©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cellular Nucleic Acid Binding Protein Regulates the CT Element of the Human c- myc Protooncogene (*)

Emil F. Michelotti , Takeshi Tomonaga , Henry Krutzsch , David Levens (§)

From the (1) Department of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The CT element of the c- myc gene is required for promoter P1 usage and can drive expression of a heterologous promoter. Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Although significant levels of CT activity persisted following Sp1 immunodepletion, EGTA totally abolished transactivation, thus implicating another metal requiring factor in CT element activity. As hnRNP K binds to one strand of the CT element, but has no metal requirement, the opposite (purine-rich strand) was examined as a target for a metal-dependent protein. A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes. Two forms of CNBP differed in their relative binding to the CT- or sterol-response elements. CNBP was shown to be a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulated expression of a CT-driven but not an AP1-dependent reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element.


INTRODUCTION

Proper regulation of the c- myc gene has been shown to be important for the execution of several aspects of cellular metabolism (for review, see Marcu et al. (1) ). Induction of terminal differentiation is, in general, accompanied by a decrease in c- myc transcription (2, 3) . Due to its rapid induction following treatment of numerous cell lines with a variety of mitogenic signals, c- myc has been classified as an immediate early response gene (4, 5) . c- myc expression may also dictate cell death as high levels of c- myc can lead to apoptosis under some conditions (6) . Because the physiological and pathological consequences of c- myc expression are profound, it must be tightly regulated; indeed, simply a 2-fold decrease in c- myc mRNA levels lengthens the time required for Gcells to enter S phase by 3-4 h and prolongs the cell cycle by 12% in actively dividing cells (7) .

The response of c- myc to a wide variety of growth and differentiation factors suggests that its expression integrates stimuli from several pathways. Two features of the c- myc gene presumably enhance the cellular ability to fine tune c- myc transcription rates. First, there are two major start sites, P1 and P2, that are located 176 base pairs apart and can be regulated independently (8) . Second, nuclear run-on experiments have recently demonstrated that there is a holdback to RNA synthesis at the P2 promoter (9, 10) .

Perhaps because of such complexities, the molecular mechanisms regulating c- myc expression have remained enigmatic. Early studies of c- myc identified six candidate regulatory regions hypersensitive to DNase I digestion (11, 12) . In several cell lines, the hypersensitivity to DNase I digestion within three of these regions correlates with active transcription of the c- myc gene. One of these three sites, termed III, is situated 125 base pairs upstream of P1 and consists of five imperfect direct repeats of the sequence CCCTCCCCA (termed the CT element); four repeats are in tandem, while the fifth downstream repeat is separated by nine base pairs.

Previous studies have demonstrated that the CT element confers a 5-10-fold stimulation of transcription upon a heterologous promoter both in vivo and in vitro (13, 14, 15) . hnRNP() K, through binding to single-stranded CT DNA (13) , and Sp1, through binding to the double-stranded CT element (15) , have each been proposed to be regulators of promoter activity. If hnRNP K binds to the pyrimidine-rich strand, are there factors that bind to the purine-rich strand? We present here the purification and cloning of such a binding activity, and identify it as the previously studied cellular nucleic acid binding protein (16) .


MATERIALS AND METHODS

In Vitro Transcription and RNase Protection Assays-HeLa cells were grown in suspension in spinner-modified minimal essential medium supplemented with 10% fetal calf serum and nonessential amino acids to a density of approximately 1 10/ml. Nuclear extracts were prepared as described previously (17) . In vitro transcription reactions were performed in 50 µl and contained 500 µ M of each rNTP, 50 µ M NaCl, 1 m M dithiothreitol, 1.5 m M MgCl, 20 m M Tris 8.0, 2 m M spermidine, and 100 ng of each template. The amount of nuclear extract given in the figure legends is in micrograms of protein. Reactions were incubated at room temperature for 1.5-2 h, terminated by the addition of 50 µl of 10% SDS, phenol extracted, and precipitated. In vitro synthesized RNA was hybridized with P-labeled antisense RNA in 30 ml of 0.5 M NaCl, 20 m M Tris 7.5, and 1 m M EDTA at 65 °C for 3 h. RNA probes were made according to SP6 RNA polymerase manufacturer's (Promega Biotec) specifications. RNase Tdigestion was performed by addition of 300 ml of 0.3 M NaCl, 20 m M Tris 7.5, 7% formamide, and 1000 units of T(Life Technologies Inc.). After 1 h, digestions were extracted with a 1:1 (v/v) mixture of phenol-chloroform and then ethanol precipitated. Products were separated on 6% denaturing polyacrylamide gels.

