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INTRODUCTION |
Homeodomain proteins are a family of transcription factors that
are important regulators of gene expression in all eukaryotes (1). The
homeodomain is a 60-amino acid motif that mediates sequence-specific
binding to DNA (2). The homeodomain can also make protein-protein
contacts that modulate the ability of the homeodomain proteins to enter
the nucleus (3), bind DNA (4), and regulate transcription (5, 6). PRH
(proline-rich homeodomain protein)
was first identified in differentiated avian hematopoietic and liver
cells (7) and was subsequently found to be conserved in human,
Xenopus, mouse, and rat where it has also been called XHex
or Hex (8-10). PRH is expressed in the anterior endoderm in developing
Xenopus and mouse embryos (11, 12) and is present in fetal
liver, thyroid, and lung (13). It is a member of the tinman family of
homeodomain-containing proteins that includes several transcription
factors essential for the development of cardiac tissue (14). Within
the hematopoietic compartment PRH mRNA is expressed in B-cells,
myelomonocytic cells, and erythroid cells but not in T-cell lineages
(7, 15), and in general, PRH mRNA levels are down-regulated as
hematopoietic cells differentiate (15, 16). Several studies suggest a
role for PRH in the regulation of cell proliferation and
differentiation. PRH is transiently expressed in vascular endothelial
cells in Xenopus embryos and overexpression of
Xenopus PRH disrupts developing vascular structures and
brings about an increase in the number of these cells (8). In addition,
overexpression of PRH in Myb-Ets transformed multipotential hematopoietic cells inhibits cell growth, whereas the expression of
truncated PRH derivatives alters the ability of these cells to
differentiate (16). Finally, PRH interacts with PML
(promyelocytic leukemia protein), a
growth control protein, in several leukemic cell lines (17) and is
up-regulated in some B-cell leukemias (18).
PRH has recently been reported to repress transcription in liver cells
(10). However, the mechanism or mechanisms whereby this protein brings
about transcriptional repression have yet to be determined.
Transcriptional repression mechanisms can be broadly classified into
four main categories: steric hinderance, quenching, direct repression,
and the modulation of chromatin structure. Many repressor proteins
hinder sterically the binding of transcription activators or general
transcription factors, and this is often referred to as passive
repression. For example, the Drosophila homeodomain protein
Engrailed (En) blocks the action of the activator protein Fushi tarazu
by competing for a common binding site on DNA (19). In vitro
studies have demonstrated that En can also repress transcription by
directly competing with the TATA box-binding protein
(TBP)1 for binding to a TATA
box (20). Similarly, in vitro studies with Even-skipped
(Eve), another Drosophila homeodomain protein, have
suggested that Eve can repress transcription by a mechanism that
involves the cooperative binding of Eve protein to low affinity Eve
binding sites that lie adjacent to TATA box sequences or activator protein binding sites. This results in Eve sterically blocking DNA
binding by TBP or the activator protein Zeste (21, 22). In quenching
repressor proteins mask the activity of locally bound activator
proteins. For example, the Drosophila protein Kruppel can
bind to Sp1 and prevent this protein activating transcription (23). In
addition, repressor proteins can interfere with the targets of the
activators, such as components of the core transcription machinery, a
process known as direct repression. Kruppel has been shown to bind
in vitro to the small subunit of TFIIE (24), and a number of
repressor proteins have been shown to bind to TBP including: Eve (25),
the mouse homeodomain protein Msx-1 (26), the unliganded thyroid
hormone receptor (27), and the global repressor proteins Dr1 (28)
and Mot1 (29).
Several repressor proteins interact with chromatin stabilizing factors
or chromatin assembly complexes. The best understood chromatin
stabilizing proteins are probably the histone deacetylases. Histone
deacetylation is thought to tighten the nucleosome-DNA interaction with
the consequence that the access of transcription factors to their
binding sites is hindered. The mammalian protein YY1 interacts directly
with a number of proteins involved in transcription, including mRPD3, a
histone deacetylase (30). Many other transcriptional repressors
interact with deacetylases indirectly via corepressor proteins. For
example, the Max-Mad repressor complex and the unliganded thyroid
hormone receptor protein interact with mSin3a/mSin3b and the closely
related corepressors N-CoR and SMRT, respectively. These corepressors
then effect the repression of transcription through the
recruitment of histone deacetylases (31, 32).
Several repressor proteins have been shown to use more than one
mechanism to bring about transcriptional repression. For example, the
En homeodomain protein appears to utilize at least four different mechanisms: En can compete with activators (19), compete with TBP for
binding to DNA in vitro (20), interact directly with the
Drosophila corepressor protein Groucho (33, 34), and, in
addition, En contains a repression domain that is separate from the
Groucho interacting region and that works by an as yet unknown
mechanism (35). In this study we examine the ability of PRH to regulate
transcription in hematopoietic cells, we investigate the PRH repression
domains, and we explore the mechanisms whereby PRH might bring about
transcriptional repression.
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MATERIALS AND METHODS |
Plasmid DNAs Used in This Study
Reporters--
The pTK luciferase reporter construct has been
described previously (36). The pTK-GAL luciferase construct was made by
cloning five GAL4 binding sites between the EcoRI and
BamHI sites of pTK. The pTK-PRH luciferase construct was
made by cloning five PRH binding sites identified in site selection
experiments (7) between the HindIII and SmaI
sites of pTK. The HS1-luciferase reporter is described in (37) and was
supplied by Dr. K. Gaston (University of Bristol, Bristol, UK). The
SV40 promoter-luciferase reporter is the pGL-3 promoter vector from
Promega. The transfection control plasmids are pSV-
-galactosidase
control vector (pSV-lacZ) from Promega and the
pRSV-
-galactosidase (pRSV-lacZ), which contains the Rous
sarcoma virus long terminal repeat placed upstream of the
lacZ gene.
Expressors--
pCMV-PRH was made by cloning the 1.5-kilobase
PRH cDNA fragment described in Ref. 7 into the EcoRI
site of pCMV (Promega). pSG424 has been described previously (38). The
N-terminal glutamine-rich Sp1 activation domain (base pairs 1-510) and
full-length HIV-1 TAT (base pair 831-1091 of the HIV genome) were
cloned in frame with GAL4 DBD into the multiple cloning site of pSG424.
