(Received for publication, October 6, 1995; and in revised form, December 19, 1995)
From the
The peripherin gene, encoding a neuron-specific intermediate filament protein, is transcriptionally induced when PC12 cells begin to terminally differentiate into neurons in response to nerve growth factor. Previously we identified two regulatory sequences of the peripherin gene: a proximal negative element (centered at -173), which prevents peripherin expression in undifferentiated PC12 cells, and a distal positive region (-2660 to -2308) necessary for full induction of peripherin in differentiated PC12 cells (Thompson, M., Lee, E. Lawe, D., Gizang-Ginsberg, E., and Ziff, E.(1992) Mol. Cell. Biol. 12, 2501-2513). Here we define a distal positive element (DPE, -2445 to -2337) within the distal positive region. Methylation interference footprinting of the DPE identified DNA-protein contact points at a novel inverted repeat sequence (AACCACTGGTT) and an Ets-like recognition sequence (CAGGAG). Functional analysis using site-directed mutagenesis demonstrates that both sites are necessary for the activity of the DPE. In addition, ternary complex formation at the DPE is dependent on both sites. Antibody competition assays confirm that an Ets family member participates in the DNA-protein complex. We have indirect evidence that the inverted repeat binding protein and the Ets-related protein interact directly with each other. Finally, we demonstrate that the DPE is constitutively active and that neuron-specific regulation of peripherin expression may be due to interaction with distal and proximal negative regulatory elements.
The neuron-specific intermediate filament proteins are a major
component of the neuronal cytoskeleton. They include NF-L, NF-M, and
NF-H, as well as peripherin, -internexin, and nestin (reviewed in (2, 3, 4) ). They are expressed in different
regions of the nervous system: NF-L, NF-M, and NF-H are found in all
neurons, whereas
-internexin is expressed in the central nervous
system(5) , nestin in neuroepithelial stem cells(6) ,
and peripherin in the peripheral nervous system and a subset of central
nervous system
neurons(7, 8, 9, 10) . During
development, the expression of these genes is closely associated with
the terminally differentiated neuronal
phenotype(5, 6, 8, 11, 12) .
Identification of regulatory proteins that control the expression of
these neuronal structural genes may lead to understanding of the
cellular mechanism underlying neuronal differentiation.
We have
studied the regulation of the peripherin gene, which encodes a
neuron-specific type III intermediate filament
protein(9, 10, 13, 14) . The
peripherin gene is a late response gene expressed approximately 12 h
after initiation of nerve growth factor (NGF) ()treatment of
PC12 cells, corresponding to the time when PC12 cells begin to exhibit
a neuronal
phenotype(9, 15, 16, 17) . In
vivo, peripherin expression is limited to sympathetic,
parasympathetic, and sensory ganglia of the peripheral nervous system
as well as a small subset of neurons in the central nervous
system(7, 9, 10) . Peripherin is first
expressed at day 11.5 of rat embryogenesis in the newly formed
sympathetic ganglia(8) . Belecky-Adams et al.(18) demonstrated that a transgene containing 5.8
kilobases of peripherin 5`-flanking sequence achieved correct temporal
and nervous system-specific expression in transgenic mice, although
intragenic sequences contribute to correct expression in certain
neuronal subtypes. These in vivo data imply that the
regulatory elements located in the 5`-flanking region are capable of
directing neuron-specific expression of the peripherin gene.
In order to identify the regulatory factors necessary for the cell-specific expression of peripherin, we have previously dissected its 5`-flanking sequence and localized a negative regulatory element (NRE) centered at -173 whose deletion results in elevated basal expression of the gene(1) . In addition, there are two positive regulatory regions required for full induction in PC12 cells treated with NGF: a distal positive region approximately 2450 bp upstream of the transcription start site and a proximal constitutive region within 111 bp of the transcription start site(1) . We proposed a two-step model of transcriptional activation of peripherin by NGF in which dissociation of a repressor from the protein complex at the NRE, coupled with a positive signal from the distal positive element, results in complete activation of the gene. We also showed that a multiprotein complex containing a member of the CTF/NF-1 family as the DNA-binding core protein interacts with the NRE(19) . The composition of this complex changes after NGF treatment, coincident with derepression of the peripherin gene.
