Cloning of human NCU-G1
Previous studies of the hCRBP1 gene promoter identified a DNA-element (FP1, +66/+96) that comprises target sites for both the nuclear factor 1 (NF1) and specificity protein 1 (Sp1) transcription factors [17]. Further studies, using SDS-PAGE fractionation and partial renaturation of nuclear proteins, enabled us to detect binding of two unknown proteins, designated Bp1 and Bp2, to the FP1-element [18]. Bp1 and Bp2, which compete with both Sp1 and NF1 for binding to the Fp1 element, could be revealed by exploiting the fact that neither Sp1 nor NF1 renature after SDS-PAGE fractionation and hence do not bind FP1 in electrophoretic mobility shift assay (EMSA) [19].
In order to study these proteins in more detail, we used the One-Hybrid cloning strategy to clone their corresponding cDNAs from an expression library prepared from human placenta. The FP1 element was used as bait after some modifications to avoid recognition by NF1 and Sp1. Screening of the placenta expression library resulted in isolation of three unique clones one of which contained an insert of 1.7 kb. DNA sequencing of this clone revealed an open reading frame of 1221 base pairs yielding a protein of 406 amino acids (figure 1A). Blast analysis identified it as kidney predominant protein, alias NCU-G1 or C1orf85 homologous to the previously published mouse NCU-G1 [20]. A search of the human genome database showed that NCU-G1 is located on chromosome 1, 1q23.1, locus 112770, and has an open reading frame spanning 6 exons. No human homologues were identified, indicating that NCU-G1 is unique.
Comparison with NCBI GenBank [21] showed a number of previously submitted sequences corresponding to human NCU-G1 (GenBank: NM_144580, BC018757, BC011575), however, no functional characterization of this protein has been reported. Database searches including other species revealed that mammals, birds, frogs and certain mosquitos all have an NCU-G1 gene (figure 1A), whereas Drosophila melanogaster does not. In rodents (mouse, rat) the open reading frame (ORF) is 404 amino acids. The human gene has inserted a leucine after amino acid 52 and a lysine after amino acid 139. The NCU-G1 gene appears to be highly conserved. Complete conservation of more than 25% of amino acids is seen from mosquito to man, and conservative conservation is found in all available sequences from worm to man. At the amino acid level, homologies between man/chimpanzee is 99.8%, man/rat 79%, man/mouse 79% and rat/mouse 82%. Figure 1A shows that there are highly conserved regions and distinct areas where variations can occur. The protein has a high content of proline (9.1%) but no specific proline-rich regions. However, the proline positions are well conserved.
To search for information on the structure and function of the NCU-G1 gene product, a variety of data mining tools were used. NCU-G1 has a predicted MW of 43.8 kDa and an acidic pI of 6.1 [22]. The molecular weight was verified by Western analysis using a polyclonal antibody which we raised against a 15-amino acid peptide covering the C-terminal sequence (see below, figure 2A). No homologies to known DNA-binding motifs, transcription activation domains or regions with enzymatic activity were found. Somewhat surprising, NCU-G1 has four NR-boxes with the LXXLL signature sequence [23, 24]. Such NR-boxes are found in a number of transcriptional co-activators, such as the p160-family of co-activators for which the LXXLL sequences are characteristic [25, 26]. The presence of an NR-box has been determined as necessary and sufficient for interaction with nuclear receptors, although both recruitment and effect on transcription are influenced by other factors such as the sequences flanking the NR-box, the ligand types of the nuclear receptor itself and the DNA response elements present on the target genes. As shown in figure 1B, the LXXLL-motifs were distributed over the entire protein. The first NR-box (aa 21–25) is conserved in all NCU-G1 sequences from man to frog. The second NR-box (aa 53–57) is unusual being preceded by a proline residue. This NR-box is found only in the NCU-G1 protein from man and chimpanzee. One of the inserted amino acids (aa 53), making up part of the difference between rodents and man, is the first leucine in NR-box no. 2 of man. The third NR-box (aa 267–271) is present in all mammals while the fourth NR-box (aa 390–394) is found in all mammals except in pig.
Four potential Src-homology 3 (SH3) interacting domains with the signature sequence PXXP were also identified in NCU-G1 (figure 1B). According to Cesareni et al. [27] motif no. 1 (aa 203–206) is a class II motif, whereas motif no. 4 (aa 298–301) belongs to class I. As shown for the modulator of non-genomic action of estrogen receptor (MNAR), these motifs may bind SH3 domains in c-Src, stabilizing its interaction with a nuclear receptor and leading to further activation of the ligand-bound receptor [28]. It may also activate the MAP kinase signal transduction pathway.
NCU-G1 was initially identified in nuclear fractions and therefore might be expected to have a nuclear localization signal (NLS), however, analysis of the NCU-G1 sequence failed to identify any candidate sequence for an NLS [18]. In spite of this, and as figure 1B indicates, two potential nuclear export signals (NES) conforming to the L(X1–3)L(X2–3)LXL formula characteristic of CRM1-mediated transport were found [29]. NES1 is localized in the N-terminus comprising amino acids 50 to 59 while NES2 is localized in the C-terminus comprising amino acids 383 to 392. Interestingly, both NES1 and NES2 overlap with an NR-box, no. 2 and no. 4, respectively.
