Expression Cloning and Characterization of PREB (Prolactin Regulatory Element Binding), a Novel WD Motif DNA-Binding Protein with a Capacity to Regulate Prolactin Promoter Activity
Makiko Suzuki Fliss1,
Patricia M. Hinkle and
Carter Bancroft
Department of Physiology and Biophysics (M.S.F., C.B.) Mount
Sinai School of Medicine New York, New York 10029
Department of Pharmacology and Physiology and the Cancer
Center (P.M.H.) University of Rochester School of Medicine and
Dentistry Rochester, New York 14642
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ABSTRACT
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Previous studies have implied that a transcription
factor(s) other than Pit-1 is involved in homeostatic regulation of PRL
promoter activity via Pit-1-binding elements. One such element, 1P, was
employed to clone from a rat pituitary cDNA expression library a novel
417-amino acid WD protein, designated PREB (PRL regulatory element
binding) protein. PREB contains two PQ-rich potential transactivation
domains, but no apparent DNA-binding motif, and exhibits
sequence-specific binding to site 1P, to a site nonidentical to that
for Pit-1. The PREB gene (or a related gene) is conserved, as an
apparently single copy, in rat, human, fly, and yeast. A single
approximately 1.9-kb PREB transcript accumulates in
GH3 rat pituitary cells, to levels similar to
Pit-1 mRNA. PREB transcripts were detected in all human tissues
examined, but the observation of tissue-specific multiple transcript
patterns suggests the possibility of tissue-specific alternative
splicing. RT-PCR analysis of human brain tumor RNA samples suggested
region-specific expression of PREB transcripts in brain. Western and
immunocytochemical analysis implied that PREB accumulates specifically
in GH3 cell nuclei. Transient transfection
employing PREB-negative C6 rat glial cells showed that PREB is as
active as, and additive with, Pit-1 in transactivation of a PRL
promoter construct; and that PREB, but not Pit-1, can mediate
transcriptional activation by protein kinase A (PKA). Expression in
GH3 cells of a GAL4-PREB fusion protein both
strongly transactivated a 5XGAL indicator construct and yielded a
further stimulation of expression of this construct by coexpressed PKA,
implying that PREB can mediate both basal and PKA-stimulated
transcriptional responses in pituitary cells. These observations imply
that PREB will prove to play a significant transcriptional regulatory
role, both in the pituitary and in other organs in which transcripts of
its gene are expressed.
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INTRODUCTION
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The PRL gene is expressed in a cell type-specific fashion in
pituitary lactotropic cells. The pituitary-specific transcription
factor Pit-1 has been show to play an important role in expression by
the pituitary of the PRL gene (and also of the genes for GH and TSH),
both during development (1) and in the mature organism (2). The PRL
promoter contains multiple binding sites for Pit-1, which have been
implicated not only in basal PRL gene expression (3), but also in
kinase-mediated hormonal regulation of gene expression of this gene.
For example, the most proximal of these Pit-1 binding sites, spanning
positions -66 to -32 and termed 1P, has been reported to direct a
response to a number of external agents, including TRH and
Ca2+ (4), cAMP (2, 5), and constitutively active forms of
G-
s (6) and G-
q (7) and of two negatively
acting G proteins,
i and
o (8). In
addition, a more distal site, termed 3P, has been reported to direct a
response to cAMP (9), while both site 3P (9) and the more distal site
4P (10) have been implicated in the Ras responsiveness of the PRL
promoter.
The observation that treatment of GH3 rat pituitary cells
with activators of either protein kinase A or protein kinase C
stimulated Pit-1 phosphorylation (11) suggested that Pit-1
phosphorylation might mediate many of the hormonal responses that
localize to PRL promoter Pit-1 binding sites. However, reports that a
number of these responses do not require Pit-1 phosphorylation (2, 12, 13) implied that factors other than Pit-1 are required for regulation
of PRL gene expression directed by binding sites for Pit-1. Indeed, it
has been known for some time that two ubiquitously expressed
factors can bind to site 1P and activate expression via this site:
Oct-1 (14) and thyrotroph embryonic factor (TEF) (15). However,
since protein kinase A (PKA)-mediated phosphorylation of Oct-1 strongly
decreases its binding, either to an octamer consensus site (16) or to
site 1P (R. Ashton and C. Bancroft, unpublished observations), this
protein is unlikely to direct kinase-mediated transcriptional
activation. TEF mRNA levels are low in GH cells, and this
protein is believed to exert its major action in thyrotroph development
and regulation of TSHß expression in this pituitary cell type (15).
More recently, a possibly ubiquitous factor has been implicated in a
Pit-1-independent transcriptional action of PKA that is mediated by PRL
promoter site 1P in nonpituitary cells (5), but this factor has
apparently not yet been further characterized. Finally, the studies
described above of Ras responsiveness of the PRL promoter via Pit-1
binding sites (9, 10) have provided evidence that this response
involves binding of an Ets-1-like factor within or adjacent to such a
site.
To search for factors that might play a role in regulation of PRL gene
expression via the proximal Pit-1 binding site 1P, we employed this DNA
element as a probe to screen a bacteriophage cDNA expression library
prepared from the GC rat pituitary cell line. We report here the
identification and characterization of PREB (PRL regulatory element
binding), a WD motif transcription factor. PREB binds specifically to
PRL promoter site 1P but contains no discernable DNA-binding motif in
its sequence; exhibits a nuclear accumulation in pituitary cells; and
can both activate PRL promoter activity, alone or in the presence of
Pit-1, and transmit the transcriptional action of PKA to this
promoter.
We also report evidence that PREB is encoded by a single-copy
gene in human and rat, while single-copy PREB gene homologs are also
detected in Drosophila and yeast; and that PREB transcripts
accumulate in multiple tissues in human, exhibiting patterns consistent
with tissue-specific splicing. We propose that PREB represents a novel
type of evolutionarily conserved transcription factor that may
contribute to regulation of PRL gene expression in the mammalian
pituitary, and that this protein and/or its isomers or analogs may also
play a more widespread transcriptional role in various tissues and
organisms.
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RESULTS
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Expression Cloning of PREB
We carried out Southwestern screening, in which a filter-bound,
denatured-renatured expression library is screened with a concatenated
probe, as described (17, 18). [32P]-labeled PRL promoter
element 1P DNA was employed to screen a GC rat pituitary cell (19)
ZAPII cDNA library (Stratagene, La Jolla, CA). Screening of 1.5
x 106 plaques yielded several presumptive positive clones
that bound to labeled site 1P. However, only one of these clones
exhibited DNA-sequence specificity: binding to element 1P, but not to
either a mutated element 1P or a canonical cAMP-response element (CRE)
(data not shown). The properties of this clone, designated PREB (PRL
regulatory element binding) protein, was further investigated. The
pBLUESCRIPT vector containing PREB cDNA was excised in vivo,
and the cloned cDNA was sequenced, yielding a 1.28-kb sequence
containing a large (243-amino acid) open reading frame (Fig. 1
, amino acids 175417).

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Figure 1. Structure of PREB
At the top is a schematic structure of the protein
sequence predicted from the PREB cDNA clone, indicating the motifs
identified: three WD repeats plus two PQ-rich regions. Shown
below are the nucleotide sequence and the predicted
amino acid sequence of PREB. Three sequences exhibiting extensive
similarity to the consensus WD motif are underlined, and
the P or Q residues in the two PQ-rich regions are
circled. In the 3'-untranslated region, two ATTTA
potential mRNA destabilizing elements and a potential polyadenylation
signal are indicated by, respectively, underlining and
capital letters. The PREB cDNA sequence has been
deposited in the GenBank database under accession no. AF061817.