We have now finely mapped the distal positive element (DPE) necessary for the full expression of peripherin in NGF-treated PC12 cells. The isolated DPE is constitutively active in PC12 cells and retains significant activity in non-neuronal cells as well. We show that a distal negative element 5` to the DPE (between -3710 and -2660) may restrict the activity of the DPE in non-neuronal cells.
Here we present a detailed analysis of the minimal cis-acting sequences within the DPE that are necessary for its activity. Methylation interference footprinting of the DPE demonstrates two protein binding sites, a unique inverted repeat sequence as well as a sequence 38 bp upstream that resembles an Ets protein binding site. The ets protooncogene family is a novel class of eukaryotic sequence-specific DNA-binding proteins ((20) , reviewed in (21, 22, 23) ). The Ets proteins are related to each other based on sequence similarity in the ETS domain, which is the DNA binding domain. The Ets family proteins recognize purine-rich sequences characterized by an invariant GGA core sequence(23, 24, 25) . Several members of the Ets family function as transcriptional activators, and a subset of these Ets family proteins are thought to require cooperation with other DNA-binding proteins for their activity ((26) , reviewed in (21) and (23) ). In this report we present evidence that an Ets-related protein interacts with a novel inverted repeat binding protein to form a transcriptionally active complex at the DPE.
Plasmids with mutations in the Ets-like site, the inverted repeat site, or both sites were generated by site-directed mutagenesis according to the method of Kunkel et al.(32) . Briefly, a 138-bp HindIII DNA fragment containing the peripherin sequence (-2446 to -2308) was subcloned into M13MP19 and used as a template for mutagenesis. In vitro mutagenesis was then performed with the following oligonucleotides: oligonucleotide I, 5`-CAATGGAGAAGGTCATGAATACTCAGTCACCTTCC-3` (mutant inverted repeat site); and oligonucleotide II, 5`-CCCTTTAACACAGACAGTAATACTTTGCTG-3` (mutant Ets-like site). The underlined nucleotides are the mutated ones. Clones containing a single mutation (I or II) or both mutations (I and II) were confirmed by dideoxynucleotide DNA sequencing, and the -2445 to -2308 HindIII insert was subcloned back into -2308-CAT to generate the mutant -2445-CAT constructs. Orientation of the mutant insert was confirmed by dideoxynucleotide DNA sequencing. Wild type -2445-CAT and the mutant -2445-CAT mutant constructs were used as templates for PCR to generate wild type and mutant 109-bp fragments (-2445 to -2337) with HindIII ends for use as probes and competitors in EMSAs.
The wild type 109-bp fragment (-2445 to -2337) with HindIII ends was subcloned into the pBLCAT2 vector (33) to generate a hybrid peripherin-TK-CAT reporter construct. PBLCAT2 contains nucleotides -105 to +51 of the thymidine kinase (TK) promoter. The orientation and sequence of the inserted fragment was confirmed by dideoxynucleotide sequencing before use.
Figure 1:
5` and 3` fine mapping of the distal
positive region of the peripherin promoter. The peripherin-CAT reporter
constructs containing 5` and 3` deletions within the distal positive
region, shown schematically (not to scale), were constructed as
detailed under ``Experimental Procedures.'' For each
experiment, equimolar amounts of plasmid DNA were transfected by
electroporation into PC12 cells, and parallel plates were either
untreated or treated with NGF for 46 h. The promoter activity of each
deletion construct is expressed as the CAT activity (normalized to
-galactosidase activity) relative to that of -2660-CAT in
NGF-treated cells, which is defined as 1.0. Results represent the
average of at least three experiments, with less than 25% variation
between experiments.
