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
|
---|
Neurosteroids are important endogenous
regulators of
-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.
SETs 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
|
---|
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
-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
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
-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
|
---|
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. 1A
). 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. 1B
) and transcriptionally activated basal activity from this
region of the rat P450c17 gene in both Leydig (Fig. 1C
, 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. 1C
, 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).

View larger version (32K):
[in this window]
[in a new window]
|
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 1 ). 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
58 (Fig. 2A
), 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 2426. 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 2629. 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.

View larger version (45K):
[in this window]
[in a new window]
|
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 58 (arrow); some StF-IT-1 protein
was retained on the column and eluted in fractions 2629 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 1776), human PHAP II (amino acids 1776), and human
I2PP2A (amino acids 1776). The alignment of the sequence was
performed by using GCG FASTA search program.
|
|
As fractions 58 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. 2B
).
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. 2C
). 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. 3A
). 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. 3B
).
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.

View larger version (55K):
[in this window]
[in a new window]
|
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 1 ) 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. 3C
, left) as
efficiently as the StF-IT-1 protein from MA-10 cells (Fig. 3C
, right); furthermore, wild-type, but not mutant
-418/-399 oligonucleotides would compete for human SET binding (Fig. 3C
, left). This binding pattern of human SET could be
duplicated by incubating -418/-399 with MA-10 cell extract (Fig. 3C
, 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. 4A
). 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.

View larger version (17K):
[in this window]
[in a new window]
|
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 -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 -Luc plus SET is
10 x -476rP450c17 -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. 4B
). 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
luciferase (-476
-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
-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
-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. 5
, panels A, C, and
D), the structure of the rhombencephalon from E11.5 [Fig. 5D
, mesencephalon (Mes) and metencephalon (Met)] but not in the developing
rhombencephalon at E10.5 (which is negative in Fig. 5B
), the
prosencephalon, rhombencephalon (mesencephalon and metencephalon) and
diencephalon at E13.5 (Fig. 5E
), and in the basal diencephalon,
pituitary, and hindbrain (shown at E18.5 in Fig. 6K
). 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).

View larger version (109K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (101K):
[in this window]
[in a new window]
|
Figure 6. Composite Lightfield Microphotographs of in
Situ Hybridizations of SET mRNA in E18.5 Mouse Embryos (AJ)
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 AJ are at the same
magnification. The bar under E = 100 µm (panels
AJ); 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. 5F
). 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. 6
). Sites of expression included the cochlea,
paraspinal muscle, thymus, whiskers, skin, intestine, spinal cord,
dorsal root ganglia, cartilage, and trigeminal ganglia (Fig. 6
). SET
mRNA was also expressed in sites related to hematopoiesis and
development of the vasculature, such as the developing liver, the
branchial arches (Fig. 5A
, 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. 5G
), hypothalamus, septum, and
in both the granular layer and Purkinje cells of the cerebellum (Fig. 6K
), 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. 6A
; smooth
muscles of the back, Fig. 6B
; and thymic cells, Fig. 6C
) as well as
from the trunk neural crest (dorsal root ganglia, Fig. 6H
; skin, Fig. 6E
) 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. 5D
) and in the vertebral cartilage (Fig. 6H
).
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 c
ruleus nucleus (not shown), trigeminal
ganglia (Fig. 7
, A and B), in the pontine
nucleus (Fig. 7
, C and D) as well as in the cortical subplate (E18.5,
Fig. 5F
). SET mRNA was also detected in the cortex (Fig. 5F
) 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.

View larger version (125K):
[in this window]
[in a new window]
|
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
|
---|
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
and SETß, that differed at their 5'-ends. SET
cDNA has an open reading frame of 867 nucleotides, while SETß cDNA
has an open reading frame of 831 nucleotides. SET
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
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 1724 of SETß and not
of SET
.
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
|
---|
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
[
-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 (00.5 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 23 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 4550 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 1
). Oligonucleotide probes were end labeled
using [
-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.