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PREB Exhibits DNA Sequence-Specific Binding to PRL Promoter Element
1P
Electrophoretic mobility shift was employed to examine the DNA
sequence- binding specificity of recombinant PREB(175417) (Fig. 2A
). PREB(175417) plus the element 1P
probe that was employed to clone PREB(175417) yielded a major shifted
band, plus weaker lower mol wt bands that probably correspond to
partially degraded PREB(175417) (lane 2), all of which were competed
by excess cold element 1P (lanes 35). Use of element 1P mutants
(illustrated in Fig. 2B
) showed that PREB(175417) binding to element
1P was also inhibited by an excess of the 1P1 mutant (lanes 911), but
not of the 11P mutant (lanes 68). The inability of 11P to compete
implies that the PREB binding site in element 1P is located at least
partially on the 5'-side of this element (Fig. 2B
). Since x-ray
analysis has recently shown that the Pit-1 POU domain binds to element
1P as a dimer by contacting the bases bracketed in Fig. 2B
(20), the ability of 1P1 to compete PREB binding implies that Pit-1 and
PREB possess different (but possibly overlapping) binding sites within
element 1P. The observation (Fig. 2A
) that PREB(175- 417) binding was
not competed by excess amounts of oligonucleotides corresponding either
to other Pit-1 binding sites, PRL-3P (lanes 1214) or GH-GHF1 (lanes
1517), or to two unrelated DNA sequences, an SP1-binding site (lanes
1820) or the PRL promoter CLE (CRE-like element) sequence (21) (lanes
2123), further confirms the DNA binding specificity of PREB. Since no
known DNA-binding motif can be detected in the PREB(175417) amino
acid sequence shown in Fig. 1
, this specificity is likely to be
conferred by a novel DNA-binding motif.

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Figure 2. The DNA Target Site for PREB
A, Partially purified poly-His-tagged PREB(175417) ( 40 ng) was
incubated with a [32]P-site 1P probe, plus or minus
excess (50- to 250-fold) of the indicated competitors, and analyzed on
polyacrylamide gels. Lane 1 received no added protein, and lanes 111
and 1223 were analyzed on separate gels. To improve resolution, the
free probe was run off the end of the gel. The single major shifted
band observed with PREB(175417) is indicated. B, Structure of site 1P
and sequences mutated in competitors *1P and 1P* and region predicted
to form part of the binding site for PREB. The bases shown by x-ray
analysis to be contacted by Pit-1 POU domain dimers (20 ) are
bracketed.
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Isolation of the Full-Length PREB-Coding Sequence
Successive applications of 5'-RACE (rapid amplification of DNA
ends) to GH3 cell RNA, employing initially primers
corresponding to the 5'-terminus of PREB(175417), yielded an
additional 636 bases of 5'-terminal DNA sequence, resulting in the PREB
cDNA sequence shown in Fig. 1
, containing in-frame methionine codons at
positions 1 and 217. The ATG at base 217 is preceded by ACC, matching
well the Kozak consensus (ACCAUGG), while the ATG at base 1
is preceded by the poorly matching sequence GGG. The sequence after
each methionine encodes PREB containing, respectively, either 345 amino
acids (
38 kDa) or 417 amino acids (
46 kDa). Putative
polyadenylation (AATAAA) and AU-rich mRNA shortened half-life (ATTTA)
elements in the cDNA 3'-UT are also noted.
Structural Motifs in PREB
Comparison of the PREB cDNA coding sequence with GenBank yielded
no match with any previously cloned protein cDNA sequence, implying
that PREB is a novel protein. However, portions of the PREB cDNA
sequence are similar to several expressed sequence tags of unknown
function posted on the Web by the Washington University-Merck EST
Project, identified in human infant brain (R12741), and in various
mouse tissues: AA111707 (thymus), AA184937 (lymph node), W30204
(19.5-day whole fetus), and AA250620 (tissue not specified).
BLASTN analysis of the PREB sequence revealed three regions
(underlined in Fig. 1
) exhibiting extensive (
39%)
identity to the WD repeat consensus (ß-transducin motif) (22) (Fig. 3
), identifying PREB as a member of the
WD-repeat protein family (23). The highest degree of similarity between
PREB and other known proteins was observed in the PREB region
containing WD repeats I and II, which exhibits the following similarity
with the corresponding region in two yeast WD-repeat gene regulators:
HIR1 (24) (30% identity, 50% similarity), and Tup1 (25) (34%
identity, 52% similarity).

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Figure 3. Sequence Alignments of WD Repeats in PREB
Amino acid positions are shown on the right. Shown
above the line are the three regions in PREB that
exhibit the indicated identities (indicated by shading)
to the ß-transducin consensus (22 ) shown below the
line.
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PREB also contains two amino acid stretches (residues 86134 and
223279) that are unusually rich in both proline and glutamine.
Proline-rich and glutamine-rich transactivation motifs have been
identified in transcription factors (26). In addition, protein regions
rich in both proline and glutamine have been shown to mediate
the transcriptional repression function of the tumor repressor WT1 (27)
and to form part of the transcriptional activation domain of YY1 (28).
Either or both of the proline/glutamine-rich domains may thus be
responsible for the transactivational capabilities of PREB described
below.
PREB Is Encoded by an Evolutionarily Conserved Single-Copy Gene
That Is Expressed Both in Rat Pituitary Cells and in Multiple Human
Organs
Southern blot analysis with a cDNA probe was employed to
investigate the structure and number of PREB genes in various organisms
(Fig. 4
). Digestion of rat DNA with
EcoRI and HindIII or XhoI and
PvuI yielded, upon hybridization with a PREB cDNA probe and
hybridization, single or double bands, respectively. The double bands
detected with the latter two enzymes probably correspond to cleavage
within an intron, since the PREB cDNA sequence contains neither
recognition site. Single bands were detected upon hybridization of PREB
cDNA to human DNA digested with any of four enzymes. Thus, PREB is
apparently a single-copy gene that is well conserved between rat and
human. Restriction enzyme digestion of either Drosophila or
yeast DNA also yielded either one or two bands that hybridized with rat
PREB cDNA, implying that the PREB gene (or a related gene) has been
highly conserved during evolution.

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Figure 4. Southern Blot Analysis of the PREB Gene in Various
Organisms
DNA isolated from the indicated organisms was digested with the
indicated restriction enzymes and subjected to Southern blot analysis,
employing a PREB cDNA probe. Hybridization was at reduced stringency
(39 C), except for rat DNA, where 42 C was employed. The sizes of
internal DNA mol wt markers are indicated.
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We next investigated PREB gene expression, beginning with examination
of accumulation of PREB mRNA in the GH3 rat pituitary cells
(Fig. 5
). Northern analysis of total
cellular RNA yielded a single PREB mRNA band comigrating with 18S
ribosomal RNA (hence
1.9 kb in size). GH3 cells thus
produce a single predominant molecular form of PREB mRNA, with an
apparent size in good agreement with that predicted by the PREB cDNA
clone (Fig. 1
). Moreover, it can be seen that Northern analysis of the
mRNAs for PREB and Pit-1 mRNA under equivalent conditions of RNA input,
probe amounts, and specific activities yielded a PREB mRNA band and a
major Pit-1 mRNA band exhibiting comparable intensities. PREB and Pit-1
mRNA thus accumulate to similar levels in the GH3 cells,
consistent with an important role for PREB in these cells in regulation
of PRL promoter activity.

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Figure 5. Northern Analysis of PREB mRNA in GH3
Cells
Upper panel, RNA was isolated from GH3
cells, and two aliquots (15 µg) of the same sample were analyzed on
the same gel, transferred to nitrocellulose, and hybridized with a cDNA
probe for either PREB or Pit-1. To permit comparison of the signals
obtained for the two mRNAs, cDNA probes of equal size were labeled to
equivalent specific activities, and equal amounts of each probe were
employed for hybridization. The positions of the two ribosomal RNA
bands are indicated. Lower panel, Stained ribosomal RNAs
from the same samples.
