(Received for publication, October 28, 1994; and in revised form, December 27, 1994)
From the
The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor stimulates PC12 cells to differentiate into neurons. We have defined a negative regulatory element (NRE) that has a functional role in repressing peripherin expression in undifferentiated and nonneuronal cells. Nerve growth factor-induced derepression of peripherin gene expression is associated with alterations in proteins binding to a GC-rich DNA sequence in the NRE as detected by the DNA electrophoretic mobility shift assay (EMSA). We have utilized DNA affinity chromatography to purify from rat liver a 33-kDa DNA-binding protein that specifically recognizes the NRE. Microsequencing reveals identity with NF1-L, a member of the CTF/NF-1 transcription factor family. This protein forms a single complex when incubated with the NRE probe using EMSA analysis. The more slowly migrating complexes characteristic of crude undifferentiated PC12 cell extract are reconstituted by mixing the purified protein with the flow-through from the DNA affinity column, thereby demonstrating that protein-protein interactions are involved in complex formation. Supershift experiments incubating anti-CTF-1 antibody with undifferentiated PC12 cell extract prior to EMSA analysis confirm that NF1-L, or a closely related family member, is the DNA-binding protein component of the multiprotein complex at the NRE.
We have examined the regulation of the peripherin gene, which encodes a neuronal-specific type III intermediate filament protein (1, 2, 3) . The peripherin gene is transcriptionally induced by nerve growth factor in PC12 cells(4) . In vivo, it is limited in expression to sympathetic, parasympathetic, and sensory ganglia of the peripheral nervous system, as well as a small subset of neurons in the central nervous system(1, 5, 6) . During development, peripherin is first expressed at day 11.5 of rat embryogenesis in the newly formed sympathetic ganglia(7) . Therefore, expression of the peripherin gene is temporally associated with acquisition of the terminally differentiated neuronal phenotype.
NGF ()is
one of the environmental factors that influence the differentiation and
survival of sympathetic adrenergic neurons during
development(8, 9, 10, 11, 12, 13) .
An approximation of this differentiation process can be studied in
vitro with PC12 cells, a cell line derived from a rat
pheochromocytoma that behaves similarly to pluripotent neural crest
cells(14, 15, 16) . After approximately 18 h
of exposure in culture to NGF, PC12 cells begin to extend neurites and
acquire biochemical and membrane properties of sympathetic neurons (for
review, see (15) ). Phosphorylation events in the NGF signal
transduction pathway lead to a cascade of gene activation, beginning
with the rapid and transient activation of immediate early gene
transcription ((17) , for review, see Refs. 18 and 19),
followed by delayed early gene transcription. The third wave in the
cascade of gene activation, the so-called late genes (for review, see (18) and (19) ), are neuronal structural genes induced
hours to days after initiation of NGF treatment, coincident with
acquisition of the neuronal phenotype. The peripherin gene is one such
late gene(6, 20) . It is not until the late gene
activation stage that the signal transduction pathways of NGF and
epidermal growth factor diverge, even though epidermal growth factor
has a mitogenic but not differentiative
effect(17, 21, 22, 23) . Therefore,
identifying factors that control the transcription of neuronal-specific
late genes may lead to understanding the mechanism by which NGF
triggers neuronal-specific gene activation.
We have previously described the mapping of transcriptional regulatory regions in the 5`-flanking sequence of the peripherin gene by 5`-deletion mapping(4) . Two positive regulatory elements are necessary for full induction by NGF: a distal positive element approximately 2400 bp upstream of the transcription start site and a proximal constitutive element within 111 bp of the transcriptional start site. In addition, there is a negative regulatory element (NRE) centered at -173 whose deletion results in elevated basal expression of the gene. We have 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 have previously shown that a unique sequence, GGCAGGGCGCC, in the NRE, is specifically recognized by a DNA-binding protein. Methylation interference footprinting of the NRE demonstrates that the specific nucleotides contacted by protein are identical in undifferentiated and differentiated PC12 cell nuclear extracts. Mutagenesis of the footprinted nucleotides in a peripherin promoter-chloramphenicol acetyltransferase reporter construct results in increased expression of the reporter gene in undifferentiated PC12 cells and in 3T3 cells. This result is consistent with the proposed function of the NRE as the binding site of a repressor. DNA mobility shift assays using an oligonucleotide probe containing the footprinted sequence demonstrate prominent DNA-protein complexes in nuclear extracts from undifferentiated PC12 cells that migrate with slower mobility than the complexes produced using differentiated PC12 cell nuclear extract. The slowly migrating complexes are also formed in EMSA assays using nuclear extract from nonneuronal cell lines (liver and mouse erythroleukemia cells). In summary, the proteins binding at the NRE are altered during the process of NGF-induced PC12 cell differentiation, whereas the methylation interference footprinting pattern is invariant. These observations taken together suggest the following hypothesis: protein(s) directly contacting the NRE are similar in undifferentiated and differentiated PC12 cells, whereas a repressor protein participates in the complex via protein-protein interactions in undifferentiated PC12 cells and nonneuronal cell types(4) .