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 manufacturers 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) 227682]
(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 316; SP-2, amino acids 4456;
and SP-3, amino acids 169181. 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
-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% Hams 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 Alzheimers Association (to S.H.M).
1 Both authors contributed equally and should be considered co-equal
first authors. 
Received for publication November 16, 1999.
Revision received February 18, 2000.
Accepted for publication February 24, 2000.
 |
REFERENCES
|
---|
-
Baulieu EE 1991 Neurosteroids: a new function in the
brain. Biol Cell 71:310[CrossRef][Medline]
-
Mellon SH, Deschepper CF 1993 Neurosteroid bio-synthesis:
genes for adrenal steroidogenic enzymes are expressed in the brain.
Brain Res 629:283292[CrossRef][Medline]
-
Mellon SH 1994 Neurosteroids: biochemistry, modes of action,
and clinical relevance. J Clin Endocrinol Metab 78:10031008[Medline]
-
Compagnone NA, Bulfone A, Rubenstein JLR, Mellon SH 1995 Expression of the steroidogenic enzyme P450scc in the central and
peripheral nervous systems during rodent embryogenesis. Endocrinology 136:26892696[Abstract]
-
Compagnone NA, Bulfone A, Rubenstein JLR, Mellon SH 1995 Steroidogenic enzyme P450c17 is expressed in the embryonic central
nervous system. Endocrinology 136:52125223[Abstract]
-
Compagnone NA, Salido E, Shapiro LJ, Mellon SH 1997 Expression of steroid sulfatase during embryogenesis. Endocrinology 138:47684773[Abstract/Free Full Text]
-
Majewska MD, Harrison NL, Schwartz RD 1986 Steroid hormone
metabolites are barbiturate-like modulators of the GABA receptor.
Science 232:10041007[Medline]
-
Harrison NL, Simmonds MA 1984 Modulation of GABA receptor
complex by a steroid anesthetic. Brain Res 323:284293
-
Lambert JJ, Belelli D, Hill-Venning C, Callachan H, Peters JA 1996 Neurosteroid modulation of native and recombinant GABAA receptors.
Cell Mol Neurobiol 16:15574[Medline]
-
ffrench-Mullen JM, Spence KT 1991 Neurosteroids clock Ca+2
channel current in freshly isolated hippocampal CA1 neurons. Eur J
Pharmacol 202:269272[CrossRef][Medline]
-
Wu FS, Gibbs TT, Farb DH 1991 Pregnenolone sulfate: a positive
allosteric modulator at the N-methyl-D-aspartate receptor. Mol
Pharmacol 40:333336[Abstract]
-
Fahey JM, Lindquist DG, Pritchard GA, Miller LG 1995 Pregnenolone sulfate potentiation of NMDA-mediated increases in
intracellular calcium in cultured chick cortical neurons. Brain Res 669:183188[CrossRef][Medline]
-
Bergeron R, de Montigny C, Debonnel G 1996 Potentiation of
neuronal NMDA response induced by dehydroepiandrosterone and its
suppression by progesterone: effects mediated via sigma receptors.
J Neurosci 16:1193202[Abstract]
-
Compagnone NA, Mellon SH 1998 Dehydroepiandro-sterone: a
potential signalling molecule for neocortical organization during
development. Proc Natl Acad Sci USA 95:46784683[Abstract/Free Full Text]
-
Monnet FP, Debonnel G, Bergeron R, Gronier B, de Montigny C 1994 The effects of sigma ligands and of neuropeptide Y on
N-methyl-D-aspartate-induced neuronal activation of CA3 dorsal
hippocampus neurones are differentially affected by pertussin toxin.
