Novel Role for the Nuclear Phosphoprotein SET in Transcriptional Activation of P450c17 and Initiation of Neurosteroidogenesis

Nathalie A. Compagnone1, Peilin Zhang1, Jean-Louis Vigne and Synthia H. Mellon

Center for Reproductive Sciences Department of Obstetrics & Gynecology & Reproductive Sciences and The Metabolic Research Unit University of California San Francisco, California 94143-0556


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Neurosteroids are important endogenous regulators of {gamma}-aminobutryic acid (GABAA) and N-methyl-D-aspartate (NMDA) receptors and also influence neuronal morphology and function. Neurosteroids are produced in the brain using many of the same enzymes found in the adrenal and gonad. The crucial enzyme for the synthesis of DHEA (dehydroepiandrosterone) in the brain is cytochrome P450c17. The transcriptional strategy for the expression of P450c17 is clearly different in the brain from that in the adrenal or gonad. We previously characterized a novel transcriptional regulator from Leydig MA-10 cells, termed StF-IT-1, that binds at bases -447/-399 of the rat P450c17 promoter, along with the known transcription factors COUP-TF (chicken ovalbumin upstream promoter transcription factor), NGF-IB (nerve growth factor inducible protein B), and SF-1 (steroidogenic factor-1). We have now purified and sequenced this protein from immature porcine testes, identifying it as the nuclear phosphoprotein SET; a role for SET in transcription was not established previously. Binding of bacterially expressed human and rat SET to the DNA site at -418/-399 of the rat P450c17 gene transactivates P450c17 in neuronal and in testicular Leydig cells. We also found SET expressed in human NT2 neuronal precursor cells, implicating a role in neurosteroidogenesis. Immunocytochemistry and in situ hybridization in the mouse fetus show that the ontogeny and distribution of SET in the developing nervous system are consistent with SET being crucial for initiating P450c17 transcription. SET’s developmental pattern of expression suggests it may participate in the early ontogenesis of the nervous, as well as the skeletal and hematopoietic, systems. These studies delineate an important new factor in the transcriptional regulation of P450c17 and consequently, in the production of DHEA and sex steroids.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nervous system makes steroids, generally termed neurosteroids (1), utilizing the same steroidogenic enzymes found in the adrenals and gonads (2, 3, 4, 5, 6). Neurosteroids act through cell surface receptors to exert novel functions not associated with the classical adrenal and gonadal steroids acting through nuclear receptors. For example, allopregnanolone, a derivative of progesterone, binds to {gamma}-aminobutyric acid (GABAA) receptors to exert anesthetic and anxiolytic activities (7, 8). Other neurosteroids, such as pregnenolone sulfate, dehydroepiandrosterone (DHEA), and DHEA-sulfate (DHEAS), act as antagonists of the GABAA receptor (9). These same steroids may have agonistic effects on N-methyl-D-aspartate (NMDA) receptors (10, 11, 12, 13, 14) and may have additional effects on type 1 {varsigma} receptors whose endogenous ligand(s) are unknown, but which are known to bind haloperidol (15). Thus, some neurosteroids affect GABA- and NMDA-associated behaviors such as anxiety, learning, and memory (16, 17, 18, 19). We have recently (14) shown that DHEA specifically promoted axonal growth while DHEAS specifically promoted dendritic growth in fetal rodent neurons, and that DHEA increased the morphological indices of synaptic contacts within neocortical neurons (14). Some of the effects of DHEA, but not of DHEAS, were mediated via NMDA receptor activation (14). Thus, the neuronal regulation of DHEA synthesis is of substantial interest in understanding fetal brain development.

The key enzyme in the biosynthesis of DHEA is P450c17, a microsomal enzyme that has both 17{alpha}-hydroxylase and 17,20-lyase activities, and hence converts pregnenolone to DHEA (20, 21). P450c17 expression is developmentally and regionally regulated in the nervous system (5) by factors other than those found in the adrenal and gonad (22, 23, 24). Expression and transcriptional regulation of the gene for P450c17 [formally termed CYP17 (25)] are regulated by ACTH in the adrenals and LH in the gonads via the cAMP/protein kinase A signaling pathways, but is not mediated through binding of CREB (cAMP response element binding protein) to a consensus cAMP- responsive DNA element (22, 26, 27, 28).

We previously identified a region of the rat P450c17 gene between -447/-399 bp upstream from the transcriptional start that is important for both basal and cAMP-regulated transcriptional activities in the adrenal and testis (29). This region was regulated by several members of the orphan nuclear receptor family, including SF-1 (steroidogenic factor-1), NGF-IB (nerve growth factor inducible protein B), and COUP-TF (chicken ovalbumin upstream promoter transcription factor). We also identified the binding sites for two additional nuclear proteins operationally termed steroidogenic factor inducer of transcription-1 and -2 (StF-IT-1 and StF-IT-2) that are important for rat P450c17 gene transcription (29). We have now demonstrated that the StF-IT-1 site is also transcriptionally active in human neuronal precursor NT2 cells, purified StF-IT-1, and identified it as the product of the protooncogene SET, a protein not previously implicated in transcriptional regulation. Our present results now show that this novel transcription factor plays a role in the regulation of P450c17 in the developing nervous system.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Purification of StF-IT-1 from Porcine Testes
Several members of the orphan nuclear receptor family bound to the -447/-399 region of the rat P450c17 gene, including COUP-TF, SF-1, and NGF-IB (Fig. 1AGo). Two additional factors, which we called StF-IT-1 and StF-IT-2, also bound to this same region (29). StF-IT-1 bound specifically to the -418/-399 region (Fig. 1BGo) and transcriptionally activated basal activity from this region of the rat P450c17 gene in both Leydig (Fig. 1CGo, left graph) and adrenocortical cells, when this region was ligated to a heterologous promoter, strongly suggesting that StF-IT-1 is important for endogenous P450c17 transcription. This same -418/-399 region could also be transcriptionally activated when transfected into human neuronal precursor NT2 cells (Fig. 1CGo, right graph). This region was only minimally affected by cAMP in MA-10 and NT2 cells and is therefore considered to be a strong basal element, rather than a cAMP-regulated element. StF-IT-1 appeared to be a novel DNA binding protein that shared no similarity to other known transcriptional factors in its DNA sequence specificity for binding or activity, although the binding sequence of StF-IT-1 contained a variant (AGGAGA) of the estrogen receptor half-site sequence AGGTCA (29).



