extfieldsofstudy: Biology Medicine

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cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis

Promoters, including neither TATA box nor initiator, have been frequently found in testicular germ cell-specific genes in mice. These investigations imply that unique forms of the polymerase II transcription initiation machinery play a role in selective activation of germ cell-specific gene expression programs during spermatogenesis. However, there is little information about testis-specific core promoters, because useful germ cell culture system is not available. In this study, we characterize the regulatory region of the haploid-specific Oxct2b gene in detail by using in vivo transient transfection assay in combination with a transgenic approach, with electrophoretic mobility shift and chromatin immunoprecipitation assays. Expression studies using mutant constructs demonstrate that a 34 bp region, which extends from −49 to −16, acts as a core promoter in an orientation-dependent manner. This promoter region includes the cAMP-responsive element (CRE)-like sequence TGACGCAG, but contains no other motifs, such as a TATA box or initiator. The CRE-like element is indispensable for the core promoter activity, but not for activator in testicular germ cells, through the binding of a testis-specific CRE modulator transcription factor. These results indicate the presence of alternative transcriptional initiation machinery for cell-type-specific gene expression in testicular germ cells.

INTRODUCTION
Spermatogenesis is a cyclic and continuous process through which spermatogonia undergo proliferation and differentiation, to give rise to mature spermatozoa. Each differentiation step requires the expression of a specific set of precisely regulated genes, some of which are expressed exclusively in testicular germ cells. Many testicular germ cell-specific genes are paralogues of genes that are expressed ubiquitously (1). It has also been reported that more functional intronless paralogues are expressed in pachytene spermatocytes and spermatids than in any other tissues (2)(3)(4). Intronless genes are considered to be retroposons derived from paralogues, or ancestral genes, that carry introns. It is generally the case for retroposons that the inserted cDNA which is reverse-transcribed from the mRNA cannot be expressed in any tissue, because the mRNAs lack a promoter in the 5 0 -flanking sequence of the genomic DNA. This indicates that an upstream sequence, which has no intrinsic promoter activity, has been adapted to function as a promoter. This prediction implies that testicular germ cells provide the appropriate environment for retroposon transcription, which facilitates gene expression from promoter-like sequences. It appears that the rules that govern gene transcription during later stages of spermatogenesis are drastically different from those in other cell types.
The expression of certain haploid-specific genes depends on TATA-containing core promoters (5)(6)(7)(8)(9). RNA polymerase II (Pol II), TFIIB and TATA-binding protein (TBP) accumulate in round spermatids, while the amount of mRNA increases in the testis (10,11). Although the overexpression of the TBP gene has been implicated in the transcription of all the TATAdependent genes in haploid germ cells, it does not account for the specificity of haploid-specific gene expression. In this context, the cAMP-responsive element (CRE) is present upstream of the TATA box in these TATA-dependent haploid-specific promoters. The testis-specific activator isoform of the CRE modulator (CREMt) upregulates promoter activity in spermatids through CRE binding (12), and spermatogenesis in male CREM-null mice stops at the stage of early spermatids (13,14). The ACT co-activator binds and modulates CREMt, and this complex activates transcriptional initiation around the TATA box (15). Recently, it is demonstrated that CREMt can upregulate the TATA-less promoter through association with TBP-like factor (TLF/TRF2/TLP) and TFIIA general transcription factors (GTFs) (16). In addition, many gene that are transcribed specifically in haploid spermatids use CRE-containing TATA-independent promoters, which include Tact1, Tact2, actin-capping protein a and OAZt (17)(18)(19). The initiator (Inr) is also capable of directing accurate transcription initiation of the Pol II promoter, in combination with the Sp1-binding site or the downstream promoter element in TATA-less promoters (20,21). However, some TATA-less haploid-specific promoters lack Inr, which implies that the machinery of initiation complex formation is unique to haploid-specific transcription.
In this study, we used the gene that encodes the testicular isoform of succinyl CoA 3-oxoacid CoA transferase (Oxct2b) as a model system for haploid spermatid-specific regulation. The Oxct2b gene consists of a single exon and is expressed exclusively during spermatid differentiation (22,23). No TATA or CCAAT boxes, Sp1-binding motif and typical Inr element were found in the upstream region of the gene (23). The promoter structure of the gene enables the study of the unique molecular mechanisms that underlie the regulation of postmeiotic gene expression. We show that the À49/À16 region, which contains a CRE-like motif (Oxct2b-CL), is indispensable as a core promoter but not as an enhancer element for Oxct2b gene transcription. This core promoter binds testis-specific CREM factor through Oxct2b-CL. We propose that the combination of CRE-like motif in the core promoter and testicular CREM plays an essential role for transcriptional initiation in the haploid germ cells.

Reporter constructs
For the transgenic analysis, the 600 bp genomic DNA fragment, that extends 566 bp upstream and 35 bp downstream of the transcriptional start site of the Oxct2b gene, was amplified by PCR from a genomic clone (23), using an upstream BglII site-containing primer and a downstream HindIII site-containing primer (Table 1). PCR was performed with 200 ng of template DNA in 100 ml of reaction mixture [1 mM of each primer, 800 mM dNTPs, 1· reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)]. The PCR conditions consisted of an initial denaturation step at 95 C for 3 min, followed by 30 cycles at 94 C for 1 min, at 60 C for 1 min and at 72 C for 1.5 min. After digestion with BglII and HindIII, the amplified fragment was subcloned into the BglII and HindIII sites of the pEGFP-1 vector (Clontech), to generate the À566/+35EGFP construct. The À103/À16EGFP construct was cloned in a similar manner into the pEGFP-1 vector, using a primer pair that introduced BglII and HindIII sites at the 5 0 and 3 0 ends, respectively (Table 1).
The DÀ49/À16Luc construct was generated by introducing a synthetic oligonucleotide that contained HindIII restriction sites at either end and extended from À15 to +35 into the HindIII site of À566/À50Luc. The À389/À49/ À16Luc, À389/À16/À49Luc and CLmut constructs were generated by introducing a synthetic oligonucleotide that contained HindIII restriction sites at either end and extended from À49 to À16 into the HindIII site of À389/À50Luc. The À49/À16(À389/À49/À16)Luc and À16/À49(À389/À49/À16) Luc constructs, or the À49/À16(CLmut) and À16/À49 (CLmut) constructs, were generated by introducing a synthetic oligonucleotide that contained BglII restriction sites at either end and extended from À49 to À16 into the BglII site of À389/ À49/À16Luc or CLmut, respectively. For the standardization of the in vivo electroporation, the OAZt-pRL construct was generated by cloning the testis-specific ornithine decarboxylase antizyme (OAZt) promoter into the pRL-Null vector (Promega) (18). All the constructs were confirmed by restriction enzyme digestion and DNA sequencing. Plasmid DNA was purified using QIAGEN columns (Qiagen).

Generation of transgenic mice
The enhanced green fluorescent protein (EGFP) constructs described above were digested using BglII and AflII. The inserted fragments, which included the Oxct2b upstream region, the EGFP gene and the SV40 polyadenylation [poly(A)] signal, were resolved by agarose gel electrophoresis and purified using glass beads (Qiaex; Qiagen). The excised DNA fragments were injected into the male pronuclei of C3HxC57BL/6 F1 eggs. Transgenic founders from the microinjected eggs were identified by PCR using the primer pair À103TG/EGFP-N (Table 1), and the founders were mated with the C57BL/6 mice. The positive male offspring from this cross were used in subsequent experiments.

Histological analysis
Fluorescent stereoscopic photographs were taken of whole testes from transgenic adult mice. Histological analyses were performed as described previously (24).

In vivo electroporation assay
The reporter DNA fragments that contained the Oxct2b upstream region, firefly luciferase gene and SV40 poly(A) signal were amplified from the recombinant luciferase constructs by PCR using oligonucleotide pair 5 0 -CTAGCAAA-ATAGGCTGTCCCC-3 0 (RV primer3) and 5 0 -GACGATAG-TCATGCCCCGCG-3 0 (RV primer4), which correspond to regions 4760-4779 and 2061-2080 of the pGL3-Basic vector, respectively. DNA fragments for standardization, which contained the OAZt promoter, the Renilla luciferase gene and the SV40 poly(A) signal, were amplified from OAZt-pRL by PCR using the primer pair 5 0 -TTTGCTCACATGGCTCGAC-3 0 (RL5) and 5 0 -CGCACATTTCCCCGAAAAGT-3 0 (RL3), which correspond to regions 3291-3310 and 1511-1530 of the pRL-Null vector, respectively. The PCR conditions were as described above except for annealing condition, which was performed at 56 C for 1 min. The PCR products were purified using SUPRECÔ-PCR (TaKaRa Biomedical). For each transfection, 12 ml of a mixture [that contained 5 mg of each PCR reporter product, 5 mg of the OAZt-pRL PCR product, 100 mM of general caspase inhibitor (Z-VAD-FMK; R&D Systems, Inc.) and 0.1 mg/ml of trypan blue (Nacalai Tesque)] was injected through the efferent duct into the seminiferous tubules (25). Electron pulses were delivered by an electric pulse generator (electroporator CUY-21; Tokiwa Science). The testes were immobilized between a pair of tweezers-type electrodes (CUY-650; Tokiwa Science) and square-form electric pulses were applied eight times in four different directions at 40 V for 50 ms, at intervals of 950 ms. The transfected testes were collected for the luciferase assay 16-20 h after this operation. The preparation of the germ cell fraction and the luciferase assays were performed as described previously (25). All assays were performed at least three times.

Electrophoretic mobility shift assay
The mouse testicular germ cell fraction was collected essentially as described previously (25,26). The germ cells were suspended in 3 ml of buffer A [10 mM HEPES-KOH (pH 7.8), 0.1 mM EDTA, 10 mM KCl, 0.1% NP-40, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethlysulfonyl fluoride (PMSF), 2 mg/ml aprotinin, 2 mg/ml pepstatin and 2 mg/ml leupeptin] before being incubated for 15 min at 4 C. The solution was mixed by gentle vortexing at 4 C for 5 min and was centrifuged at 2300 g for 5 min at 4 C. Nuclear protein extracts were prepared from testis germ cell fraction and liver cells, essentially as described previously (27). The nuclear pellets were washed in buffer A and resuspended in 1 ml (testis) or 2 ml (liver) of buffer C [50 mM HEPES-KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl 2 , 2% glycerol, 1 mM DTT, 0.5 mM PMSF, 2 mg/ml aprotinin, 2 mg/ml pepstatin and 2 mg/ ml leupeptin]. The suspension was rotated slowly for 30 min at 4 C. The resultant nuclear lysates were centrifuged at 13 000 g for 15 min (testis) or 30 min (liver) at 4 C. The supernatants of the nuclear extracts were aliquoted, frozen in liquid nitrogen and stored at À80 C until use. Protein concentrations were 3-10 mg/ml.
The synthetic oligonucleotides (10 nmol; Table 2) were annealed to produce a double-stranded DNA fragment with extruding GG-dinucleotides at the termini. Each DNA fragment was labeled with 5-amino-propargyl-2 0 -deoxycytidine 5 0 -triphosphate coupled to Cy5 Electrophoresis was performed at 4 C at 250 V for 40 min. Gel imaging was carried out using the Odyssey Infrared Imaging System (LI-COR) at a wavelength of 700 nm.

Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed with modifications to the protocol described by the manufacturer (Upstate Biotechnology) and Sakakibara et al. (28). The collected germ cell fraction described above was cross-linked in vivo in 1% formaldehyde in phosphate-buffered saline (PBS) at 37 C for 10 min with slow rotation. The cross-linked germ cells were pelleted by centrifugation at 2300 g for 4 min at 4 C and were washed twice in ice-cold PBS containing 0.5 mM PMSF, 2 mg/ml The underlined GG are recessed dinucleotides at 5 0 end used for Cy5.5 labeling.
aprotinin, 2 mg/ml pepstatin and 2 mg/ml leupeptin, incubated on ice for 10 min, and were then disrupted by Dounce homogenization. The nuclei were collected by low-speed centrifugation and were then incubated with 50 U of micrococcal endonuclease (TaKaRa Biomedical) in 400 ml of micrococcal nuclease buffer for 20 min at 37 C to digest chromosome DNA into fragment <1 kb. Digested chromatin was immunoprecipitated with 1 mg of the anti-CREM or anti-CRE-binding (anti-CREB) antibodies overnight with rotation at 4 C. To collect the immune complexes, a salmon sperm DNA/protein Aagarose slurry was added to the mixture, which was incubated for 1 h with rotation at 4 C. The precipitated matrix was washed with various washing buffers according to the manufacturer's recommendations. The immune complexes were eluted from the protein A-agarose beads by the addition of elution buffer (1% SDS in 0.1 M NaHCO 3 ) and rotation at room temperature. The eluted solution (500 ml) was treated with 20 ml of 5 M NaCl before being heated to 65 C for 5 h to reverse CREM-or CREB-DNA cross-linking. DNA was purified using proteinase K treatment followed by phenol/ chloroform/isoamyl alcohol (25:24:1) extraction and ethanol precipitation. Input DNA and immunoprecipitated DNA were analyzed by PCR using the primer pair 5 0 -GCTCACTGC-CATTTGTCAGC-3 0 (À181/À162) and 5 0 -AGCTCCGGGA-CAGAGAGAGA-3 0 (+35/+16), which are specific for the Oxct2b promoter sequence. The PCR products were analyzed by electrophoresis on 2% agarose gels.

RESULTS
The 0.6 kb 5 0 -upstream sequence of the Oxct2b gene is responsible for haploid spermatid-specific expression in transgenic mice The Oxct2b gene is expressed in late spermatids in the testis (22). To determine the genomic region that is responsible for haploid-specific Oxct2b expression, transgenic mouse lines were generated by using À566/+35EGFP ( Figure 1A). Of the six founder lines that were established, almost all (49/52) of the transgenic male pups expressed the EGFP reporter gene. Green fluorescence was observed in the seminiferous tubules of the testes of the transgenic mice, but not in the other tissues examined ( Figure 1B and C). No EGFP signals were observed in non-transgenic littermates. To determine which cell type expressed the reporter gene, the testes of À566/+35EGFP transgenic mice were examined histologically. The EGFP signals were faint in the inner layers of the seminiferous tubules at stage IX, but were strong at other stages ( Figure 1D). These strong signals were restricted to elongating and elongated spermatids, and were not observed in spermatogonia, spermatocytes, round spermatids or in any other non-spermatogenic cells within the testis ( Figure 1D). The EGFP expression pattern was in good agreement with the expression pattern of the endogenous Oxct2b transcripts described previously (22). These results confirm that the 0.6 kb, 5 0 -upstream sequence is sufficient for haploid-specific expression of the Oxct2b gene. The two Oxct2 genes, Oxct2a and Oxct2b, are virtually identical and are expressed simultaneously in the haploid spermatids of mice (23), which indicate that regulatory mechanisms of these two genes are equivalent. The 5 0 -flanking sequences of the two murine Oxct2 genes are highly homologous and encompass several putative regulatory elements, although they lack the TATA box, CCAAT box and canonical Inr ( Figure 2).

Identification of a core Oxct2b promoter by in vivo transient transfection
Detailed functional analyses of promoters are usually carried out using cultured cells that transcribe the gene of interest. However, a culture system for spermatogenic cells is not currently available. In this study, to identify the essential elements for Oxct2b transcription in the male germ cells, we used an in vivo transient transfection technique in testicular germ cells, as described previously (24,25). We introduced PCR products that contained Oxct2b promoters fused to a luciferase reporter into testicular germ cells using a combination of DNA injection into the seminiferous tubules and subsequent in vivo electroporation. We made a series of 5 0 -deletion constructs and tested their promoter activities. As shown in Figure 3A, deletions that extended up to À932 or to À389 resulted in no significant reduction in promoter activity compared to À566/ +35Luc, which is the upstream region that was used for the transgenic analysis. Further deletions from À389 to À256, À256 to À103 and À103 to À50 resulted in stepwise reductions of the luciferase activity of À566/+35Luc to 61, 37 and 16%, respectively. These data suggest that the 339 bp region from À389 to À50 is required for high-level promoter activity in testicular germ cells. In addition, À50/+35Luc showed basal promoter activity, because luciferase activity produced by this construct was 2-fold higher than that of the promoter-less pGL3-Basic vector. The Oxct2b promoter contains the CCAAAGT motif, which is similar to the Inr consensus sequence of PyPyANA/TPyPy (29) that surrounds the transcription initiation site, but lacks a TATA box (Figure 2). The functions of several Inr-containing promoters that lack TATA boxes are supported by important downstream sequences (25). To investigate whether the intergenic region of the Oxct2b gene also contributes to the promoter activity of this gene, we generated several 3 0 -deletion constructs and examined their promoter activities using in vivo transient transfection. As shown in Figure 3B, the construct that contained a 51 bp deletion to position À16, which included the downstream region and transcription initiation site of the gene, showed no significant reduction in promoter activity. A further 34 bp deletion, which removed the À16 to À49 region, reduced the reporter gene activity to basal levels. These results indicated that the intergenic region of the Oxct2b gene does not contribute to promoter activity, whereas the sequence from À49 to À16 is necessary for Oxct2b promoter activity.
We further verified the functional importance of this fragment, using the internal deletion construct DÀ49/À16Luc. As shown in Figure 4A, the DÀ49/À16Luc construct induced a level of luciferase activity that was not significantly higher than that of the À566/À50Luc construct. This result indicates that the À49/À16 sequence is indispensable for Oxct2b promoter activity. We postulate that the À49/À16 sequence  Luciferase activity, which was normalized to that of Renilla luciferase driven by the OAZt promoter (25), is presented as the mean values (-SEM) of three independent experiments that were performed in duplicate. *P < 0.05, **P < 0.01 compared with À566/+35Luc (A) and À389/+35Luc (B) (ANOVA followed by Bonferroni analysis). The luciferase activities of -566/+35Luc (A) and -389/+35Luc (B) were set at 1.0. includes a core promoter, i.e. a site for the assembly of the pre-initiation complex and for the determination of transcription orientation. We examined whether the direction of the À49/À16 region was critical for Oxct2b promoter activity. Figure 4B shows that the level of luciferase activity of the inverted orientation of the À49/À16 sequence (À389/À16/ À49Luc) was reduced to that of the À389/À50Luc construct, which lacks the À49/À16 region. These results demonstrate that the À49/À16 region contains a core promoter to direct transcription of the gene. To examine whether the À49/À16 region contains cis-regulatory elements, such as an enhancer, we positioned the À49/À16 sequence at the 5 0 end of À389/ À49/À16Luc and performed in vivo transient transfection into testicular germ cells. Figure 4C shows that both orientations of the re-positioned À49/À16 fragment failed to upregulate the luciferase activity of the À389/À49/À16Luc construct. These results suggest that the À49/À16 sequence does not act as a cis-regulatory element. The 20 bp sequence that encompasses the CRE-like motif (Oxct2b-CL) in the promoter region of the Oxct2b gene is conserved in the mouse paralogue Oxct2a and human orthologue OXCT2 ( Figure 4D), which implies that this element plays an essential role in Oxct2 gene expression in spermatids.
To confirm that the Oxct2b promoter directs the reporter gene in spermatids, we generated transgenic mice using the À103/À16EGFP construct, which contains the core promoter and an additional 5 0 -upstream sequence ( Figure 5A) that affects promoter activity but not cell type-specificity. Three transgenic lines were established. EGFP expression was detected in the testes of the transgenic, but not in non-transgenic littermates ( Figure 5B and C). The EGFP signal in sections of testes from the À103/À16EGFP transgenic line was detected in the innermost layers of the seminiferous tubules and was restricted to elongating and elongated spermatids ( Figure 5D), as seen in the À566/+35EGFP transgenic lines (Figure 1). None of the transgenic lines showed EGFP signals in any organ other than the testis. These results demonstrate that the sequences upstream from À103 and downstream from À16 are not necessary for the accurate transcription of the EGFP reporter gene in the spermatids of transgenic mice. . The À49/À16 region of the Oxct2b promoter functions as a core promoter. (A) An internal deletion that extends from À49/À16 in the À566/ +35Luc construct abolishes Oxct2b promoter activity. In vivo transient transfection of testicular germ cells was performed with DÀ49/À16Luc and À566/ À50Luc. (B) Inversion of the À49/À16 region abolishes the transcriptional activity of the Oxct2b promoter. The constructs À389/À49/À16Luc and À389/ À16/À49Luc, which carry the correct orientation and inverted orientation of the indicated region, respectively, were transiently transfected into testicular germ cells. **P < 0.01 compared with À389/À50Luc (ANOVA followed by Bonferroni analysis). (C) The À49/À16 region acts as a core element but not as an activator element. The À49 to À16 fragment that contains Oxct2b-CL was ligated to the 5 0 end of À389/À49/À16Luc to re-position the core promoter element, and the reporter gene assays were carried out via in vivo transient transfection of testicular germ cells. Data are presented as described in the legend to Figure 3. The luciferase activities of -566/À50Luc (A), -389/À50Luc (B) and -389/À49/À16Luc (C) were set at 1.0. (D) Alignment of the À49/À16 sequence of the mouse Oxct2b gene with those of the mouse Oxct2a and human OXCT2 (hOxct2) genes. Identical nucleotides are represented by asterisks; deletions are indicated by dashes. The numbers are relative to the transcription start site at +1. The Oxct2b-CL consensus is shown in boldface. The highly conserved region among these genes is boxed. The Oxct2b-CL motif within the promoter binds the CREM protein in testicular germ cell nuclei The in vivo transient transfection experiments in combination with the transgene experiments show that the À49/À16 region is necessary for Oxct2b gene expression in haploid spermatids. We identified the CRE-like motif (Oxct2b-CL) TGACGCAG in the À29 to À22 region (Figure 2 and 4D), in which 3 nt in the 3 0 -half of the motif differ from the corresponding sites in the CRE consensus sequence TGACGTCA. To examine whether the transcription factor(s) in the testis nuclear extract could bind the Oxct2b promoter, an electrophoretic mobility shift assay (EMSA) was performed using an oligonucleotide of the À50/À10 region as the probe. Some DNA-protein complex bands were observed using nuclear extracts from the testis ( Figure 6A, lane 3). In addition, the most prominent band was not observed in the liver sample ( Figure 6A, lane 2). The most prominent gel-shift band was eliminated when the non-labeled À50/À10 oligonucleotide and fragments 3 and 4, all of which contained Oxct2b-CL, were used as competitors ( Figure 6A, lanes 5-7). In contrast, fragment 2 (À50 to À31), which lacks Oxct2b-CL, did not compete for this band ( Figure 6A, lane 4). Furthermore, a mutant competitor, in which all eight nucleotides of Oxct2b-CL were substituted, could not compete for complex formation ( Figure 6B). These results indicate that the testicular nuclear protein binds directly to the Oxct2b-CL motif. The CRE or CRE-like motif is a well-known binding site for the CREB protein and CREM. We tested the specific interactions between CREB or CREM and Oxct2b-CL by supershift analysis with specific antibodies ( Figure 6C). The specific band of the complex was removed by the anti-CREM antibody, but not by either the CREB or AP-2a antibody, which indicates that the trans-acting factor involved in this DNA-protein complex is a member of the CREM family. To test whether CREM directly binds to Oxct2b-CL in the core promoter within testicular germ cells, ChIP assays were performed. Testicular germ cell nuclei were cross-linked by formaldehyde and the lysates were immunoprecipitated with CREM or CREB antibodies. PCR amplification was performed using primers designed to amplify a fragment containing the Oxct2b-CL within the Oxct2b promoter. The predicted 215 bp PCR product was amplified from samples derived from CREM immunoprecipitates, but not from samples immunoprecipitated with CREB ( Figure 6D). These data demonstrate that CREM interacts with Oxct2b-CL within the testicular germ cell nuclei.
To define in greater detail the binding sequence within the motif, several Oxct2b-CL mutants were used as competitors in EMSA with testis nuclear extracts. In this experiment, a fragment of the À40/À20 region was used as the probe. Substitution mutations in the TGACG sequence abolished the binding capability (m1, m2 and m3 in Figure 6E), whereas mutations in the CAG sequence did not affect binding capability (m4 and m5 in Figure 6E). These results indicate that the 5 0 -half of the Oxct2b-CL sequence is crucial for proteinbinding activity. The binding specificity of Oxct2b-CL was examined further in a competition experiment with the CRE-like motif of the testis-specific angiotensin-converting enzyme (ACEt), the promoter activity of which has been demonstrated to be CRE-like sequence (TGAGGTCA)dependent in male germ cells (30). The fragment that includes The arrow shows testis-specific protein binding. (B) The Oxct2b-CL element in the Oxct2b promoter (À50/À10) binds a testis-specific nuclear protein. A 100fold molar excess of the unlabeled wild-type (wt) or mutated (mut) promoter fragment was used as the competitor. (C) The CREM factor binds to the Oxct2b promoter (À40/À20) in vitro. EMSAs were performed with testis nuclear extracts in the presence of anti-CREM, anti-CREB or anti-AP-2a antibodies, using a labeled promoter fragment that spanned À40 to À20. the ACEt CRE-like motif also competed for the testis-specific Oxct2b-CL-protein complex ( Figure 6E). These data indicate that CREM binds haploid-specific promoters via the TGAC/GG sequence, regardless of whether the motif is included in a core promoter or regulatory elements.
Oxct2b-CL in the core promoter is responsible for transcription in testicular germ cells EMSA and ChIP showed that the Oxct2b-CL motif is the binding site in the Oxct2b promoter that is recognized by testis nuclear proteins, including CREM. To confirm that the Oxct2b-CL sequence is necessary for core promoter activity, we generated a mutant construct (CLmut) in which the Oxct2b-CL sequence (TGACGCAG) was changed to CCTTTGGC, which could not bind CREM in the EMSA ( Figure 6B). In vivo transient transfection of CLmut into testicular germ cells showed that mutation of Oxct2b-CL diminished promoter activity, with results similar to those obtained with the À389/À50Luc construct, which lacks the core promoter ( Figure 7). These results indicate that Oxct2b-CL is essential for promoter activity through binding the CREM transcription factor. Several studies have demonstrated that CRE and CRE-like motifs act as activators or enhancers, but not as core promoter elements in the regulation of testis-specific gene transcription (31). If Oxct2b-CL is a cisactivator, additional Oxct2b-CL upregulates the promoter activity of the CLmut construct. To examine this possibility, we re-positioned the À49/À16 sequence to the 5 0 end of CLmut and performed in vivo transient transfection of testicular germ cells. As shown in Figure 7, the levels of luciferase activity of the À49/À16CLmut and À16/À49CLmut constructs were similar to those seen with CLmut and were significantly lower than those seen with the À389/À49/À16Luc construct. These results indicate that Oxct2b-CL does not function as a cis-activator and that no other core promoter elements are present within the À49/À16 region.

