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Volume 270, Number 12, Issue of March 24, 1995 pp. 6975-6983
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
NF1-L Is the DNA-binding Component of the Protein Complex at the Peripherin Negative Regulatory Element (*)

(Received for publication, October 28, 1994; and in revised form, December 27, 1994)

Allen D. Adams (§),  ,  Donna M. Choate ,  Mary Ann Thompson (¶)

From the Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.

INTRODUCTION

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 (^1)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.

EXPERIMENTAL PROCEDURES

Cell Culture and Treatment

Stock cultures of PC12 cells were maintained as described previously(24) . PC12 cells grown for nuclear extract preparation were plated on plastic culture dishes coated with collagen (Celtrix Laboratories, Palo Alto, CA) at a density of 3 times 10^6 cells/150-mm dish. NGF was added 24 h after plating (50 ng/ml; 2.5 S, Bioproducts for Science, Indianapolis, IN). NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% defined and supplemented calf serum (HyClone, Logan, UT).

Nuclear Extract Preparation

Rat liver extracts were prepared according to the method of Gorski et al.(25) with the following modifications. Each buffer in the preparation contained the following additives, added immediately before use: 0.5 mM dithiothreitol, 20 µM 4-amidinophenylmethanesulphonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 10 mM NaF. Frozen rat livers from male Sprague-Dawley rats (average weight, 10 g; Harlan, Indianapolis, IN) were thawed on ice in treated homogenization buffer, blended in a Waring blender, homogenized using a motor-driven Teflon homogenizer, and then filtered through cheesecloth. After purification and lysis of the nuclei, the KCl concentration was adjusted to 0.42 M by the addition of nuclear lysis buffer + 1 M KCl. After 45 min shaking at 4 °C, the chromatin was pelleted by centrifugation in a TLA100.3 rotor at 100,000 times g for 45 min. The supernatant was dialyzed against BC100 (20 mM HEPES, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM NaF) for 4 h with one change of buffer.

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.

Gel Retardation Assays (EMSA)

The conditions for the DNA-protein binding reactions were essentially as described previously (4) . Poly(dI-dC) was reduced to 0.1-0.5 µg/reaction when partially purified protein fractions were used. Competition experiments included 100-fold molar excess of unlabeled double-stranded oligonucleotide unless otherwise indicated. DNA-protein complexes were resolved on 5 or 7% polyacrylamide gels (30:0.8, acrylamide/bis-acrylamide), using conditions described previously(27) . Supershift assays were performed by preincubating nuclear extract with 1 µl of anti-NF1 antibody (partially purified by elution from a protein A-Sepharose column) or preimmune serum for 1 h at room temperature prior to the addition of the DNA probe.

Photoactivated Protein-DNA Cross-linking

Protein-DNA binding reactions were performed as reported previously (50) except that reactions were scaled up to a 45-µl final volume, with 90,000 cpm (0.5 ng) of P-labeled DNA probe. The DNA probe was generated by primed synthesis using [P]dCTP (800 Ci/mmol) from a single-stranded M13 template into which the WTRS oligonucleotide (see Table 1) had been cloned. After cleavage with HindIII, the P-labeled probe was purified by polyacrylamide gel electrophoresis and eluted overnight at 37 °C in 0.5 M ammonium acetate, 1 mM EDTA, followed by two ethanol precipitations.

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.

Methylation Interference Footprinting

Methylation interference footprinting with the purified protein fraction was performed essentially as described previously(4) . Briefly, a BamHI-StyI 147-bp restriction fragment containing the NRE (-245 to -98 of the peripherin promoter) was used as probe. Reactions contained 1 µl (approximately 20 fmoles) of purified protein. After EMSA, DNA was eluted from gel pieces containing bound or free DNA in 0.5 M ammonium acetate, 1 mM EDTA. The rest of the procedure was performed as described by Baldwin (28) .

Synthetic DNA Oligonucleotides

Oligonucleotides used in DNA mobility shift assays were synthesized on a Milligen Biosearch Cyclone Plus DNA synthesizer and gel purified.