Human NCU-G1 is a nuclear protein
Our previous studies have shown that NCU-G1 is present in nuclear extracts but we could not exclude the possibility that it may also be present in other subcellular compartments [18]. The small size of NCU-G1 would predict an ability to shuttle between cytosol and nucleus. This hypothesis was examined by analyzing nuclear and cytosolic fractions from several cell types by Western blotting.
A polyclonal antibody was raised against a peptide representing the last fifteen amino acids in the C-terminus of NCU-G1. Its specificity was verified by Western analysis of transiently expressed NCU-G1 in Drosophila Schneider S2 cells which do not express this protein. The predicted size of NCU-G1 is 43.8 kDa. As shown in figure 2 (panel A, lane 6) a protein of approximately 45 kDa in size is detected with this antibody. In contrast, a transiently expressed truncated version of NCU-G1 lacking exon 6 comprising the C-terminal end of the protein could not be detected (figure 2A, lane 7). Using this antibody, we found that NCU-G1 is primarily localized in the nuclei of JEG3, RPE and 293 cells (figure 2B; lanes 2, 4 and 6, respectively). The traces of NCU-G1 detected in the cytosol were probably due to leakage from the nuclei during preparation. In order to test this assumption, the membranes were stripped and analyzed with an antibody against Sp1 as a marker of nuclear proteins. Figure 2C shows that some Sp1 is found in the cytosolic fractions, supporting the notion that partial nuclear rupture during preparation may account for the NCU-G1 detected in cytosol.
Human NCU-G1 binds specifically to the FP1-oligonucleotide
The initial identification of NCU-G1 was based on its ability to bind FP1 in EMSA. In order to show that our cloned NCU-G1 represents the proteins we set out to clone, its ability to bind DNA was studied. Figure 3 shows complex formation in EMSA between 32P-labeled FP1-oligonucleotide and GST-NCU-G1 fusion protein recombinantly expressed in E. coli. A major band (lane 2) shows specific binding which is inhibited in the presence of 50-fold molar excess of unlabeled FP1 oligonucleotide (lane 3). Unlabeled AP1-, Sp1- or NF1-oligonucleotides at 50-fold molar excess (lane 4, 5 and 6, respectively) are unable to inhibit complex formation. Control GST did not bind the FP1 oligonucleotide (data not shown).
Human NCU-G1 stimulates transcription from the hCRBP1 promoter
The ability of NCU-G1 to interact in EMSA with the FP1-element of the human CRBP1 promoter prompted us to analyse whether such interaction might entail an ability to modulate transcription. The human CRBP1 promoter (-567/+104 bp) was inserted upstream of the chloramphenicol acetyl-transferase (CAT) gene in a reporter vector (pCAT3) and transiently co-transfected with an expression vector for NCU-G1 into Drosophila Schneider S2 cells (S2 cells). These cells were chosen in order to avoid interference with Sp1 and NF1 which also bind to the FP1 element of the hCRBP1 promoter. Although these cells are not mammalian, S2 cells express transcription factors similar to those found in mammalian cells, but they often do not recognize mammalian proteins or mammalian DNA consensus elements, resulting in lower background and less interference [30]. Figure 4A shows that expression of low levels of hNCU-G1 leads to a 17-fold increase of CAT expression.
In order to test whether transcriptional induction was mediated by the FP1 element, we created deletion mutants of the CRBP1 promoter controlling reporter gene expression. The expression vector (pOCAT) used in these experiments has a somewhat higher basal activity resulting in the induction by NCU-G1 appearing smaller [17]. Expression of NCU-G1 increased reporter gene expression by 7.5-fold (fig. 4B). Deletion of FP1 from the full-length reporter construct (phCRBP1(-483/+69)OCAT) abrogated the induction of reporter gene expression by NCU-G1. Removing most of the promoter region upstream of the transcription start site but leaving FP1 intact (phCRBP1(-20/+104)OCAT) did not affect the ability of NCU-G1 to induce reporter gene expression. Again, a deletion that removes the FP1 element from this construct abolishes this induction (phCRBP1(-20/+69)OCAT). These results demonstrate that the FP1-element is an absolute requirement for NCU-G1's ability to induce reporter gene expression from the CRBP1 promoter.
In order to eliminate the possibility of any nuclear receptor (NR)-box contribution to the CRBP1 promoter controlled reporter gene expression, we created mutated versions of NCU-G1. Single or multiple NR-boxes were mutated from LXXLL to LXXAA to inhibit interaction with possible NR-box binding proteins. Figure 4C shows that mutation of one or more NR-boxes does not affect NCU-G1's ability to activate reporter gene transcription from the CRBP1 promoter, confirming that the NR-boxes are not involved.