Br J Pharmacol 112:709715[Abstract]
-
Roberts E, Bologa L, Flood JF, Smith GE 1987 Effects of
dehydroepiandrosterone and its sulfate on brain tissue in culture and
on memory in mice. Brain Res 406:357362[CrossRef][Medline]
-
Flood JF, Morley JE, Roberts E 1995 Pregnenolone sulfate
enhances post-training memory processes when injected in very low doses
into limbic system structures: the amygdala is by far the most
sensitive. Proc Natl Acad Sci USA 92:1080610810[Abstract]
-
Li PK, Rhodes ME, Jagannathan S, Johnson DA 1995 Reversal of
scopolamine induced amnesia in rats by the steroid sulfatase inhibitor
estrone-3-O-sulfamate. Brain Res Cogn Brain Res 2:251254[Medline]
-
Vallee M, Mayo W, Darnaudery M, Corpechot C, Young J, Koehl M,
Le Moal M, Baulieu EE, Robel P, Simon H 1997 Neurosteroids: deficient
cognitive performance in aged rats depends on low pregnenolone
sulfate levels in the hippocampus. Proc Natl Acad Sci USA 94:1486514870[Abstract/Free Full Text]
-
Geller DH, Auchus RJ, Mendonca BB, Miller WL 1997 The genetic
and functional basis of isolated 17,20-lyase deficiency. Nat Genet 17:201205[Medline]
-
Auchus RJ, Lee TC, Miller WL 1998 Cytochrome b5 augments the
17,20-lyase activity of human P450c17 without direct electron transfer.
J Biol Chem 273:31583165[Abstract/Free Full Text]
-
Givens C, Zhang P, Bair S, Mellon S 1994 Transcriptional
regulation of rat cytochrome P450c17 expression in mouse Leydig MA-10
and adrenal Y-1 cells: identification of a single protein that mediates
both basal and cAMP-induced activities. DNA Cell Biol 13:10871098[Medline]
-
Zhang P, Mellon SH 1996 The orphan nuclear receptor
steroidogenic factor-1 regulates the cAMP-mediated transcriptional
activation of rat cytochrome P450c17. Mol Endocrinol 10:147158[Abstract]
-
Ikeda Y, Shen WH, Ingraham HA, Parker KL 1994 Developmental
expression of mouse steroidogenic factor-1, an essential regulator of
the steroid hydroxylases. Mol Endocrinol 8:654662[Abstract]
-
Nebert DW, Adesnik M, Coon MJ, Estabrook RW, Gonzales FJ,
Guengerich FP, Gunsalus IC, Johnson EF, Kemper B, Levin W, Phillips IR,
Sato R, Waterman MR 1987 The P450 gene super-family: recommended
nomenclature. DNA 6:111[Medline]
-
Kagawa N, Ogo A, Takahashi Y, Iwamatsu A, Waterman MR 1994 A
cAMP-regulatory sequence (CRS1) of CYP17 is a cellular target for the
homeodomain protein Pbx1. J Biol Chem 269:1871618719[Abstract/Free Full Text]
-
Picado-Leonard J, Miller WL 1987 Cloning and sequence of the
human gene for P450c17 (steroid 17
-hydroxylase/17, 20
lyase): similarity to the gene for P450c21. DNA 6:437448
-
Brentano ST, Picado-Leonard J, Mellon SH, Moore CCD, Miller WL 1990 Tissue-specific, cyclic adenosine 3',5'-monophosphate-induced, and
phorbol ester-repressed transcription from the human P450c17 promoter
in mouse cells. Mol Endocrinol 4:19721979[Abstract]
-
Zhang P, Mellon SH 1997 The rat P450c17 gene is regulated by
multiple orphan nuclear receptors. Mol Endocrinol 11:891904[Abstract/Free Full Text]
-
von Lindern M, van Baal S, Wiegant J, Raap A, Hagemeijer A,
Grosveld G 1992 Can, a putative oncogene associated with myeloid
leukemogenesis, may be activated by fusion of its 3' half to different