Figure 2:
DNA-protein complex formation at the DPE. A, EMSAs were performed with a P-labeled DPE
probe containing 109 bp of peripherin sequence (-2445 to
-2337) and nuclear extract from PC12 cells treated with NGF for
12 h. A 100-fold molar excess of unlabeled competitor oligonucleotides
was included as indicated. Competitor in lane 7 is an unrelated E box
oligonucleotide. B, the nucleotide sequence of the DPE.
Nucleotides in the DPE that conform to the Ets core consensus sequence
are underlined. The inverted repeat sequence is marked by arrows.
Figure 3:
Sequence requirements for formation of the
DPE-protein complex. Panel A, methylation interference
footprinting of the DPE. The -2445 to -2337 BamHI-HindIII DPE fragment of the peripherin promoter
was asymmetrically end-labeled and partially methylated with dimethyl
sulfate prior to incubation with nuclear extracts prepared from
NGF-treated PC12 cells. The DNA-protein complexes were separated on a
nondenaturing polyacrylamide gel. Bands containing complexed DNA (B) or free probe (F) were cut out, and the DNA was
electroeluted. After piperidine cleavage, samples of DNA containing
equal counts per minute were loaded and run on an 8% sequencing gel.
DNA in lanes labeled G+A are the corresponding
Maxam-Gilbert sequencing ladder. The peripherin promoter sequence is
indicated to the left or right of each gel with arrowheads marking guanines whose methylation interferes with
protein binding. Data are summarized at the bottom. G residues
whose methylation interferes with protein binding are boxed. B, EMSA competition analysis was performed using the P-labeled DPE as probe and nuclear extracts from PC12
cells treated with NGF for 5 days. PCR was used to generate competitors
of identical length to the DPE probe, which contain mutations in the
Ets-like site, the inverted repeat site, or both sites. The mutations
introduced in each site are identical to those shown in the mutant
oligonucleotide sequences in Table 1. A 100-fold molar excess of
the following unlabeled competitors was added as indicated. Lane
1, no competitor; lane 2, the wild type DPE
(E+I+); lane 3, wild type Ets-like site and mutated
inverted repeat site (E+I-); lane 4, mutated
Ets-like site and wild type inverted repeat site (E-I+); lane 5, mutations in both sites (E-I-). The
specific DPE-protein complex is indicated with an arrowhead.
In order to confirm that formation of the specific DNA-protein complex depends just upon the inverted repeat site and the Ets-like site, EMSAs were performed using the same radiolabeled probe as in Fig. 2B (DPE; -2445 to -2337) and nuclear extracts from PC12 cells treated with NGF for 5 days. Competitor fragments identical in length to the DPE probe but containing mutated nucleotides in the Ets-like site, the inverted repeat site, or both sites were generated by PCR using mutant templates created by site-directed mutagenesis. Mutations introduced into the Ets-like site or the inverted repeat site were as listed for the mutant oligonucleotides (E-, I-) in Table 1. Each nucleotide shown by methylation interference footprinting to contact protein was mutated; in addition, the 3` GGAA was mutated in the E- oligonucleotide and additional palindromic nucleotides were mutated in the I- oligonucleotide. As expected, the unlabeled DPE competitor efficiently competed away proteins binding to the radiolabeled DPE probe; however, the 109-bp fragment with mutations in both footprinted sites (E-I-) is an ineffective competitor (Fig. 3B). Both fragments mutated in only one site were able to compete for the protein complex. However, the 109-bp fragment mutated in the Ets-like site (E-I+) alone was a much more effective competitor than the 109-bp fragment mutated in the inverted repeat site alone (E+I-). This competition data confirms the dependence of specific complex formation on the two sites identified by methylation interference footprinting.