DISCUSSION
In vivo electroporation is a useful tool for functional analyses of testicular germ cell-specific gene promoters The highly ordered process of spermatogenesis, in turn, requires a precise and well-coordinated programme to regulate constantly changing patterns of gene expression. The lack of conventional tissue and cell culture systems that re-capitulate precisely the coordinated processes of spermatogenesis has hindered the elucidation of the molecular mechanisms that govern gene expression during spermatogenesis. In this study, we have demonstrated the feasibility of using in vivo transient transfection, which is a combination of intratubular DNA injection and subsequent in vivo electroporation, for the functional analysis of testicular germ cell-specific promoters. In this technique, foreign DNA sequences are introduced into Sertoli and germ cells (32). Thus, the values obtained for the reporter activities in experiments using whole testis represent the accumulated activities of both cell types, although Oxct2b promoter activity could not be detected in the somatic cell line (P. Somboonthum and M. Nozaki, unpublished data), and the haploid-specific promoter is dominantly activated in the spermatids in this system (24). To exclude potential reporter activity in Sertoli cells, we collected the germ cell fraction and performed luciferase assays. Since linearized DNA is more unstable than circular DNA in Sertoli cells (32), we introduced linear PCR products as reporters. Data validation was performed by transgenic mouse analysis. From these data, we conclude that this system functions as a simple assay system for the analysis of the regulatory elements of haploid spermatid-specific genes.
A CRE-like element regulates the Oxct2b core promoter In this study, we demonstrated that the À49 to À16 region of the Oxct2b gene functions as a core promoter in testicular germ cells, because this 34 bp fragment had 2-fold greater luciferase activity than the promoter-less control, and the inversion of this sequence abolished transcriptional activity in the in vivo transient transfection assays (Figures 3 and 4A and B). The 34 bp region, which contains a CRE-like element (Oxct2b-CL) but lacks any basal promoter elements, such as a TATA box or Inr sequence ( Figure 4D), had no cis-activator activity ( Figure 4C). Mutation of Oxct2b-CL also abolished the promoter activity, which was not restored by repositioning the À49/À16 region to the 5 0 end (Figure 7). In EMSA, the Oxct2b-CL unit in this fragment bound exclusively to a protein in the testis nuclear extract ( Figure 6). These results indicate that Oxct2b-CL plays an essential role as a core promoter element in haploid germ cell-specific expression. CRE is a conserved cis-acting element that usually comprises the 8 bp palindromic sequence 5 0 -TGACGTCA-3 0 (33), although variations of this sequence exist. CRE-like sequences are present in the regulatory regions of many spermatid-specific genes, including those for transition proteins (TP1 and TP2), Tact1, Tact2, Oazt, ACEt, protamines (P1 and P2) and RT7 (5,6,17,18,(34)(35)(36)(37). In these genes, the CRE-like motif acts as an activator to enhance promoter activity. In contrast, Oxct2b-CL in the Oxct2b core promoter cannot function as a cis-activator ( Figures 4C and 7). Therefore, Oxct2b is a very rare example of how the CRE-like motif is essential for basal promoter activity. Figure 7. Oxct2b-CL is indispensable for Oxct2b core promoter activity in testicular germ cells. Reporter gene assays with the 5 0 -upstream constructs, which comprise À389/À49/À16, mutated Oxct2b-CL in À389/À49/À16 (CLmut), and the correct (À49/À16CLmut) and inverted (À16/À49CLmut) sequences of À49/À16 ligated to the 5 0 end of the CLmut and À389/À50, were carried out via in vivo transient transfection of testicular germ cells. The results are presented as described in the legend to Figure 4. **P < 0.01 compared with the À389/À49/À16Luc construct (ANOVA followed by Bonferroni analysis). The luciferase activity of À389/À49/À16Luc was set at 1.0.
Transcription initiation of protein-encoding genes is a complex process that requires the precise positioning of Pol II on the core promoter DNA. This is accomplished via a series of interactions between Pol II and at least six accessory proteins,  namely TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, which have been termed GTFs (38,39). Nucleation of the initiation complex occurs by recognition of a specific core element. To date, the TATA box has been considered to be the primary core element responsible for positioning the basal transcription machinery on the promoter. Among the GTFs, TFIID is a multisubunit protein complex that contains the TBP at its core and TBP-associated factors (TAFs). It is generally accepted that the binding of TBP to the TATA box nucleates the formation of the transcription pre-initiation complex (PIC), either through stepwise assembly of other basal factors or through recruitment of the holoenzyme (40,41). Following the characterization of more promoters of protein-coding genes, it has been noted that many genes lack the TATA box (42). A second core element, Inr, which encompasses the transcription start site, is sufficient for the positioning of the basal transcription machinery in the absence of a TATA box (43). It has also been postulated that the sequence-specific transcription factor Sp1 tethers the PIC to TATA-less promoters by interacting with a component of TFIID (21,44). However, the Oxct2b promoter lacks the TATA box, Sp1-binding site, and Inr, which suggests that Oxct2b-CL plays an essential role in PIC formation in testicular germ cells. If this is true, Oxct2b-CL-binding factor(s) should be involved in the formation of the functional PIC through Oxct2b-CL, that is, Oxct2b-CL at À29/À22 may be a platform for the assembly of GFTs and for Pol II to initiate accurate haploid spermatid-specific transcription.