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The isolation from various human and mouse organs of ESTs homologous to
PREB mRNA, described above, suggests that the PREB gene may be
expressed in multiple tissues. To investigate this question, a human
multiple tissue Northern blot was probed with a rat PREB cDNA probe
(Fig. 6A
). PREB transcripts were detected
in all tissues examined, with the strongest signals seen in heart,
skeletal muscle, and pancreas. Significantly, three different bands
were detected, corresponding to mRNA sizes of approximately 2.2, 1.9,
and 1.5 kb. Most tissues yielded two bands, but different tissues
yielded different patterns, suggesting the possibility of
tissue-specific alternative splicing of the PREB gene transcript. Thus,
all tissues examined except lung yielded the 2.2-kb species (the kidney
band is detectable upon prolonged exposure), while the 1.5-kb species
was detected only in heart, muscle, and pancreas, and the 1.9-kb
species only in brain, placenta, and lung. Finally, lung and liver
yielded only, respectively, the 1.9- and 2.2-kb species. The 1.9-kb
mRNA band may correspond to the 1.9-kb PREB mRNA species we have
detected in the rat GH3 pituitary cells (see Fig. 5
).

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Figure 6. Analysis of PREB mRNA in Various Tissues and Cell
Lines
A, PREB mRNA expression in various human organs. A human multiple
tissue Northern blot (Clontech 7760, 2 µg polyA+ RNA from each
tissue) was probed with a PREB cDNA probe (upper panel),
then stripped and reprobed with ß-actin cDNA (lower
panel). In the upper panel, a shorter exposure
of lane 1 is shown in lane 1'. Open circles,
closed circles, and arrowhead mark bands
corresponding to transcripts approximately 2.2, 1.9, and 1.5 kb,
respectively, in size. B, PREB mRNA expression in cell lines.
Poly(A)-enriched mRNA (1 µg) prepared from either GH3 or
C6 cells was subjected to Northern blot analysis with a PREB cDNA
probe. EtBr staining indicated that each lane received equivalent
amounts of undegraded ribosomal RNA (not shown). The PREB band in the
GH3 lane is purposefully overexposed to determine whether
the C6 cell lane contains detectable PREB mRNA, and its size is
estimated from migration of the 18S rRNA band. C, PREB mRNA expression
in various human brain tumors and cells. Lanes 27, An aliquot of cDNA
(2 µl) from each of the indicated brain tumors was PCR-amplified
employing gene-specific primer sets for either PREB
(top) or HGPRT (bottom). Shown are the
sizes of an internal 1-kb DNA Ladder (GIBCO), and an
arrow indicating the major product amplified with PREB
primers. Insufficient amounts of cDNA were available for HGPRT analysis
of the lane 3 sample. Lane 9 (top), PCR amplification
products of PREB cDNA; lanes 10 and 11 (top), RT-PCR
amplification products of 100 ng poly(A)-enriched mRNA from C6 cells.
Astro, Astrocytoma; gliom, glioma; br tum, brain tumor (metastasis into
brain); neuroep, neuroepithelioma; RT, reverse transcriptase.
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For the characterization of the regulatory properties of PREB, it was
desirable to attempt to identify a PREB-negative cell line, for use
both as a negative control for studies of cellular PREB expression and
as a recipient for transfection analysis of PREB activity. The
widespread distribution of PREB transcripts among human organs
suggested that it might be difficult to identify a PREB-negative cell
line. However, we have found previously that the rat C6 glial cell line
exhibits virtually undetectable expression of transfected PRL promoter
constructs in the absence of cotransfected transcription factors (29),
suggesting that this cell line is deficient in factors capable of
stimulating PRL promoter activity. Northern analysis of C6 cell
polyA+ RNA (Fig. 6B
) yielded no detectable PREB mRNA band,
under conditions where GH3 cell mRNA yielded a strong
signal for this mRNA; and RT-PCR analysis of C6 cell polyA+
RNA (Fig. 6C
) yielded a readily detectable control HGPRT signal, but no
detectable PREB signal. In addition, PREB protein was undetectable in
the C6 cells, both by Western blot analysis (data not shown) or by
immunocytochemistry (see below). Hence, the C6 cells were employed as a
PREB-negative cell line in our further studies.
The above observations that PREB transcripts accumulate in a
total brain preparation but not in glial cells suggested that PREB
expression might be restricted to specific brain regions. To
investigate this concept, primers specific for PREB or HGPRT were
employed for RT-PCR analysis of cDNA prepared from various human brain
tumors (Fig. 6C
). An amplified PREB signal was detected in cDNA
prepared from two astrocytomas (lanes 2 and 3) and a metastasis into
the brain (lane 6). By contrast, no such signal was detected in cDNA
from either of two gliomas (lanes 4 and 5) or from a neuroepithelioma
(lane 7), even though an HGPRT signal was detectable in both glioma
samples and in the neuroepithelioma sample. These observations with
human tumor samples imply that PREB transcripts accumulate in
astrocytes but not in glial or neuroepithelial cells, and thus that the
PREB gene exhibits region-specific expression within the brain.
PREB Can Function as a Transcriptional Activator
To begin to investigate whether PREB might serve as a pituitary
cell transcription factor, we investigated the expression and
intracellular location of PREB protein in the GH3 rat
pituitary cells. Nuclear or cytosolic proteins isolated from an equal
number of cells were subjected to Western blot analysis (Fig. 7A
). Anti-PREB antiserum, but not control
preimmune serum, detected a major approximately 45-kDa band that
accumulates preferentially in nuclei. This observation implies that
PREB is a nuclear protein in pituitary cells. In addition, the size of
the protein detected suggests both that synthesis of this protein is
initiated by the more N-terminal methionine encoded by the PREB cDNA
sequence (i.e. amino acid 1 in Fig. 1
) and that the entire
PREB cDNA coding sequence has been cloned.

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Figure 7. Size and Nuclear Accumulation of PREB in
GH3 Cells
A, Nuclei and cytosol were prepared, and the nuclei extracted with 0.42
mM KCl. Nuclear and cytosolic proteins from an equal number
of cells were then analyzed on a Western blot, employing anti-PREB
antiserum. Sizes of marker proteins on the same gel are indicated. B,
The same antiserum was employed for immunocytochemistry of
GH3 cells or C6 cells. Ab, anti-PREB antiserum; Pre,
preimmune serum from the same animal.
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The intracellular location of PREB was examined further by
immunocytochemical analysis (Fig. 7B
). With anti-PREB antiserum, the
pituitary GH3 cells yielded a strong signal that was
located specifically over the nuclei, while control rat glial C6 cells
yielded only a faint diffuse background signal. Preimmune serum also
yielded only a background signal with GH3 cells (Fig. 7B
)
or C6 cells (data not shown). The observation with both techniques
that PREB cross-reacting material exhibits a substantial nuclear
accumulation in pituitary cells is consistent with a role for PREB as a
cellular transcription factor.
To investigate directly a possible role for PREB as a PRL gene
transcription factor, we examined the ability of this protein to
transactivate PRL promoter activity in rat glial C6 cells which, as
described above, do not exhibit detectable PREB expression. We first
examined the ability of PREB to regulate expression of a construct
(-1957)PRL-CAT (chloramphenicol acetyl transferase), which contains
the first 1957 bp upstream of the PRL gene body and thus covers both
the promoter and enhancer regions (30) (Fig. 8
). As expected from previous studies
(29), this PRL-CAT construct alone was inactive in the C6 cells, but
was activated by coexpression of Pit-1. Coexpression of PREB was also
observed to activate (-1957)PRL-CAT expression, showing that this
protein can act as a transcriptional activator. Although in the
experiment illustrated, PREB was more active than Pit-1, in other
similar experiments, the two proteins exhibited equivalent activation
of (-1957)PRL-CAT expression (data not shown). This result
demonstrates that PREB can transactivate PRL promoter/enhancer
activity. This region of the PRL-regulatory region contains at least
seven binding sites for Pit-1 (30). The similar levels of activity
observed for Pit-1 and PREB on this construct thus suggests that the
PRL promoter/enhancer region may also contain multiple functional
PREB-binding sites.