Templates for in vitro transcription were constructed by inserting four repeats of the CT element (-149 to -122) upstream of the same promoter used for the basal transcription control (the c- fos promoter from -56 to +109) so that any influence of the vector on transcription would affect both activated and basal transcription equally. A 10-base pair deletion 109 base pairs downstream of the transcription start site was introduced into p56CT so that RNase protection assay of the CT-activated transcription products yielded a molecule that was 10 bases smaller than the protected transcription product of the basal promoter. Details of plasmid construction will be given in a subsequent article.()

Chromatography and Protein Purification

Oligonucleotide was coupled to CNBr-Sepharose according to manufacturer's (Pharmacia Biotech Inc.) specifications at 0.5 mg/ml. Typically, 1.5 column volumes of extract was loaded on the affinity column at 1 column volume/h in 20 m M Tris, pH 8.0, 0.05% Tween 20 (Bio-Rad), 50 µ M ZnCl, 50 µ M EDTA, 20% glycerol, plus the indicated concentration of NaCl. For purification of CNBP, extracts were loaded onto the purine oligonucleotide column at 0.1 M NaCl and step eluted with the same buffer containing 0.5 M NaCl, 1.0 M NaCl, and finally 5 M guanidine HCl. For the second round of affinity purification, the guanidine eluate was dialyzed against 0.1 M NaCl buffer and fractionated again on a column one-fifth the size of the first affinity column. Prior to each cycle of affinity purification, extract was first passed through a nonspecific single strand DNA oligonucleotide column at 0.1 M NaCl to remove nonspecific single strand binding proteins.

EMSA

One nanogram of oligonucleotides phosphorylated with P-gamma-ATP (10,000 cpm) were incubated with the indicated proteins in a final volume of 7 µl containing 50-70 m M NaCl, 20 m M Tris, pH 8.0, 0.25 mg/ml bovine serum albumin, and 0.02% Tween 20. Binding reactions proceeded for 30 min on ice and were loaded on prerun 4% nondenaturing polyacrylamide gels and then run at 15-20 V/cm, dried, and exposed to autoradiography.

Protein Sequencing: Preparation of Fragments for Internal Sequence Analysis

To generate peptides for internal sequence analysis, the protein was first reduced and alkylated with N-isopropyliodoacetamide and then cleaved with cyanogen bromide and digested with trypsin; the resulting peptides were separated by HPLC chromatography, all as described previously (18) . The sequences of purified peptides were determined with a Porton/Beckman 2090 online sequencer using standard program 1. Phenylthiohydantoin amino acid identification was accomplished with a Beckman System Gold system with a modified sodium acetate gradient program and a Hewlett-Packard narrow bore C-18 column.

Transfection and Chloramphenicol Acetyltransferase Assays

Hela cells were cultured in Dulbecco's modified minimal essential medium supplemented with 10% fetal calf serum. 5 10cells were resuspended in 250 µl of Dulbecco's modified minimal essential medium, and incubated on ice for 10 min with plasmid DNA. Electroporation was performed with Cellporator (Life Technologies, Inc.) at 230 V, 1180 millifarads setting. After electroshock, cells were incubated on ice for an additional 10 min. Transfected cells were added to 10 ml of medium and incubated for 48 h before harvesting for chloramphenicol acetyltransferase assays (19) . Expression constructs were generated via PCR using the following oligonucleotides: amino-terminal CNBP TGA GAT GGA TCC ATG AGC AGC AAT GAG TGC TTC AAG TGT GGA and carboxyl-terminal CNBP TAA AGC GGA TCC TAA GGC TGT AGC CTC AAT TGT GCA TTC CCG. PCR products were digested with BamHI and cloned into the BamHI site of PC Amp DNA I (Invitrogen).