CREM involvement in PIC formation in testicular germ cells
In this study, we found that Oxct2b-CL (TGACGCAG) is bound specifically to testis-specific CREM via the 5 0 -half of TGACG. The CREM isoform is the same as the CREM that is bound to the CRE-like motif of the testis-specific ACEt promoter ( Figure 6). The ACEt promoter is activated by the CREM testicular isoform through the CRE-like motif (45). CREM is a member of the CREB family and is involved in the regulation of gene expression in response to several signaling pathways. CREM proteins are produced as activators or repressors by the process of alternative splicing. The N-terminal half of the CREM activator contains an activator domain (AD) that is made up of glutamine-rich Q1 and Q2 domains and the P-box, which is phosphorylated by various kinases that regulate the transcriptional potential of these proteins (46). The testis isoform of CREM, which contains the exons that encode AD, is highly expressed after meiosis during spermatogenesis (47,48). However, the testis CREM isoform is not phosphorylated and its transcriptional activity can be stimulated by interaction with the ACT co-activator in spermatids (15). Thus, Oxct2b-CL should be bound to the non-phosphorylated CREM testicular isoform. The Oxct2b promoter has no TATA box, which suggests that a certain scaffold of PIC formation is required for Oxct2b promoter activity. In this regard, full-length CREM and CREMDQ2, which lack the Q2 domain, were able to bind to TBP and TAF II 130 (TAF4) in GST-pull down assays (49). Therefore, we postulate a possible mechanism whereby the Oxct2b promoter is activated by a CREM activator, which is mediated by the recruitment of TFIID to Oxct2b-CL via its interaction with TAF4 and TBP. In this case, PIC formation does not necessarily require a TATA box. Alternatively, the AD of CREM interacts with TFIIAa, as shown by two-hybrid and co-immunoprecipitation analyses. This interaction is restricted to the activator isoform of CREM and does not require phosphorylation. CREM and TFIIA co-localize intracellularly (16). These data indicate that CREM regulates transcription through binding to TFIIA in spermatids. PIC formation in TATA-less Pol II promoters is postulated to require specific interactions between various factors, including TFIIA (50,51). TFIIA has been shown to interact with TBP-related factor TRF2 (TLF/TLP) and TBP (52)(53)(54)(55)(56), and TRF2 has been suggested to operate on TATA-less promoters (57)(58)(59)(60)(61). Furthermore, in vivo experiments in which the association of TFIIA and TRF2 were studied indicated that the TRF2-TFIIA complex is more stable than the TBP-TFIIA complex, which implies that TRF2 initiates transcription more efficiently through TFIIA binding than does TBP (62). TRF2 is encoded by two mRNAs with common protein-coding sequences, but distinct 5 0 -untranslated regions. One of these mRNAs is expressed ubiquitously, whereas the other shows a restricted expression pattern and is abundant in the testis (59,61). TRF2-deficient male mice are viable, but sterile due to late arrest of spermatogenesis (63,64). Although the expression of CREM and many other postmeiotic genes is unaltered in TRF2-null mice, several spermiogenesis genes that are transcribed in late-round spermatids appear to be under the control of TRF2 (63). Based on our results, we propose a model in which a testicular CREM-dependent TFIIA-TRF2 complex is formed on Oxct2b-CL, which serves as an efficient scaffold for the remainder of the PIC in spermatids.


extfieldsofstudy: Medicine Biology

text:
Purification and kinetic studies of recombinant gibberellin dioxygenases

Background The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in the metabolism of a major plant hormone. The activity of recombinant GA 2ODDs from many species has been characterised in detail, however little information relates to enzyme purification. Native GA 2ODDs displayed lability during purification. Results Two GA 2ODDs were expressed in Escherichia coli and purified to homogeneity. The GA 2-oxidase from Pisum sativum L., PsGA2OX1, was expressed as a glutathione s-transferase (GST) fusion. It was purified in the three steps of affinity chromatography, GST removal and gel filtration. Highly pure PsGA2OX1 was obtained at a yield of 0.3 mg/g of cells. It displayed a Km of 0.024 μM and a Vmax of 4.4 pkat/mg toward [1β,2β,3β-3H3]GA20. The GA 3-oxidase from Arabidopsis thaliana, AtGA3OX4, was expressed as a poly(His)-tagged thioredoxin fusion. It was purified by Immobilised Metal Affinity Chromatography followed by gel filtration. Cleavage of the fusion took place between the two purification steps. Highly pure AtGA3OX4 was obtained at a yield of 0.01 mg/g of cells. It displayed a Km of 0.82 μM and Vmax of 52,500 pkat/mg toward [1β,2β,3β-3H3]GA20. Conclusion Fusion tags were required to stabilise and solubilise PsGA2OX1 and AtGA3OX4 during E. coli expression. The successful purification of milligram quantities of PsGA2OX1 enables mechanistic and structural studies not previously possible on GA 2ODDs. A moderate yield of pure AtGA3OX4 requires the further optimisation of the latter stages of the enzyme purification schedule. PsGA2OX1's action in planta as deduced from the effect of the null mutation sln on GA levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.

Background
The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in plant hormone metabolism. Three principal classes of 2ODDs control the later stages of the pathway where bioactive GA is produced and inactivated ( Figure 1). Activation and deac-tivation of the GA molecule are performed by GA 3-oxidase and GA 2-oxidase enzymes, respectively.
The genes encoding GA 2ODDs have been described from many species. They belong to multigene families which show spatial and temporal patterns of expression [1]. Factors including hormone levels and light have been shown to influence GA 2ODD gene expression [2], suggesting that the enzymes are important in regulating the overall GA pathway. The genetic manipulation of GA levels for agricultural purposes has often involved 2ODD-catalysed steps.
The GA 2ODDs are well suited to in vitro studies because they retain good activity when expressed in Escherichia coli and their natural substrates are mostly known and available in synthetic form. Thus, a reasonable amount of data relates to the activity of recombinant GA 2ODDs, e.g. [3][4][5][6][7]. Their catalysis generally appears to reflect that of the native enzymes.
Unfortunately, the excellent literature on the activity of recombinant GA 2ODDs is accompanied by a lack of progress on their purification and structural characterisation. In native form the enzymes displayed a lability that hindered their complete purification [8][9][10][11]. The prob-lems associated with this lability have ostensibly not been overcome through using recombinant expression methods. The sole report on a pure GA 2ODD relates to a native GA 20-oxidase from pumpkin endosperm [12].
Herein, we report the successful purification of a recombinant GA 2-oxidase from Pisum sativum L. (pea) and a GA 3-oxidase from Arabidopsis thaliana (Arabidopsis), findings that represent a significant step for an important group of enzymes.

Expression and purification of the GA 2-oxidase, PsGA2OX1
The pea GA 2-oxidase, PsGA2OX1, accumulated intact and in soluble form in E. coli when expressed as a GST fusion in pGEX. The expression conditions employed were determined to be optimal with respect to incubation temperature and induction conditions. Trials with a GST fusion expression system driven by the stronger T7 promoter did not improve expression levels (results not shown).
Affinity chromatography of GST-PsGA2OX1 from the soluble cell fraction gave a high degree of purification ( Figure  2A). Subsequent removal of the GST tag yielded PsGA2OX1 with only two major contaminants ( Figure  2A). Gel filtration removed these contaminants without significant loss of PsGA2OX1 and increased its purity to > 95% as estimated from SDS-PAGE analysis ( Figure 2B). Pure PsGA2OX1 was obtained in milligram quantities and was concentrated to 10 mg/mL ( Figure 2B, Table 1).
PsGA2OX1 was active at all stages of purification (Table  1). EDTA had an inhibitory effect on activity that was removed most effectively by dialysis rather than dilution. However, when crude samples were dialysed, activity diminished, presumably because of enzyme inactivation or degradation. In addition, endogenous components of E. coli lysates are suspected to inhibit GA 2ODDs [7]. Therefore, the activity measurements after the first two stages of purification (Table 1) gave apparent values.
Concentrated highly pure PsGA2OX1 retained activity after storage at -70°C.

Kinetic analysis of PsGA2OX1
The activity of this pure PSGA2OX1 displayed a saturation curve of the Michaelis Menten type in response to increasing concentration of substrate [ 3 H 3 ]GA 20 ( Figure 3). The K M was calculated to be 0.024 µM and the V max 4.4 pkat/ mg. The latter steps of biosynthesis of non-13-hydroxylated/13-hydroxylated GAs Figure 1 The latter steps of biosynthesis of non-13-hydroxylated/13hydroxylated GAs. The steps surrounding production of bioactive GAs in higher plants are catalysed by the 2-oxoglutarate-dependent dioxygenases (2ODDs); 1 GA 20-oxidase, 2 GA 3-oxidase and 3 GA 2-oxidase. In pea the 13-hydroxylated pathway predominates; in Arabidopsis the non-13hydroxylated pathway generally predominates, however developing seeds contain comparable levels of non-13hydroxylated and 13-hydroxylated GAs [18]. The sln phenotype of pea arises from a null mutation affecting the predominant GA 2-oxidase (PsGA2OX1) of maturing seeds (steps 3). In this study the activities of a pea GA 2-oxidase (PsGA2OX1) and arabidopsis GA 3-oxidase (AtGA3OX4) are both assayed using the substrate [1β,2β,3β-3 H 3 ]GA 20 . It should be noted that in addition both enzymes are likely to use GA 9 as a natural substrate.  Bioactive

Expression and purification of the GA 3-oxidase, AtGA3OX4
The Arabidopsis GA 3-oxidase, AtGA3OX4, accumulated intact and in soluble form when expressed as a poly(His)tagged TRX fusion in pET32 to a level that was discernible by SDS-PAGE analysis of the soluble cell fraction ( Figure  4A).
During purification AtGA3OX4 tended to precipitate, although precipitation could be minimised during the first step by dilution of the soluble cell lysate and the use of a buffer of pH 6.3 with 10 mM β-ME.
TRX-AtGA3OX4 was purified to a high degree by Immobilised Metal Affinity Chromatography ( Figure 4A, Table 2). Cleavage of the TRX tag from AtGA3OX4 was enabled by a thrombin site engineered into the expression construct -the Factor Xa site encoded by pET32 failed to cleave.  (pkat/mg) AtGA3OX4 was difficult to separate from TRX, remaining strongly associated with it subsequent to thrombin cleavage. The single factor identified that most obviously mitigated this association was β-ME, with low pH further aiding separation. Resolution of the two proteins was achieved by gel filtration in a buffer of pH of 5.8 with 10 mM β-ME. Highly pure AtGA3OX4 was thus obtained in microgram quantities ( Figure 4B, Table 2).
Once pure, AtGA3OX4 resisted concentration. The problem was partially overcome by the addition of Triton X-100. However, the maximum concentration of AtGA3OX4 achieved was 1.5 mg/mL and its activity did not tolerate freezing.
Otherwise, AtGA3OX4 was active at all stages of purification, permitting a kinetic study on highly pure AtGA3OX4.
Removal of the TRX tag had no obvious affect on AtGA3OX4 activity levels. Triton X-100 had an inhibitory effect but this seemed to disappear with dilution. The use of low pH buffers during purification did not obviously dampen AtGA3OX4's activity. Indeed, we found AtGA3OX4 displayed 80 % activity when assayed in the phosphate buffer of pH 6.3 used for the first purification step, providing the reducing agent DTT was present (results not shown). However, the buffer of pH 5.8 in the second step was not compatible with activity assays.
In light of the difficulties associated with obtaining AtGA3OX4 in high yield, further purification of the fusion, TRX-AtGA3OX4, was conducted. Milligrams of highly pure TRX-AtGA3OX4 were obtained. The protein was amenable to concentration ( Figure 4B) and could be frozen at -70°C without activity loss.