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Figure 1. Nuclear Proteins Binding to the -447/-399 Region of the Rat P450c17 Gene

A, Cartoon of the nuclear proteins binding to this region, determined by gel shift analysis (29 ). This region of the rat P450c17 is bound by at least five factors in vivo, including COUP-TF, SF-1, NGF-IB, and two factors that we termed StF-IT-1 and StF-IT-2. The factors and the regions to which they bind are depicted by the circles and ovals on the P450c17 sequence. Binding of StF-IT-1 and StF-IT2 are detected when COUP-TF binding is inhibited, indicating that the relative abundance of COUP-TF is greater than that of StF-IT-1 and StF-IT-2, or that the binding affinity of COUP-TF is greater than that of StF-IT-1 and StF-IT-2. We readily detected StF-IT-1 and StF-IT-2 binding when the COUP-TF site was physically eliminated by restriction endonuclease digestion of the -447/-399 oligonucleotide. The arrows indicate the sequences bound by orphan nuclear receptors, ERE half-sites (AGGTCA-like sequences) found in the rat P450c17 gene. B, Gel shift analysis of binding of nuclear proteins from MA-10 cells. StF-IT-1 from MA-10 cell nuclear extracts binds to the -418/-399 rat P450c17 oligonucleotide, and its binding is competed by a 50-fold molar excess of wild-type oligonucleotide (+ -418/-339) but not by mutant oligonucleotide (mutant 2, Table 1Go). C, Functional analysis of the -418/-399 rat P450c17 construct. Wild-type and mutant oligonucleotides were cloned into a TKLUC expression vector, transfected into mouse Leydig MA-10 cells (left) or human neuronal precursor NT2 cells (right), and luciferase activity was determined after cells were treated without (-cAMP, open bars) or with 1 mM 8-Br-cAMP for 6 h (+cAMP, black bars).

 
Using gel mobility shift assays to monitor the purification of StF-IT-1, we purified this nuclear factor from immature porcine testes, an abundant source of tissue that expressed StF-IT-1. After cell fractionation, we tried a variety of procedures to enrich StF-IT-1 before fast pressure liquid chromatography (FPLC). Salting out procedures with ammonium sulfate did not provide an enrichment, but precipitation of cellular proteins at various pH values showed that StF-IT-1 remained soluble at acid pH, whereas many other proteins precipitated. Hence, we dialyzed our cellular extract at pH 5.5 and used the dialysate for further purification. We tried a variety of FPLC columns, including size exclusion and anion and cation exchange chromatography, and found the best purification of a protein associated with StF-IT-1 binding activity using a cation exchange Mono-S FPLC column. We therefore applied the pH 5.5 dialysate to a Mono-S FPLC column, where we detected StF-IT-1 in the flow through in fractions 5–8 (Fig. 2AGo), while the majority of the protein was retained on the column. An additional protein binding to the -418/-399 rP450c17 oligonucleotide was eluted from the column in fractions 24–26. This protein-DNA complex was slightly larger than the StF-IT-1-DNA complex seen in the starting material and was not analyzed further. Some StF-IT-1 was also retained on the Mono-S column and was eluted in fractions 26–29. However, this retention of StF-It-1 was only seen when a large amount of protein (>20 mg) was applied to the Mono-S column.



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Figure 2. Purification and Identification of the Porcine SET Protooncogene Product

A, DNA binding activities of the fractions from the cation-exchange Mono-S column. The porcine testicular extract was dialyzed against MES buffer (pH 5.5) and was applied onto a Mono-S FPLC column. The flow through and the column-eluted fractions were tested for DNA binding activity using the oligonucleotide probe of -417/-399 of the rat P450c17 gene (StF-IT-1 binding site). The last lane represents the original starting material (SM). The majority of StF-IT-1 protein eluted in fractions 5–8 (arrow); some StF-IT-1 protein was retained on the column and eluted in fractions 26–29 when large amounts of protein (>20 mg) were applied to the column. B, Proteins purified by Mono-S FPLC and by DNA affinity chromatography were separated by SDS-PAGE on 12% acrylamide gels, electroblotted onto a PVDF membrane, and stained by Coomassie brilliant blue. The arrows indicate the two bands about 24K StF-IT-1 and 16K streptavidin, respectively. C, Amino acid sequence of 60 amino acids of the purified 24K StF-IT-1 protein and comparison with GenBank database sequences. The StF-IT-1 protein sequence is 95% identical to human SET (amino acids 17–76), human PHAP II (amino acids 17–76), and human I2PP2A (amino acids 17–76). The alignment of the sequence was performed by using GCG FASTA search program.