genes: characterization of the set gene. Mol Cell Biol 12:33463355[Abstract]
-
Adachi Y, Pavlakis GN, Copeland TD 1994 Identification and
characterization of SET, a nuclear phosphoprotein encoded by the
translocation break point in acute undifferentiated leukemia. J
Biol Chem 269:22582262[Abstract/Free Full Text]
-
Adachi Y, Pavlakis GN, Copeland TD 1994 Identification of
in vivo phosphorylation sites of SET, a nuclear
phosphoprotein encoded by the translocation breakpoint in acute
undifferentiated leukemia. FEBS Lett 340:231235[CrossRef][Medline]
-
Ruggiero-Lopez D, Manioc C, Geourjon C, Louisot P, Martin A 1994 Purification and partial amino acid sequence of fuctinin, an
endogenous inhibitor of fucosyltransferase activities. Eur J
Biochem 224:4755[Abstract]
-
Vaesen M, Barnikol-Watanabe S, Gotz H, Awni LA, Cole T,
Zimmermann B, Kratzin HD, Hilschmann N 1994 Purification and
characterization of two putative HLA class II associated proteins:
PHAPI and PHAPII. Biol Chem Hoppe Seyler 375:113126[Medline]
-
Matsumoto K, Nagata K, Ui M, Hanaoka F 1993 Template
activating factor I, a novel host factor required to stimulate the
adenovirus core DNA replication. J Biol Chem 268:1058210587[Abstract/Free Full Text]
-
Matsumoto K, Okuwaki M, Kawase H, Handa H, Hanaoka F, Nagata K 1995 Stimulation of DNA transcription by the replication factor from
the adenovirus genome in a chromatin-like structure. J Biol Chem 270:96459650[Abstract/Free Full Text]
-
Mellon SH, Miller WL, Bair SR, Moore CCD, Vigne JL, Weiner RI 1994 Steroidogenic adrenocortical cell lines produced by genetically
targeted tumorigenesis in transgenic mice. Mol Endocrinol 8:97108[Abstract]
-
Kim EG, Choi ME, Ballermann BJ 1994 Spatially restricted
expression of set mRNA in developing rat kidney. Am J Physiol
266:F155F161
-
Li M, Makkinje A, Damuni Z 1996 The myeloid
leukemia-associated protein SET is a potent inhibitor of protein
phosphatase 2A. J Biol Chem 271:1105911062[Abstract/Free Full Text]
-
Pallas DC, Shahrik LK, Martin BL, Jaspers S, Miller TB,
Brautigan DL, Roberts TM 1990 Polyoma small and middle T antigens
and SV40 small t antigen form stable complexes with protein phosphatase
2A. Cell 60:167176[Medline]
-
Walter G, Ruediger R, Slaughter C, Mumby M 1990 Association of
protein phosphatase 2A with polyoma virus medium tumor antigen. Proc
Natl Acad Sci USA 87:25212525[Abstract]
-
Kawabe T, Muslin AJ, Korsmeyer SJ 1997 HOX11 interacts with
protein phosphatases PP2A and PP1 and disrupts a G2/M cell-cycle
checkpoint. Nature 385:454458[CrossRef][Medline]
-
Adler HT, Nallaseth FS, Walter G, Tkachuk DC 1997 HRX leukemic
fusion proteins form a heterocomplex with the leukemia-associated
protein SET and protein phosphatase 2A. J Biol Chem 272:2840728414[Abstract/Free Full Text]
-
Ito T, Bulger M, Kobayashi R, Kadonaga JT 1996 Drosophila NAP-1 is a core histone chaperone that functions
in ATP-facilitated assembly of regularly spaced nucleosomal arrays. Mol
Cell Biol 16:31123124[Abstract]
-
Chang L, Loranger SS, Mizzen C, Ernst SG, Allis CD, Annunziato
AT 1997 Histones in transit: cytosolic histone complexes and
diacetylation of H4 during nucleosome assembly in human cells.