Kinetic analysis of AtGA3OX4
The activity of highly pure AtGA3OX4 displayed a saturation curve of the Michaelis Menten type in response to increasing concentration of [ 3 H 3 ]GA 20 ( Figure 5). The K M was calculated to be 0.82 µM and the V max 52, 500 pkat/ mg. (pkat/mg) [ 3 H 3 ]GA 20 concentration ( µM)

Discussion
High levels of activity are frequently obtained for GA 2ODDs in E. coli expression systems in the absence of accumulated soluble enzyme [13]. The instability generally reported in native 2ODDs appears to be a feature of the recombinant enzymes. In early trials of GA 2ODDs, the pea GA 2-oxidase, PsGA2OX1, and the Arabidopsis GA 3-oxidase, AtGA3OX4, showed promise as candidates for purification (D.R. Lester, unpublished). In both cases, a N-terminal fusion tag was needed to obtain soluble intact protein.
The purification of the pea GA 2-oxidase, PsGA2OX1, was straightforward. No problems were encountered during chromatography, tag removal or enzyme concentration. All steps of purification were performed in a buffer that preserved enzyme activity. The pH was within physiological range when the chelator EDTA and the reducing agent DTT were present. (The latter were considered desirable because PsGA2OX1 is Fe ++ -dependent and its active site is likely to be susceptible to metal-catalysed oxidation). The milligram amounts of PsGA2OX1 that we obtained are unprecedented for a highly pure GA 2ODD.
The gene encoding PsGA2OX1 is affected by the null mutation, sln, which causes the accumulation of GA 20 within maturing pea seed [14,15]. The mutation has a lesser effect on GA 20 levels in the stem, presumably because other GA 2-oxidases compensate for its loss in this tissue. Mature seeds contain at least two GA 2-oxidases [11,14]. The sln phenotype indicates that PsGA2OX1 is the main catalyst for the conversion GA 20 to GA 29 at latter stages of seed maturation, when 2-oxidation of GA 20 takes place at high levels. Although the sln mutation is not deleterious to developing seeds, the mature seeds contain abnormally high amounts of GA 20 , which, after germination, is converted to the bioactive GA 1 resulting in the slender (overgrowth) phenotype of the seedling [16]. The levels of GA 20 present in mature SLN and sln seeds are 11 ng/g and 4,118 ng/g, respectively [16]. The difference can be solely attributed to the action of PsGA2OX1 in planta.
Multiple GA 2-oxidases have been previously partially purified in low yield from pea seeds of unspecified maturity [11]. The incomplete purification was attributed to a low abundance of the enzymes in the tissue, and instability during purification. It can be assumed that native PsGA2OX1 was among these GA 2-oxidases because its gene is expressed throughout seed maturation [15]. Recombinant expression methods have now enabled us to complete the successful purification of PsGA2OX1.
At 0.024 µM, the K M of recombinant PsGA2OX1 toward GA 20 was much lower than that obtained for the native pea seed GA 2-oxidases (1.55 µM) [11]. The comparison of K M results is complicated, however, because the native preparation contained multiple activities. Certainly, a high substrate affinity of PsGA2OX1 is consistent with its ability to clear the maturing seed of GA 20 .
The gene encoding PsGA2OX1 is expressed relatively strongly throughout the latter weeks of pea seed maturation [15]. That the enzyme is abundant in mature seeds was supported by a study of GA 2-oxidases from this tissue in the related species Phaseolus vulgaris [8]. If present in abundance, it is plausible that PsGA2OX1 (V max of 4.4 pkat/mg) could account for the difference in GA 20 levels between SLN and sln seeds.
The V max of the partially pure native GA 2-oxidases toward GA 20 was 24.8 pmol/h mg (0.0068 pkat/mg) [11]. The higher figure of the recombinant enzyme suggests that its purity and/or retention of activity was enhanced compared with the native preparation. Interestingly, the V max of PsGA2OX1 was several orders of magnitude lower than the V max of AtGA3OX4 toward GA 20 ( Figure 5). However, this difference is difficult to interpret considering the two enzymes are from different species and are likely to be expressed differentially within the plant alongside isoenzymes.
With their role in GA activation, the GA 3-oxidases are particularly interesting to study, but in native form the enzymes are difficult to purify. It required 6,661 immature seeds for Kwak et al. [9] to obtain 0.086 mg of Our experience provides valuable insight for future work.
The first step we used in the purification of the Arabidopsis GA 3-oxidase, AtGA3OX4, was IMAC chromatography. This method does not permit the inclusion of DDT or EDTA in buffers and therefore has been considered unsuitable for purifying 2ODDs [17]. Nonetheless, AtGA3OX4 with high specific activity was obtained using this method. Evidently, 10 mM β-ME was able to protect the enzyme active site from metal-catalysed oxidation during chromatography. The result suggests that this widely used and highly efficient purification strategy does not necessarily compromise the activity of a 2ODD.
AtGA3OX4 showed a tendency to precipitate, one that became more pronounced when it was cleaved, but not physically separated, from TRX. This diminished the yield of the protein, particularly in the final step. A beneficial effect from low pH and β-ME implied that an exposed cysteine was causing the precipitation. However, a combination of factors were probably to blame because the aforementioned conditions did not fully overcome the problem. Furthermore, replacement by site-directed mutagenesis of the cysteine predicted as most likely to be exposed (residue 9) brought no improvement. Certainly, AtGA3OX4 displayed hydrophobic behaviour when pure, sticking to tubes unless Triton X-100 was present. Hydrophobicity was also a feature of the GA 3-oxidase of bean [9].
Good scope exists for further optimisation of the AtGA3OX4 purification method. Efficient separation of AtGA3OX4 and TRX will lead to a greatly increased yield. Hydrophobic interaction chromatography is a possible alternative to gel filtration in separating the two proteins.
AtGA3OX4 was amenable to concentration and freezing in the presence of TRX, whether or not it was chemically attached to it. The identification of buffer ingredients that replace TRX's stabilising role, such as alternative detergents, salts or organic solvents may improve the behaviour of purified AtGA3OX4.
AtGA3OX4 is encoded by a member of a family of four GA 3-oxidase genes in Arabidopsis. Generally the non-13hydroxylated GA pathway predominates in Arabidopsis, however recent studies found comparable levels of non-13-hydroxylated and 13-hydroxylated GAs in developing seeds [18]. This tissue is the main site of expression of the gene encoding AtGA3OX4 (AtGenExpress microarray data), therefore GA 20 is likely to be a natural substrate of AtGA3OX4. The high V max gained here shows that AtGA3OX4 can efficiently metabolise GA 20 . The value of 52,500 pkat/mg is higher than the V max of 62 nmol/min mg (1,033 pkat/mg) reported for a purified GST fusion of another Arabidopsis GA 3-oxidase, AtGA3OX1 [7]. AtGA3OX4's K M of 0.82 µM toward GA 20 was lower than that of AtGA3OX1 (10 µM) [6,7], yet higher than the value for the bean seed GA 3-oxidase (0.29 µM) [9]. Accumulated kinetic information with respect to both 13hydroxylated and non-13-hydroxylated GAs on each of the GA 3-oxidases of Arabidopsis will give valuable insight into the control of active hormone levels in this model species.

Conclusion
The previous lack of reports on the complete purification of GA 2-oxidases and GA 3-oxidases is probably due to a combination of their lability and their low abundance in most plant tissues. One might speculate that a crucial role in the control of active hormone levels requires a short enzyme half-life. This study on PsGA2OX1 and AtGA3OX4 shows that GA 2-oxidases and GA 3-oxidases are not necessarily inherently unstable and can be purified active from E. coli expression systems under standard conditions. Indeed, the GA 2-oxidase, PsGA2OX1, was exceptionally stable throughout purification. Problems were encountered with the Arabidopsis GA 3-oxidase, AtGA3OX4, due to its hydrophobicity and the presence of hydrostatic charges, rather than metal-catalysed autocleavage or proteolytic breakdown.
Indeed, plant 2ODDs from secondary metabolic pathways are also problematic during purification [17,19]. Yet in some cases, they have been purified in high yield permitting detailed structural and mechanistic studies [20,21]. We note the recent elucidation of the structure of the labile 2ODD-related enzyme, ACC-oxidase, of ethylene biosynthesis [22]. Our work opens the door to similar studies on the GA 2ODDs.

Expression of PsGA2OX1
The cDNA PsGA2ox1 (GenBank AF100954) was ligated into the EcoRI site of the tac promoter-based E. coli expression vector pGEX4T2 (Amersham Biosciences).
A culture of 100 mL LB broth with 100 µg/mL ampicillin was inoculated with E. coli BL21 (DE3) pGEX4/PsGA2ox1 and grown overnight at 37°C with shaking. Thirty mL of starter culture was used to inoculate each of 3 flasks containing 1 L of LB broth with 100 µg/mL ampicillin. Flasks were shaken at 220 rev/min on a circular orbit of 2.54 cm at 37°C. Protein expression was induced when cells reached the OD 600 of 1.0. After 2 h cells were harvested, washed with 1X PBS and frozen at -70°C.

Purification of PsGA2OX1
The GST-PsGA2OX1 purification was performed with glutathione agarose beads (Amersham Biosciences) at 4°C according to the manufacturer's instructions with the following additional details. The buffer used for the lysis of cells and washing of beads was 50 mM Tris-HCl pH 8, 300 mM NaCl, 5 mM EDTA, 5 mM DTT and 0.4 mM PMSF. Cells (approximately 6 g) were resuspended in 120 mL of buffer and lysozyme was added to 0.1 µg/mL. After incubation at room temperature for 15 min, cells were lysed with a Vibra Cell sonicator at 65% amplitude for a total of 60 sec. The cell lysate was spun at 20,000 g and then GST-PsGA2OX1 was extracted from the supernatant using a bed volume of 2 mL of beads in batch mode. After a final wash, the beads were resuspended in 2 mL of buffer containing 10 U of bovine thrombin (Sigma) and incubated at 4°C overnight. Thrombin was extracted from the supernatant containing cleaved PsGA2OX1 using benzamidine agarose beads (Amersham Biosciences).
The protein solution was concentrated to a volume of 300 µL using a Vivascience spin concentrator and applied to a Superdex 75 column (Amersham Biosciences). Gel filtration was performed at 6°C in the same buffer as was used for affinity chromatography. After separation, fractions containing PsGA2OX1 were pooled and dialysed versus 10 mM Tris-HCl pH 8.0, 1 mM DTT.
Pure PsGA2OX1 protein was concentrated to 10 mg/mL using a Vivascience spin concentrator and frozen.
The cDNA was inserted into the T7 promoter-based expression plasmid pET32 Xa/LIC (Novagen) using ligation independent cloning according to the manufacturer's instructions. The resulting construct expressed AtGA3OX4 with an N-terminal tag of both poly(His) and thioredoxin (TRX). A thrombin site was adjacent to the N-terminal of AtGA3OX4, encoded by the forward PCR primer.
A culture of 100 mL LB broth with 100 µg/mL ampicillin was inoculated with E. coli BL21 (DE3) pET32/AtGA3ox4 and grown overnight at 37°C with shaking. Thirty mL of starter culture was used to inoculate each of 3 flasks containing 1 L of LB broth with 100 µg/mL ampicillin. The flasks were incubated at 37°C with shaking at 220 rev/ min on a circular orbit of 2.54 cm until the OD 600 reached 1.0. IPTG was added to 1 mM and the cultures were grown for a further 3 h. Cells were harvested by centrifugation, washed with 1X PBS and frozen at -20°C.