 
As fractions 5–8 contained protein that gave a single protein-DNA band by gel shift analysis, they were pooled and used directly for DNA affinity chromatography. Affinity-purified proteins were separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane, and stained with Coomassie blue, revealing a major protein of about 24 kDa and a minor protein of about 16 kDa (Fig. 2BGo). The N-terminal sequence of 20 amino acids from the ~16-kDa protein identified it as streptavidin that came from the affinity column. The N-terminal sequence of 60 amino acids from the ~24-kDa protein shared 95% identity with the human SET protein (GenBank accession no. Q01105), protein phosphatase inhibitor 2A (I2PP2A) (GenBank accession no. U60823), and PHAP II (HLA-DR associated protein II, EMBL accession no. X75091) (Fig. 2CGo). The high degree of amino acid sequence identity among porcine StF-IT-1, human SET, human I2PP2A, and human PHAP II suggests that all three are closely related and possibly identical. However, it is not certain that StF-IT-1 is identical to SET, or simply encoded by a very similar gene. The absence of the first 16 amino acids of human SET in our purified porcine protein may reflect proteolytic processing, alternate splicing, protein degradation during the purification, a species-specific difference, or the possibility that SET and StF-IT-1 are encoded by highly homologous, but not identical, genes.

SET Is a Site-Specific DNA Binding Protein
The identification of human SET and its name derive from the description of a chromosomal translocation found in a patient with acute undifferentiated leukemia (patient "SE translocation") (30). In this patient, the gene set (located on chromosome 9q34, centromeric of c-abl) was fused to the gene can, also found on chromosome 9q34. SET is an acidic nuclear phosphoprotein (pI 3.9) of 277 amino acids, with a total of 33% acidic amino acids at the carboxy terminus (31, 32). SET was previously purified from several sources, and its expression was associated with roles other than transcription, including its role as a phosphatase 2 inhibitor and activator of adenovirus replication (30, 33, 34, 35, 36).

To determine whether the StF-IT-1 protein binding to -418/-399 was immunologically related to SET, we obtained antisera to human SET peptides (generously provided by Dr. Terry Copeland, National Cancer Institute). Using the -418/-399 rat P450c17 oligonucleotide as probe and mouse testicular Leydig MA-10 cells or adrenocortical Y-1 cells as a source of nuclear extract, addition of anti-SET antisera blocked the formation of the complex whereas preimmune serum did not (Fig. 3AGo). Similar results were obtained with nuclear proteins prepared from mouse adrenocortical ST-R cells (37) and rat C6 glioma cells (data not shown). When the SET-related protein(s) found in the nuclear extracts were depleted by immunoprecipitation with SET antiserum, the immunodepleted extract did not form the StF-IT-1-DNA complex with the -418/-399 rat P450c17 oligonucleotide (Fig. 3BGo). Thus, the StF-IT-1-DNA complex seen in vivo with the -418/-399 region of the rat P450c17 gene is due to binding of a protein immunologically related to SET.



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Figure 3. SET Protein Binds to Specific DNA Sequences

A, Effect of SET antibody on gel shift assays of nuclear proteins binding to the -418/-399 rat P450c17 oligonucleotide. Anti-human SET antisera blocked the binding of the proteins from mouse Leydig MA-10 and mouse adrenocortical Y-1 cells to the -418/-399 rat P450c17 DNA sequence (StF-IT-1 binding site). B, Gel shift assay of untreated and SET-immunodepleted MA-10 cell nuclear extracts binding to -418/-399 rat P450c17 DNA. Nuclear extracts were treated with 3 µl nonimmune mouse serum (Pre-imm), or with 3 µl of human SET antibody (+Ab), and antibody/antigen complexes were precipitated with protein A Sepharose. The proteins remaining in the supernatant were incubated with the -418/-399 rat P450c17 oligonucleotide. Immunodepletion with anti-SET antisera diminished the protein-DNA complex seen with the MA-10 cell extract and the DNA sequence from the rat P450c17 gene (StF-IT-1 binding site) (MA-10). The lane "+cold" contains MA-10 cell extract, -418/-399 oligonucleotide probe, and 50-fold molar excess of unlabeled oligonucleotide. C, Competition gel shift assays. Bacterially expressed recombinant human SET (left panel) or MA-10 cell extracts (right panel) were incubated with the wild-type -418/-399 rat P450c17 oligonucleotide, and 50-fold molar excess of unlabeled wild-type (+cold) or three mutant oligonucleotides (Table 1Go) were added. When nucleotides -404/-402 (Mut 1), -407/-405 (Mut 2), or -410/-408 (Mut 3) were mutated, the cold -418/-399 probe could not compete with recombinant SET or with StF-IT-1 (MA-10 cells) for binding to the wild-type DNA.

 
To determine whether authentic human SET would bind to -418/-399 of the rat P450c17 promoter, we used the known human SET cDNA sequence (30) and RT-PCR of human fetal adrenal RNA to clone human SET cDNA into a bacterial expression vector. Bacterially expressed human SET bound to the -418/-399 oligonucleotide (Fig. 3CGo, left) as efficiently as the StF-IT-1 protein from MA-10 cells (Fig. 3CGo, right); furthermore, wild-type, but not mutant -418/-399 oligonucleotides would compete for human SET binding (Fig. 3CGo, left). This binding pattern of human SET could be duplicated by incubating -418/-399 with MA-10 cell extract (Fig. 3CGo, right). Thus, human SET binds to -418/-399 indistinguishably from MA-10 cell StF-IT-1.