We are particularly interested in understanding how NGF-triggered events relieve the repression of the peripherin gene during the NGF-induced differentiation of PC12 cells. As a first step in understanding this mechanism, we have purified the DNA-binding protein forming the basis of the protein complex at the NRE. In this paper, we report the purification of a 33-kDa protein that specifically recognizes the NRE. Microsequencing of this protein reveals identity with NF1-L, a member of the CTF/NF-1 transcription factor family. In addition, fractionation experiments suggest that non-DNA binding proteins interact with NF1-L to form a multiprotein complex at the NRE.
Nuclear extracts were prepared from PC12 cells according to the method of Dignam (26) as described previously(4) . Differentiated PC12 cells were treated with NGF (50 ng/ml) for 6 days prior to harvesting for nuclear extract preparation.
The binding reactions were incubated at room temperature for 30 min and then irradiated with the output of a frequency-quadrupled, 266-nm pulsed Nd:YAG laser (Coherent, Palo Alto, CA). The net energy output of the laser was 9 mJ/s. Cross-linking was performed with 405 mJ, after which the samples were immediately placed on ice. This laser was made available to us by Dr. Joseph Beecham, Department of Molecular Physiology and Biochemistry, Vanderbilt University School of Medicine. The reactions were then boiled in SDS loading buffer and DNA-protein adducts separated on a 12% SDS-PAGE gel.
The PPRS affinity column was prepared by coupling concatenated double-stranded PPRS oligonucleotides to CNBr-activated Sepharose CL2B as described by Kadonaga and Tjian(29, 30) , yielding 75 µg of coupled DNA/ml resin. The pooled fractions from the calf thymus DNA-Sepharose column were incubated with 0.2 µg of poly(dI-dC)/5 µg of protein (Pharmacia) for 10 min at 4 °C and spun at 10,000 rpm. This protein was then incubated with 2 ml of the PPRS-Sepharose resin for 30 min with gently rocking at 4 °C. The extract-resin slurry was then allowed to settle in a 10-ml column. The flow-through was collected and passed over the affinity column a second time. The column was washed with 10 CV of BC200 and eluted with 10 CV of BC1500, followed by 5 CV of BC3000. 1.5-ml fractions from the wash step and 1-ml fractions from the high salt elution were collected in siliconized tubes. Aliquots of every other fraction were tested for activity by EMSA, and active fractions were pooled, concentrated with Centricon-10 microconcentrators (Amicon), and adjusted to 150 mM KCl with BC00. The pooled active fractions were passed over the PPRS affinity column a second time as above except that the preincubation was with 2.5 µg of poly(dI-dC)/ml of protein, and the protein was loaded directly onto the preformed PPRS affinity column. Elution was performed with 12 CV BC2000 and 4 CV of BC3000. Eluent fractions were assayed by EMSA with the PPRS probe, pooled, concentrated, and diluted to 150 mM KCl. Aliquots of protein at various stages of purification were analyzed by SDS-PAGE (12% gel), and protein bands were visualized by silver staining (Bio-Rad Silver Stain Plus).
Figure 1:
A and B, effect of increasing salt on DNA-protein complexes formed
with undifferentiated and differentiated PC12 cell extract. EMSA was
performed with standard binding reactions containing 0.25 ng of P-labeled PPRS oligonucleotide and 20 µg of nuclear
extract from undifferentiated PC12 cells (Undif) or PC12 cells
differentiated in the presence of NGF for 1 week (Dif).