These plasmids were kindly supplied by Dr. M. Dickens (University of Leicester, Leicester, UK). pMLV-GAL147plink has been described previously (39). GAL4-VP16 was made by cloning a polymerase chain
reaction fragment encoding the viral VP16 acidic activation domain
(base pairs 1680-1923) in frame between the SalI and
SpeI sites of pMLV-GAL147plink. The plasmid pcDNA3-TLE1
expresses human TLE1 (40) and was kindly supplied by Dr S. Stifani
(McGill University, Montreal, Canada). To construct the pBlueScript-TBP
plasmid used for in vitro transcription and translation
experiments, an EcoRI-HindIII fragment encoding
the TBP cDNA was isolated from the plasmid pGEXKG-TBP (41) kindly
supplied by Dr D. Hornby (University of Sheffield, Sheffield, UK). This
fragment was cloned between the EcoRI and HindIII
sites of pBlueScript II (Stratagene).
GAL4-PRH Fusion Proteins--
The PRH cDNA described in Ref.
7 was cloned into the EcoRI site in pBlueScript (Stratagene)
to create pBSK-PRH16.3. This plasmid was cut with SalI
(which is located 5' to the first PRH ATG codon) and NarI
(98 bp after the ATG), and an oligonucleotide corresponding to the
coding region from the ATG to the NarI site was cloned
between the SalI and NarI sites. A
SmaI-SmaI fragment was then removed from the
3'-noncoding region, and the vector was self-ligated to create
pBSK-PRH18.3. For GAL4-PRH 1-277, a SalI-SpeI
fragment from pBSK-PRH18.3 was cloned into pMLV-GAL147plink between the
SalI and SpeI sites. GAL4-PRH 1-141 was
constructed from pBSK-PRH18.3 by cutting with NotI and
cloning in a linker with a stop codon and a SpeI site to
create pBSK-PRH18.341; the 423-bp SalI-SpeI
fragment was then transferred to pMLV-GAL147plink. For GAL4-PRH
28-141, plasmid GAL4-PRH 1-141 was cut with SalI and
PstI (removing bp 1-84) blunted with T4 polymerase and
religated. For GAL4-PRH 81-141, an AccI fragment was cut
from pBSK-PRH18.341. This corresponds to amino acids (aa) 81-141 and
some pBlueScriptKII vector sequence. The AccI sites were
filled in with Klenow and cloned into the pMLV-GAL147plink
SalI and SpeI sites that had been cut and filled
in by T4 polymerase. This recreates the SalI site, but the
SpeI site is lost. For GAL4-PRH 107-141, GAL4-PRH 1-141
was cut with SalI (aa 1) and filled in and then cut with ApaI (aa 105), trimmed back with T4 polymerase, and
religated. Thus, the 1-107 SalI to ApaI fragment
was removed, leaving the 107-141 PRH fragment and the vector on
religation recreated the 5'SalI site. For GAL4-PRH 1-105,
an ApaI fragment from pBSK-PRH18.3 was cloned into the
SmaI site of pBSK to make pBSK-PRH18.305. The
ApaI fragment contained the 5'SalI site and aa
1-105 of PRH. The 315-bp SalI-SpeI PRH fragment
from this vector was cloned into pMLV-GAL147plink. For GAL4-PRH
28-105, pBSK-PRH18.305 was cut with PstI and
SalI, blunted with T4 polymerase, and religated to itself,
thus removing aa 1-28 of PRH and the 5'SalI site to create
pBSK-PRH18.3205. An XhoI-SpeI PRH fragment from
this vector was cloned into pMLV-GAL147plink. The
XhoI-SpeI PRH fragment has three extra codons 5'
to aa 1 in PRH, so GAL4-PRH 28-105 contains three extra codons between
the GAL4 DBD and aa 1 of PRH. For GAL4-PRH 1-125, an ApaI
(aa 105-141)-SpeI fragment was removed from GAL4-PRH 1-141
leaving aa 1-105. An oligonucleotide linker coding for amino acids
105-125 (from the ApaI site) and including a
SpeI site was cloned into this plasmid. For GAL4-PRH
107-277, the ApaI SpeI 510-bp PRH fragment from
pBSK-PRH18.3 was isolated after the ApaI site was blunted
with T4 polymerase. This fragment was cloned into the SalI
SpeI site of pMLV-GAL147plink after the SalI site in the vector was filled in with Klenow. This creates GAL4-PRH 107-277
and recreates the SalI site at the 5' end. For GAL4-PRH 49-141, GAL4-PRH 143-277, GAL4-PRH 143-210, and GAL4-PRH 202-277 were made by polymerase chain reaction from GAL4-PRH 1-277 using 5'
oligonucleotides carrying a SalI site and a 3'
oligonucleotides carrying a SpeI site. The polymerase chain
reaction fragments were cloned into the SalI and
SpeI sites of pMLV-GAL147plink: 49-56,
5'-GTCGACCCGGCCCCCCACTCCCTGCCCGCC-3'; 61-65,
5'-GTCGACACGCTGCCGTCGCCCAAC-3'; 143-148,
5'-GTCGACAAGAGGAAGGGTGGCCAG-3'; 202- 208, 5'-GTCGACCTGAAGCAGGGAGAACCCCCAGG-3'; 125-120,
5'-ACTAGTGTCCTGGCGGATCAGTGC-3'; 141-136,
5'-ACTAGTCAGCGGCCGCTGGATGA-3'; 210-204,
5'-ACTAGTGGTGGCCTGGGGGTTCTCCTG-3'; and 277-270,
5'-ACTAGTGCGTGTGGCGCTGTAGAAGCCTTT-3'.
Bacterial Expression Plasmids--
The histidine-tagged PRH
expression vectors were created by cloning the
NotI-EcoRI fragment from pBSK-PRH16.3 containing
sequences encoding the PRH homeodomain and C terminus, together with an XhoI-NotI linker, between the XhoI and
EcoRI sites of pTrcHisA (Invitrogen) to create pTrcHisA-PRH
137-277. A NotI-SmaI PRH fragment carrying the
N194A mutation described below was transferred from the
pBSK-PRHHDM 18.3 clone into pTrcHisA-PRH 137-277,
replacing the wild type PRH sequence and creating
pTrcHisA-PRHHDM 137-277.