Purification of AtGA3OX4
Cells (approximately 7 g) were thawed and resuspended at room temperature in 120 mL of 5 mM imidazole, 10 mM β-ME, 1X PBS pH 6.3 with 100 µg/mL lysozyme. Cell lysis was performed in a tube on ice using a Vibra Cell sonicator at 65% amplitude for 6 bursts of 10 s. The lysate was centrifuged at 20,000 g at 4°C and the soluble fraction was retained and kept on ice. Two more 10 s bursts of sonication were performed to remove high molecular weight DNA. The soluble fraction was diluted to a total volume of 300 mL with 1X PBS pH 6.3, 5 mM imidazole, 10 mM β-ME.
All column chromatography steps in the purification of AtGA3OX4 were performed at 6°C.
The crude protein solution was passed over a 5 mL HiTrap Chelating column (Amersham Biosciences) loaded with Ni ++ using an Äkta Purifier chromatography system. The column was washed with 50 mL of 5 mM imidazole, 10 mM β-ME, 1X PBS pH 6.3 and the poly(His) TRX-AtGA3OX4 was eluted with a linear gradient of 0 -200 mM imidazole, 10 mM β-ME, 1X PBS pH 6.3. Fractions containing TRX-AtGA3OX4 were pooled and bovine thrombin (Sigma, 20 U) was added.
After incubation at 6°C overnight, cleaved protein was concentrated and loaded onto a Superdex 75 column equilibrated with 1X PBS pH 5.8, 10 mM β-ME. Fractions containing AtGA3OX4 were pooled and concentrated to 1.5 mg/mL with 1% Triton X-100.
In parallel experiments, the intact fusion TRX-AtGA3OX4 was further purified by gel filtration on Superdex 200 with 1X PBS pH 5.8, 10 mM β-ME. The purest fractions were pooled, protein was concentrated to 10 mg/mL and dialysed against 10 mM Tris-HCl pH 8.0, 2 mM β-ME.
Protein concentrations were measured with the Bradford method.

Activity assays on the substrate GA20
The substrate [1β,2β,3β-3 H 3 ]GA 20 (approx. 0.4 TBq/ mmole [23]), enabled the measurement of activity from both PsGA2OX1 and AtGA3OX4 via the formation of tritiated water. Quantitative estimates of product formation were possible because the 3 H was incorporated at a ratio of 1:4:4 at the 1β, 2β and 3β positions [23]. The conversion GA 29 to GA 29 -catabolite was assumed to make a relatively insignificant contribution to 3 H release by PsGA2OX1 under the conditions of assay. This assumption was based on previous findings [4,14,15]. Similarly, it was assumed that AtGA3OX4 produced insignificant amounts of GA 5 [4]. It should be noted that the kinetic parameters may be influenced by the presence of the label in the substrate due to a tritium isotope effect.
The standard assays were conducted using cofactors concentrations of established methods e.g. [14]. Reactions were in a volume of 200 µL with 100 mM Tris-HCl pH 7.9, 4 mM ascorbate, 4 mM DTT, 4 mM 2-oxoglutarate, 0.5 mM FeSO 4 , 2 mg/mL BSA and 1 mg/mL catalase. Substrate (5 µL in methanol) was added to a concentration of 0.02 µM for PsGA2OX1 assays and 0.1 µM for AtGA3OX4 assays. The enzyme solution was added in volumes ranging from 1 to 50 µL. Highly active solutions were serially diluted using 1 mg/mL BSA before addition. The reactions took place at 30°C and were stopped with 25 µL acetic acid after 1 h. Labelled GA 20 was precipitated by adding 780 µL of 25 mM EDTA containing 50 mg/mL activated charcoal and centrifuging at 5,000 rev/min for 5 min. The release of tritiated water was measured by radiocounting a 300 µL reaction aliquot in scintillation fluid with tritium standards alongside. The specific activity figures for each purification stage were derived from enzyme samples (or dilutions of samples) showing similar rates of product conversion.
The substrate, [ 3 H 3 ]GA 20 , was varied in the assays for kinetic studies as shown in Figures 3 and 5. At higher substrate concentrations the maximum volume of methanol added was 10 µL (the extra methanol was shown not to be inhibitory). The amount of enzyme added was 1 µg for PsGA2OX1 and 0.1 µg for AtGA3OX4. Results were analysed using Origin by MicroCal.

Authors' contributions
DRL designed and performed most of the experimental work. She also drafted the manuscript. AP and PH developed the vector that enabled expression and purification of AtGA3OX4. IA conceived of the project, participated in experimental design and helped draft the manuscript. All authors read and contributed to the final draft of the manuscript.


text:
Nrf2 Transcriptionally Activates the mafG Gene through an Antioxidant Response Element*

Nrf2 accumulates in nuclei upon exposure to oxidative stress, heterodimerizes with a small Maf protein, and activates the transcription of stress target genes through antioxidant response elements (AREs). We found that diethyl maleate (DEM), a well known activa-tor of Nrf2, induces one of the small Maf genes, mafG . To elucidate roles MafG might play in the oxidative stress response, we examined transcriptional regulation of the mouse mafG gene. MafG utilizes three independent first exons that are each spliced to second and third coding exons. Among the small maf genes, mafG showed the strongest response to DEM, and of the three first exons, the highest -fold induction was seen with the proximal first exon (Ic). Importantly, one ARE (Ic-ARE) is conserved in the promoter flanking exon Ic of the human and mouse mafG genes. The Nrf2/MafG heterodimer bound the Ic-ARE and activated transcription, whereas DEM failed to activate mafG in nrf2 -null mutant cells. Chromatin immunoprecipitation further revealed that both Nrf2 and small Maf proteins associate with the Ic-ARE in vivo . These results demonstrate that mafG is itself an ARE-dependent gene

Nrf2 accumulates in nuclei upon exposure to oxidative stress, heterodimerizes with a small Maf protein, and activates the transcription of stress target genes through antioxidant response elements (AREs). We found that diethyl maleate (DEM), a well known activator of Nrf2, induces one of the small Maf genes, mafG. To elucidate roles MafG might play in the oxidative stress response, we examined transcriptional regulation of the mouse mafG gene. MafG utilizes three independent first exons that are each spliced to second and third coding exons. Among the small maf genes, mafG showed the strongest response to DEM, and of the three first exons, the highest -fold induction was seen with the proximal first exon (Ic). Importantly, one ARE (Ic-ARE) is conserved in the promoter flanking exon Ic of the human and mouse mafG genes. The Nrf2/MafG heterodimer bound the Ic-ARE and activated transcription, whereas DEM failed to activate mafG in nrf2-null mutant cells. Chromatin immunoprecipitation further revealed that both Nrf2 and small Maf proteins associate with the Ic-ARE in vivo. These results demonstrate that mafG is itself an ARE-dependent gene that is regulated by an Nrf2/small Maf heterodimer and suggest the presence of an autoregulatory feedback pathway for mafG transcriptional regulation.
Cells have the ability to adapt to oxidative stress or to exposure to xenobiotics. By inducing a battery of antioxidant and xenobiotic metabolizing enzymes, such as glutathione S-transferase, heme oxygenase-1 (HO-1), 1 and NAD(P)H:quinone oxidoreductase 1 (NQO1), cells protect themselves from oxidative stress and xenobiotics (1,2). It has long been known that the genes encoding these enzymes are often coordinately regulated through AREs in their gene-regulatory regions (2). The ARE is a Maf (musculo-aponeurotic fibrosarcoma) recognition element (MARE)-related sequence, with strong similarity to the MARE (3) (see Fig. 4A). Basic region-leucine zipper transcription factors, including CNC (Cap'n'Collar protein) family (Nrf1, Nrf2, Nrf3, and p45 NF-E2) and Bach (BTB and CNC homology) proteins (Bach1 and Bach2), form heterodimeric partners with a small Maf protein (MafG, MafK, and MafF), and then associate with various MARE-related sequences (including AREs).
CNC and Bach proteins possess transcriptional activation and repression domains, respectively. Therefore, ARE-mediated transcription is either positively or negatively regulated, depending on which basic region-leucine zipper factors bind to the ARE(s). Whereas small Maf proteins lack any recognizable transcriptional effector domains (other than the basic regionleucine zipper motif), they are nonetheless regarded as critical regulators of ARE-mediated transcription, since CNC and Bach proteins require them as obligatory partners for site-specific association with the ARE. Moreover, it is well documented that homodimers and heterodimers composed exclusively of small Maf proteins can act as repressive competitors to small Maf/ CNC and small Maf/Bach heterodimers. One implication of these data is that small Maf proteins can participate in both positive and negative regulation of MARE-dependent genes. Additionally, the amount of small Maf proteins could be an important determinant of the transcriptional activity of target genes (for reviews, see Refs. 4 and 5).
The positive contributions of CNC proteins and the negative contributions of Bach1 to ARE-mediated transcription have been validated by gene targeting strategies. ARE-dependent gene induction was greatly impaired in nrf2-null mutant mice (6), and Nrf1 is also reported to contribute to the activation of ARE-dependent genes (7,8). The ARE-dependent HO-1 gene was significantly derepressed in bach1-null mutant mice, suggesting that HO-1 is negatively regulated by Bach1 (9). Although nrf3-null and bach2-null mutant mice have been reported (10,11), the contribution of these factors to AREmediated regulation is still to be determined.
We recently observed that a fraction of ARE-dependent genes (e.g. NQO1 and thioredoxin reductase 1) are not normally induced in either small maf compound mutant mice or in nrf2null mutant mice. 2 Therefore, we concluded that small Maf proteins act cooperatively with Nrf2 to activate the transcription of these genes. In contrast, HO-1 transcription is derepressed in small maf compound mutant mice, similarly to the bach1-null mutant (16), suggesting that small Maf proteins are also important for collaboratively repressing HO-1. In this way, we genetically confirmed that small Maf proteins act as required cofactors in both positive and negative MARE-dependent regulation of gene transcription.
In the course of previous gene expression analyses, we found that diethyl maleate (DEM), a well known inducer of Nrf2/AREdependent genes, induced the expression of mafG. 3 This observation suggested that mafG is an Nrf2/ARE-dependent gene. However, the mechanisms that regulate mafG transcription are only poorly understood. Here, we show that a functional promoter ARE controls mafG induction. By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analyses, we demonstrate that Nrf2 and small Maf proteins bind specifically to the mafG ARE. We also confirm that induction of mafG is severely impaired in nrf2-null mutant cells. Thus, mafG is indeed an Nrf2/ARE-dependent gene.