RESULTS

A Purine Strand-specific Binding Factor Requires Zinc

hnRNP K binds the pyrimidine-rich strand of the CT element. What is the state of its partner purine strand? The displaced strand might be naked; alternatively it might be complexed with other macromolecules. Examination of the native c- myc CT element in vivo using potassium permanganante modification as a single strand-specific probe revealed prominent reactivity at the margins of the CT region (data not shown). The relative lack of reactivity within the CT repeats suggested that bound factors were protecting this region from permanganate reactivity, which predicts the existence of a purine strand-specific binding factor. To test this, EMSAs were performed using HeLa cell nuclear extracts and the purine single strand as probe. An abundant purine strand binding activity was discovered (Fig. 1, lane 1). This binding activity was specific since increasing amounts of the purine strand effectively competed the probe, whereas two nonspecific oligonucleotides at identical concentrations had no effect (Fig. 1, lanes 2-13). Additional characterization revealed that the purine strand binding activity was dependent upon divalent cations, since binding was abolished by EGTA (Fig. 2, compare lanes 2 and 3). Zinc, and to a lesser extent manganese, conferred resistance to EGTA inhibition (compare lanes 5 and 6). The addition of magnesium, zinc, manganese, or calcium in the absence of EGTA had no effect on binding ( lanes 8-11). It seemed that this zinc-dependent purine strand binding activity might contribute to CT-mediated transcription activation.


Figure 1: A purine strand binding activity is sequence-specific. Three micrograms of nuclear extract was incubated with 1 ng of P-labeled purine strand probe alone ( lane 1) or with 1, 5, 25, or 125 ng of specific competitor ( lanes 2-5) or with two different nonspecific competitors ( lanes 6-9 and 10-13). Arrow to the right indicates the specific mobility shift




Figure 2: Purine strand binding activity is EGTA sensitive. Nuclear extract was preincubated either without ( lanes 1, 2, 8-11), or with ( lanes 3-7) 4 m M EGTA. To block EGTA inhibition, some preincubations included 100 µ M MgCl( lane 4), ZnCl( lane 5), MnCl( lane 6), or CaCl( lane 7). The effects of each of these metals alone is shown in lanes 8-11. Samples were then assayed by EMSA as described under ``Materials and Methods'' using the purine-rich single strand as probe. (Following EGTA treatment, probe was bound by EGTA-insensitive factors, which yielded complexes of slower electrophoretic mobility and are not shown; see lanes 3, 4, and 7)



Zinc-requiring Factors Mediate CT Element Transactivation

When located upstream of the c- fos minimal promoter, four tandem CT repeats drove increased levels of specific transcription in vitro using HeLa cell nuclear extracts (Fig. 3 a, lane 1, compare basal- and CT-mediated bands). Chelation of HeLa extracts with EGTA totally eliminated CT element-stimulated transcription ( lane 2), implicating a divalent cation requiring protein in CT-mediated transactivation. The level of RNA synthesis supported by the minimal fos promoter was unperturbed by EGTA, indicating that the affected proteins were not required for basal transcription. Zinc was the only divalent cation that protected extracts from the inhibitory effects of EGTA (Fig. 3 b), implicating a zinc metalloprotein in CT element activity. Although the CT element has been considered simply as a noncanonical Sp1 site, extracts completely immunodepleted of Sp1 retain considerable ability to transactivate through CT repeats in vitro, revealing the contribution of additional factors to CT-activated transcription (data not shown). It therefore seemed that a second zinc metalloprotein, in addition to Sp1, contributed to up-regulation through this element.


Figure 3:a, CT-activated transcription in vitro is sensitive to EGTA. Nuclear extract was preincubated either without ( lane 1) or with ( lane 2) 4 m M EGTA at 30 °C for 10 min. Remaining components required for in vitro transcription were then added as described under ``Materials and Methods,'' and transcripts were quantitated by RNase protection. Bands corresponding to basal and CT-activated transcripts are indicated to the right. b, CT-mediated transcription requires zinc. Nuclear extract was preincubated with 4 m M EGTA ( lanes 1-4). In order to block EGTA inhibition, either 100 µ M of CaCl( lane 2), MgCl( lane 3), or ZnCl( lane 4) were included in the preincubation. In vitro transcription products directed by basal or CT-bearing templates were assayed by RNase protection and are indicated to the right. (Less EGTA inhibition was noted than in Fig. 1, because reactions were preincubated at 4 instead 30 °C; the metals alone had no effect on in vitro transcription, data not shown).