The GST-PRH 1-141 expression vector was created by cloning the DNA
sequence encoding the PRH N terminus (amino acids 1-141), as a
SalI-SpeI fragment from pGAL4-PRH 1-141 into
pGEX20T that had been cut with XhoI and SpeI,
creating pGEX20T-PRH 1-141. pGEX20T is a derivative of pGEX2T
(Amersham Pharmacia Biotech) and contains unique XhoI and
SpeI restriction sites in the polylinker downstream of the
GST moiety. The GST-tagged human PRH N terminus was a gift from Dr. G. Manfioletti (University of Trieste, Trieste, Italy). Briefly, the human
PRH N terminus (amino acids 1-131) was cloned as an EcoRI
fragment into pGEX3X (Amersham Pharmacia Biotech). All constructs were
checked by DNA sequencing.
Cell Culture and Transient Transfection Assays
BM2 cells were grown in RPMI 1640 (Life Technologies, Inc.) 25 mM HEPES medium supplemented with 10% tryptose phosphate,
10% glutamine, 10% fetal calf serum, and 5% chicken serum to a
density of ~1 × 106 cells/ml. The cells were
collected by centrifugation and then resuspended in RPMI 1640 25 mM HEPES plus 10% fetal calf serum to a density of 5 × 107 cells/ml. 1 × 107 cells plus 5 µg each of the luciferase and lacZ reporter plasmids and the amount
of expressor plasmid indicated under "Results" were electroporated
using a Bio-Rad Genepulser (0.25 V, 960 microfarads). Cells were rested
for 10 min and then incubated overnight in 10 ml of supplemented
medium. After 24 h the cells were harvested, and luciferase
activity was assayed using the Promega luciferase assay system
according to the manufacturer's instructions.
-Galactosidase assays
were performed as an internal control for transfection efficiency.
After subtraction of background, the luciferase counts were normalized
against the
-galactosidase value. QT6 cells were grown in
Dulbecco's modified Eagle medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum and 2% chicken serum. 3 × 106 QT6 cells were transfected with 5 µg of each of
the reporter plasmids and 1 µg of expressor plasmid by calcium
phosphate precipitation. Cell extracts were made 18 h after
transfection as described for BM2 cells. At least two independent
batches of expressor and reporter constructs were assayed in multiple
experiments, and the results were averaged.
Site-directed Mutagenesis
Mutagenesis of PRH asparagine 194 to alanine was carried out in
pBSK-PRH18.3 using a mutant oligonucleotide:
5'-AAAACGTGGTTCCAGGCCCGCAGAGCCAAATG-3' and a QuikChange
mutagenesis kit (Stratagene) according to the manufacturer's
instructions. The underlined positions mismatch the template and
introduce the desired sequence change. After sequencing the mutated
clone (pBSK-PRHHDM 18.3), a NotI-SpeI
PRH fragment carrying the mutation was transferred from
pBSK-PRHHDM 18.3 into pMLV-GAL147plink-PRH (GAL4-PRH),
replacing the wild type PRH sequence and creating
pGAL4-PRHHDM.
Proteins Used in This Study
Histidine-tagged fusion proteins were purified from bacterial
lysate by chromatography on a nickel (Ni2+-NTA-agarose)
column (Qiagen) essentially according to the manufacturer's instructions. The eluted proteins were assayed for purity by SDS-PAGE followed by staining with Coomassie Blue and quantified using the
Bio-Rad phosphoric acid protein assay. GST fusion proteins were
purified over glutathione-Sepharose 4B beads (Sigma) and assayed for
purity and concentration as above.
Circular Dichroism
Circular dichroism spectra of pTrcHisA-PRH 137-277 and
pTrcHisA-PRHHDM 137-277 proteins (3.3 and 6.4 µM, respectively, in 25 mM phosphate buffer,
pH 7.9) were obtained using a JY CD6 CD spectrometer with
5-mm-pathlength cells. Spectra were collected at 1-nm increments, using
a 20-s integration time. The spectra have been corrected for
concentration differences.
Electrophoretic Mobility Shift Assays
Single stranded oligonucleotides (100 ng) were 5'-end labeled
with [
32P]ATP using T4 polynucleotide kinase. After
annealing to the complementary oligonucleotide free label was removed
using Sephadex G50 spin columns. Labeled oligonucleotides (20,000 cpm)
were incubated with purified proteins in the quantities indicated in
the figures in binding buffer (10% glycerol, 1 mM
MgCl2, 20 mM Tris, pH 8.0, 1 mM
dithiothreitol, 50 mM NaCl, 0.04 mg/ml dI-dC, 0.1 mg/ml
bovine serum albumin). After 30 min on ice, complexes were resolved on 6% nondenaturing polyacrylamide gels run in 1× TBE and visualized using a PhosphorImager.
In Vitro Binding Assays
Transcription and translation was carried out using a TNT kit
(Promega) according to the manufacturer's protocol. Approximately 50-100 µg of affinity-purified histidine-tagged fusion protein (PRH
137-277 or PRHHDM 137-277) was bound to 100 µl of
nickel-NTA beads (Qiagen) under the conditions recommended in the
manufacturer's protocol. To assay for specific interactions 10 µl of
[35S]methionine-labeled in vitro translated
TBP was added and incubated in binding buffer (20 mM HEPES,
pH 7.8, 200 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 0.5% Nonidet P-40, 50 ng/µl bovine
serum albumin) with gentle agitation for 60 min at 4 °C. The beads
were washed six times with 1 ml of binding buffer. Bound proteins were eluted with elution buffer (250 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4)
and analyzed by SDS-PAGE and fluorography.