Protein Purification

For protein purification, BCxx buffers were used where x denotes mM KCl in the following buffer: 20 mM HEPES, 0.2 mM EDTA, 20% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 10 mM NaF. Approximately 700 mg of liver nuclear extract was passed over a 120-ml bed volume column of heparin-Sepharose (Pharmacia Biotech Inc.) in two separate runs. The column was loaded at 1 ml/min and washed with 2 column volumes (CV) of BC150 buffer. Step elution of proteins was performed with 2.5 CV of BC350 buffer, and 5 ml fractions were collected. 5-µg aliquots of flow-through and eluent fractions were assayed for PPRS DNA-binding activity by EMSA (see Table 1). Positive fractions were pooled, adjusted to 100 mM KCl with BC00, and concentrated in Centriprep-10 concentrators (Amicon). Aliquots of the positive heparin-Sepharose column eluent fractions (30 mg each) were loaded onto a 10 ml of calf thymus DNA-Sepharose column at 0.2 ml/min. The calf thymus DNA-Sepharose was prepared by coupling calf thymus DNA to cyanogen bromide-activated Sepharose-CL2B, according to the procedure described by Kadonaga and Tjian(29, 30) . Proteins were eluted from the column with a linear gradient of KCl (0.1-1.5 M; 36 ml), and 2-ml fractions were collected. The majority of PPRS-binding activity eluted between 200 mM KCl and 700 mM KCl. Active fractions were pooled and brought to 150 mM KCl with BC00, and insulin was added to a final concentration of 250 µg/ml.

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).

RESULTS

Evidence for Common Constituents of the Protein Complex at the NRE in Undifferentiated and Differentiated PC12 Cells

In order to test the hypothesis that the DNA-binding core of the complex at the NRE is common to undifferentiated and differentiated PC12 cells, we performed EMSA assays under conditions that might disrupt protein-protein but not protein-DNA interactions. We reasoned that such conditions might dissociate the unique components of the slowly migrating complexes found in undifferentiated cells, producing the differentiated pattern of complexes. Deoxycholate was used in a first attempt to disrupt protein-protein interactions, with no effect (data not shown). Next we asked whether high salt would differentially affect protein-protein interactions. This experiment was performed with a mutant NRE oligonucleotide in which three point mutations outside the contact nucleotides make the binding site a perfect palindrome (PPRS; Table 1). DNA-protein complexes formed with the PPRS oligonucleotide are identical to those formed with the wild-type oligonucleotide (WTRS; Table 1) in EMSA analysis (compare Fig. 1A, lanes1-2 with 1B, lanes1-2). However, the DNA-protein interaction can withstand higher salt conditions (compare Fig. 1A, lane3 with 1B, lane5). This enables the salt titration shown in Fig. 1A to be performed. Under normal EMSA conditions (67 mM KCl), a pattern of complexes A, B, and C is obtained with undifferentiated PC12 cell extract, whereas complexes C, D, and E predominate in differentiated PC12 cell extract (Fig. 1A, compare lanes1 and 2). As the KCl concentration is increased above 250 mM KCl in reactions performed with undifferentiated PC12 cell extract, complexes A and B disappear concomitant with the appearance of complexes D and E. At 750 mM KCl, the complexes formed with both extracts migrate similarly (lanes7 and 8). Thus high salt conditions selectively disrupt the slowly migrating complexes unique to undifferentiated PC12 cell extract, leaving intact a constitutively present protein core.

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.


A Perfect Palindrome NRE Mutant Binds Proteins with Higher Affinity Than the Wild-type NRE

In order to facilitate purification of the protein(s) directly binding to the NRE, we considered that mutation of the NRE binding sequence from an imperfect to a perfect palindrome might increase the affinity of the DNA-binding protein for this site. This approach was successfully used by Treisman in the purification of SRF(31) . The PPRS oligonucletide (Table 1) shown above to form complexes resistant to high salt was used for this purpose. Off-rate analysis was performed to obtain a qualitative measure of the relative stability of complexes formed with the wild-type or perfect palindromic DNA oligonucleotides. Complexes formed with undifferentiated PC12 cell nuclear extract and either labeled WTRS or PPRS probes were incubated with 100-fold molar excess of unlabeled WTRS or PPRS competitor DNA, respectively, for varying times before analysis of DNA-protein complex formation by EMSA. Densitometric analysis of the resultant autoradiograph demonstrated that after 30 min of incubation with cold competitor, only 23% of the labeled WTRS-protein complexes remain, whereas 86% of the labeled PPRS-protein complexes remain (data not shown).

