters and probed with an anti-Flag M2 monoclonal antibody (Sigma) at 30 μg/ml. Immunoreactive bands were detected by autoradiography on X-ray film. MultiMark pre-stained, multi-colored proteins (NOVEX) were used to assess protein transfer and as molecular weight standards.
Mammalian Cell Transcription Assay
Transient transfections were conducted with either the human embryonal kidney cell line 293, or the human hepatocellular carcinoma cell line, HepG2. The cells were transiently transfected by the method of calcium phosphate as previously described . Following a 6 hr incubation with transfecting plasmids in 10 cm plates, cells were replated into 96 well plates, plus or minus activating ligands. After 42–48 hours, induced luciferase and normalizing βGal activities were determined as previously described . The amount of plasmid used per 10 cm plate transfection was as follows: βRE-tk-luc, 2 μg; ERE(5)-MTV-luc, 4 μg; E2FRE-tk-luc, 5 μg; pRShER, 1 μg; pCMVrNPDC-1 or pCMVhNPDC-1, 4 μg in βRE and E2FRE experiments and 2 μg in ERE experiments; pRSβGal, 4 μg in βRE and ERE experiments and 5 μg in E2FRE experiments. The total amount of plasmid per 10 cm plate transient transfection was brought to 20 μg with pUC19 DNA.
These studies were supported through grants to DJN from the Kentucky Lung Cancer Research Program and the National Institutes of Health (HL67321)
- 1.Sporn MM, Roberts AB, Goodman DS: The Retinoids: Biology, Chemistry, and Medicine. Edited by: Roberts MB and Goodman AB. 1984, New York, Raven Press, Ltd., 1Google Scholar
- 3.Sporn MB, Roberts AB, Goodman DS: The Reinoids: Biology, Chemistry and Medicine. 1994, New York, NY, Raven Press, 2ndGoogle Scholar
- 25.Henry Kenneth, O'Brien Michelle L., Clevenger William, Jow Lilly, Noonan Daniel J.: Peroxisome proliferator-activated receptor response specificities as defined in yeast and mammalian cell transcription assays. Toxicol. Appl. Pharmacol. 1995, 132: 317-324. 10.1006/taap.1995.1113.CrossRefPubMedGoogle Scholar
- 26.Allegretto Elizabeth A., McClurg Michael R., Lazarchik Steven B., Clemm David L., Kerner Sandra A., Elgort Marc G., Boehm Marcus F., White Steven K., Pike J. Wesley, Heyman Richard A.: Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast. J. Biol. Chem. 1993, 268: 26625-26633.PubMedGoogle Scholar
- 29.Dupont E, Sansal I, Evrard C, Rouget P: Developmental pattern of expression of NPDC-1 and its interaction with E2F-1 suggest a role in the control of proliferation and differentiation of neural cells. J. Neurosci. Res. 1998, 51: 257-267. 10.1002/(SICI)1097-4547(19980115)51:2<257::AID-JNR14>3.3.CO;2-D.CrossRefPubMedGoogle Scholar
- 33.Leid M, Kastner P, Lyons R, Nakshatri H, Saunders M, Zacharewski T, Chen JY, Staub A, Garnier JM, Mader S, et al.: Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently [published erratum appears in Cell 1992 Nov 27;71(5):following 886]. Cell. 1992, 68: 377-395.CrossRefPubMedGoogle Scholar
- 39.Kawashima H, Takano H, Sugita S, Takahara Y, Sugimura K, Nakatani T: A novel steroid receptor co-activator protein (SRAP) as an alternative form of steroid receptor RNA-activator gene: expression in prostate cancer cells and enhancement of androgen receptor activity. Biochem J. 2003, 369: 163-171. 10.1042/BJ20020743.PubMedCentralCrossRefPubMedGoogle Scholar
- 43.Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, et al.: Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor [see comments]. Nature. 1995, 377: 397-404. 10.1038/377397a0.CrossRefPubMedGoogle Scholar
- 46.Zhou D, Quach KM, Yang C, Lee SY, Pohajdak B, Chen S: PNRC: a proline-rich nuclear receptor coregulatory protein that modulates transcriptional activation of multiple nuclear receptors including orphan receptors SF1 (steroidogenic factor 1) and ERRalpha1 (estrogen related receptor alpha-1). Mol Endocrinol. 2000, 14: 986-998. 10.1210/me.14.7.986.PubMedGoogle Scholar
- 48.Medin JA, Minucci S, Driggers PH, Lee IJ, Ozato K: Quantitative increases in DNA binding affinity and positional effects determine 9-cis retinoic acid induced activation of the retinoid X receptor beta homodimer. Mol Cell Endocrinol. 1994, 105: 27-35. 10.1016/0303-7207(94)90032-9.CrossRefPubMedGoogle Scholar
- 50.Chiba H, Muramatsu M, Nomoto A, Kato H: Two human homologues of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid receptor. Nucleic Acids Res. 1994, 22: 1815-1820.PubMedCentralCrossRefPubMedGoogle Scholar
- 61.Lee HY, Dohi DF, Kim YH, Walsh GL, Consoli U, Andreeff M, Dawson MI, Hong WK, Kurie JM: All-trans retinoic acid converts E2F into a transcriptional suppressor and inhibits the growth of normal human bronchial epithelial cells through a retinoic acid receptor- dependent signaling pathway. J Clin Invest. 1998, 101: 1012-1019.PubMedCentralCrossRefPubMedGoogle Scholar
- 66.Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. A Laboratory Manual. 1989, Cold Spring Harbor, NY, Cold Spring Harbor Press, secondGoogle Scholar
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