Western Blotting
QT6 cells were harvested after transient transfection and lysed
in luciferase assay lysis buffer (Promega). The supernatant (which
contains no detectable signal with antibodies against GAL4 1-147) was
removed and used for 
galactosidase and luciferase assays. The
chromatin pellet was resuspended in 20 µl of 100 mM HEPES, pH 7.9, 5 mM MgCl2, and 1 mM
CaCl2 containing sufficient DNase I to digest the sample in
~5 min at 20 °C. After the DNase I digestion, 20 µl of 2× SDS
loading buffer was added, and the sample was boiled for 2 min. A
proportional amount of each sample was separated by SDS-PAGE and
blotted onto an Immobilon membrane. GAL4 fusion proteins were then
detected using a mixture of two monoclonal antibodies 2GV3 and 3GV2 (a
gift from Professor P. Chambon, (42) and an ECL kit (Amersham
Pharmacia Biotech).
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RESULTS |
PRH Is a Repressor of Transcription in Hematopoietic
Cells--
The PRH protein consists of three regions (Fig.
1A). The N-terminal region (aa
1-143) is 20% rich in proline residues and 13% rich in alanine
residues and is separated by the homeodomain (aa 144-203) from a
C-terminal region (aa 204-277) 25% rich in acidic residues. Both
proline/alanine rich regions and acidic regions are commonly found in
the regulatory domains of transcription factors. The PRH cDNA was
originally cloned from an avian cDNA library derived from BM2
cells, an avian myeloblastosis virus-transformed hematopoietic
cell line (7). To investigate whether PRH functions as a transcription
factor in BM2 cells, we performed transient cotransfection assays.

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Fig. 1.
PRH is a repressor of transcription.
A, the top line is a schematic representation of
avian PRH. The second and third lines are
schematic representations of the reporter plasmids pTK and pTK-PRH,
respectively. The reporter plasmids contain the minimal TK promoter
(from 109 to +52) located upstream of the luciferase gene. pTK-PRH
contains five PRH binding sites upstream of the TK promoter.
B, the graph shows the relative promoter activity
found in BM2 cell extracts 24 h after transient cotransfection
with increasing amounts of the PRH expression plasmid pCMV-PRH, 5 µg
of the pTK-PRH reporter plasmid, and 10 µg of the -galactosidase
expression plasmid pSV-lacZ. The amount of PRH expression
plasmid added is shown on a logarithimic scale. Relative promoter
activity is the ratio of luciferase activity in the presence of
pCMV-PRH to luciferase activity in the presence of an equal amount of
the empty pCMV vector. The luciferase values were normalized with
respect to transfection efficiency using the cotransfected
-galactosidase plasmid. Each transfection was performed a minimum of
three times, and the values shown represent the means and S.D.
C, BM2 cells were transiently cotransfected with increasing
amounts of pCMV-PRH, 5 µg of the pTK reporter plasmid, and 10 µg of
pSV-lacZ. Details are as in B.
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Previous site selection experiments using a truncated PRH protein
(consisting of the PRH homeodomain and C terminus) determined the
consensus PRH binding site to be 5'-(C/T)(A/T)ATT(A/G)-3' (7). Five
copies of a PRH binding site identified in these experiments were
placed upstream of the luciferase gene under the control of the
thymidine kinase minimal promoter (TK) to create the reporter plasmid
pTK-PRH (Fig. 1A). Plasmid pCMV-PRH expresses PRH under the
control of the cytomegalovirus promoter. This expression plasmid was
transiently cotransfected into BM2 cells along with pTK-PRH. The effect
of pCMV-PRH on luciferase activity was compared with the effect of the
empty expression vector pCMV. To normalize for transfection efficiency,
a constant amount of a control plasmid containing the lacZ
gene under the control of the SV40 promoter and enhancer was also
cotransfected. As can be seen from the data shown in Fig.
1B, increasing amounts of the pCMV-PRH plasmid bring about a
dramatic decline in luciferase activity. As little as 10 ng of the PRH
expression plasmid is sufficient to bring about a 50% decrease in TK
promoter activity, suggesting that PRH is a potent repressor of transcription.
A number of homeodomain proteins have been shown to repress
transcription even in the absence of their cognate binding sites. To
determine whether repression of the pTK-PRH promoter by PRH is
dependent upon the presence of PRH binding sites, we cotransfected pCMV-PRH with the pTK reporter lacking PRH binding sites. Increasing amounts of pCMV-PRH bring about increasing repression of this reporter
(Fig. 1C). However, in this case a 10-fold higher amount of
pCMV-PRH (100 ng) is required to bring about a 50% repression of
promoter activity. Thus, PRH is also capable of repressing transcription in the absence of PRH binding sites.
PRH Represses Activated Transcription--
In the experiments
described above PRH was able to repress transcription from a TK
promoter lacking PRH binding sites. To establish whether PRH could
repress activated transcription from this promoter, five copies of a
GAL4 binding site were inserted upstream of the minimal TK promoter to
create pTK-GAL (Fig. 2A). Plasmid pSG424 carries the yeast GAL4 DBD and was used to express a
number of activator-GAL4 fusion proteins (38). Transcription activation
domains from the Sp1 and HIV-1 TAT proteins were fused in frame with
the GAL4 DBD present in pSG424. The HSV-1 VP16 acidic activation domain
was also expressed as a GAL4 DBD fusion using the expression vector
pMLV-GAL147plink (Fig. 2B). Between 50 and 200 ng of each
expression plasmid or equivalent amounts of the empty vectors were
transiently cotransfected into BM2 cells along with the pTK-GAL
reporter (Fig. 2C). In each case plasmids expressing the
activator-GAL4 fusion proteins increase luciferase activity. Activation
levels range between 6- and 85-fold above basal TK-GAL promoter
activity depending on the activator (Fig. 2C). Increasing amounts of pCMV-PRH were then cotransfected into BM2 cells along with
each activator-GAL4 expression plasmid. In each case, PRH is able to
repress activated transcription (Fig. 2C). Because each
fusion protein contains a different class of activation domain (Sp1 is
glutamine-rich, VP16 is acidic, and TAT does not contain a predominance
of a single kind of amino acid), these data suggest that binding
site-independent repression by PRH is not activator-specific.

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Fig. 2.
PRH represses activated transcription.
A, a schematic representation of the pTK-GAL reporter
plasmid. Five GAL4 binding sites were placed upstream of the TK
promoter in plasmid pTK to create pTK-GAL. B, a schematic
representation of the GAL4-activator fusion proteins used in this
study. Activation domains from VP16, Sp1, and HIV TAT were cloned in
frame with the GAL4 DBD (for details see "Materials and Methods").