Binding conditions were adjusted so that the final KCl concentration
was as indicated. DNA-protein complexes were resolved on a 8%
nondenaturing polyacrylamide gel. B, conditions were as in A except that
P-labeled WTRS oligonucleotide was
used as probe. In lanes1 and 2, complexes
were resolved on an 8% nondenaturing gel. In lanes3-5, complexes were resolved on a 5% nondenaturing
gel. C, specificity comparison of complexes formed with WTRS
or PPRS probes. Binding reactions contained 20 µg of
undifferentiated PC12 cell extract and approximately 0.04 ng of WTRS (lanes1-4) or PPRS (lanes5-8)
P-labeled probe. Cold competitor
oligonucleotides were included in the reaction as indicated: 25 ng in lanes1-4 and 100 ng in lanes5-8.
DNA-protein complexes formed with the PPRS probe exhibit identical specificity in competitor analysis to those formed with the wild-type NRE (Fig. 1C). Complexes formed with either the WTRS or PPRS probe and PC12 cell nuclear extract are ineffectively competed away by an unlabeled competitor oligonucleotide that has been mutated in four of the contact nucleotides determined by methylation interference footprinting (RM3; Table 1; (4) ). Unlabeled PPRS and WTRS oligonucleotides effectively compete for protein binding to either probe. Since the PPRS is GC-rich (Table 1), we additionally tested several GC-rich competitors (SP1 binding site (32) and epidermal growth factor receptor GCF binding site(33) ) in EMSA with the PPRS probe. These competitors could not effectively compete for specific protein binding either to the WTRS or the PPRS oligonucleotides (data not shown).
Figure 2:
Comparison of WTRS-binding activity
present in rat liver and PC12 cell nuclear extracts by EMSA. Increasing
amounts of undifferentiated PC12 (lanes1-3) or
rat liver (lanes4-6) nuclear extract were
incubated with 0.05 ng of P-labeled WTRS probe in a
standard binding assay. Complexes were resolved by electrphoresis on a
nondenaturing 5% polyacrylamide gel.
Purification of the NRE binding protein was accomplished by DNA affinity chromatography(29, 30) . The EMSA assay (34, 35) was used to follow DNA-binding activity through the purification steps. Rat liver nuclear extract was applied to a heparin-Sepharose column and step-eluted at 0.35 M KCl, as described under ``Experimental Procedures.'' Active fractions were then passed over a calf thymus DNA-Sepharose CL2B column in order to remove high affinity nonspecific DNA-binding proteins.
In order to purify the proteins binding specifically to the NRE, we prepared an affinity matrix consisting of concatemerized PPRS oligonucleotides coupled to activated Sepharose CL2B(29) . Pooled active fractions eluted from the calf thymus DNA column were incubated with poly(dI-dC) as nonspecific competitor DNA prior to loading on the PPRS oligonucleotide affinity column. Flow-through fractions were collected, followed by a low salt wash at 200 mM KCl, and high affinity proteins were eluted at 1.5 M and 3 M KCl. Aliquots of each fraction were assayed for DNA binding activity. Active fractions were pooled, adjusted to 150 mM KCl, and passed over the PPRS affinity column a second time to achieve greater enrichment for the DNA-binding protein.
Fig. 3demonstrates EMSA analysis of fractions from the first and second pass over the PPRS affinity column. The flow-through and low-salt wash are negative for specific NRE binding activity, whereas the majority of the DNA binding activity elutes at 1.5 M KCl. A single DNA-protein complex is formed with the protein eluted from the affinity column. This complex migrates with similar mobility to band E, the fastest migrating complex formed with crude differentiated PC12 cell extract. The specific activity of protein fractions at each stage of purification is shown in Table 2. An approximate 15,000-fold purification was achieved.
Figure 3:
DNA affinity chromatography of rat liver
nuclear extract. The activity of fractions specifically eluted from the
first (leftpanel) and second (rightpanel) pass over the PPRS affinity column were assayed by
EMSA using a P-labeled PPRS probe and standard binding
conditions, except for reduction of poly(dI-dC) to 0.2 µg/reaction.
The fraction number and the KCl concentration of each step elution are
indicated. The following amounts of each fraction were assayed:
flow-through (FT), 20 µl; 200 mM wash, 15 µl;
1.5 M elution, 3 µl; 2 M elution, 2.25 µl; 3 M elution, 1.5 µl. In the leftpanel, 5
µg of the pooled active fractions eluted from the heparin Sepharose
column (HS; lane1) and from the calf thymus
DNA column (CT; lane2) are assayed. In the rightpanel, 20 µg of crude PC12 nuclear extract (lanes8-9) was run on the same gel as the
second pass eluent fractions.