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).

Purification of the NRE-binding Protein by DNA Affinity Chromatography

In order to characterize the proteins comprising the repressor complex at the NRE, we decided to first purify the protein that directly contacts the DNA. To facilitate the purification of the NRE binding protein, we utilized the findings that the PPRS sequence has higher affinity for the protein complex, and binding is resistant to high salt. In addition, we tested whether rat liver, a tissue in which peripherin expression is repressed, would be a convenient source for large scale purification of the repressor proteins. Fig. 2shows that proteins in liver extract produce the same slowly migrating complexes (A and B) that are present in undifferentiated PC12 cell nuclear extract, consistent with the repressed state of the peripherin gene in both of these cell types (compare lanes2 and 3 with 4-6). It is of interest that liver nuclear extract also forms the faster migrating complexes that predominate in differentiated PC12 cells. (A possible explanation for this is that the multiprotein complexes might partially dissociate in the longer procedure of preparing nuclear extract from tissue.) Additionally, the titration of binding activity with increasing protein concentration in the EMSA assay in Fig. 2suggests a greater abundance of NRE-binding proteins in rat liver.

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.


UV Cross-linking Identifies a 33-kDa Protein Binding Directly to the NRE

In order to prove that the 33-kDa protein is indeed the protein responsible for the specific DNA binding activity of the extensively purified protein fraction, we performed UV cross-linking analysis(36) . An aliquot of protein eluted from the second affinity column was incubated with the radiolabeled WTRS oligonucleotide probe followed by irradiation with a 266-nm pulsed Nd:YAG laser. This technique forms irreversible cross-linked adducts between thymidine nucleotides and adjacent protein contacts(37) . The protein-DNA adducts were separated by SDS-PAGE and identified by autoradiography. Fig. 5demonstrates that only one complex, migrating with an apparent MW of at 45 kDa, displays the proper DNA binding specificity. The presence of 100-fold molar excess of the unlabeled mutant RM3 oligonucleotide (see Table 1) in the cross-linking reaction does not prevent formation of the radiolabeled DNA-protein complex migrating at 45 kDa. However, 100-fold molar excess of the unlabeled PPRS oligonucleotide successfully prevents formation of the 45-kDa complex. In determining the molecular weight of the protein component of the protein-DNA adduct, we have assumed that during denaturation in boiling SDS buffer, one strand of the oligonucleotide probe (38 bp) remains cross-linked to the protein, contributing 12 kDa in mass. Therefore, the 45-kDa DNA-protein cross-linked complex, when adjusted for DNA contribution, appears to correspond to the 33-kDa protein visualized on silver-stained gels.

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.


The Footprint Obtained with the Purified Protein Is Identical to That of Crude PC12 Cell Nuclear Extract

Further corroboration of the binding specificity of the 33-kDa protein was obtained by performing methylation interference footprinting with the extensively purified protein. A 147-bp peripherin promoter fragment spanning the NRE was used as probe. As shown in Fig. 6, the extensively purified protein bound an 11-bp region defined by 3 guanosines located at positions -170, -177, and -178 on the coding strand and 3 guanosines at positions -168, -169, and -176 on the noncoding strand. The methylation interference footprinting pattern obtained with crude undifferentiated and differentiated PC12 cell extract (4) is identical to that obtained with the extensively purified protein.

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 Me(2)SO(4) 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.