Figure 4:
Evidence that an Ets protein binds to the
Ets-like site in the DPE. EMSAs were performed with differentiated PC12
cell nuclear extract and P-labeled 109-bp DPE probe (panel A and panel B, lanes 1-4) or
P-labeled Sp1 probe (panel B, lanes
5-6). Extract was preincubated with 2 µl of either
anti-ETS-domain antibody (ETS; panel A, lane
2, and panel B, lane 6), pre-immune rabbit serum (pre; panel B, lane 3), anti-HMGI(Y)
antibody (HMG; panel B, lane 2), or
anti-interleukin 8 antibody (IL8; panel B, lane
4) for 1 h prior to the addition of the
probe.
Figure 5: Dependence of DNA-protein complex formation on an intact inverted repeat sequence. A, EMSAs were performed with nuclear extracts from differentiated PC12 cells and a series of wild type and mutated 109-bp DPE probes (used as competitors in Fig. 3B). The probe used in each binding assay was as follows: the wild type DPE (E+I+) (lanes 1 and 2); the DPE mutated in the Ets-like site (E-I+) (lanes 3-5); the DPE mutated in the inverted repeat site (E+I-) (lanes 6-8); the DPE mutated in both the Ets-like site and the inverted repeat site (E-I-), lanes 9-11. Mutations in each binding site are as listed in Table 1. Unlabeled DPE (E+I+) was included as a competitor in lanes 2, 5, 8, and 11, and mutant unlabeled competitors were included as indicated in lanes 4, 7, and 10. B, measurement of the rate of association of protein with the wild type DPE probe (lanes 1-4) or the E-I+ mutant DPE probe (lanes 5-8). Binding reactions were incubated at room temperature for the indicated times (in minutes) prior to loading onto a 5% nondenaturing polyacrylamide gel. C, comparison of the migration of the complex formed on the wild type DPE probe or E-I+ mutant DPE probe. Equal amounts of differentiated PC12 nuclear extract were incubated with wild type DPE probe (lane 1) or E-I+ mutant DPE probe (lane 2) at room temperature for 40 min and analyzed on a 4% nondenaturing polyacrylamide gel for 3.5 h.
To further investigate requirements for interaction of protein with the two sites in the DPE, an EMSA was performed using oligonucleotides containing a dimer of either site. Previously we have observed that radiolabeled oligonucleotide probes containing a single copy of the inverted repeat site or the Ets-like site alone were unable to form a stable complex (data not shown). However, a stable complex does form on probes containing a dimer of either the Ets-like site or the inverted repeat site. Complex formation with each dimer probe (Fig. 6, lanes 1 and 6) is specifically competed by unlabeled oligonucleotides representing the wild type site (Fig. 6, lanes 2 and 7) but not by the corresponding mutant oligonucleotides (Fig. 6, lanes 3 and 8). Surprisingly, the complex formed with the inverted repeat site probe can be effectively competed by an Ets-like site oligonucleotide, and the complex formed with the Ets-like site can be effectively competed by an inverted repeat site oligonucleotide (Fig. 6, lanes 4 and 9). The corresponding mutant oligonucleotides (Fig. 6, lanes 5 and 10) do not compete significantly. In addition, complex formation is effectively competed by the 109-bp DPE fragment but not a DPE fragment mutated at both sites (data not shown). These data suggest that the DPE complex consists of two proteins that can be tethered at either site, leaving the DNA-binding site of the associated protein free to interact with competitor DNA.
Figure 6:
Cross-competition of complexes formed on
Ets-like site or inverted repeat site dimers. EMSAs were performed with
nuclear extract from differentiated PC12 cells and a P-labeled oligonucleotide probe containing two copies of
the inverted repeat site separated by a 10-bp HindIII linker (INV DIMER, lanes 1-5) or two copies of the
Ets-like site separated by a 10-bp HindIII linker (ETS
DIMER, lanes 6-10). The following competitor
oligonucleotides were added in 150-fold molar excess: no competitor DNA (lanes 1 and 6); wild type inverted repeat site
monomer (I+) (lanes 2 and 9); oligonucleotide
with one mutated inverted repeat site (I-) (lanes 3 and 10); wild type Ets-like site monomer (E+) (lanes 4 and 7); oligonucleotide with one mutated Ets-like site
(E-) (lanes 5 and 8). The nucleotide sequence of the
competitor oligonucleotides are listed in Table 1.