To determine whether authentic SET protein exerts the same biological effects as those we have previously shown for StF-IT-1 (29), we assessed the ability of rat SET to transactivate the rat -418/-399 sequence in human NT2 neuronal precursor cells (Fig. 4AGo). Rat SET cDNA, cloned by RT-PCR, was inserted into a eukaryotic expression vector, and NT2 cells were cotransfected with this vector and with a luciferase reporter gene under the control of the -418/-399 oligonucleotide linked to the 32-bp minimal promoter of the herpes simplex virus thymidine kinase gene (-418/-399TK32LUC). Because NT2 cells express low levels of SET endogenously, the wild-type -418/-399TK32LUC vector shows luciferase activity in the absence of the SET expression vector. A mutant -418/-399TK32LUC vector in which the variant of the estrogen receptor half -site was changed, however, shows no more activity than the TK32LUC construct alone, consistent with endogenous NT2 SET acting through the wild-type rat -418/-399 sequence. When the cells are cotransfected with the rat SET expression vector, activity from wild-type -418/-399TK32LUC increased 550% above the level without the rat SET vector, but the mutant -418/-399TK32LUC still had no more activity than the TK32LUC control. Thus SET specifically binds to the TCTCCTCAA sequence of the rat P450c17 promoter to elicit a profound increase in basal transcription.



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Figure 4. Transactivation of Rat P450c17 by Rat SET

A, A eukaryotic expression vector (PCR3) expressing rat SET cDNA (1 µg) was transfected into neuronal precursor NT2 cells together with the wild-type (WT, 1 µg) or mutant 1 (M1, 1 µg) -418/-399 rat P450c17 TK32LUC reporter constructs, and luciferase activity was determined. Data, reported as luciferase activity per microgram of protein, are means ± SE of three or four experiments each done in triplicate. The control (Tk32LUC alone), M1, and M1 coexpressed with SET are all indistinguishable. The WT plus SET is 6 x WT. B, SET-PCR3 (1 µg) was transfected into neuronal precursor NT2 cells together with the 5'-deletional construct -476rP450c17 {Delta}-Luc (called "-476", 1 µg), and luciferase activity was determined. Data are reported as luciferase activity per microgram of protein and are means ± SE of experiments done in triplicate. The -476rP450c17 {Delta}-Luc plus SET is 10 x -476rP450c17 {Delta}-Luc alone.

 
We also assessed the ability of rat SET to transactivate the -418/-399 sequence in the context of its natural environment within the 5'-flanking DNA of the rat P450c17 gene (Fig. 4BGo). We previously made a deletional construct containing 476 bp of the 5'-flanking region of the rat P450c17 DNA that contained the -418/-399 region, ligated to the reporter gene {Delta} luciferase (-476 {Delta}-Luc), and showed that this construct was transcriptionally active in mouse Leydig MA-10 and adrenocortical Y-1 cells (22). This vector was transfected into N2A cells, in the absence or presence of the SET expression vector. The -476 {Delta}-Luc vector has some activity in the absence of the SET expression vector, but it is less than the -418/-399TK32LUC vector because it contains a binding site for the transcriptional inhibitor COUP-TF (29). Nevertheless, when the SET expression vector was cotransfected into the N2A cells, activity from the -476 {Delta}-Luc vector increased 10-fold, indicating that SET could increase transcription from the rat P450c17 gene in its natural DNA context.

Developmental Analysis of SET Expression in the Nervous System
We have previously shown that P450c17 is expressed in specific regions of the developing fetal rodent brain, even though SF-1, which appears to be required for adrenal and gonadal expression of P450c17, is not expressed in the fetal brain where P450c17 is expressed (5, 24). To determine whether SET is expressed in the same brain regions that express P450c17, we used immunocytochemistry to colocalize SET and P450c17 protein, and in situ hybridization to colocalize SET mRNA.

Early in brain development, SET mRNA was expressed from E10.5 in the prosencephalon (Fig. 5Go, panels A, C, and D), the structure of the rhombencephalon from E11.5 [Fig. 5DGo, mesencephalon (Mes) and metencephalon (Met)] but not in the developing rhombencephalon at E10.5 (which is negative in Fig. 5BGo), the prosencephalon, rhombencephalon (mesencephalon and metencephalon) and diencephalon at E13.5 (Fig. 5EGo), and in the basal diencephalon, pituitary, and hindbrain (shown at E18.5 in Fig. 6KGo). These data suggest that SET is expressed in the brain in an antero-posterior gradient. At this same time in development (E11), SET protein was coexpressed with P450c17 in the developing neural tube in the lateral motor column (data not shown). In addition, SET mRNA expression was restricted to the dorsal and ventral segments of the neural tube. SET protein was also expressed in the notochord at E11 although P450c17 protein was not expressed there (data not shown).



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Figure 5. Expression of SET and P450c17 in the Developing Mouse Nervous System

Darkfield in situ hybridization of SET mRNA on embryonic day (E) 10.5 to postnatal day 9. Panels A, B, and C are from embryonic day 10.5; panel D is from embryonic day 11.5, panel E is from embryonic day 13.5, panel F is from embryonic day 18.5, and panel G is from postnatal day 9. Panels A and C are coronal sections; panels B, D, E, F, and G are sagittal sections. The structures indicated within the panels are: eye; ba, branchial arches; t, trigeminal neural crest; liver; neural tube; Pros, prosencephalon; Di, diencephalon; Mes, mesencephalon; Met, metencephalon; rhombencephalon; DRGs, dorsal root ganglia; genital ridge; sp, spinal cord, tongue; neocortex; (neocortical) subplate; hippocampus; thalamus. The bars under panels A/B, D, E = 1 mm; the bars under panels C, F, and G = 100 µm.

 


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Figure 6. Composite Lightfield Microphotographs of in Situ Hybridizations of SET mRNA in E18.5 Mouse Embryos (A–J) and a Postnatal Day 9 Mouse Brain (K)

Positive signals for SET mRNA appear as black dots. A, Cochlea, B, paraspinal muscle, C, thymus, D, whiskers, E, skin, F, intestine, G, cervical spinal cord, H, dorsal root ganglia and vertebral cartilage, I, trigeminal ganglia, J, lumbar spinal cord, K, postnatal day 9 mouse brain showing SET mRNA highly expressed in the cortex (ctx) and in the Purkinje and granular cell layers of the cerebellum (Crb), and to a lesser extent in the thalamus (Th), septum (Sep) nuclei hypothalamus, and amygdala. Panels A–J are at the same magnification. The bar under E = 100 µm (panels A–J); the bar under K = 1 mm.