C, BM2 cells were transiently cotransfected with increasing
amounts of pCMV-PRH, 5 µg of the pTK-GAL reporter plasmid, 10 µg of
pSV-lacZ, and either GAL4-VP16 (100 ng), GAL4-Sp1 (50 ng),
GAL4-TAT (200 ng), or GAL4 DBD (200 ng). Relative promoter activity is
shown on a logarithmic scale. All other details are as described in
Fig. 1B.
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PRH Strongly Represses TATA Box-dependent
Promoters--
In vitro the homeodomain protein En can bind
directly to the TATA box and block the binding of TBP (20). To
investigate whether the TATA box is an important element in binding
site-independent transcriptional repression by PRH, we examined the
effect of PRH on transcription from the two promoters shown in Fig.
3A. The enhancer-less SV40
early promoter consists of a TATA box and six Sp1 binding sites (43)
and is present upstream of the luciferase gene in the pGL-2 Promoter
vector (Promega). The human Surf-1 promoter (HS-1) is a TATA-less
housekeeping promoter, which consists of an Sp1 binding site, two
binding sites for members of the ETS family of transcription factors,
and a YY1 binding site and is present upstream of the luciferase gene
in the pGL2-Basic vector (37). These two reporters were transiently
transfected into BM2 cells along with increasing amounts of pCMV-PRH
(Fig. 3, B and C, respectively). As in the case
of the TATA box containing TK promoter, 100 ng of pCMV-PRH is
sufficient to bring about a 50% repression of SV40 promoter activity.
In contrast, the same amount of pCMV-PRH has no effect on the TATA-less
HS1 promoter and even 50-100-fold higher amounts of pCMV-PRH bring
about only partial repression. Taken together with the data shown in
Fig. 1, these experiments show that PRH is capable of strongly
repressing two different TATA box containing promoters but is very
inefficient at repressing a promoter that lacks a TATA box. These
results suggest that the sensitivity of the TK and SV40 promoters to
repression by PRH might be due to the TATA element.

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Fig. 3.
PRH strongly represses TATA box dependent
promoters. A, the top line is a schematic
representation of the pGL-2 Promoter vector (Promega). This reporter
plasmid contains the SV40 promoter upstream of the luciferase gene. The
bottom line is a schematic representation of the HS1
reporter plasmid. This construct contains sequences from the HS1
promoter (from 180 to +20) cloned upstream of the luciferase gene in
pGL-2 basic. B, BM2 cells were transiently cotransfected
with increasing amounts of pCMV-PRH, 5 µg of the pGL-2 Promoter
vector (SV40), and 10 µg of the -galactosidase expression plasmid
pRSV-lacZ. Details are as in Fig. 1B.
C, BM2 cells were transiently cotransfected with increasing
amounts of pCMV-PRH, 5 µg of the HS1 reporter plasmid, and 10 µg of
pRSV-lacZ. Details are as in Fig. 1B.
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PRH Binds to the TK TATA Box--
An alignment of the consensus
PRH binding site with the TK and SV40 TATA boxes shows that these
sequences are very similar (Fig.
4A). The TK and SV40 TATA
boxes deviate from the core consensus PRH binding site at 1 of 7 and at
2 of 7 positions, respectively. To determine whether PRH is capable of
binding to the TK TATA box, we expressed a histidine-tagged fragment of
PRH comprising the homeodomain and C-terminal region (amino acids
137-277) in bacterial cells. The histidine-tagged PRH137-277 protein
was purified by chromatography over a nickel column (see "Materials
and Methods"). Fig. 4B shows the results of an
electrophoretic mobility shift assay in which PRH137-277 was added to
labeled DNA carrying the TK TATA box. After 30 min at 4 °C,
protein-DNA complexes were separated from free DNA by electrophoresis
on a 6% nondenaturing polyacrylamide gel and visualized by
autoradiography. The addition of PRH results in the formation of a
protein-DNA complex (Fig. 4B, lane 1). To
investigate the specificity of this complex, we added competitor
oligonucleotides to the binding reaction. Addition of an unlabeled
oligonucleotide carrying the TK TATA box abolishes the binding of PRH
to the labeled DNA (Fig. 4B, lanes 2 and
3). In contrast, addition of an unlabeled oligonucleotide
carrying an unrelated DNA sequence did not compete away the PRH-TATA
box complex (Fig. 4B, lanes 4 and 5).
These data show that PRH is capable of binding to the TK TATA box and
is thus capable of binding to the TK promoter even in the absence of
upstream PRH binding sites.

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Fig. 4.
PRH binds to the TK TATA box.
A, an alignment of the core consensus PRH binding site
(top line) with the TATA boxes from the TK promoter
(middle line) and SV40 promoter (bottom line).
B, a labeled oligonucleotide carrying the TK TATA box was
incubated with histidine-tagged PRH137-277 under the conditions
described in the text. Free and bound DNA was resolved on a 6%
polyacrylamide gel and visualized using a PhosphorImager. The addition
of 100 ng PRH results in the formation of a retarded complex
(PRH-DNAC). The complex was competed away by the addition
of 500 ng or 1000 ng of unlabeled TK TATA box (lanes 2 and
3, respectively) but not by equal amounts of an unrelated
oligonucleotide carrying an HPV 16 E2 binding site (lanes 4 and 5, respectively).
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The Homeodomain Mediates PRH Binding Site-independent
Repression--
The experiments described above strongly implicate the
homeodomain in the ability of PRH to repress transcription from the TK
promoter in the absence of upstream PRH binding sites. To determine whether the binding of PRH to the TATA box brings about transcriptional repression, the homeodomain of PRH was mutated to abrogate its DNA
binding activity. One of the invariant amino acids in all homeodomains
is asparagine 51. This amino acid in the En homeodomain makes bidentate
hydrogen bonds to adenine 13 of the En binding site, and this contact
is crucial for DNA binding (44). The equivalent residue within the PRH
homeodomain (asparagine 194) was mutated to an alanine using
site-directed mutagenesis. To confirm that the N194A
mutation blocks DNA binding, we first introduced this mutation into the
histidine-tagged PRH137-277 protein. The resulting histidine-tagged
PRHHDM137-277 protein was expressed in bacteria and
purified exactly as described above (Fig.