Figure 7:
Dependence of the activity of the DPE in
PC12 cells on the inverted repeat site and the Ets-like site. Mutations
were generated by site-directed mutagenesis and correspond to those
analyzed by EMSA for complex formation in Fig. 5. Equimolar
amounts of plasmid DNA were cotransfected with 4 µg of
RSV--galactosidase plasmid DNA into PC12 cells by transient
transfection using electroporation. Parallel plates were untreated or
treated with NGF for a total of 46 h. CAT activity was normalized to
-galactosidase activity and expressed as activity relative to the
activity of the -2308-CAT construct in NGF-treated cells, defined
as 1.0. For each construct, the mean of the results from three or more
experiments is shown. Error bars indicate one standard
deviation.
In light of the data from the site-directed mutagenesis, it was initially perplexing that the activity of the -2396-CAT 5` deletion construct is comparable with that of the -2445-CAT construct, which contains the Ets-like site in addition to the inverted repeat site (Fig. 1). Careful inspection revealed a DNA sequence in the pUC-derived vector 5` of the junction with the peripherin sequence, which matches the peripherin Ets-like site in five contiguous nucleotides out of eight comprising the Ets consensus sequence. Treisman et al.(42) report a similar artifactual result in which a plasmid-derived Elk-1/Sap-1 site interfered with an inserted SRF site. Since vector sequences can influence the promoter activity of test plasmids, we believe that examination of the relative activity of each site by site-directed mutagenesis in the context of an equal length of peripherin sequence yields the most accurate results.
Figure 8:
The DPE is constitutively active. A, a 109-bp DPE fragment containing the peripherin sequence
(-2445 to -2337) was cloned into the TK-CAT vector
pBLCAT2(35) . Equimolar amounts of plasmid DNA were
co-transfected with 4 µg of RSV--galactosidase plasmid into
PC12 cells by electroporation. Parallel plates were treated with or
without NGF for 46 h prior to harvesting the cells. A representative
CAT assay performed with protein extracts from the transfected cells is
shown. B, EMSAs were performed using
P-labeled
DPE as a probe and nuclear extracts from undifferentiated PC12 cells (lanes 1 and 2), differentiated PC12 cells treated
with NGF for 6 days (lanes 3 and 4), or 3T3 cells (lanes 5 and 6). 100-fold molar excess of unlabeled
DPE DNA was included in lanes 2, 4, and 6 as
competitor.
We have identified a DPE in the 5`-flanking region of the
peripherin gene, which is required for the full induction of peripherin
in PC12 cells. Methylation interference footprinting of the DPE
identifies two DNA-protein binding sites separated by 38 bp: a novel
inverted repeat site and an Ets-like site. Functional data based on
site-directed mutagenesis of these two sequences demonstrates that the
combination of the Ets-like site and the inverted repeat site is
necessary for significant activity of the DPE. Our results suggest that
both the Ets-like site binding protein and the inverted repeat binding
protein form a ternary complex at the DPE and act synergistically to
achieve efficient activation of the peripherin promoter. There is a
growing list of positive elements in which synergy exists between an
Ets family member and a transcription factor of another class that
binds to an adjacent site. This list includes Ets-1 and Sp1 at the
HTLV-1 LTR(43) , PU.1 and NF-EM5 at the immunoglobulin 3`
enhancer (44) and the immunoglobulin
2-4
enhancer(45) , Ets-2 and Myb at the mim-1 promoter(46) , and Elk-1 or Sap-1 and SRF at the serum
response element of the fos promoter(47, 48) . The nature of the interactions
among the Ets protein, the adjacent transcription factor, and their
cognate DNA binding sites is variable (reviewed in (21) and (23) ).