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Figure 8. PREB Can Transactivate Expression Directed by the
PRL Promoter Plus Enhancer
C6 rat glial cells (2.6 x 106) were electroporated
with (-1957)PRL-CAT (10 µg) plus RSV-ß gal (2 µg), plus 5 µg
either RcRSV ("None"), or RSV-Pit-1 or RSV-PREB, divided into three
60-mm dishes, incubated 2 days, and then assayed for CAT and
ß-galactosidase activity. Shown is the mean ± SD of
CAT activity, corrected for ß-galactosidase activity, observed with
triplicate dishes.
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Since element 1P is the only presently known PREB-binding site in
the PRL promoter (Fig. 2
), we concentrated on this site in our further
functional studies of PREB. To study element 1P in its natural context
within the PRL promoter, we investigated the ability of exogenously
expressed PREB to trans-regulate construct (-113)PRL-CAT. As
illustrated in Fig. 9
(top),
the only known PRL promoter elements in this truncated construct are
the CLE, element 1P, and a TATA box. The observation that the CLE does
not bind PREB (Fig. 2
) implies that any effects of PREB (and/or Pit-1)
on this PRL-CAT construct are mediated via element 1P.

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Figure 9. PREB and Pit-1 Yield Equivalent Activation of
Expression of a PRL Promoter Construct
Top, Structure of plasmid (-113)PRL-CAT, illustrating
the positions of the known PRL promoter elements.
Bottom, C6 cells (2.6 x 106) were
electroporated with (-113)PRL-CAT (10 µg) and RSV-ß gal (2 µg),
plus the indicated amounts of either RSV-Pit-1 or RSV-PREB, and treated
thereafter as in Fig. 8 . Shown is the mean ± SD of
corrected CAT activity observed with triplicate dishes.
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A comparison of the abilities of various inputs of RSV-based expression
vectors for Pit-1 and PREB to transregulate (-113)PRL-CAT expression
(Fig. 9
) yielded equivalent stimulation by the two proteins. This
result again demonstrates that PREB can act as a transcriptional
activator of a PRL promoter-regulatory region. Furthermore, since Pit-1
is known to be a powerful transactivator of PRL gene expression (31, 32), the observation of equivalent activities for Pit-1 and PREB on two
PRL-regulatory region constructs (see Figs. 8
and 9
) suggests that PREB
may also represent a significant regulator of expression of this
gene.
The results of electrophoretic shift mobility analysis described
above imply that the PREB- and Pit-1-binding sites within element 1P
are centered over different regions. We thus investigated the ability
of PREB to regulate PRL promoter activity in the presence of Pit-1
(Fig. 10
). As before, transfection of an equal amount
(2.5 µg) of an expression construct for either protein alone yielded
similar levels of transactivation of (-113)PRL-CAT. Cotransfection of
2.5 µg of expression vectors for each protein yielded a level of
transactivation that was approximately additive over that yielded by
either expression vector alone, suggesting that these two proteins
exert additive actions on element 1P. Doubling the input of each
expression vector, to close to maximal activity levels (see Fig. 9
),
increased expression approximately 3-fold, again consistent with an
approximately additive action of PREB and Pit-1. These observations
suggest that, at least in the basal cellular state, these two proteins
may exert actions on element 1P that are of approximately equal
strength, but are largely independent.

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Figure 10. PREB and Pit-1 Exhibit Additive Stimulation of
Expression of a PRL Promoter Construct
C6 cells were electroporated with (-113)PRL-CAT and RSV-ß gal as in
Fig. 9 , plus the indicated amounts of RSV-PREB and/or RSV-Pit-1
("1" = 2.5 µg plasmid), and treated thereafter as in Fig. 8 .
Shown is the mean ± SD of corrected CAT activity
observed with triplicate dishes.
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PREB Can Mediate Transcriptional Stimulation by PKA in Either
GH3 Pituitary Cells or Heterologous C6
Cells
As described above, previous studies have implied that Pit-1 is
not the direct functional target of PKA action on the PRL promoter.
Inspection of the predicted PREB amino acid sequence revealed a number
of potential PKA phosphorylation sites (data not shown). This, together
with the observations described above that PREB can bind specifically
to PRL element 1P and exhibits transcriptional activity, suggested that
PREB may represent the cellular protein that directly mediates PKA
action on the PRL promoter via element 1P. To begin to investigate this
possibility in GH3 cells, under conditions that are
independent of both endogenous PREB and PREB-binding sites, we
investigated the ability of a GAL4-PREB fusion protein to transmit PKA
action to a cotransfected GAL4 indicator construct (Fig. 11
). As expected, control
GAL(1147) was unable to transactivate 5XGAL4-CAT expression, in
either the presence or absence of an RSV-PKA expression vector. In the
absence of RSV-PKA, GAL4-PREB alone strongly transactivated 5XGAL4-CAT,
demonstrating again that PREB contains a transcriptional activator
domain. Cotransfection of RSV-PKA yielded an approximately 3-fold
increase in the ability of GAL4-PREB to transactivate 5XGAL4-CAT. This
observation demonstrates that PREB can support a PKA-mediated
transcriptional response in pituitary cells, possibly in the absence of
any change in its ability to bind DNA.

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Figure 11. A GAL4-PREB Fusion Protein Can Transmit Basal and
PKA-Stimulated Reporter Gene Activation
GH3 rat pituitary cells were electroporated with 5XGAL4-CAT
(10 µg) and RSV-ß gal (2 µg), plus 5 µg of either pGAL4(1147)
or GAL4-PREB, plus 5 µg of either RcRSV (PKA-) or RSV-PKA (PKA+),
and treated thereafter as in Fig. 8 . Shown is the mean ±
SD[ of corrected CAT activity observed with triplicate
dishes.
|
|
We next investigated whether PREB can transmit a PKA transcriptional
signal to the PRL promoter, in the absence of other pituitary cell
signals. To do this, we examined the effect of expression of RSV-PKA in
C6 glial cells on transactivation by either Pit-1 or PREB of indicator
construct (-113)PRL-CAT (Fig. 12
). As before,
(-113)PRL-CAT alone exhibited minimal activity, which was only
slightly increased by coexpression of PKA. Coexpression of Pit-1
strongly transactivated CAT activity. However, this activity was not
further increased by coexpression of PKA, in agreement with previous
observations (2). In contrast, the transactivation of CAT activity by
PREB was strongly increased by coexpression of PKA. Thus, PREB (but not
Pit-1) can support a PKA-mediated transcriptional response directed by
element 1P in the context of the PRL promoter.

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Figure 12. PREB but not Pit-1 Can Mediate Regulation by PKA
of Expression of a PRL Promoter Construct
C6 cells were electroporated with (-113)PRL-CAT and RSV-ß gal as in
Fig. 9 , plus 2.5 µg of either RcRSV (None), RSV-Pit-1, or RSV-PREB,
plus 5 µg of either RcRSV (PKA-) or RSV-PKA (PKA+), and treated
thereafter as in Fig. 8 . Shown is the mean ± SD of
corrected CAT activity observed with triplicate dishes.
|
|
 |
DISCUSSION
|
---|
We report here the identification of an 1805-bp rat cDNA encoding
a 417- amino acid protein, PREB. This protein binds directly to a
regulatory element in the PRL promoter, element 1P, previously shown to
represent a target for the pituitary-specific transcription factor
Pit-1, and can mediate both basal and PKA-activated expression of the
PRL promoter.