 
At E18.5, SET mRNA was seen in the upper layers of the neocortex and in a layer of cells called the subplate (Fig. 5FGo). SET mRNA and P450c17 protein colocalized within the subplate at this time (data not shown). At this same time in development, SET was widely expressed in the embryo (Fig. 6Go). Sites of expression included the cochlea, paraspinal muscle, thymus, whiskers, skin, intestine, spinal cord, dorsal root ganglia, cartilage, and trigeminal ganglia (Fig. 6Go). SET mRNA was also expressed in sites related to hematopoiesis and development of the vasculature, such as the developing liver, the branchial arches (Fig. 5AGo, ba), and the walls of vessels (spinal artery and vessels in the adult ovary, not shown). SET protein and mRNA expression were developmentally regulated and were largely reduced around birth. Nevertheless, at P9, SET mRNA could still be detected in the hippocampus, cortex, thalamus (Fig. 5GGo), hypothalamus, septum, and in both the granular layer and Purkinje cells of the cerebellum (Fig. 6KGo), but its expression disappeared in the adult.

Both SET and P450c17 were expressed in structures derived from the migration of neural crest cells. P450c17 protein was found mainly in structures derived from the cranial neural crest (5) while SET mRNA was found in structures derived from the cranial neural crest (cranial-facial bones and cartilage such as cochlea, Fig. 6AGo; smooth muscles of the back, Fig. 6BGo; and thymic cells, Fig. 6CGo) as well as from the trunk neural crest (dorsal root ganglia, Fig. 6HGo; skin, Fig. 6EGo) and cardiac neural crest (walls of the large arteries, e.g. spinal artery, data not shown). SET mRNA was also expressed in mesodermally derived structures such as the sclerotomes (E10.5 and E11.5, Fig. 5DGo) and in the vertebral cartilage (Fig. 6HGo). SET mRNA was expressed in developing bone, including primordial cartilage and limb bud, and later in the skeleton in the ribs, femur, skull, and jaws.

SET mRNA and protein were found in the same neurons that express P450c17: in the peri-locus coeruleus nucleus (not shown), trigeminal ganglia (Fig. 7Go, A and B), in the pontine nucleus (Fig. 7Go, C and D) as well as in the cortical subplate (E18.5, Fig. 5FGo). SET mRNA was also detected in the cortex (Fig. 5FGo) and in mesencephalic, hypothalamic, thalamic, and septal nuclei (not shown) where P450c17 was not expressed, suggesting that SET may regulate other genes in those structures. However, P450c17 was never expressed in regions that did not express SET.



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Figure 7. Colocalization of P450c17 Protein and SET mRNA

Trigeminal ganglia (A and B) and mesopontine nuclei (C and D), embryonic day 18.5. P450c17 detected by immunocytochemistry (A and C) and SET mRNA detected by darkfield in situ hybridization (B and D) colocalize in the trigeminal ganglia (A and B) and in several meso-pontine nuclei (C and D), including the pontine nucleus. SET mRNA is also expressed in a nucleus (arrow, panel D) that is not P450c17-positive; P450c17 is expressed in fibers (arrow, panel C) that are not SET-positive. The bar under panel A = 100 µm.

 
Thus our developmental analysis of SET expression shows: 1) SET is expressed in cell tissues that express P450c17; 2) SET is also expressed in some tissues that do not express P450c17; 3) P450c17 is not expressed in tissues that do not express SET; 4) where SET and P450c17 are coexpressed, SET expression always precedes P450c17 expression; 5) SET expression in the developing central nervous system follows an antero-posterior gradient; 6) sites of SET expression indicate it may play a role in organogenesis of the neural tube, differentiation of blood cells, and development of the skeleton. These observations are consistent with the transcriptional data that show that SET activates (and hence precedes) P450c17 expression and also suggest additional key roles for SET in the early development of the nervous, hematopoietic, and skeletal systems.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
One Protein, Many Functions
Human (30), rat (38), and mouse (GenBank direct submission, accession no. AB015613) SET cDNAs have been cloned, revealing similarities and differences. These cDNAs are more than 94% identical to each other in nucleotide sequence, and more than 97% identical in amino acid sequence, indicating that both their nucleotide and amino acid sequences are extremely conserved across species. Northern blots detect two human SET mRNAs of about 2.7 and 2.0 kb, most likely due to differential polyadenylation, as two polyadenylation sites were identified in the 3'-untranslated region of human SET cDNA (30). Northern blots of mouse RNA detect the 2.7- and 2.0-kb mRNAs as the major transcripts and also detect two minor mRNAs of about 1.8 and 1.5 kb (30). In situ hybridization of chromosomal DNA detected the set gene on chromosome 9, centromeric of c-abl. Cloning of rat SET cDNA identified two cDNAs, given the names SET{alpha} and SETß, that differed at their 5'-ends. SET{alpha} cDNA has an open reading frame of 867 nucleotides, while SETß cDNA has an open reading frame of 831 nucleotides. SET{alpha} cDNA encodes a protein that has 36 amino acids at its amino terminus that differ from the first 24 amino acids of the protein encoded by SETß cDNA; the remaining cDNA sequences are identical between the two SET species. These differences may arise from alternative splicing or may represent sequences from two different genes. SET{alpha} mRNA was much less abundant than SETß mRNA in all tissues examined, including rat brain, heart, lung, and kidney and thus may represent only a minor transcript. The sequence of our porcine StF-IT-1 corresponds with the sequences found in SETß, as the first eight amino acids of our porcine protein correspond to those predicted for amino acids 17–24 of SETß and not of SET{alpha}.