There are several novel features of the Ets-related protein interaction at the peripherin DPE. First, the Ets-like site and the inverted repeat site are separated by 38 bp. In the above listed examples, the location of the Ets site is only 1-3 nucleotides away from the site of its synergistic partner. Treisman et al.(42) have shown that ternary complex formation between Elk-1 and SRF occurs on synthetic oligonucleotides in which spacing between the sites varies from the wild type (2 bp) to as far as 27 bp, suggesting that there is flexibility in the spacing requirements for ternary complex formation. Our data provide evidence for a functional interaction between an Ets protein and a transcription factor that binds to a site that is not directly adjacent.
The second novel feature of the Ets protein interaction at the DPE is that the Ets-like site (AGGAG) deviates from the Ets canonical recognition motif ((C/A)GGA(A/T)) at the fifth position(22, 23, 24, 25) . However, EMSAs using an anti-ETS domain antibody confirm that the DNA-protein complex formed at the DPE contains an Ets family member. It has been proposed that the Ets protein interaction with another transcription factor may influence its ability to bind preferentially to a subset of possible Ets sites (reviewed in (21) ). Therefore, interaction with the inverted repeat protein at the DPE may result in the binding of the Ets-like protein to a noncanonical site.
The third novel feature of the ternary complex is that the inverted repeat sequence is recognized by a novel transcription factor. A computer search performed with the Quest program of the Intelligenetics Suite (Intelligenetics, Inc., Mountain View, CA) demonstrated that the inverted repeat sequence does not match any known sites in the Transcription Factors Database (TFD7.3). We have utilized the yeast one-hybrid screen to obtain candidate cDNA clones from a rat olfactory epithelium cDNA library (generously donated by Dr. R. Reed, Johns Hopkins University; (49) ) that will activate a reporter plasmid via multiple inverted repeat sites.
Our data demonstrate that the proteins binding to the Ets-like site and the inverted repeat site interact to form a specific DNA-protein complex at the DPE. We have shown that specific complex formation still occurs on a DPE probe containing the mutated Ets-like site and the wild type inverted repeat site (E-I+). However, the complex formed on this mutated probe behaves differently from the one on the wild type DPE. The on rate of complex formation on the mutated DPE is 4-fold slower than that of complex formation on the wild type DPE (See Fig. 5B). The relative rate of complex formation reflects the activation energy that must be overcome for the DNA-protein interaction to occur, especially if a conformational change must occur(50) . In addition, in vivo experiments indicate that the complex on the mutated DPE is less active in transactivation of the peripherin promoter. Taken together, these data suggest that when the Ets-related protein and the inverted repeat binding protein both interact with DNA in the ternary complex, the activation energy is reduced due to formation of a more stable complex. This results in increased transcriptional activity of the DPE.
Our data suggest that the Ets-related protein cannot bind to the DPE autonomously. A subset of Ets family proteins that display little or no independent DNA binding (e.g. Ets-1 and Ets-2 (26, 51) and Elk-1 or Sap-1(42, 47, 52) ) contain an inhibitory domain that may hinder interaction with DNA. However, this inhibition may be relieved by interaction with an adjacent factor. For example, Elk-1 cannot bind independently to the Ets site at the serum response element but rather requires the presence of SRF at an adjacent site(24, 48, 53) . It will be interesting to determine whether the Ets-related protein binding at the DPE contains a similar inhibitory domain.