Analysis of the primary sequence of PREB revealed that it is a novel
protein. However, PREB contains three segments (Fig. 3
, WD I, WD II,
and WD III) possessing a significant degree of homology to the WD
repeat consensus and is thus identified as a member of the WD-repeat
protein family (23). It is interesting that the highest degree of
similarity between PREB and known proteins occurs in segments WD I and
WD II, which resemble regions in the two yeast WD-repeat gene
regulators, HIR1 (24) and Tup1 (25). Two significant features are
shared by most known members of the subset of WD-repeat proteins that
are gene regulators [HIR1 and Tup1, plus the Drosophila
proteins Groucho (33) and dTAFII80 (34), yeast Met30p (35),
and plant COP1 (36)]: 1) Of the proteins whose regulatory functions
are known (all but dTAFII80), each is a repressor of its
target gene. 2) With one possible exception, none is a DNA-binding
protein; instead they all repress their target genes by interacting
with a DNA-bound transcription factor. The possible exception is COP1,
which may bind directly to DNA via a novel zinc-binding motif (36). The
first common feature above suggests that PREB might prove, under some
conditions, to represent a repressor of target gene expression. The
second common feature implies that PREB may represent the first member
of a new family of mammalian WD-repeat gene regulatory transcription
factors that act by binding directly to a DNA target.
PREB(175417) exhibited DNA sequence-specific binding to site 1P.
However, the amino acid sequence of this portion of PREB exhibits no
homology to known DNA-binding motifs, implying that this region of PREB
will prove to contain a DNA-binding region(s) representing a novel
member of this class of regulatory domains. Analysis by electrophoretic
mobility shift assay of interaction of PREB(175417) with site
1P mutants (Fig. 3
) implied that at least a portion of the PREB-binding
site resides upstream of the well defined binding site for Pit-1 in
this element (20). This observation suggests the possibility that PREB
and Pit-1 can co-occupy element 1P, which could represent the
structural basis for the ability of PREB and Pit-1 to exhibit additive
activity on the PRL promoter via this element (see below).
A number of observations suggest that PREB plays a role as a pituitary
cell transcription factor. Antiserum raised against recombinant PREB
detects a major protein that localizes to the nuclei of GH3
cells (Fig. 7
). It is conceivable that the protein detected corresponds
to a cross-reacting protein. However, the observation that the apparent
size of the detected protein, 45 kDa, corresponds to that predicted for
PREB, and that PREB mRNA is expressed in GH3 cells (Fig. 5
), strongly suggests that this protein represents endogenous PREB, and
that PREB is thus a nuclear protein in pituitary cells. The observation
that PREB exhibits sequence-specific binding to a specific element in
the PRL promoter (Fig. 3
) suggested that PREB might be capable of
regulating transcription of this promoter. The ability of PREB to
stimulate PRL promoter activity was demonstrated directly by the
observation that this protein can stimulate activity of cotransfected
PRL-CAT constructs in heterologous cells (
Figs. 810

and 12). In these
investigations, PREB yielded transactivation of a truncated PRL
promoter that was equivalent to that yielded by Pit-1. The
interexperimental variability in the relative basal transactivation
activities of the two proteins on either the full-length PRL
promoter/enhancer region (see Results text) or on this
truncated promoter (e.g. compare Figs. 9
and 10
with Fig. 12
) does not permit a more precise delineation of the relative
activities of PREB and Pit-1 on PRL promoter activity. However, the
observation that the two proteins exhibited approximately additive
transactivational activities on the truncated PRL promoter (Fig. 10
) is
consistent with the concept that PREB can strongly regulate basal PRL
promoter activity independently of Pit-1. Although these experiments,
together with the transactivational properties of a GAL4-PREB fusion
(Fig. 11
), clearly imply that PREB can act as a transcriptional
activator, it is not presently known which region of the protein is
responsible for transactivation. However, as noted above, it seems
possible that such a domain may encompass either or both of the PREB
proline/glutamine-rich regions between residues 86134 and 223279.
The observation that a region containing this type of motif has been
shown previously to mediate the ability of the tumor repressor WT1 to
repress transcription (27) suggests the interesting possibility that
PREB may, in the appropriate context, act as a transcriptional
repressor.
What might be the physiological role of PREB in pituitary cells? The
ability of PREB to transactivate cotransfected PRL constructs in
heterologous cells suggests a role for this protein in basal PRL gene
expression. Similarly, the ability of PREB (Fig. 12
), but not Pit-1
(Fig. 12
; see also Refs. 2, 12), to mediate PKA stimulation of a
cotransfected PRL-CAT construct in heterologous cells suggests that
PREB may play a role in cAMP-mediated transcriptional actions of
extracellular stimulators of PRL promoter activity such as PACAP, which
is apparently completely dependent upon cellular PKA activity for its
transcriptional activation of the PRL promoter (21). The ability of a
PREB fusion protein to direct either basal or PKA-stimulated
transcriptional effects to a heterologous target promoter in a
pituitary cell context (Fig. 11
) lends further support to this
suggestion. However, it should be emphasized that a determination of
whether PREB does play a significant physiological role in either basal
or PKA-stimulated regulation of PRL gene expression will require
further investigations, involving examination of the consequences of
repression of PREB activity in pituitary cells.
The present studies imply that PREB has the capacity to mediate PKA
action on the PRL promoter in the absence of Pit-1 (Fig. 12
). This
property and the observations that PREB exhibits sequence-specific
binding to element 1P (Fig. 2
) and that its mRNA sequences are
expressed in multiple tissues (Fig. 6
) are consistent with the
functional properties of a factor detected by Gutierrez-Hartmann and
co-workers (5) in HeLa cells, which has been reported to exhibit
Pit-1-independent regulation of the PRL promoter via element 1P.
Although the HeLa cell factor has apparently not yet been further
characterized, it is thus possible that PREB corresponds to this
factor. The studies to date of both PREB and the HeLa cell factor imply
that PKA-mediated regulation of PRL promoter activity can occur in the
absence of Pit-1. However, it seems possible that, although Pit-1 alone
cannot mediate PKA transcriptional actions, an interaction between
Pit-1 and PREB on PRL promoter element site 1P could yield an enhanced
response by pituitary cells to PKA-mediated stimuli. A possible model
for this type of cellular role for PREB would involve PREB activation
via PKA-mediated phosphorylation. Although it is not yet known whether
PREB can serve as a PKA substrate either in vitro or
in vivo, the predicted sequence of this protein (Fig. 1
)
does contain a number of sequences resembling PKA phosphorylation
consensus sites (37). Finally, a very recent report that the cofactor
CBP (CREB-binding protein) can interact with Pit-1 during a
PKA-mediated transcriptional response (38) suggests the interesting
possibility of an anologous functional interaction between CBP and
PREB.
A number of observations suggest that PREB may also play a
transcriptional role in organs other than the pituitary. The
observation that a GAL4-PREB construct can transmit both basal and
PKA-stimulated transcriptional effects to a GAL4 promoter construct
(Fig. 11
) implies that PREB can exert these actions independently of
its ability to bind to the PRL promoter. Consistent with a wider role
for this protein, PREB transcripts were observed in all human tissues
examined (Fig. 6A
). It is of course conceivable that some of the PREB
transcript size variants detected in specific tissues in this
experiment arise from modifications in either transcript initiation
site or poly(A) tail size. However, the tissue-specific transcript size
patterns observed does suggest the possibility of alternative splicing
of the initial PREB transcript, and thus of the production in different
tissues of isoforms of PREB potentially possessing unique
transcriptional properties. It is worth noting in this connection that
the PREB cDNA sequence (Fig. 1
) contains a 306-bp (102-amino acid) open
reading frame that starts at position 1333 and thus slightly overlaps
the terminus of the major PREB open reading frame, suggesting that
domain shuffling arising from alternative splicing might generate a
PREB isoform containing some or all of this peptide sequence at its N
terminus. Finally, the detection of PREB transcripts in human brain
tumors arising from astrocytes, but not from glial or neuroepithelial
cells (Fig. 6C
), suggests that the PREB gene exhibits region-specific
brain expression, and thus the intriguing possibility that PREB serves
a specific transcriptional function within restricted portions of the
brain.