SET is a potent and specific noncompetitive inhibitor of protein phosphatase 2A (PP2A) (39), a protein involved in the regulation of normal cell growth (40, 41, 42). SET may also be involved in the alteration of chromatin structure to promote increased gene transcription. The human homolog, HRX, of the Drosophila trithorax protein interacts with SET and protein phosphatase 2A (43). Experiments have suggested that HRX and associated proteins may affect nucleosome assembly, alter chromatin structure, and hence alter access of transcription factors to DNA. SET also has amino acid sequence similarity to the Drosophila nucleosome assembly protein NAP-1 (44, 45), a core histone shuttle that delivers histones H2A and H2B from the cytoplasm to the chromatin-assembly machinery in the nucleus in a cell cycle-dependent manner. Yeast NAP-1 can stimulate binding of transcription factors by a mechanism involving nucleosome displacement (46). A 43-kDa Xenopus homolog of SET, as well as a 60-kDa Xenopus NAP-1 protein, both interact specifically with B-type cyclins (47). Hence, in Xenopus, NAP/SET proteins may regulate cell cycle. Finally, a 39- or 41-kDa protein purified from HeLa cells, called template activating factor-1, or TAF-1 (48) was shown to be identical to SET and was shown to stimulate adenovirus core DNA replication (35, 36). Thus in this context, SET and SET-like proteins may be involved in chromatin remodeling and direct transcriptional activation, perhaps inappropriately, leading to leukemogenesis.

Our data now demonstrate that, in addition to the other functions attributed to SET or SET-like proteins, SET is a DNA binding protein and transcriptional activator, that plays a role in transcription of the gene for P450c17, and possibly other genes, as we find SET expressed in cells not expressing P450c17. The structure of SET protein resembles no known class of transcription factor identified so far. It is a protein of 277 amino acids, with a long acidic tail of 53 amino acids at its carboxy terminus. This acidic region of TAF-1, which is identical to SETß, is essential for stimulation of replication from adenovirus DNA and for interaction with cellular histones (48, 49, 50). In addition, the replication activity of TAF-1 is dependent upon dimerization (51), and it is presumed that the acidic tail plays a role in this dimerization. It is unknown whether these regions may play roles in transcriptional activation as well.

The sites of SET expression suggest that P450c17 and other target genes may be involved in neural induction and more generally in cell differentiation. Thus the set-can gene fusion that initially identified the set gene may cause undifferentiated leukemia by creating a SET-CAN fusion protein that alters SET protein function, rather than by altering CAN protein function. Removal of the seven carboxy-terminal amino acids by fusion with CAN may alter the transactivating function of SET. By reducing SET activity, cells may not enter the last stages of differentiation and may be propagated as abnormal pluripotent stem cells that result in undifferentiated leukemia.

In another type of leukemia, acute nonlymphocytic leukemia, the can gene is fused to the dek gene, resulting in leukemia-specific, chimeric dek-can mRNA and fusion protein. Recent experiments have shown that DEK also binds to specific DNA sequences to increase gene transcription (52). The DNA binding site for DEK is not similar to the binding site we identified for SET. Also, it has not been demonstrated whether DEK increases or decreases gene transcription. Thus, the genetic mechanism of leukemogenesis may be similar for both set-can and dek-can translocations, but the downstream genes affected by these translocations are likely to be different. With the identification of SET as a DNA binding protein, it may now be possible to search for other SET-target genes. Searching the GenBank database has not yet produced other genes.

Recent experiments have proposed a novel mechanism for leukemogenesis in chromosomal translocation-generated oncoproteins (53). Both DEK-CAN and SET-CAN encode nuclear fusion proteins, called nucleoporins, that contain a Phe-Gly (FG) repeat region within CAN protein. This repeat region has been shown to interact with the transcriptional coactivator CREB binding protein (CBP) and p300. As a result, transcriptional activators fused to proteins such as CAN show increased transcriptional activity. As we have now shown that SET is a potent transcriptional activator, the fusion of the set to the can gene in patients with acute undifferentiated leukemia may result in a SET-CAN fusion protein that is able to recruit additional coactivators to increase the transactivation function of SET further, thus leading to oncogenesis.

Role of SET in Nervous System Development
The pattern of expression of SET in the developing neural tube supports the hypothesis that SET may activate genes involved in the organogenesis of the spinal cord. SET is expressed both in the developing neural tube in restricted dorsal and ventral areas as well as in the notochord, suggesting that its expression may be involved in turning on genes involved in regulating neuronal induction in these regions. One of these genes is P450c17, whose expression lags behind SET in the developing motor neurons. From the sites of SET expression and from roles determined for SET homologs, we believe that SET expression in regions where P450c17 is expressed, and in other regions where P450c17 is not expressed, may be related to determination of cell fate. For example, SET is expressed in cells derived from the migrating neural crest and is found in cranio-facial cartilage. SET is also present from the early stages of the determination of somites to the formation of bones.