We have presented indirect evidence that the Ets-related protein may interact with the inverted repeat binding protein through protein-protein interactions (See Fig. 6). The protein complex formed on a dimer of either the Ets-like site or the inverted repeat site can be specifically competed by excess unlabeled competitor oligonucleotides containing either binding site. This cross-competition suggests that the Ets-related protein and the inverted repeat protein may be tethered together and potentially interact with either the Ets sequence or the inverted repeat sequence via the DNA binding domain of the appropriate member of the complex. The other DNA binding domain would be free to be contacted by competitor DNA. This model predicts that the Ets-related protein is present in the DNA-protein complex formed on the mutated DPE (E-I+), thereby explaining the similar migration of the complex formed on the wild type DPE and the mutated DPE (See Fig. 5C). Based on our findings, we propose a mechanism of ternary complex formation at the DPE in which the Ets-related protein cannot bind to the Ets-like site autonomously but rather is recruited to the DPE by interaction with the inverted repeat protein. Formation of this ternary complex in which the Ets-related protein and inverted repeat protein both interact with DNA, as well as each other, results in a stable, transcriptionally active DNA-protein complex.
Finally, we have found that the DPE is not an NGF response element.
It is constitutively active in PC12 cells, as shown by the activity of
the DPE-TK-CAT reporter transfected into cells with and without
subsequent treatment with NGF. In addition, it is moderately active in
3T3 cells. In light of these results, how does one explain the
NGF-inducible and tissue-specific pattern of expression of the
peripherin gene? This specific pattern of expression appears to be the
result of the action of two separate negative regulatory elements in
the 5`-flanking region modulating the constitutive activity of the DPE:
the proximal negative element (NRE, -173) and the distal negative
region (DNR, -3710 to -2660). Several results suggest that
the proximal NRE rather than the DNR is able to repress DPE activity in
undifferentiated PC12 cells. First, if the DPE is inserted at the 5`
boundary of a -305-CAT peripherin construct containing the NRE,
its activity is regulated in response to NGF. ()Second, a
hybrid peripherin reporter construct containing the DNR as well as the
DPE, -3710/-2308-TK-CAT, is equally active in PC12 cells
with or without NGF treatment.
However, the activity of DPE
appears to be repressed by the DNR rather than by the NRE in 3T3 cells
(see Fig. 9). Therefore, the relative activity of each negative
element may differ in neuronal precursor cells versus non-neuronal cells. We are currently testing the relative in
vivo importance of the NRE and the DNR in transgenic mice.
Figure 9:
Mapping of a distal negative region 5` of
the DPE. Equimolar amounts of test plasmids indicated were
co-transfected with 4 µg of an RSV--galactosidase internal
control plasmid into PC12 cells (via electroporation) or 3T3 cells (via
calcium phosphate precipitation). In PC12 cell transient transfections,
parallel plates were untreated or treated with NGF for a total of 46 h.
The difference in transfection efficiency between the two cell types is
corrected for by normalization of the CAT activity to
-galactosidase activity in the protein extracts from the
transfected cells. Units of normalized activity are calculated as
(percentage of CAT activity per microgram of heated protein
assayed)/(units of
-galactosidase activity per microgram of
protein assayed). Results represent the average of at least two
experiments, with error bars representing one standard
deviation from the mean.
The
regulation of neural-specific gene expression by the interaction of
negative regulatory elements with a constitutively positive element is
in keeping with the hypothesis that the neural state is the
``default'' state in development(54, 55) .
Several neural-specific structural genes, including
SCG10(56, 57, 58) , the type II sodium
channel gene(59) , and dopamine -hydroxylase (60) have constitutively active promoter/enhancers whose
expression is regulated by a upstream silencer element (reviewed in (61) ). The silencer element in SCG10 and the type II sodium
channel gene is bound by a protein expressed in both non-neuronal cells
and neuronal precursors(62, 63) . A unique feature of
peripherin gene regulation may be the differential effect of the two
negative elements in different cell types.
This report is the first to demonstrate a functional role for an Ets protein in the activation of a mammalian neural-specific structural gene. Interestingly, an Ets-like recognition sequence has been noted in the NF-L promoter(64) . Elucidation of the mechanism by which the ternary complex at the DPE activates transcription should reveal how its action is modulated by proximal and distal repressors, resulting in neural-specific expression.