In summary, we have cloned from a pituitary cell cDNA library a
transcription factor, termed PREB, that can bind to a regulatory
element in the PRL promoter, and has the capacity to mediate both basal
and PKA-stimulated activity, either of the PRL promoter, or as a fusion
protein of a heterologous promoter. Further investigations should
delineate the regions of PREB that direct binding to, and
transcriptional activation of, the PRL promoter and also illuminate the
physiological role of this novel transcription factor, both within the
pituitary and in other tissues that express the PREB gene
transcript.
 |
MATERIALS AND METHODS
|
---|
Imaging of Gels
Autoradiograms of gel blots or photographs of gels were recorded
electronically using an AGFA (ARCUS II) Scanner
(Agfa-Gevaert Group, Mortsel, Belgium).
Electrophoretic Mobility Shift Assay
Partially purified recombinant his-tagged PREB (PREB amino acids
175417, preceded by six histidines) was prepared from
Escherichia coli by solubilization of an insoluble pellet in
binding buffer [5 mM imidazole, 0.8 M NaCl, 10
mM Tris (pH 7.9), 8 M urea], application to a
nickel affinity column, gradual refolding in the column by progressive
dilution of the urea as described (17), elution in buffer containing
0.51 M imidazole, followed by desalting and concentration
in an Amicon (Beverly, MA) spin column. We have previously described
the sequences of double-stranded oligonucleotides corresponding to PRL
promoter sites 1P, 11P, 1P1, and 3P (39) and the CLE (29). The sequence
of the double-stranded SP1-binding site oligonucleotide probe
(containing a 5' SalI site) is 5'-TCGACGGGGCGGGGCC-3', and
of the double-stranded rat GH pGHF-1 site is
5'-TCGACTGGCTCCAGCCATGAATAAATGTATAGGGAAAG-3'. All procedures were
performed at 4 C in the presence of protease inhibitors [1
mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml
aprotonin, and 5 µg/ml leupeptin]. The partially purified his-tagged
PREB was then incubated 10 min at room temperature in 9 µl containing
10 mM Tris (pH 7.9), 60 mM KCl, 1
mM EDTA, 0.03% NP40, 4% Ficoll, 1 mM
dithiothreitol, 5 µg poly(dI-dC), 1 µg BSA, with or without
unlabeled DNA competitors, and then an additional 10 min after addition
of 1 ng 32P-end-labeled site 1P probe, followed by analysis
on a 5% polyacrylamide gel in 0.25x Tris-borate-EDTA (TBE) at
4 C. The dried gel was then autoradiographed 13 h at -70 C with
intensifying screens.
Cell Lines
GH3 rat pituitary cells (19) were propagated in
suspension culture as described (40). C6 rat glial cells (41) were
maintained in monolayer culture in DMEM containing 5% FCS, 100 mg/ml
streptomycin, and 100 U/ml penicillin at 37 C in 10% CO2,
and cultures were split 1:5 every other day.
Southern Blot Analysis
Standard procedures were employed to prepare genomic DNA from
rat GH3 cells and from Drosophila (provided by
Dr. M. Frasch of Mount Sinai). Human DNA was provided by Dr. D. Bishop
(Mount Sinai School of Medicine). Yeast genomic DNA was prepared
employing a PureGene kit obtained from GENTRA (Minneapolis, MN).
Southern blot analysis was performed as described previously (42),
employing a 32P-labeled probe prepared by either PCR or
random primer labeling from a PREB cDNA template, followed by
autoradiography for 316 h as described above.
Northern Blot and PCR Analysis
Total or poly(A)-enriched RNA was prepared from the
GH3 or C6 cell lines using the Oligotex column affinity
purification system (Qiagen, Inc., Chatsworth, CA) without or with
addition of oligo-d(T)-linked beads. For Northern analysis, RNA was
analyzed by 1% formaldehyde agarose gel electrophoresis as described
(43). After transfer of size-separated RNA to nitrocellulose, baking,
and prehybridization (42), the nitrocellulose was hybridized for
15 h to a 32P-labeled PREB cDNA probe prepared as
described above, and the membranes were then autoradiographed for 13
days as described above. For PCR analysis of RNA samples, RNA was
denatured (70 C, 5 min), annealed with oligo(dT), and subjected to
first-strand synthesis in the presence of 100 U reverse transcriptase
(SuperSCRIPT II), according to the directions of the manufacturer (Life
Technologies, Inc., Gaithersburg, MD). The resultant cDNA, or cDNA
prepared from tumor sample RNA, was then subjected to nested PCR using
Taq polymerase (Perkin-Elmer Corp., Norwalk, CT). The first
and second amplification employed primer sets corresponding,
respectively, to positions 10811097/17761761 and
13001318/16541636 of the PREB cDNA sequence shown in Fig. 1
. The
first amplification began with a hot start (94 C, 5 min; 80 C, 5 min),
followed by 35 cycles [94 C, 1 min; 41 C, 1 min; 72 C, 40 sec (10 min
in the last cycle)]. The second amplification employed the same
conditions, except that in each cycle a 60 C annealing temperature was
employed instead of 41 C, and the 40-sec extension time was replaced by
30 sec.
Preparation of anti-PREB Antiserum
Inclusion bodies were prepared from E. coli
expressing recombinant his-tagged PREB (PREB amino acids 175417,
preceded by six histidines), as described (43). Briefly, cells were
lysed with lysozyme and deoxycholic acid in the presence of 50
mM PMSF, treated with DNAase (1 mg/ml) at room temperature
1530 min, and subjected to centrifugation. The pellet was then
suspended, washed with 6.5 M urea in 0.1 mM
Tris, pH 8.5, after which PREB was extracted with elution buffer (8
M urea, 50 mM Tris, pH 8.0, 1 mM
EDTA, 100 mM NaCl, 0.1 mM PMSF), and subjected
to SDS-PAGE. A gel fragment containing the major PREB band was excised,
frozen, ground with a mortar and pestle, and supplied frozen to
Cocalico Biologicals, Inc. (Reamstown, PA) for preparation of antiserum
in rabbits according to their standard protocol. Before use in Western
blots and immunocytochemistry, either anti-PREB antiserum or preimmune
serum from the same animal was preadsorbed with extracts of host
E. coli. An equal volume of 2x SDS sample buffer was added
to a pelleted 50-ml bacterial culture, mixed thoroughly, incubated at
6570 C for 10 min, fractionated on a 4.5% SDS-PAGE minigel employing
a 1.5-mm preparative comb, and transferred to nitrocellulose. After
soaking the filter in a 5% solution of Carnation brand nonfat milk,
the filter was incubated on a rocking platform either 1 h at room
temperature or 4 C overnight, with anti-PREB antiserum diluted 1:250
into TBST buffer (20 mM Tris, pH 7.6; 137 mM
NaCl, 0.1% Tween-20) containing 3% BSA, and the adsorbed antiserum
was employed for analysis of PREB.
Western Blot Analysis of Cellular Fractions
Nuclei and cytosol (i.e. the soluble fraction of the
postnuclear supernatant) were prepared from GH3 cells as
described (44), in the presence of the protease cocktail described
above. Preliminary SDS-PAGE analysis revealed no gross degradation of
proteins in either fraction (data not shown), and that, on a per cell
basis, the cytosol contained a considerably higher protein content than
the nucleus. Samples were subjected to SDS-PAGE, transferred to
nitrocellulose on a semidry transfer apparatus (Hoeffer Scientific
Instruments, San Francisco. CA), employing Towbin buffer (25
mM Tris, 192 mM glycine, 0.0372% SDS, 20%
methanol) for 3060 min at 100 mA. Filters were blocked for 1 h
with 5% Carnation nonfat dry milk in TBST, incubated with anti-PREB
antibody (1:250), followed by washing with TBST. The filter was then
exposed to secondary antibody (conjugated to horseradish peroxidase and
diluted 1:5000) in TBST containing 3% BSA for 3060 min. After
washing with TBST (four times for 10 min each time), immunoreactive
proteins were visualized by enhanced chemiluminescence according to the
directions of the manufacturer (Amersham Corp., Arlington Heights,
IL).