5A). CD was used to
determine whether the N194A mutation altered the folding of the
PRHHDM137-277 protein. The CD spectra for PRH137-277 and
PRHHDM137-277 are very similar, implying that the mutation has no effect on protein folding or stability (Fig. 5B).
Increasing amounts of PRH137-277 and PRHHDM137-277 were
added to labeled DNA carrying the TK TATA box, and DNA binding activity
was assayed as described above. The PRHHDM137-277 protein
shows little DNA binding activity (Fig. 5C, lanes
2-5), whereas at equal protein concentrations, PRH137-277 binds
tightly to the labeled fragment (Fig. 5C, lanes
7-10).

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Fig. 5.
The homeodomain mediates repression in the
absence of upstream PRH binding sites. A,
histidine-tagged PRH 137-277 (WT) and
histidine-tagged PRHHDM 137-277 containing the
N194A mutation (HDM) were purified over an
Ni2+-NTA-agarose column and the eluted proteins analyzed by
SDS-PAGE. M, markers; RM, rainbow markers. B, circular
dichroism was used to determine whether the presence of the N194A
mutation affected the folding or stability of PRHHDM
137-277. C, a labeled oligonucleotide carrying the TK TATA
box was incubated with 16, 80, 400, or 2000 ng of histidine-tagged
PRHHDM 137-277 (lanes 2-5) or equal amounts of
the histidine-tagged PRH137-277 (lanes 7-10) under the
conditions described in the text. Free and bound DNA was resolved on a
6% polyacrylamide gel and visualized using a PhosphorImager.
D BM2 cells were transiently cotransfected with increasing
amounts of pGAL-PRH (empty circles) or
pGAL-PRHHDM (filled circles), along with 5 µg
of the pTK reporter plasmid and 10 µg of pSV-lacZ. Other
details are as in Fig. 1B. E, the experiment
shown in D was repeated using the pTK-GAL reporter plasmid.
F, in vitro transcribed and translated TBP
(lane 1) was incubated with Ni2+-NTA-agarose
beads carrying histidine-tagged PRH 137-277 (lane 2),
Ni2+-NTA-agarose carrying histidine-tagged
PRHHDM 137-277 (lane 3), or
Ni2+-NTA-agarose beads alone (lane 4) under the
conditions described in the text. After extensive washes, bound TBP was
removed from the beads using imidazole, run on an SDS-PAGE gel, and
visualized using a PhosphorImager.
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To determine the effects of the N194A mutation on the ability of PRH to
repress transcription from the TK promoter, this mutation was
introduced into the full-length PRH cDNA in the context of a
GAL4-PRH fusion. The resulting GAL4-PRHHDM and GAL4-PRH
expression plasmids were cotransfected into BM2 cells along with the TK
reporter. The effects of GAL4-PRHHDM and GAL4-PRH on
transcription were compared with the effect of the GAL4 DBD alone, and
transfection efficiency was measured as before. GAL4-PRH brings about a
significant repression of transcription from the TK promoter when as
little as 50 ng of expression plasmid is cotransfected (Fig.
5D). In contrast, equivalent amounts of the
GAL4-PRHHDM expression plasmid have little or no effect on
the TK promoter (Fig. 5D). However, with higher amounts of
cotransfected GAL4-PRHHDM expression plasmid (100 ng), TK
promoter activity is partially repressed. These data are consistent
with the idea that PRH represses transcription at the TK promoter by
binding to the TK TATA box. It is possible that the transcriptional
repression seen in the presence of high levels of the
GAL4-PRHHDM expression plasmid results from the low level
of TATA box binding activity shown by the mutated homeodomain (Fig.
5C, lane 5).
To determine whether binding to the TATA box is the only mechanism
whereby PRH can repress transcription, we next looked at the ability of
the GAL4-PRHHDM protein to repress transcription when
tethered upstream of the TATA box at GAL4 binding sites. The GAL4-PRH
and GAL4-PRHHDM expression plasmids were cotransfected into
BM2 cells along with the TK-GAL reporter, and promoter activity was
assayed as before. As can be seen from the data shown in Fig. 5E, there is little if any difference in the ability of
these proteins to repress the TK-GAL promoter. Therefore, PRH must
repress transcription using at least two mechanisms, one of which is
dependent upon the DNA binding activity of the homeodomain and one of
which is independent of the homeodomain-DNA interaction. In addition, because both of these GAL4 fusion proteins repress the TK-GAL promoter
equally, this experiment shows that the wild type protein and the
mutated protein are probably expressed at equivalent levels.
The Eve homeodomain is involved in binding to TBP (45). To determine
whether the PRH homeodomain can interact with TBP, we performed
in vitro binding assays. The PRH137-277 and
PRHHDM137-277 proteins described above were incubated with
in vitro transcribed and translated TBP. Labeled TBP binds
strongly to beads coated with either PRH137-277 or
PRHHDM137-277 but binds only very weakly to uncoated beads
(Fig. 5F). Thus, PRH can bind directly to the TATA box and
can also bind TBP. Furthermore, although the N194A mutation
significantly reduces the DNA binding activity of the PRH homeodomain,
this mutation has no effect on the ability of PRH137-277 to bind TBP.
The ability of PRHHDM137-277 to bind TBP might be another
explanation for the transcriptional repression observed when high
amounts of the GAL4-PRHHDM expression plasmid are
cotransfected with the TK promoter (Fig. 5D).
Mapping the PRH Repression Domains--
The N194A mutation
abolishes the DNA binding activity of the PRH homeodomain and
significantly reduces the ability of PRH to repress transcription from
the TK promoter. However, this mutation does not affect the ability of
PRH to repress transcription when tethered upstream of the TK promoter
by the GAL4 DNA-binding domain. This suggests that when bound upstream
of the promoter, PRH might repress transcription via protein-protein
interactions with TBP and/or via homeodomain-independent mechanisms. To
delineate the regions of PRH that function as repression domains, a
number of deletions mutants of PRH were assayed for their ability to
repress transcription in transient transfection experiments. The
GAL4-PRH expression plasmid described above was cotransfected into BM2 cells along with either the TK or TK-GAL reporter plasmids. The addition of 1 µg of the GAL4-PRH expression plasmid brings about 100% repression of the TK-GAL promoter, and 95% repression of the TK
promoter (Fig. 6, first line).