Biochemistry 36:469480[CrossRef][Medline]
-
Walter PP, Owen-Hughes TA, Cote J, Workman JL 1995 Stimulation
of transcription factor binding and histone displacement by nucleosome
assembly protein 1 and nucleoplasmin requires disruption of the histone
octamer. Mol Cell Biol 15:61786187[Abstract]
-
Kellogg DR, Kikuchi A, Fujii-Nakata T, Turck CW, Murray AW 1995 Members of the NAP/SET family of proteins interact specifically
with B-type cyclins. J Cell Biol 130:661673[Abstract]
-
Nagata K, Kawase H, Handa H, Yano K, Yamasaki M, Ishimi Y,
Okuda A, Kikuchi A, Matsumoto K 1995 Replication factor encoded by a
putative oncogene, set, associated with myeloid leukemogenesis. Proc
Natl Acad Sci USA 92:42794283[Abstract]
-
Kawase H, Okuwaki M, Miyaji M, Ohba R, Handa H, Ishimi Y,
Fujii-Nakata T, Kikuchi A, Nagata K 1996 NAP-I is a functional
homologue of TAF-I that is required for replication and transcription
of the adenovirus genome in a chromatin-like structure. Genes Cells 1:10451056[Abstract/Free Full Text]
-
Okuwaki M, Nagata K 1998 Template activating factor-I remodels
the chromatin structure and stimulates transcription from the
chromatin template. J Biol Chem 273:3451134518[Abstract/Free Full Text]
-
Miyaji-Yamaguchi M, Okuwaki M, Nagata K 1999 Coiled-coil
structure-mediated dimerization of template activating factor-I is
critical for its chromatin remodeling activity. J Mol Biol 290:547557[CrossRef][Medline]
-
Fu GK, Grosveld G, Markovitz DM 1997 DEK, an autoantigen
involved in a chromosomal translocation in acute myelogenous leukemia,
binds to the HIV-2 enhancer. Proc Natl Acad Sci USA 94:18111815[Abstract/Free Full Text]
-
Kasper LH, Brindle PK, Schnabel CA, Pritchard CE, Cleary ML,
van Deursen JM 1999 CREB binding protein interacts with
nucleoporin-specific FG repeats that activate transcription and mediate
NUP98-HOXA9 oncogenicity. Mol Cell Biol 19:764776[Abstract/Free Full Text]
-
Lu XP, Salbert G, Pfahl M 1994 An evolutionary conserved
COUP-TF binding element in a neural-specific gene and COUP-TF
expression patterns support a major role for COUP-TF in neural
development. Mol Endocrinol 8:17741788[Abstract]
-
Hagenbuchle O, Wellauer PK 1992 A rapid method for the
isolation of DNA-binding proteins from purified nuclei of tissues and
cells in culture. Nucleic Acids Res 20:35553559[Abstract]
-
Manley JL, Fire A, Cano A, Sharp PA, Gefter ML 1980 DNA-dependent transcription of adenovirus genes in a soluble whole-cell
extract. Proc Natl Acad Sci USA 77:38553859[Abstract]
-
Mellon SH, Vaisse C 1989 cAMP regulates P450scc gene
expression by a cycloheximide-insensitive mechanism in cultured mouse
Leydig MA-10 cells. Proc Natl Acad Sci USA 86:77757779[Abstract]
-
Lin D, Black SM, Nagahama Y, Miller WL 1993 Steroid 17
-hydroxylase and 17,20-lyase activities of P450c17: contributions of
serine106 and of P450 reductase. Endocrinology 132:24982506[Abstract]
-
Ascoli M 1981 Characterization of several clonal lines of
cultured Leydig tumor cells: gonadotropin receptors and steroidogenic
responses. Endocrinology 108:8895[Abstract]
-
Brasier AR, Tate JE, Habener JF 1989 Optimized use of the
firefly luciferase assay as a reporter gene in mammalian cell lines.
Biotechniques 7:11161122[Medline]