To evaluate the purity of the fractions eluted from the PPRS affinity column, aliquots of active fractions were taken after each successive round of purification, loaded onto a SDS-PAGE gel, and visualized with silver stain. Fig. 4demonstrates the selective enrichment of a single 33-kDa protein. Maximum enrichment of the 33-kDa protein and diminution of nonspecific proteins occurs after elution of protein fractions from the second pass over the PPRS affinity column (compare lanes5 and 6).
Figure 4: Enrichment of a 33-kDa protein by PPRS affinity chromatography of rat liver nuclear extract. Protein present in fractions from each step of the purification procedure were separated by SDS-PAGE using a 12% gel, and bands were detected by silver stain. The following amounts of each fraction were loaded: crude liver nuclear extract (C), 5 µg; 0.35 M eluent from heparin-Sepharose column (HS), 5 µg; 0.2-0.5 M eluent from calf thymus DNA-Sepharose column (CT), 5 µg; flow-through from first pass over PPRS affinity column (FT), 10 µl; 1.5-3 M eluent from first pass over PPRS affinity column (1), 10 µl; 2-3 M eluent from second pass over PPRS affinity column (2), 8 µl. The marks to the side of the gel indicate migration of 106-, 80-, 49.5-, 32.5-, 27.5-, and 18.5-kDa protein molecular mass standards.
Figure 5:
The UV cross-linking assay identifies a
33-kDa DNA-binding protein with correct DNA-binding specificity. UV
cross-linking of the P-labeled WTRS probe with the protein
eluted from the second pass over the PPRS affinity column (3 µl)
was performed in the presence of 100 molar excess of PPRS
oligonucleotide (lane1), RM3 oligonucleotide (lane2), or no competitor (lane3). The purified protein used corresponds to that loaded
in lane6 of Fig. 5. DNA-protein adducts were
boiled in SDS-loading buffer and separated on a 12% SDS-PAGE gel
immediately following UV cross-linking. The marks to the right of the gel indicate migration of 106-, 80-, 49.5-,
32.5-, 27.5-, and 18.5-kDa protein
standards.
Figure 6:
Methylation interference footprinting of
the NRE with purified protein. A -245/-98 BamHI-StyI peripherin fragment containing the NRE was
asymmetrically end-labeled and partially methylated with
MeSO
prior to incubation with 20 fmol of
purified protein. 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
eluted. After piperidine cleavage, samples of DNA containing equal
counts/min were loaded and run on an 8% sequencing gel. DNA in lanes
labeled P was partially methylated and cleaved with piperidine
but was not incubated with protein. The peripherin promoter sequence is
indicated to the left of each gel with arrowheads marking guanines whose methylation interferes with protein
binding.
Figure 7:
Reconstitution of DNA-protein complexes by
mixing of flow-through and eluent fractions from the PPRS affinity
column. A, EMSA was performed to compare complexes formed by
protein eluted from the second pass PPRS affinity column in the
presence (lanes3 and 5) or absence (lanes2 and 4) of flow-through protein from
the first pass PPRS affinity column. Standard binding conditions were
used with 0.25 ng of P-labeled PPRS probe, 2 µl of 1.5 M, or 3 M eluent protein and 10 µl of
flow-through in lanes3 and 5. Lane1 shows that 10 µl of flow-through alone has no
DNA-binding activity. Complexes were resolved on a 8% nondenaturing
polyacrylamide gel. B, specificity of complexes reconstituted
with the flow-through fraction. Cold competition assays were performed
using either
P-labeled WTRS (lanes1-6) or PPRS (lanes7-12)
as probe. Protein used in the reactions was the 1.5 M eluent
from the second pass over the PPRS affinity column (2 µl) with (lanes4-6 and 10-12) or without (lanes1-3 and 7-9) 10 µl of
flow-through. Standard EMSA binding conditions were used with the
addition of 100-fold molar excess of unlabeled WTRS oligonucleotide (lanes2, 5, 8, and 11),
RM3 oligonucleotide (lanes3, 6, 9,
and 12) or no competitor (lanes1, 4, 7, and 10). DNA-protein complexes were
resolved on a 8% nondenaturing polyacrylamide
gel.