Immunocytochemistry of Cultured Cells
Cells were plated on glass coverslips in serum-containing media
the night before use (C6 cells, DMEM plus 5% FCS; GH3
cells in Hams F10 plus 15% horse serum plus 2.5% FCS). For use with
GH3 cells, the coverslips were first coated with CellTak
(14 µg/25 mm coverslip) (Collaborative Biomedical Products, Bedford,
MA). The cells were washed twice with PBS, fixed 20 min at room
temperature in 2% paraformaldehyde, incubated 20 min in blocking
buffer (0.6% Tween 20 in DMEM containing 5% FCS) and then incubated
overnight at 4 C with either 1:250 dilutions of preimmune serum or
anti-PREB, each preadsorbed as described above. The dishes were then
washed three times with PBS and incubated 30 min with rhodamine-labeled
goat antirabbit IgG (American Qualex, LaMiranda, CA) at 1:500 in
blocking buffer, rinsed three times, and mounted in Mowiol. Images were
captured using a 40x oil objective on a Nikon (Melville, NY)
inverted microscope with a Cohu (San Diego, CA) CCD4910 camera
in conjunction with a Colorado video integrator unit. Digital data were
processed with Metamorph software from Universal Imaging (Media,
PA).
Plasmids
We have previously described construction of plasmids
(-1957)PRL-CAT and (-113)PRL-CAT (45, 46). RSV-PKA was prepared by
employing PCR primers to amplify the murine PKA catalytic subunit
described by Uhler and McKnight (47), followed by
HindIII/Xba digestion of the amplified product, and ligation
into the corresponding site in plasmid RcRSV (Invitrogen, San Diego,
CA). RSV-Pit-1 (32) and RSV-ß gal were kindly supplied by Dr. H.
Samuels (New York University). GAL4 constructs pSG424 (48) [referred
to in the present paper as pGAL4(1147)] and 5XGAL4-CAT (49) were
kindly supplied by Dr. M. Ptashne (Sloan-Kettering Institute).
GAL4-PREB was constructed by cloning the PREB coding sequence (amino
acids 1417) upstream of and in register with the GAL4(1147)
sequence in pSG424. RSV-PREB was constructed by cloning the PREB-coding
sequence into the HindIII/XbaI site of RcRSV.
Transient Cotransfection Assays
For each treatment group, approximately 0.5 x
108 GH3 cells or 2 x 106 C6
cells were subjected to electroporation. Preliminary experiments were
performed with PRL-CAT constructs and a known PRL promoter regulator,
Pit-1, to optimize transfection efficiency for each cell line (data not
shown). For transfection, cells were resuspended in 0.75 ml DMEM
containing 10% FBS plus the indicated plasmids and subjected to
electroporation at 960 µFarads and either 300 V (C6 cells) or 240 V
(GH3 cells), divided among three 60-mm tissue culture
dishes, and incubated 48 h as described above for each cell line.
One day after transfection, each dish was examined microscopically, and
any experiment that exhibited gross differences in cell survival among
different treatment groups was discarded. Cells were then harvested
with a rubber policeman, lysed by sonication (four times for 2 min each
time) in 0.25 M Tris, pH 7.8, 10 mM EDTA, and
heated 65 C for 10 min to inactivate deacetylases. Half of each cell
extract was assayed for CAT activity as described previously (12),
employing [3H]chloramphenicol (0.01 µCi/µl) and
butyryl CoA (5 mg/ml) and a 4-h incubation, which yielded results in
the linear range of the assay. The remainder of the cell extract was
employed for assay of ß-galactosidase activity as described (42). For
each experimental condition, the average CAT assay result was divided
by the average ß-galactosidase activity under that condition,
relative to a value of 1 assigned to the average ß-galactosidase
activity in the controls. It may be noted that this procedure always
yielded a correction of
2%. Each experiment reported here has been
repeated a total of at least three times, with results similar to those
shown in the figures.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. S. Aaronson and A. Chan (Mount Sinai School of
Medicine, New York, NY) for generously supplying cDNA samples prepared
from mRNA isolated from various human brain tumors. We also thank Drs.
M. Ptashne (Harvard University, Boston, MA) and H. Samuels (New York
University, NY) for kindly supplying the plasmids indicated in the
text, and Drs. M. Frasch and D. Bishop (Mount Sinai School of Medicine)
for providing, respectively, Drosophila and a sample of
human DNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Carter Bancroft, Department of Physiology and Biophysics, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029. E-mail:
cbancro{at}smtplink.mssm.edu
This work was supported by NIH Grant GM-36847 and GM-056186 (to C.B.)
and NIH Grant DK-19974 and Cancer Center Core Research Grant CA-11098
(to P.M.H.). M.S.F. was supported by NIH Cellular and Molecular
Endocrinology Training Grant DK-07645.
1 Present address: Department of Otolaryngology, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205. 
Received for publication October 19, 1998.
Revision received December 1, 1998.
Accepted for publication December 16, 1998.
 |
REFERENCES
|
---|
-
Andersen B, Rosenfeld MG 1994 Pit-1 determines cell types
during development of the anterior pituitary gland. A model for
transcriptional regulation of cell phenotypes in mammalian
organogenesis. J Biol Chem 269:2933529338[Free Full Text]
-
Okimura Y, Howard PW, Maurer RA 1994 Pit-1 binding sites
mediate transcriptional responses to cyclic adenosine
3',5'-monophosphate through a mechanism that does not require inducible
phosphorylation of Pit-1. Mol Endocrinol 8:15591565[Abstract]
-
Iverson RA, Day KH, dEmden M, Day RN, Maurer RA 1990 Clustered point mutation analysis of the rat prolactin promoter. Mol
Endocrinol 4:15641571[Abstract]
-
Yan G, Bancroft C 1991 Mediation by calcium of
thyrotropin-releasing hormone action on the prolactin promoter via
transcription factor Pit-1. Mol Endocrinol 5:14881497[Abstract]
-
Rajnarayan S, Chiono M, Alexander LM, Gutierrez-Hartmann A 1995 Reconstitution of Protein Kinase A regulation of the rat prolactin
promoter in HeLa nonpituitary cells: identification of both
GHF-1/Pit-1-dependent and -independent mechanisms. Mol Endocrinol 9:502512[Abstract]
-
Tian J, Chen J, Bancroft C 1994 Expression of constitutively
active GS alpha subunits in GH3 pituitary cells
stimulates prolactin promoter activity. J Biol Chem 269:3336[Abstract/Free Full Text]
-
Tian J, Ma HW, Bancroft C 1995 Constitutively active
Gq-alpha stimulates prolactin promoter activity via a
pathway involving Raf activity. Mol Cell Endocrinol 112:249256[CrossRef][Medline]
-
Lew AM, Yao H, Elsholtz HP 1994 Gi
- and
Go
-mediated signalling in the Pit-1-dependent inhibition
of the prolactin gene promoter. J Biol Chem 269:1200712013[Abstract/Free Full Text]
-
Howard PW, Maurer RA 1995 A composite Ets/Pit-1 binding site
in the prolactin gene can mediate transcriptional responses to multiple
signal transduction pathways. J Biol Chem 270:2093020936[Abstract/Free Full Text]
-
Bradford AP, Conrad KE, Tran PH, Ostrowski MC, Gutierrez
Hartmann A 1996 GHF-1/Pit-1 functions as a cell-specific integrator of
Ras signaling by targeting the Ras pathway to a composite Ets-1/GHF-1
response element. J Biol Chem 271:2463924648[Abstract/Free Full Text]
-
Kapiloff MS, Farkash Y, Wegner M, Rosenfeld MG 1991 Variable
effects of phosphorylation of Pit-1 dictated by the DNA response
elements. Science 253:786789[Medline]
-
Fischberg DJ, Chen X-h, Bancroft C 1994 A Pit-1
phosphorylation mutant can mediate both basal and induced prolactin and
growth hormone promoter activity. Mol Endocrinol 8:15661573[Abstract]
-
Howard PW, Maurer RA 1994 Thyrotropin releasing hormone
stimulates transient phosphorylation of the tissue-specific
transcription factor, Pit-1. J Biol Chem 269:2866228669[Abstract/Free Full Text]
-
Voss JW, Wilson L, Rosenfeld MG 1991 POU-domain proteins Pit-1
and Oct-1 interact to form a heteromeric complex and can cooperate to
induce expression of the prolactin promoter. Genes Dev 5:13091320[Abstract]
-
Drolet DW, Scully KM, Simmons DM, Wegner M, Chu KT, Swanson
LW, Rosenfeld MG 1991 TEF, a transcription factor expressed
specifically in the anterior pituitary during embryogenesis, defines a
new class of leucine zipper proteins. Genes Dev 5:17391753[Abstract]
-
Segil N, Roberts SB, Heintz N 1991 Mitotic phosphorylation of
the Oct-1 homeodomain and regulation of Oct-1 DNA binding activity.