Next, we studied the effect of deleting exons or segments of NCU-G1 on its ability to control reporter gene expression from the CRBP1 promoter. Figure 4D shows that deletion of exon 6 actually potentiates NCU-G1's stimulatory effect on CAT expression. This exon comprises a stretch of hydrophobic amino acids (aa 372–394) that may be involved in attenuating NCU-G1 stimulation of CAT expression. A deletion mutant lacking just this hydrophobic region, however, did not stimulate CAT expression to the same extent. Deletion of exon 5 and 6, exon 4–6, exon 3–6 or amino acid 1–81 reduces NCU-G1 stimulatory activity to approximately 50% of that of wild-type NCU-G1. In contrast, deletion of exon 2 results in a complete loss of stimulatory activity, indicating that this region comprising amino acids 42–126 is important for NCU-G1 activity.
NCU-G1 mRNA is highly expressed in human liver, kidney and prostate
The tissue specific expression of NCU-G1 in man was investigated at the mRNA-level. Most tissues tested were found to express NCU-G1 mRNA, however, it was most abundant in liver, kidney and prostate, with somewhat lower levels in placenta, ovary and adrenal. In contrast, expression was low in heart, lung, and skeletal muscle (data not shown). These results are corroborated by data reported on the SAGE-express web-site [31].
Human NCU-G1 acts as a co-activator for PPAR-alpha
The presence of four NR-boxes in NCU-G1 prompted us to study its possible capacity to function as a co-regulator for nuclear receptors, here represented by PPAR-alpha, in transient transfections of S2 cells. The rationale for choosing PPAR was the identification of a number of potential peroxisome proliferator-activated receptor response elements (PPARE) in the human CRBP1 promoter region, using "MatInspector" from Genomatix [23]. In addition, PPAR-beta agonists have been reported to stimulate CRBP1 expression [15]. The ability of NCU-G1 to stimulate PPAR transcriptional activity may therefore be relevant to CRBP1 expression.
We chose to use a reporter gene construct expressing CAT under control of the rat acyl-CoA oxidase gene promoter [32], a well-known PPAR-alpha target gene [33]. We first determined the optimal level of PPAR-alpha to be co-expressed with NCU-G1 in S2 cells. As shown in figure 5A, expression of CAT was very low when PPAR-alpha was expressed only in the presence of the PPAR-alpha agonist WY14643, nor did expression of NCU-G1 lead to any increase in reporter gene expression. When co-expressed, however, a strong induction of reporter gene expression was observed, indicating that NCU-G1 may function as a co-activator for PPAR-alpha. Maximal induction using 150 ng of the PPAR-alpha expressing vector increased reporter gene expression by 11-fold.
Interaction between co-activator NR-boxes and nuclear receptor AF-2 domains have been shown to require the ligand-induced conformational change of the latter [28]. Hence, we next sought to determine whether NCU-G1's ability to co-activate PPAR-alpha controlled reporter gene expression was ligand-dependent. Figure 5B shows that this is indeed the case. When both PPAR-alpha and NCU-G1 were expressed in the absence of the ligand, no CAT activity beyond the level directed by the pACO-CAT plasmid alone could be observed. However, addition of the agonistic ligand WY14643 increased CAT activity by approximately 11-fold. This result demonstrates that NCU-G1's ability to co-activate PPAR-alpha requires ligand activation of the nuclear receptor.
We next sought to determine which, if indeed any, of the four NR-boxes in NCU-G1 is responsible for the observed co-activation. As shown in figure 5C, mutation of NR-boxes 2, 3, or 4 lowered the co-activating capacity of NCU-G1 to approximately 50% of that observed with wild-type NCU-G1. Mutation of NR-box 1, however, completely abolished NCU-G1's ability to co-activate PPAR-alpha induced reporter gene expression, supporting our hypothesis that stimulation of PPAR-alpha controlled reporter gene expression by NCU-G1 is NR-box mediated. A similar effect was observed when all four NR-boxes were mutated. In summary, these results suggest that the mechanism of NCU-G1 co-activation of PPAR-alpha transcriptional activity requires an intact NR-box 1 and ligand mediated activation of the nuclear receptor.
NCU-G1 co-localises with PPAR-alpha in the nucleus
Nuclear receptor co-activators may be divided into a primary and a secondary group where members of the former interact directly with nuclear receptors and the latter interact with members of the primary group [34]. In an effort to determine which of these groups NCU-G1 may belong to, its ability to interact with PPAR-alpha was studied in mammalian as well as yeast two-hybrid systems. No interaction was detected indicating that NCU-G1 is not a primary co-activator of this nuclear receptor (data not shown). The possibility of NCU-G1 being a secondary co-activator was analysed by confocal fluorescence microscopy. HeLa cells were transiently transfected with expression vectors for EGFP-tagged NCU-G1 and RFP-tagged PPAR-alpha. As shown in figure 6B RFP-PPAR-alpha is predominantly localised in the nucleus as expected. In contrast, as shown in figure 6A the fusion protein NCU-G1-EGFP is mostly cytosolic. However, some NCU-G1-EGFP was found in the nucleus at distinct spots which might indicate transport into the nucleus. Once within the nucleus, as shown in figure 6C, the few NCU-G1-EGFP spots co-localise with RFP-PPAR-alpha showing potential cellular interaction domains for NCU-G1 and PPAR-alpha.