EXPERIMENTAL PROCEDURES
Animals-Germ line mutagenesis of mouse nrf2 was described previously (6). The mice used in this study were on a mixed genetic background of 129SvJ and ICR.
Mouse Embryonic Fibroblasts (MEF)-MEFs were prepared from individual embryos at embryonic day 13.5. The head and internal organs were removed, and the torso was minced and dispersed in 0.25% trypsin/EDTA. MEFs were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum and antibiotics. To induce ARE-dependent genes, MEFs were treated with 50 M DEM (Wako Pure Chemicals, Osaka, Japan) for 12 h.
Quantitative RT-PCR-Total RNA was prepared from mouse liver or from mouse embryonic fibroblasts using Isogen (Nippon Gene, Japan) following the recommended protocol. Random cDNA was synthesized from the isolated RNAs, and real time PCR (ABI PRISM 7700) was performed as described (14). Sequences of the primers and probes used for detecting total mafG transcript have been described previously (14), and those used for detecting each mafG transcript are shown in Table  I. The mafG-III-II primers and mafG-P TaqMan probe were designed at a region common to all four transcripts.
RNA Blot Analysis-Total RNA was electrophoresed on formaldehyde-agarose gels and transferred to a nylon membrane (Zeta probe; Promega). 32 P-Labeled probes were prepared from the cDNAs by random primer labeling. References for the sequences of the cDNAs used in this study are as follows: HO-1 (17), NQO1 (6), and peroxiredoxin 1 (PRDX1) (17).
Rapid Amplification of cDNA Ends (RACE)-RACE was performed with total RNA from a mouse embryonic day 16 Fig. 5C (m1, m2, and m3 competitors) were annealed. An increasing amount of competitors, from 10-to 100-fold or a 300-fold excess of competitors was added. Incubation of the probe and recombinant proteins with or without unlabeled competitors was carried out as described previously (3). The protein-DNA complexes and free probe were resolved by electrophoresis on a 5% polyacrylamide (79:1) gel in 1ϫ TBE buffer.
Transfection-293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics and seeded in a 12-well plate 24 h before transfection. Mouse Nrf2 cDNA was inserted into pEF-BssHII (3) and used for transient expression of Nrf2 (pEF-Nrf2). pIM-MafG (20) was used for transient expression of mouse MafG. To make pNQO1-ARE-Luc, the pair of oligonucleotides NQO1-ARE-F and NQO1-ARE-R (above) were annealed and inserted into pRBGP3 (21). To generate pMafG-ARE-Luc and their mutant derivatives, a pair of oligonucleotides MafG-ARE-F and MafG-ARE-R (above), either bearing or without any introduced mutation (Fig. 5C), were annealed and inserted into pRBGP3. These expression vectors and the luciferase reporter vectors were transfected into the cells using FuGENE6 (Roche Applied Science). 12 h after transfection, cells were harvested, and the lysates were used for luciferase assays. The expressions of both firefly and sea pansy luciferase were quantified using a dual luciferase reporter assay (Promega); firefly luciferase activity was normalized to co-transfected sea pansy luciferase activity for transfection efficiency.

RESULTS
MafG Gene Expression Is Induced by DEM-In an earlier study, we found that DEM, a well known inducer of Nrf2/ARE-   The 5Ј-ends of the RACE clones are indicated by solid circles. The translational initiation ATG codon of mafG is boxed. B, structure of the mouse mafG gene and the four MafG mRNA splice isoforms identified by RACE. C, tissue distribution of the four MafG mRNA splice isoforms. cDNAs were synthesized from total RNAs prepared from the various tissues of adult animals, as indicated. The relative copy numbers of the MafG mRNA splice isoforms Ia, Ib, Ic, and Ic-int were determined by quantitative RT-PCR using a plasmid containing the cDNA for each splice isoform as the abundance standard. The relative copy number of the Ia splice isoform in brain was used as a reference and set to 1. The error bars represent the S.D. values (n ϭ 3).
Nrf2 Activates mafG through an ARE 4485 dependent genes, induced the expression of mafG in MEF. 3 To confirm this observation, we treated wild type MEFs with DEM and examined the expression of small maf genes by quantitative RT-PCR. Among the small maf genes, mafG was the most significantly induced by DEM (Fig. 1A). Induction of mafG peaked 3 h after DEM treatment before gradually decreasing (Fig. 1B). Thus, mafG is a DEM-responsive gene induced at a relatively early stage in the course of the response. The HO-1 gene showed a similar induction profile (Fig. 1B). Notably, the induction of other ARE-dependent genes, such as NQO1 and PRDX1, peaked only after 12 h following DEM treatment (Fig. 1B).
The mafG Gene Has Three Alternative First Exons-To decipher the mechanisms controlling mafG induction, we examined transcriptional regulation of the mafG gene. We previously isolated phage clones containing the mouse mafG locus and mapped coding exons II and III but did not determine the nature of the noncoding first exon(s) (13). Therefore, we first performed 5Ј-RACE to determine first exon(s) utilized by the mafG gene and identified three (designated Ia, Ib, and Ic) ( Fig.  2A). We also identified one splice isoform, designated "Ic-int" (Fig. 2B), which has an Ic first exon and intron sequence lying between the Ic and second exon but lacks the intron between exons II and III, demonstrating that this sequence was not due to genomic DNA contamination. We asked whether these splice isoforms (Ia, Ib, Ic, and Ic-int) are also found in the adult mouse and whether or not they are differentially utilized. To address the latter question, we designed a quantitative RT-PCR assay system which allowed specific transcript identification. Furthermore, to compare the relative abundance of each isoform, we prepared plasmid vectors containing each PCR product and used them as a copy number standard for quantitative RT-PCR. These results show that each splice isoform is differentially utilized (Fig. 2C). In the tissues tested, the Ib and Ic isoforms were relatively abundant (Fig. 2C). Whereas the Ia isoform was used relatively more rarely, this transcript seems to be preferentially expressed in hematopoietic tissues, such as spleen and bone marrow (Fig. 2C). The expression of the Ic-int isoform was utilized differently than the Ic isoform (Fig. 2C). In conclusion, accumulation of the four distinct MafG transcripts differs in a manner that is dependent on tissue type, suggesting that alternative transcriptional regulatory mechanisms differentially control expression from each of the three mafG promoters.
An ARE Consensus Sequence in the Promoter-proximal Region of mafG Exon Ic-We next asked whether the distinct mafG transcripts are utilized differentially in response to stress. We performed quantitative RT-PCR using cDNA recovered from cells treated or untreated with DEM. The Ia, Ib, and Ic isoforms were all significantly induced by DEM, whereas the Ic-int isoform was not (Fig. 3A). After DEM treatment, the Ib isoform was still the most abundant species of the mafG transcripts (Fig. 3A). Notably, upon comparison of the induction ratios for each transcript, we found that isoform Ic was most markedly induced (Fig. 3B).
While searching through the mafG genomic locus in silico, we discovered a potential ARE sequence 5Ј to exon Ic (Ic-ARE) (Fig. 4A). The Ic-ARE partially matches the ideal ARE consensus sequence and is similar to the ARE identified in the NQO1 regulatory element (Fig. 4A). An NCBI data base search led to the identification of a putative first exon for human mafG corresponding nicely to the mouse Ic exon (Fig. 4B). The promoter-proximal region of exon Ic is highly conserved between mice and humans, including the putative mafG promoter Ic-ARE (Fig. 4B).
An Nrf2/MafG Heterodimer Binds to, and Activates Transcription from, the mafG Ic-ARE-The potential binding interaction of the Nrf2/small Maf heterodimer to the Ic-ARE was examined by EMSA. To assess the binding affinity of the mafG Ic-ARE, we compared it with the high affinity ARE site identified in the mouse NQO1 gene as a positive control. The result clearly demonstrated that the Nrf2/MafG heterodimer binds well to the mafG promoter ARE (Fig. 5A). The binding affinity of the heterodimer for the Ic-ARE appeared to be somewhat weaker than for the NQO1-ARE (Fig. 5A); in contrast, MafG homodimers seemed to prefer the MafG-ARE over the NQO1-ARE (Fig. 5A). The binding activities to the MafG-ARE were competed efficiently by a 300-fold molar excess of unlabeled wild type MafG-ARE, but not by the mutant MafG-ARE, possessing mutated nucleotides at both ends of the TPA response element contained within the MafG-ARE (m1) (Fig. 5B). These results showed that DNA binding of Nrf2 and MafG depends on the integrity of the TPA response element core sequence of the MafG-ARE. To the contrary, these binding activities were eliminated by the addition of a 300-fold molar excess of the mutated MafG-ARE possessing a mutation in the sequence lying just outside of the short ARE consensus (m2). The mutated MafG-ARE possessing both of the mutations within a single ARE (m3) behaved in a similar way with m1 mutant oligonucleotides (Fig. 5B). Further study using a lower molar excess of competitors showed that m2-type MafG-ARE was almost comparable with the wild type MafG-ARE in the ability to suppress the Nrf2/MafG-shifted complexes (Fig. 5C). These results showed that the mutated nucleotides in m2-type MafG-ARE are not