Science 254:18141816[Medline]
-
Vinson CR, LaMarco KL, Johnson PF, Landschulz WH, McKnight SL 1988 In situ detection of sequence-specific DNA binding activity
specified by a recombinant bacteriophage. Genes Dev 2:801806[Abstract]
-
Singh H, LeBowitz JH, Baldwin Jr AS, Sharp PA 1988 Molecular
cloning of an enhancer binding protein: isolation by screening of an
expression library with a recognition site DNA. Cell 52:415423[Medline]
-
Bancroft FC 1981 GH cells: functional clonal lines of rat
pituitary tumor cells. In: Sato G (ed) Functionally Differentiated Cell
Lines. Alan R. Liss, New York, p 47
-
Jacobson EM, Peng L, Leon-del-Rio A, Rosenfeld MG, Aggarwal AK 1997 Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected
arrangement and flexibility. Genes Dev 11:198212[Abstract]
-
Coleman DT, Chen X-h, Sassaroli M, Bancroft C 1996 Pituitary
adenylate cyclase-activating polypeptide regulates prolactin promoter
activity via a protein kinase A-mediated pathway that is independent of
the transcriptional pathway employed by thyrotropin-releasing hormone.
Endocrinology 137:12761285[Abstract]
-
van der Voorn L, Ploegh HL 1992 The WD-40 repeat. FEBS Lett 307:131134[CrossRef][Medline]
-
Neer EJ, Schmidt CJ, Nambudripad R, Smith TF 1994 The ancient
regulatory-protein family of WD-repeat proteins. Nature 371:297300[CrossRef][Medline]
-
Sherwood PW, Tsang SV, Osley MA 1993 Characterization of HIR1
and HIR2, two genes required for regulation of histone gene
transcription in Saccharomyces cerevisiae. Mol Cell Biol 13:2838[Abstract]
-
Komachi K, Redd MJ, Johnson AD 1994 The WD repeats of Tup1
interact with the homeo domain protein alpha 2. Genes Dev 8:28572867[Abstract]
-
Mitchell PJ, Tjian R 1989 Transcriptional regulation in
mammalian cells by sequence-specific DNA binding proteins. Science 245:371378[Medline]
-
Madden SL, Cook DM, Morris JF, Gashler A, Sukhatme VP,
Rauscher FJI II 1991 Transcriptional repression mediated by the WT1
Wilms tumor gene product. Science 253:15501553[Medline]
-
Bushmeyer S, Park K, Atchison ML 1995 Characterization of
functional domains within the multifunctional transcription factor,
YY1. J Biol Chem 270:3021330220[Abstract/Free Full Text]
-
Yan G, Chen X, Bancroft C 1994 A constitutively active form of
CREB can activate expression of the rat prolactin promoter in
non-pituitary cells. Mol Cell Endocrinol 101:R25R30
-
Nelson C, Albert VR, Elsholtz HP, Lu LI, Rosenfeld MG 1988 Activation of cell-specific expression of rat growth hormone and
prolactin genes by a common transcription factor. Science 239:14001405[Medline]
-
Mangalam HJ, Albert VR, Ingraham HA, Kapiloff M, Wilson L,
Nelson C, Elsholtz H, Rosenfeld MG 1989 A pituitary POU domain protein,
Pit-1, activates both growth hormone and prolactin promoters
transcriptionally. Genes Dev 3:946958[Abstract]
-
Fox SR, Jong MTC, Casanova J, Fe Z-S, Stanley F, Samuels HH 1990 The homeodomain protein, Pit-1/GHF-1, is capable of binding to and
activating cell-specific elements of both the growth hormone and
prolactin gene promoters. Mol Endocrinol 4:10691080[Abstract]
-
Paroush Z, Finley RLJ, Kidd T, Wainwright SM, Ingham PW, Brent
R, Ish Horowicz D 1994 Groucho is required for Drosophila
neurogenesis, segmentation, and sex determination and interacts
directly with hairy-related bHLH proteins. Cell 79:805815[Medline]
-
Dynlacht BD, Weinzierl RO, Admon A, Tjian R 1993 The dTAFII80
subunit of Drosophila TFIID contains beta-transducin
repeats. Nature 363:176179[CrossRef][Medline]
-
Thomas D, Kuras L, Barbey R, Cherest H, Blaiseau PL, Surdin
Kerjan Y 1995 Met30p, a yeast transcriptional inhibitor that responds
to S-adenosylmethionine, is an essential protein with WD40 repeats. Mol
Cell Biol 15:65266534[Abstract]
-
Deng XW, Matsui M, Wei N, Wagner D, Chu AM, Feldmann KA,
Quail PH 1992 COP1, an Arabidopsis regulatory gene, encodes a protein
with both a zinc-binding motif and a G beta homologous domain. Cell 71:791801[Medline]
-
Kennelly PJ, Krebs EG 1991 Consensus sequences as substrate
specificity determinants for protein kinases and protein phosphatases.
J Biol Chem 266:1555515558[Free Full Text]
-
Xu L, Lavinsky RM, Dasen JS, Flynn SE, McInerney EM, Mullen
T-M, Heinzel T, Szeto D, Korzus E, Kurokawa R, Aggarwal AK, Rose DW,
Glass CK, Rosenfeld MG 1998 Signal-specific co-activator domain
requirements for Pit-1 activation. Nature 395:301306[CrossRef][Medline]
-
Yan G, Pan WT, Bancroft C 1991 Thyrotropin-releasing hormone
action on the prolactin promoter is mediated by the POU protein Pit-1.
Mol Endocrinol 5:535541[Abstract]
-
Coleman DT, Bancroft C 1993 Pituitary adenylate
cyclase-activating polypeptide (PACAP) stimulates prolactin gene
expression in a rat pituitary cell line. Endocrinology 133:27362742[Abstract]
-
Benda P, Lightbody J, Sato G, Levine L, Sweet W 1968 Differentiated rat glial cell strain in tissue culture. Science 161:370371[Medline]
-
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith
JA, Struhl K (eds) 1996 Current Protocols in Molecular Biology.
John Wiley and Sons, Inc., New York
-
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY
-
Lee KA, Bindereif A, Green MR 1988 A small-scale procedure for
preparation of nuclear extracts that support efficient transcription
and pre-mRNA splicing. Gene Anal Tech 5:2231[CrossRef][Medline]
-
Lufkin T, Bancroft C 1987 Identification by cell fusion of
gene sequences that interact with positive trans-acting factors.
Science 237:283286[Medline]
-
Jackson AE, Bancroft C 1988 Proximal upstream flanking
sequences direct calcium regulation of the rat prolactin gene. Mol
Endocrinol 2:11391144[Abstract]
-
Uhler MD, McKnight GS 1987 Expression of cDNAs for two
isoforms of the catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem 262:1520215207[Abstract/Free Full Text]
-
Sadowski I, Ptashne M 1989 A vector for expressing
GAL4(1147) fusions in mammalian cells. Nucleic Acids Res 17:7539[Medline]
-
Carey M, Lin YS, Green MR, Ptashne M 1990 A mechanism for
synergistic activation of a mammalian gene by GAL4 derivatives. Nature 345:361364[CrossRef][Medline]