Because 1 µg of GAL4-PRH is sufficient to give almost complete
repression of both the TK and TK-GAL promoters, this amount of
expressor plasmid was chosen for all subsequent transfections. A series
of deletion mutants of PRH were placed in frame with the GAL4 DBD in
pMLV-GAL147plink. As can be seen from the data (Fig. 6, second
line), the N-terminal 141 amino acids of PRH (which excludes the
PRH homeodomain) are sufficient to bring about full repression of the
TK-GAL promoter. However, this fusion protein does not repress the TK
promoter. Thus, PRH contains a proline-rich N-terminal repression
domain that functions when tethered to a promoter either via the PRH homeodomain or via the GAL4 DBD. The C-terminal 134 amino acids of PRH
(aa 143-277), which includes the PRH homeodomain, brings about full
repression of both TK reporters (Fig. 6, fourth line), and a
further deletion of amino acids 210-277 shows that the PRH homeodomain
alone is sufficient to repress transcription from the TK promoter (Fig.
6, fifth line). In contrast, the C-terminal amino acids from
202-277 only weakly repress transcription (Fig. 6, sixth
line). These data confirm that the PRH homeodomain is responsible
for the repression of the TK promoter seen in the absence of upstream
PRH binding sites. The C-terminal acidic region of PRH might contribute
to repression by the homeodomain (Fig. 6, compare fourth and
fifth lines); however, the increased repression seen in the
presence of this region might be a consequence of increased protein
expression and/or stability.

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Fig. 6.
PRH contains at least two independent
transcriptional repression domains. A series of GAL4-PRH fusion
proteins were assayed for their ability to repress transcription from
the pTK reporter (solid bars) and the pTK-GAL reporter
(empty bars). BM2 cells were transiently cotransfected with
1 µg of each GAL4-PRH expression plasmid, 5 µg of the each reporter
plasmid, and 10 µg of pSV-lacZ. The difference between TK
promoter activity in the presence of the GAL4 DBD alone and TK promoter
activity in the presence of full-length PRH (PRH1-277) fused to the
GAL4 DBD was taken to represent 100% repression. All other details are
as described in Fig. 1B. NA, not assayed.
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Deletion Analysis of the Proline-rich Repression Domain--
The
proline-rich N-terminal 141 amino acids of PRH function as an
independent transferable repression domain. To investigate this
repression domain, a series of deletion fragments from the PRH N
terminus were fused in frame with the GAL4 DBD. The proline-rich domain
was deleted in ~20-amino acid intervals from the N terminus to amino
acid 107. Because we were unable to detect protein expression levels
for any of these constructs in BM2 cells (data not shown), we
transiently transfected these constructs into QT6 cells, a quail
fibroblast cell line. We first established that the PRH N terminus can
repress transcription in quail fibroblasts and is detectable by Western
experiments (Fig. 7, A,
first line, and C, first track). This
suggests that the proline-rich repression domain is functional in these
cells and that there are no essential species-specific or cell-type
requirements for the activity of this domain. Interestingly none of the
deletions that remove N-terminal amino acids greatly affect repression
(Fig. 7A). Although the GAL4-PRH 81-141 and GAL4-PRH
107-141 constructs appear to repress somewhat less well than the
GAL4-PRH 1-141 construct, the former are expressed at virtually
undetectable levels (Fig. 7C, fourth and
fifth tracks, compared with first track).
However, both GAL4-PRH 81-141 and GAL4-PRH 107-141 still bring about
at least 50% repression of the TK-GAL promoter (Fig. 7A,
fourth and fifth lines). Thus, the 34 amino acids
from 107 to 141 can function as a repression domain.

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Fig. 7.
Fine mapping of the PRH N-terminal repression
domain. A, defining the N-terminal boundary of the
proline-rich repression domain. The series of GAL4-PRH fusion proteins
shown in the figure were assayed for their ability to repress
transcription from the pTK-GAL reporter. QT6 cells were transiently
cotransfected with 1 µg of each GAL4-PRH expression plasmid, 5 µg
of pTK-GAL, and 5 µg of pSV-lacZ. The percentage
repression was determined as described in Fig. 6. B,
defining the C-terminal boundary of the proline-rich repression domain.
The series of GAL4-PRH fusion proteins shown in the figure were assayed
for their ability to repress transcription from the pTK-GAL reporter
exactly as described in A. C, a Western blot of
the GAL4-PRH N-terminal fusion proteins after transient transfection
into QT6 cells. Proteins were probed with a monoclonal antibody against
the GAL4 DBD, and specific antibody binding was detected by ECL. The
bracket marks the positions of the fusion proteins. The
other bands are endogenous proteins that cross-react with the
antibody.
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Fig. 7B shows the effect of deletions into the C terminus of
the proline-rich repression domain. Deletion of the C-terminal 16 amino
acids to create GAL4-PRH 1-125 has a minor effect on repression (Fig.
7B, second line). A further deletion of 20 amino acids to produce GAL4-PRH 1-105 completely abolishes repression (Fig.
7B, third line). However, although the GAL4-PRH
1-105 construct completely fails to repress transcription, it is
expressed in QT6 fibroblasts at a much lower level than GAL4-PRH 1-125
(Fig. 7C). Although these data suggest that amino acids
105-141 are essential for repression, two further deletion constructs
indicate that this is not the case. PRH derivatives GAL4-PRH 28-141
and GAL4-PRH 28-105 both strongly repress transcription and are both strongly expressed in these cells (Fig. 7B,
fourth and fifth lines). Thus the 77 amino acids
from 28 to 105 can also function as a repression domain. One possible
explanation for the lack of repression by GAL4-PRH 1-105 is that
although this construct is expressed, (albeit at low level), it may be
misfolded and either no longer interacts or interacts aberrantly with
the transcription apparatus. Taken together with the experiments
described in Fig. 7A, these data suggest that the N-terminal
repression domain is composed of at least two elements that can
independently bring about repression. The first element lies within the
region 28-105 of PRH, and the second element lies within the region
107-141. Thus, the PRH proline-rich repression domain is not composed
of a single discrete region that is essential for transcriptional
repression but may instead be composed of multiple regions that are
independently capable of transcriptional repression.