Nrf2 Activates mafG through an ARE
critical for the binding of Nrf2/MafG heterodimer and MafG homodimer to Ic-ARE at least in vitro (Fig. 5C).
To examine whether the mafG promoter Ic-ARE is functional, we performed transient transfection assays. Oligonucleotides containing ARE sequences were inserted into luciferase reporter genes and transfected into 293T cells. In order to make a comparison with the activity of a known ARE, we co-transfected luciferase reporter constructs containing either the ARE from mafG promoter or the ARE derived from the mouse NQO1 gene. The ARE reporter gene was strongly activated by co-transfection with the Nrf2 expression vector alone (Fig. 5D). The ARE/luciferase reporter was activated to even higher levels when co-transfected with expression vectors that simultaneously forced Nrf2 and MafG expression (Fig. 5D). The induction of the reporter gene was comparable using either the mafG promoter or NQO1 promoter ARE sites (Fig. 5D). We further dissected the Ic-ARE activity by investigating the trans-activation potential of mutant AREs tested in EMSA. Strong induction of the reporter gene that is normally elicited by the Nrf2 expression vector was no longer observed with a mutated reporter gene possessing an m1-type mutation (Fig.  5E). Consistent with the result of EMSA, this showed that the TPA response element core sequence of the Ic-ARE is critical. Whereas m2-type mutation did not affect the binding activity of Nrf2/MafG, this mutation abolished Nrf2-mediated induction of the ARE luciferase reporter gene (Fig. 5E). It is noteworthy that both m1-type and m2-type mutated reporter genes were slightly activated when both Nrf2 and MafG expression vectors were co-introduced by transfection. However, no combination of factors was able to activate the reporter gene possessing the FIG. 4. A putative ARE in the mafG gene Ic promoter. A, alignment of the MARE, the short ARE, the long ARE, the mafG Ic-ARE, and the ARE from the mouse NQO1 gene regulatory region. The MafG genomic locus is depicted at the top. The boxes represent exons, and the circle indicates the position of the mafG Ic-ARE. Each sequence was aligned, with asterisks indicating the nucleotides that are identical between the long ARE and the MafG Ic-ARE or between the MafG Ic-ARE and the NQO1 ARE. B, comparison of the first exons and the 5Ј upstream regulatory regions of human and mouse mafG genes. For A and B, the arrows indicate nucleotides corresponding to the long ARE. The TPA response elopement core sequence of the MARE is shaded in dark gray. The GC boxes of the MARE are shaded in light gray. The abbreviations follow standard IUPAC nomenclature (where M represents A or C, R is A or G, Y is C or T, W is A or T, and S is G or C). m3-type mutation (Fig. 5E). These mutational analyses suggest that the sequences outside of the classical short ARE are important for maximum induction of mafG transcription.
Nrf2 and Small Maf Interact with the Endogenous mafG Ic-ARE-In order to confirm the biological significance of the data obtained from both EMSA and reporter transfections, ChIP experiments were performed using Hepa-1c1c7 cells. To induce nuclear accumulation of Nrf2, Hepa-1c1c7 cells were treated with DEM for 3 h, fixed with formaldehyde, and used for ChIP assays.
The association of Nrf2 with the NQO1 ARE was first examined as a positive control (22). As reported, Nrf2 and small Mafs associate strongly with the NQO1 ARE in DEM-treated cells but only weakly in mock-treated cells (Fig. 6). In the absence of IgG or in the presence of preimmune rabbit IgG, immunoprecipitations failed to select the NQO1 ARE (as well as other nonspecific genomic regions), demonstrating that the sites were not enriched in nonspecific fashion (Fig. 6). The LDH gene promoter region was not immunoprecipitated with either Nrf2 or small Mafs, suggesting that the antibodies used enriched DNA in a sequencespecific manner (Fig. 6). Under such conditions, these experiments showed that both Nrf2 and small Mafs associated with the mafG Ic-ARE in DEM-treated cells (Fig. 6). These results clearly demonstrated that Nrf2 and small Mafs can associate with an endogenous genomic region containing the mafG promoter Ic-ARE. Interestingly, small Mafs also strongly associated with the mafG Ic-ARE in vehicle-treated cells, suggesting occupation of the Ic-ARE by either inactive or repressive binding complexes prior to DEM induction.

Induction of the mafG Gene Is Dependent on Nrf2-Finally,
we sought genetic confirmation to support the dependence of mafG transcription on Nrf2 expression. To this end, we prepared nrf2-null embryonic fibroblasts, treated the cells with DEM, and examined mafG mRNA accumulation. Before DEM treatment, the expression of mafG was slightly elevated in nrf2-null mutant cells (in comparison with wild type controls) by ϳ1.5-fold. However, mafG was no longer significantly activated in nrf2-null mutant cells after adding DEM, whereas a dose-dependent induction of the mafG was observed in wild type cells (Fig. 7). These results demonstrate that mafG induction is dependent on Nrf2. DISCUSSION In this study, we examined the transcriptional regulation of the mouse mafG gene and concluded that mafG is itself an ARE-dependent gene controlled by Nrf2. In agreement with the present data, other Nrf2 inducers have also been reported to induce the mafG gene. A hamster mafG homolog was identified as a hydrogen peroxide-inducible gene in a differential display analysis of gene expression (23). Another group reported that pyrrolidine dithiocarbamate, a thiol compound, induces human MAFG (24). Furthermore, it has been reported that the zebrafish mafG homolog is inducible by DEM (25). Thus, mafG seems to be a stress-inducible gene in many species.
When we initially cloned the first exons of mafG by 5Ј-RACE analysis, we found that the mouse mafG gene has at least three alternative first exons (Ia, Ib, and Ic). We previously reported that mafK and mafF also have multiple untranslated first exons (14,26). Taken together, it seems clear that all three small maf genes utilize multiple first exons. MafG does not appear to have tissue-specific first exons, such as the mafK I N exon that is employed exclusively in neurons (27). Nonetheless, expression analysis revealed that mafG first exons are utilized differentially in various tissues.
Based on the sequences derived from the 5Ј-RACE clones, we concluded that none of the alternative splice isoforms of mafG produce a change in the amino acid composition of the protein.
We inferred that utilizing multiple first exons presents itself as a solution by which small maf genes can confer distinct, broad (but not ubiquitous) expression profiles. Although we cloned a splice isoform of mafG (Ic-int) that contains the intron between exon Ic and exon II and is expressed at a significant level, its biological significance is yet to be determined.
All three first exons of mafG were induced upon oxidative stress. Whereas the Ib transcript was the most abundant before and after DEM treatment, the Ic transcript was the most strongly activated by DEM relative to its constitutive expression level. These observations show that the alternative first promoters vary in their inducible response to DEM. The critical transcription factor-binding site that could be responsible for DEM-mediated induction was anticipated to be an ARE(s). We searched for AREs in the mafG locus and identified one in the proximal promoter of exon Ic. Although the short ARE consensus sequence was originally defined as 5Ј-RGTGACnnnGC-3Ј, subsequent studies revealed that functional AREs, such as the one in the NQO1 gene, are often represented by a long ARE consensus sequence, 5Ј-TMAnnRTGAYnnnGCRwwww-3Ј (22,28). Indeed, the mafG Ic-ARE is similar to the longer consensus sequence and is also similar to the ARE found in the NQO1 gene regulatory region. Since the Ic-ARE is well conserved in the human MAFG gene, we suspect that these AREs are evolutionarily conserved for mafG regulation.
In at least two previous reports, investigators failed to observe cooperative activation of AREs by Nrf2 and small Maf proteins (29,30). Although we also failed to observe this synergistic transcriptional activation in 3T3 or COS1 cells, 2 we did detect cooperativity in 293T cells. In the reporter analysis using 293T cells, we observed that the transcription mediated by the mafG Ic-ARE was cooperatively activated by Nrf2 and MafG. In this assay, a mutation outside the short ARE consensus of the Ic-ARE ( Fig. 5C; m2) greatly inhibited induction of the reporter gene by Nrf2 and MafG. Since this mutation did not affect the binding of the Nrf2/Maf heterodimer in EMSA, unidentified factor(s), which recognize the sequence outside of ARE, might play critical cooperative roles with the Nrf2/Maf heterodimer in the transcriptional activation (31). Taken together, our observations provided support for the longer ARE consensus sequence as important for strong Nrf2-mediated induction.
ChIP analysis showed that Nrf2 actually binds to the mafG Ic-ARE in a chromosomal context. Moreover, DEM treatment of nrf2-null mutant cells led to no mafG induction. That is not to say that these data refute a possible contribution of Nrf1 or Nrf3 to mafG regulation, but in MEFs, neither Nrf1 nor Nrf3 is able to compensate for the absence of Nrf2. In fact, Nrf2 and Nrf1 have been shown to play important roles in ARE-dependent gene induction in an overlapping manner (8). ChIP analysis also showed that small Mafs bind to the mafG Ic-ARE. Although the small Maf antibody used in the ChIP assay recognizes primarily MafK, we suspect that all three small Maf proteins can bind to the mafG Ic-ARE, since this DNA binding motif is conserved among the small Maf proteins (5).
ChIP analysis showed that small Mafs bind to the mafG Ic-ARE even in untreated cells (Fig. 6), indicating that there is the small Maf binding activity independent of Nrf2. Since MafG homodimer is able to bind to the mafG Ic-ARE in EMSA, it is possible that small Maf proteins form inactive homodimers by themselves and occupy the mafG Ic-ARE in untreated cells. However, it is also possible that small Maf proteins heterodimerize with Bach1 to form a transcriptional repressor. The Bach1-MafG heterodimer may repress mafG gene transcription through mafG Ic-ARE in the same way as it represses HO-1 gene expression (9,16). The contribution of Bach1 proteins to the regulation of mafG is under investigation.


Division as modified migration

![Graphic][1]

PTEN in the middle of the cell (top) and PI3 kinase at the poles (bottom) help drive cytokinesis.

DEVREOTES/ELSEVIERA dividing cell mimics two half cells migrating away from each other, according to Chris Janetopoulos, Peter Devreotes, and colleagues (Johns Hopkins

A PTEN in the middle of the cell (top) and PI3 kinase at the poles (bottom) help drive cytokinesis.

DEVREOTES/ELSEVIER
Text by William A. Wells wellsw@rockefeller.edu cell results in contraction. This situation is maintained by membrane localization of PI kinases at the front of the cell and of the PIP phosphatase PTEN at the rear.
The team set out to investigate the migration function of these enzymes further by making knockouts. "That was about a year and a half ago," says Devreotes. "We still haven't got around to studying migration." Instead they immediately came across a striking cytokinesis defect in the mutants, which couldn't regulate PI(3,4,5)P 3 levels because of a lack of both PTEN and two PI3 kinases. Cells continued to grow but cytokinesis often failed, especially for cells growing in suspension. In wild-type cells undergoing cytokinesis, by contrast, the kinases and PI(3,4,5)P 3 were concentrated at the poles of cells and PTEN was at the furrow. This localization was blocked if spindle formation was prevented.
Before cytokinesis, cells round up. This coincided in wild-type cells with the arrival of PTEN at the plasma membrane all around the cell. "That would tend to erase the polarity from migration," says Devreotes. The spindle then may reestablish two opposing polarities, setting the two ends of the cell off in opposite directions, and simultaneously instructing the middle of the cell to contract. Dictyostelium already has a reputation for cytokinesis that relies on daughter cells pulling themselves away from each other, so it will be important to establish whether other organisms use similar mechanisms. Reference: Janetopoulos, C., et al. 2005. Dev. Cell. 8:467-477. Really outside signaling eactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) pose a potential chemical threat in cells, so it was surprising enough when they turned up as essential signaling components downstream of receptor activation. Now Garrett DeYulia, Juan Cárcamo, David Golde (Memorial Sloan-Kettering Cancer Center, New York, NY) and colleagues have found that H 2 O 2 can be generated outside cells, independent of any intracellular events, by ligand binding to cytokine receptors.
The group had earlier seen that extracellular catalase, which breaks down H 2 O 2 , inhibited signaling through the GM-CSF receptor. They now find that ligand binding to this receptor increases the amount of extracellular H 2 O 2 . This occurs even when the cells are fixed before R ligand addition or with a version of the receptor lacking intracellular signaling domains. H 2 O 2 production was also evident with EGF binding to its receptor in the presence of an inhibitor of downstream signaling.
Extracellular destruction of H 2 O 2 reduced signaling and cell survival downstream of GM-CSF binding. Addition of extracellular H 2 O 2 , by contrast, induced phosphorylation of downstream targets.
H 2 O 2 makes a good second messenger-it is small, diffusible, and can be destroyed easily. It can also inactivate proteins such as phosphatases by oxidizing active site cysteine residues. How it is generated initially is a mystery. In intracellular ROS signaling the necessary electrons are supplied by NADPH oxidase, but outside the cell the only precedent is an unusual reaction between oxygen radicals and water catalyzed by antibodies. DeYulia and colleagues plan to mutate ligand and receptor residues to track down what parts of the proteins may be responsible for ROS generation.