First, we determined whether an
oligonucleotide containing a consensus NF-1 binding site would be an
effective competitor for protein binding to the NRE oligonucleotide in
EMSA performed with PC12 cell nuclear extract. Fig. 8A
compares the consensus NF-1 site to the WTRS NRE sequence. Although the
NRE does not have the CAAT box, which is commonly found in NF-1 binding
sites, it does have the GGCNCGG motif of the NF-1 consensus
sequence(39) . The 3` side of the partially palindromic
sequence also has the TGG motif crucial for an NF-1 site in the
noncoding strand. It is now apparent that the higher affinity of the
PPRS oligonucleotide is due to the creation of a second TGGA site at
the 5` end of the NRE (see Table 1). Fig. 8B demonstrates that the NF-1 oligonucleotide completely competes for
protein binding to the labeled WTRS probe at 100-fold molar excess of
cold competitor. Significantly, the competition is equally effective
for protein forming complexes A and B unique to undifferentiated PC12
cell extract and for protein forming complexes C-E predominant in
differentiated PC12 cell nuclear extract. This result suggests that
NF1-L, or a similar member of the NF-1 transcription factor family, is
the DNA-binding protein forming the core of the protein complex at the
NRE in both undifferentiated and differentiated PC12 cells.
Figure 8:
Evidence that NF1-L is the NRE binding
activity in crude PC12 cell nuclear extract. A, comparison
between the WTRS sequence and the consensus NF-1 sequence. The sequence
of the NF-1 oligonucleotide used as competitor in EMSA is the NF-1
recognition site in the adenovirus origin of replication(39) .
This is compared with the WTRS oligonucletide and the NF-1 consensus
sequence(39) . B, competition of NRE-binding proteins
by a NF-1 oligonucleotide. EMSA analysis was performed with crude PC12
cell extract (undifferentiated or differentiated 1 week in the presence
of NGF) and a P-labeled WTRS oligonucleotide probe.
Unlabeled WTRS (lanes2-3 and 7-8) or PPRS (lanes4-5 and 9-10) competitor oligonucleotides were added to the
reaction mixtures in the molar ratios indicated. C, supershift
of NRE-binding proteins by anti-CTF-1 antibody. EMSA was performed with
15 µg of undifferentiated PC12 cell nuclear extract and
P-labeled WTRS (lanes1-3) or NF-1 (lanes4-6) oligonucleotide probe. In lanes7-9 EMSA was performed with 10 µg of HeLa
nuclear extract and a
P-labeled Sp1 probe. Extract was
preincubated with 1 µl of either BC100 (lanes1, 4, and 7), preimmune serum (pre; lanes2, 5, and 8), or anti CTF-1/NF-1
antibody (NF1; lanes3, 6, and 9) for 1 h prior to the addition of the
probe.
In order to determine whether NF1-L or another factor with identical binding specificity binds to the NRE in PC12 cells, a supershift experiment was performed with anti-CTF-1 antibody (Dr. N. Tanese, NYU Medical Center). The amino acid sequence of CTF-1 is 98% similar to the NF1-L sequence over the N-terminal 175 residues(38) . Antibody to CTF-1 or preimmune sera was incubated with undifferentiated PC12 cell nuclear extract prior to incubation with labeled WTRS probe in the EMSA assay. Fig. 8C demonstrates that incubation with anti CTF-1 antibody but not preimmune serum resulted in a supershifted band as well as the virtual disappearance of all the complexes formed with the WTRS probe and undifferentiated PC12 cell extract. The completeness of the supershift demonstrates that NF1-L or a closely related NF-1 family member is the predominant DNA-binding protein at the NRE in PC12 cells. Similar DNA-protein complexes are formed when the NF-1 oligonucleotide was used as probe; the same supershift results when anti-CTF-1 is preincubated with the extract prior to addition of the NF1 oligonucleotide probe (lanes1-3). However, preincubation with the antibody has no effect on complexes formed with a radiolabeled Sp1 site oligonucleotide probe (lanes7-9). This demonstrates the specificity of the supershift obtained with the anti-CTF-1 antibody.
We have characterized the DNA binding component of the protein complex that binds to a unique GC-rich sequence in the negative regulatory element of the peripherin promoter. We have used DNA affinity chromatography to purify a 33-kDa protein that specifically interacts with the NRE. Microsequencing reveals identity of this protein with NF1-L, a member of the CTF/NF-1 transcription factor family. Experiments reconstituting complexes by mixing protein fractions further suggest that a multiprotein complex is formed at this site by protein-protein interactions.