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DISCUSSION |
Using a series of truncated proteins, we have shown that PRH
contains two independently acting transcription repression domains. One
repression domain consists of the PRH homeodomain, whereas the other
consists of the proline-rich N-terminal region of the protein. The
homeodomain proteins Eve and Msx-1 can bind directly to TBP, and these
interactions are probably important for transcriptional repression (25,
26, 45). Although PRH is similar to Eve in that it can also bind
directly to both TBP and DNA, the mechanism of repression appears to be
different. Mutations in the Eve homeodomain that block binding to DNA
have no effect on the binding of this protein to TBP and do not prevent
Eve from repressing transcription (45). In direct contrast, a mutation
in the PRH homeodomain that blocks binding to DNA has no effect on
the binding of this protein to TBP but significantly reduces
transcriptional repression. These data suggest that PRH might repress
transcription by binding to the TATA box and sterically hinder the
binding of TBP. This mechanism of repression has been observed
previously in vitro for the En protein (20). However, this
is the first strong evidence to suggest that a homeodomain protein can
regulate transcription in intact cells using this passive repression
mechanism. Several observations support this conclusion. First,
repression by the full-length PRH protein does not require the presence
of PRH binding sites upstream of the target promoter. Second, PRH can
bind to an oligonucleotide carrying the TK TATA box in
vitro. Third, PRH is capable of repressing transcription from TATA
box containing promoters but is very inefficient at repressing
transcription from a TATA-less promoter. Fourth, the PRH homeodomain
alone is able to repress transcription in vivo.
Interestingly, although En and PRH seem to use a similar mechanism for
the repression of transcription, PRH unlike En, can interact directly
with TBP. To determine whether TBP and PRH can form a tripartite
complex with DNA, we carried out electrophoretic mobility shift assay with both proteins. However, TBP was unable to bind to the TK TATA box
under conditions that allowed the strong binding of PRH. Thus, the
functional significance of the TBP-PRH interaction could not be
assessed using this method. The possibility remains, therefore, that in
addition to the passive repression mechanism observed at this promoter,
under some circumstances the PRH homeodomain might also repress by
direct repression of the basal transcription complex by interaction
with TBP.
Proline-rich transcription repression domains have been identified in a
number of homeodomain proteins including Eve, Msx-1, and Evx-1 (35, 46,
47). The Eve proline-rich repression domain is involved in mediating
protein-protein interactions that result in the cooperative binding of
Eve to DNA (21). However, it is not yet clear how proline-rich domains
bring about repression. The proline-rich PRH N-terminal domain
represses transcription when tethered upstream of a promoter both in
the hematopoietic BM2 cell line and also in QT6 fibroblasts. Deletions
within the PRH N terminus showed that there are at least two regions
within the PRH N terminus that are capable of significantly repressing transcription. Region 28-105 of PRH is both strongly expressed in QT6
fibroblasts and shows strong repression activity. Region 107-141 of
PRH, although not detectable in Western experiments, also displays
significant repression activity. Thus, we infer that there are at least
two nonoverlapping regions within this domain that can function
independently. Han and Manley (48) have shown that a
proline-leucine-rich peptide only 27 amino acids in length is capable
of acting as a potent repressor in transient transfection assays; they
suggest that a key feature of a repression domain is that it is
relatively unstructured and hydrophobic. In keeping with this view,
region 28-105 of PRH contains a large proportion of alanine (14%),
proline (23%), and leucine (6%) residues. However, the 34-amino acid
region () contains only 11% proline residues and 11% leucine
residues. Studies on the Wilm's tumor (WT) protein repression domain
have shown that proline residues outside the WT minimal repression
domain are important for repression. These proline residues may aid the
accessibility of key amino acids from the WT minimal repression domain
with interacting proteins (49). Similarly, it is possible that the
proline and leucine residues from region 107-141 in PRH may allow
other amino acids within this region to be more accessible to any
interacting proteins.
There are two sequences within the PRH N terminus that might mediate
interactions with other transcription factors. The first sequence
LLWSPF (amino acids 131-136 in avian PRH and 124-129 in human PRH) is
located 7 amino acids upstream of the PRH homeodomain and is within the
region 107-141. Hexapeptide sequences that loosely match this motif
are located upstream of the homeodomain in several members of the HOX
family of transcription factors, and HOX proteins use these sequences
to contact members of the PBC family of homeodomain proteins
(50). The PBC proteins modulate transcription of the HOX proteins by
altering either their binding specificity (4) or their transcriptional
regulatory properties (5, 6). Similar tryptophan containing sequences
(WRPY and WRPW) are also found in proteins that recruit the
Drosophila corepressor protein Groucho (51, 52). The second
sequence TPFYIEDILGR (amino acids 33-43 in avian PRH and 30-40 in
human PRH) is present in region 28-105. This sequence strongly
resembles the eh1 motif found in En that mediates the interaction of En
with Groucho (34). Deletions in PRH that remove either this putative
eh1 motif or the LLWSPF sequence do not block transcriptional
repression. One possibility is that the PRH N terminus interacts with
multiple proteins or that any interacting proteins might make several
contacts with the PRH N terminus and that the removal of any one
contact might only partially block the interaction. Certainly in the
case of the interaction of TLE1, the human equivalent of Groucho, with the AML1 protein, several regions of AML1 are important for the interaction (40).
In summary, the PRH homeodomain can passively repress transcription by
binding to TATA box sequences. However, the proline-rich repression
domain of PRH also represses transcription and may do so by interacting
with basal transcription factors or by altering chromatin structure in
conjunction with corepressors. We have shown previously that the
proline-rich domain of PRH plays a role in the control of cell growth
and differentiation in the hematopoietic compartment (16). Future
experiments may allow us to determine which of these repression
mechanisms are important for the function of PRH in vivo.