SET expression is developmentally regulated, and its expression declines in parallel with the decline in P450c17 expression in the central nervous system. COUP-TF is a nuclear factor that competes for the SET binding site in the rat P450c17 gene, and hence is a potent repressor of P450c17 (29). COUP-TF, unlike SET, is not expressed early in development, but rather is expressed later in embryogenesis in regions that express P450c17 (54). We postulate that increased expression of COUP-TF inhibits P450c17 expression in the central nervous system. Thus SET activation of P450c17 transcription in the developing nervous system may ultimately result in increased DHEA production, which may be an important signal for modulating neurotransmission to trigger formation of neuronal circuits.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation of the Biotinylated Oligonucleotide for Affinity Chromatography
Synthetic oligonucleotides (20 µg) were annealed in 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2. The annealed, double-stranded oligonucleotide was first end-labeled by T4 polynucleotide kinase and [{gamma}-32P]ATP (50 µCi, 3000 mCi/mmol) to monitor the presence of the oligonucleotide. Then, the oligonucleotides were blunt-ended by Klenow DNA polymerase using dTTP, dGTP, and dCTP and biotin-dATP (final concentration of 20 µM each nucleotide in a volume of 100 µl). The biotin and 32P double-labeled oligonucleotides were purified by chromatography on NAP-10 columns (Pharmacia Biotech, Piscataway, NJ) and used for the protein binding reactions.

Preparation of Homogenates of Immature Porcine Testes
Immature porcine testes were collected from a commercial pig farm and shipped to the laboratory on ice. The tissue was dissected and homogenized with a Dounce homogenizer in buffer containing 60 mM KCl, 15 mM NaCl, 15 mM HEPES, pH 7.8, 14 mM mercaptoethanol, 0.3 M sucrose, and a cocktail of protease inhibitors (0.5 mM phenylmethylsulfonylfluoride, 0.5 µg/ml pepstatin, 0.5 µg/ml antipain, and 0.5 µg/ml leupeptin). The crude tissue homogenate was centrifuged at 3000 x g for 5 min to remove the large tissue debris, and the supernatant was collected and stored in aliquots at -70 C. We also made a nuclear preparation as described previously(55), tested a number of known nuclear proteins, and found that all these known nuclear proteins, including StF-IT-1, StF-IT-2, and SF-1, were present in the cytoplasmic fraction (supernatant). Therefore, we used the supernatant for further purification.

Protein Purification by Chromatography
The porcine testicular extract was dialyzed against equilibration buffer (MES 20 mM, pH 5.5) (MES is 2-[N-morpholino]ethanesulfonic acid), centrifuged for 1 h at 100,000 x g, and the supernatant was applied to a 10 ml Protein Pack SP 15 HR FPLC column (Waters Corp., Milford, MA), previously equilibrated with the same buffer. Proteins were detected by UV absorption at 280 nm using a Waters UV Detector, model 440. Proteins not retained on the column were eluted in MES buffer until the OD280 returned to 0. Proteins retained on the column were eluted with a linear gradient of NaCl (0–0.5 M NaCl) generated by a Waters Controller System, model 600E, at a flow rate of 1 ml/min. About one third of the protein was found in the flowthrough of the column, and two thirds of the protein was retained.

Fractions containing DNA binding activity (flowthrough) were pooled and dialyzed overnight against the DNA binding buffer containing 20 mM HEPES, pH 7.9, 50 mM KCl, 4 mM Tris-HCl, pH 7.9, 5 mM EDTA, pH 7.9, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride. The dialysate was centrifuged at 13,000 x g for 5 min to remove any precipitate before being used for DNA binding assays. The biotinylated oligonucleotide (20 µg) was added to the sample together with nonspecific calf thymus DNA that was previously sonicated, heated at 100 C for 5 min, and used at final concentration of 150 µg/ml. The DNA binding reaction was performed at 4 C for 2–3 h before adding the streptavidin-agarose (1 ml prewashed twice with 1 M KCl in 1x binding buffer, and then five times with 1x DNA binding buffer to remove the salt). After incubation with streptavidin at room temperature for 1 h, the sample was centrifuged at 3,000 x g for 10 min to pellet the streptavidin-biotinylated oligonucleotide conjugates. The supernatant was carefully removed by pipette, and the conjugated agarose matrix was transferred to an Eppendorf tube to remove unconjugated protein. The conjugated agarose matrix was washed ten times with 1x binding buffer (1 ml each time, mixed manually for 1 min), removing the supernatant each time. The final elution of protein specifically bound to the oligonucleotide was performed by adding 400 µl elution buffer twice (1 M KCl in 1x binding buffer), incubating the sample with the elution buffer for 5 min at room temperature, and separating from the agarose matrix by centrifugation at 3000 x g. The elute was stored at -20 C until further analysis.

Protein Sequencing
The final 1 M KCl eluate from the oligonucleotide affinity column was dialyzed against ammonium bicarbonate (50 mM, pH 8.3), dried under vacuum, resuspended in SDS sample buffer, and separated by 10% SDS-PAGE. Immediately after electrophoresis, proteins were transferred to a PVDF membrane (Bio-Rad Laboratories, Inc. Richmond, CA) in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CHAPS) buffer, pH 11, in 10% methanol. Transferred proteins were stained in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol/1% acetic acid, and were destained in 50% methanol. The band of interest was excised from the PVDF membrane and subjected to N-terminal microsequencing on a vapor phase Beckman-Porton PI 2090 sequencer (Beckman Coulter, Inc., Fullerton, CA), using the Edman degradation procedure. The Edman degradation cycles had yields of more than 90% and contained about 45–50 pmol of material per cycle. Sequences obtained were searched for homology with sequences in the SWISS-PROT database, using the FASTA search of the GCG program.