The 33-kDa protein extensively purified from liver nuclear extract has DNA binding properties that are consistent with its identity as the DNA binding component of the protein complexes formed at the negative regulatory element. The fine specificity of binding of the purified protein is identical to that of the DNA binding activity of crude PC12 cell extract. The specificity of binding was demonstrated by competition with mutant NRE oligonucleotides in the EMSA assay and confirmed by methylation interference footprinting with the extensively purified protein. It will be of interest to determine whether DNase I footprinting would indicate that a wider region of DNA is covered by the entire protein complex present in crude nuclear extract than by the DNA-binding protein alone. UV cross-linking data directly implicates the 33-kDa protein as the only protein in the extensively purified fraction that binds to the NRE with the correct specificity.
Previously, we have concluded from our footprinting data that a DNA binding protein contacts the NRE constitutively. Here we present further evidence for this hypothesis with the salt titration experiment performed with undifferentiated PC12 cell extract. Loss of slowly migrating complexes concomitant with the gain of the differentiated pattern of complexes suggests that the complexes have a protein core common to undifferentiated and differentiated PC12 cells. That NF1-L is a ubiquitous transcription factor is consistent with its role as a constitutive DNA binding protein at the NRE. The results of the supershift experiment in which the slowly migrating complexes formed at the NRE are greatly diminished by preincubation with anti-CTF-1 antibody support the hypothesis that NF1-L or a closely related NF-1 protein is the DNA-binding component of all of the complexes formed at the NRE.
In this paper, we present evidence that protein-protein interactions as well as protein-DNA interactions are necessary for formation of the complex at the NRE. The results of the salt titration suggest that the high salt disrupts protein interactions unique to the undifferentiated PC12 cell extract. However, whether these interactions are protein-DNA or protein-protein cannot be assessed by this data alone. Evidence that proteins interact with the 33-kDa DNA-binding protein rather than with DNA directly is provided by mixing experiments. Mixing of the flow-through and eluent fractions from the DNA affinity column reconstitutes the slowly migrating complexes, whereas the flow-through has no DNA binding activity alone. It is unlikely that the flow-through contains another DNA-binding protein that must interact with the 33-kDa protein before effectively binding to DNA, since the 33-kDa protein contacts every nucleotide implicated in the methylation interference footprint performed with crude PC12 nuclear extract. However, we cannot formally rule out the possibility that the flow-through contains a modifying activity that allows the 33-kDa protein to form oligomers or additional protein-protein contacts, which it is unable to do in an unmodified state.
We have
mentioned above that the slowly migrating complexes A and B are formed
using nuclear extract from cells in which peripherin expression is
repressed (undifferentiated PC12, liver, mouse
erythroleukemia(4) , 3T3()). Since mixing of the
non-DNA binding flow-through fraction with the 33-kDa protein is
necessary for formation of these complexes, our hypothesis is that the
repressor protein interacts with the constitutive complex via
protein-protein interactions. This interaction would then give a
specific modulatory capability to the constitutively acting NF1-L
protein. It will be important to confirm the relative roles of these
proteins in repression by in vitro transcription analyses and
co-transfection experiments.
Formation of a multiprotein regulatory
complex dependent on protein-protein interactions has been observed at
several other positive and negative regulatory elements. The E2F
transcriptional complex is a multiprotein complex whose effect on
transcription is dependent on non-DNA binding members of the
complex(40, 41, 42) . In contrast to the E2F
complex where E2F-1 itself is a transcriptional
activator(40, 42) , the minimal complex at the
peripherin NRE (i.e. in differentiated PC12 cells) has either
a neutral or negative transcriptional effect. Therefore, the most
analogous repressor complex may be that formed at the mating type locus
in yeast. Operators that repress a-specific genes in cells and
haploid-specific genes in diploid cells are occupied by MCM1-
2 and
a1-
2, respectively. However, these proteins cannot repress on
their own; they ``mark'' the operator for recognition by the
general repressor proteins, Tup1 and Ssn6(43) . Like the
putative repressor protein(s) forming the complete repressor complex at
the peripherin NRE, Tup1 and Ssn6 interact with the complex strictly
via protein-protein interactions.