Mixing of Purified Protein and Flow-through Fractions Reconstitutes the Full Pattern of DNA-protein Complexes

In order to assess why the extensively purified protein produces only the one fastest migrating DNA-protein complex on EMSA, we performed a mixing experiment in which fractions with DNA binding activity eluted from the second PPRS affinity column were mixed with the flow-through from the first PPRS affinity column. If formation of the additional complexes seen with crude nuclear extract is dependent on protein-protein interactions, these non-DNA-binding proteins would be present in the flow-through fraction. Fig. 7A demonstrates that combination of the flow-through fraction with fractions eluted at 1.5 M or 3 M from the second PPRS affinity column can reconstitute the full array of DNA-protein complexes seen with the crude liver extract in the EMSA assay (compare lanes2 and 3, 4 and 5). The flow-through fraction alone has no significant DNA binding activity (Fig. 7A, lane1). The specificity of the reconstituted complexes was tested by performing the EMSA assay in the presence of 100-fold molar excess of the WTRS oligonucleotide or the mutant RM3 oligonucleotide (see Table 1). Fig. 7B shows that the protein eluted at high salt from the second PPRS affinity column has the appropriate binding specificity with either the WTRS (lanes1-3) or PPRS (lanes7-9) oligonucleotide as probe. The complexes reconstituted by mixing with the flow-through fraction also are competed by the WTRS oligonucleotide but not the nonbinding mutant RM3 oligonucleotide (lanes4-6, 10-12). These results suggest that protein(s) are present in the flow-through fraction that do not bind DNA directly but do interact through protein-protein contacts with the purified DNA binding protein.

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.


Microsequencing of the 33-kDa Protein

The eluent from the second PPRS affinity column was concentrated, run on a preparative SDS-PAGE gel, and transferred electrophoretically to polyvinylidine difluoride membrane (Bio-Rad). This sample was subjected to in situ tryptic digestion, and four peptides were microsequenced (Dr. William Lane, Harvard Microchemistry Facility). N-terminal sequence was not able to be obtained. The amino acid sequences of all four peptides match identically to the sequence of rat NF1-L predicted from the nucleotide sequence of the NF1-L cDNA clone (Table 3). NF1-L, a member of the CTF/NF-1 family of transcription factors, was initially purified from rat liver by Paonessa et al.(38) and cloned from a rat liver cDNA library.

Evidence That a CTF/NF-1 Transcription Factor Is the Core of the Complex at the NRE in PC12 Cells

DNA affinity chromatography using the PPRS binding site was used to purify from rat liver a 33-kDa DNA-binding protein identical in sequence to NF1-L. NF1-L is a ubiquitously expressed transcription factor(38) , and therefore we would expect it to be present in PC12 cells as well. We performed the following experiments to prove whether the DNA-binding protein contacting the NRE in PC12 cells is indeed NF1-L. In addition, these experiments were performed using the WTRS oligonucleotide probe to confirm that NF1-L is the DNA-binding protein contacting the wild-type NRE sequence.

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.

DISCUSSION

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(^2)). 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 alpha cells and haploid-specific genes in diploid cells are occupied by MCM1-alpha2 and a1-alpha2, 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.

FOOTNOTES

*
This work was supported in part by Public Health Service Grant NS-30943 from the NINDS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Medical Scientist Training Program at Vanderbilt University.

Recipient of a Research Career Development Award from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Cell Biology, C-2210 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-0337; Fax: 615-343-4539.

(^1)
The abbreviations used are: NGF, nerve growth factor; NRE, negative regulatory element; bp, base pair(s); EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; PPRS, perfect palindrome mutant repressor site oligonucleotide; WTRS, wild-type repressor site oligonucleotide; CV, column volumes.

(^2)
L. Chang, and M. Thompson, unpublished results.

ACKNOWLEDGEMENTS

We thank Tony Weil, Roland Stein, Robin Webster, and Lufen Chang for critical review of the manuscript; Ronald Arildsen for many helpful discussions; and Margaret Thompson for help with the manuscript. We thank Naoko Tanese for the generous gift of anti-CTF-1 antibody. We thank Joseph Beecham of the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, for use of the Nd:YAG laser for UV cross-linking.

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