Gel Mobility Shift Assays
Gel mobility shift assays were performed as described previously (22, 23, 29). Whole-cell extracts from MA-10 and N2A cells were prepared according to previously published procedures (22, 56). A wild-type oligonucleotide was derived from sequences 399 to 418 bp upstream from the transcription initiation site of the rat P450c17 gene (called "-418/-399") (29). A number of mutant oligonucleotides were also used as the unlabeled competitors for the wild-type probe (Table 1Go). Oligonucleotide probes were end labeled using [{gamma}-32P] ATP and T4 polynucleotide kinase and mixed with 10 µg of the proteins in the presence of 100 µg/ml poly dI/dC, 50 µg/ml salmon sperm DNA, 5 mM dithiothreitol, and 1 mg/ml BSA and incubated at room temperature for 40 min. One quarter of the total reaction was loaded onto a 6% nondenaturing polyacrylamide gel, using 0.5x Tris-borate-EDTA as a running buffer. The dried gel was then exposed to x-ray film. In the case of monitoring the DNA binding activity from the column fractions, we used 10 µl of each column fraction in the gel shift assay reaction. The film exposure time varied from 2 h to overnight depending upon the protein concentration and the DNA binding affinity of the specific binding protein.


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Table 1. P450c17 Oligonucleotides Used in the Studies

 
Recombinant SET Protein Preparation
A human SET cDNA sequence was cloned by PCR from the human fetal adrenal total RNA, using human oligonucleotide sequences as primers (5'-primer: 5'-ACCATGTCGGCGCCGGCGGCC-3'; 3'-primer: 5'-GTCATCTTCTCCTTCATCCTCCTCTCC-3') (30). Full-length human SET cDNA was cloned into the prokaryotic expression vector pET (Novagen, Madison, WI), and SET protein was overexpressed in bacteria strain BL21 and purified as inclusion bodies. Renatured SET was quantified using BCA protein assay (Pierce Chemical Co., Rockford, IL) following the manufacturer’s instruction. Bacterially expressed SET was used for the gel mobility shift assays as described.

Full-length rat SET cDNA was also cloned into the eukaryotic expression vector pCR3 (Invitrogen, San Diego, CA), amplifying SET RNA from rat kidney RNA, using rat oligonucleotide sequences as primers (5'-primer: 5'-ATGTCTGCGCCGACGGCC-3'; 3'-primer: 5'-CTAGTCATCCTCGCCTTCATCCTC-3') (38).

Analysis of SET and P450c17 mRNAs and Proteins
A 455-bp rat SET cDNA fragment [nucleotides (nt) 227–682] (38), prepared by RT-PCR amplification of rat kidney RNA using rat-specific oligonucleotides, was cloned into pKS (Stratagene, La Jolla, CA) and generated a 529-nt probe. A 120-bp EcoRI-BamHI rat P450c17 cDNA fragment cloned into pKS generated a 171-nt probe (57). In situ hybridization of SET mRNA was performed on fresh frozen embryos (4, 5) or on paraffin sections obtained commercially (Novagen), using 35S-labeled RNA probes. Immunocytochemistry on fresh frozen tissues was performed as described previously (4, 5) using antibodies against human SET peptides (31) and recombinant human P450c17 (58), and using a fluorescein isothiocyanate-conjugated second antibody. P450c17 antibodies were used at a 1:2000 dilution for immunocytochemistry. The SET antibodies were generated against three human SET peptides: SP-1, amino acids 3–16; SP-2, amino acids 44–56; and SP-3, amino acids 169–181. Antibody SP-2 worked best in gel shift assays while antibody SP-3 worked best for immunocytochemistry and was used at a 1:3000 dilution.

Construction of the Rat P450c17 Oligonucleotide-TK-LUC Expression Plasmids
Rat P450c17 oligonucleotides were cloned into a luciferase expression vector with a minimal promoter from the TK gene of herpes simplex virus (TK32LUC) as described (22). 5'-Deletional constructs of the rat P450c17 gene, ligated to the reporter gene {Delta}-luciferase, were described previously (22). All constructs were confirmed by DNA sequencing to determine oligonucleotide copy number, orientation, and sequence. Plasmids containing only a single copy of the oligonucleotide cloned in the 5' -> 3' direction were used for transfection experiments.

Cell Culture, Transfections, and Luciferase Assays
Mouse Leydig MA-10 cells (59) were grown as described previously (22). Human neuronal NT2 precursor cells (Stratagene, La Jolla, CA) were cultured in 50% Ham’s F12/50% DME H21, 10% FBS, 1% glutamine, 1% penicillin/streptomycin. Plasmid DNAs were transfected into MA-10 or NT2 cells by lipofection, using the Fugene 6 transfection reagent (Roche Molecular Chemicals, Indianapolis, IN). When vectors expressing SET were cotransfected with reporter luciferase constructs, the molar ratio of these two plasmids was 1:1. DNA concentrations were equalized by the addition of the cloning vector pCR3. Cells stimulated with cAMP were treated with 1 mM 8-Br-cAMP for the indicated times. Luciferase assays and data analysis were as described elsewhere (60), using a Monolight 1500 luminometer (Analytical Luminescence Laboratory, San Diego, CA) and a luciferase assay system (Promega Corp., Madison, WI). Cellular protein concentrations were assayed using the BCA protein assay kit (Pierce Chemical Co.).


    ACKNOWLEDGMENTS
 
We thank Dr. Terry Copeland, National Cancer Institute, for the anti-SET antibodies and Dr. Walter L. Miller, University of California, San Francisco, for the anti-hP450c17 antibody used in this study. We also thank Mr. James Farley for procuring the pig testes.


    FOOTNOTES
 
Address requests for reprints to: Dr. Synthia H. Mellon, Center for Reproductive Sciences, Department of Obstetrics/Gynecology, University of California, San Francisco, Box 0556, San Francisco, California 94143-0556.

This work was funded by NIH Grants HD-27970 (to S.H.M.) and HD-11979 (to the Reproductive Endocrinology Center, UCSF) and by a grant from the Alzheimer’s Association (to S.H.M).

1 Both authors contributed equally and should be considered co-equal first authors. Back

Received for publication November 16, 1999. Revision received February 18, 2000. Accepted for publication February 24, 2000.


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 MATERIALS AND METHODS
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