Microsequencing reveals that the 33-kDa protein is a member of the CTF/NF-1 family of transcription factors. All four sequenced peptides are identical in sequence to NF1-L, purified from rat liver by Paonessa et al.(38) . This group demonstrated that the cDNA encoding NF1-L has an open reading frame encoding 505 amino acids that would correspond to a protein similar in molecular mass to other CTF/NF-1 family members (55-62 kDa; (44) ). However, the NF1-L that Paonessa et al.(38) purified from rat liver has a molecular mass of 30 kDa, similar to the apparent molecular mass of our purified protein. The chicken homologue to NF1-L was also purified from liver as a 30-kDa protein(45) . Paonessa et al.(38) conclude that the 30-kDa protein is a specific proteolytic degradation product of NF1-L since amino acid sequence obtained from six peptides is encoded by a contiguous region of the NF1-L cDNA corresponding to about 27 kDa. Therefore, it is likely that the 33-kDa protein that we have purified is also a degradation product of NF1-L. However, we cannot rule out that the 33-kDa protein is a minor alternatively spliced form of NF1-L. Paonessa et al.(38) report eight RNA species hybridizing to a NF1-L probe on Northern blot analysis of multiple rat tissues. Some transcripts are ubiquitous, others specific to a few tissues. Interestingly, the major species found in brain and liver are the same.
NF1-L belongs to a family of closely related transcription factors in which diversity stems not only from multiple genes (four genes in hamster(46) , but also from alternative splicing (e.g. CTF-1, -2, and -3 in human; (44) )). In addition, NF1-L interacts with DNA as a dimer(47, 48) . This raises two questions about the nature of the DNA-protein complexes formed at the NRE in PC12 cells. First, an alternative explanation for the multiple DNA-protein bands seen in the EMSA assay could be that multiple NF-1 proteins, or heterodimers thereof have affinity for the NRE in the in vitro assay. However, the additional complexes are reconstituted by mixing the flow-through fraction, which has no DNA binding activity, with the 33-kDa protein. Additional NF-1 proteins would presumably have independent DNA-binding activity. Second, it is possible that the active protein binding to the NRE in PC12 cells is not NF1-L but a related NF-1 family member. The completeness of the supershift with anti-CTF-1 antibody makes it unlikely that an antigenically unrelated protein is the actual DNA-binding protein.
We present evidence in this paper demonstrating protein-protein interactions involving a CTF-1/NF-1 family member. Comparison of the amino acid sequence of the NF-1 family members reveals extensive conservation of the N-terminal region that contains the DNA-binding domain (48) and less conservation of the C terminus. It has been proposed by Gil et al.(46) that the C terminus may be involved in protein-protein interactions. Such interactions may be important in conferring specificity of binding of particular NF-1 proteins to specific NF-1 sites. At the NRE, where there is only one complete TGG half-site, binding may be stabilized by interaction with another protein in this manner. Additionally, the presumptive C-terminal protein interaction domain may contact the putative repressor protein binding to NF1-L at the NRE.
A NF-1 site has been implicated as important for conferring repression in several other genes. Mapping of the regulatory sequences mediating the neonatal extinction of liver lipoprotein lipase identified a region containing an NF-1-like site. EMSA analysis of this site demonstrates formation of additional complexes with liver nuclear extracts from adult versus neonatal animals(49) . This is analogous to the situation at the peripherin NRE. In addition, an NF-1 site appears to be involved in repression of the hepatitis B virus enhancer(50) . Also of interest is a report that cells overexpressing c-myc suppress the transcription of multiple genes whose promoters contain NF-1 sites(51) . The mechanism appears to be c-myc-induced phosphorylation of NF-1.
Relatively few neural-specific gene promoters have been carefully analyzed for regulatory elements responsible for neural-specific expression. Of those which have been examined, a high proportion have negative regulatory elements that restrict gene expression to neuronal lineages(52) . The expression of SCG10 (16) and the brain type II sodium channel gene both are restricted to neurons by a negative regulatory element(53, 54, 55) . These elements have sequence similarity, and competitive gel shift analysis suggests that they may bind a common protein(54) . However, the sequence of this negative regulatory element is different than the peripherin NRE and does not appear to be involved in modulating the response to NGF. It will be interesting to see if the preponderance of negative regulation in the control of neuralspecific genes continues as the mechanisms of regulation of more neural-specific genes are delineated.