Purine Single Strand Binding Factors Are Involved in CT-mediated Transcription Activation

Was this purine strand binding activity a player in CT element transcriptional stimulation? If so, then the same purine-rich oligonucleotide that effectively competed the zinc-dependent binding activity (see Fig. 1) would be predicted to antagonize CT-dependent activation by titrating the same binding activity in in vitro transcription reactions. To test this, increasing amounts of either the purine oligonucleotide or a nonspecific oligonucleotide were added to in vitro transcription assays. Indeed, 90% of CT element-mediated stimulation was eliminated at the highest purine oligonucleotide concentration, whereas a nonspecific oligonucleotide at the same concentration inhibited transcription activation only 10% (Fig. 4). The concentration of oligonucleotide required to compete CT transactivation correlated with the concentration required to compete the EGTA-sensitive mobility shift. Although the simplest interpretation of this result is titration of a trans-acting factor, qualitatively similar results, previously reported by Cooney et al. (20) , were attributed to triplex formation between the purine oligonucleotide and the CT element in the template. Formation of this triplex was hypothesized to block binding of any positively acting transcription factors at the CT element.


Figure 4: The purine single strand inhibits CT-mediated transcription activation. 0.25, 0.5, 1.0, or 2.0 µg of either the purine single strand ( lanes 2-5) or a nonspecific single-stranded oligonucleotide ( lanes 6-9) were added to in vitro transcription reactions utilizing 60 µg of nuclear extract. Reaction products were analyzed by RNase protection. Bands corresponding to basal and CT-mediated transcription are indicated to the right.



If a trans-acting factor necessary for CT element function bound to the purine strand, then passage of extracts through a purine strand affinity column should subtract this factor, inactivating CT-stimulated but not basal transcription. Alternatively, if the purine strand oligonucleotide directly inactivated the CT element through triplex formation with the template, then extracts should be inert to purine strand affinity chromatography. The purine oligonucleotide was coupled to Sepharose, and nuclear extract was passed through the column at 0.1 M NaCl. The column was washed and sequentially step eluted with 0.5 M NaCl and 5 M guanidine HCl. The flow-through of the column was devoid of CT-mediated transcription activation, but still supported basal transcription (Fig. 5), proving that the mechanism of purine strand inhibition of transcription was due to interaction with trans-acting factors and did not require triplex formation. Passage of nuclear extract through a nonspecific oligonucleotide column had no effect on transcription activation (data not shown). Elution of the zinc-dependent purine strand binding activity from the affinity column required denaturation (see below), therefore it was not surprising that the high salt- and guanidine-eluted fractions failed to restore CT activation when added to the flow-through of the purine oligonucleotide column (data not shown).

Elution of the purine strand affinity column with salt concentrations as high as 2 M failed to remove any purine oligonucleotide binding activity, indicating either that chromatography inactivated the factor or that its interaction with the CT element purine strand was extremely tenacious. Tightly bound protein, eluted from the purine strand column with denaturants (urea or guanidine HCl), displayed specific purine strand binding activity following dialysis (data not shown). Using this data, the activity was purified by two successive cycles of affinity chromatography in which the 0.1 M NaCl flow-through of a nonspecific single-stranded DNA oligonucleotide column was directly loaded on a purine oligonucleotide column, washed, and step eluted with 0.5 M NaCl, 1.0 M NaCl, and 5 M guanidine HCl. Following two rounds of affinity purification, SDS-polyacrylamide gel electrophoresis showed the prominent bands to be a doublet of approximately 19 kDa (Fig. 6, lanes 4 and 5). EMSA analysis of the renatured proteins from the doublet revealed an EGTA-sensitive binding activity with a gel mobility shift identical to that seen with crude extract (data not shown).


Figure 6: Purification of the major purine strand binding activity. Nuclear extract was fractionated over a purine strand affinity column at 0.1 M NaCl, washed with 1.0 M NaCl, and eluted with guanidine HCl. The guanidine HCl eluate was dialyzed to 0.1 M NaCl and fractionated again on the affinity column. Sixty micrograms of nuclear extract ( lane 2), 10 µl of first round affinity-purified protein ( lane 3), and 2.5 ( lane 5) or 10 µl ( lane 4) of second-cycle affinity-purified protein were visualized by gold-blot analysis after electrophoresis (SDS-polyacrylamide gel electrophoresis, 15%). The molecular weights of marker protein ( lanes 1 and 6) are indicated to the right. The doublet that was electroeluted and sequenced is indicated by an arrow ( right).



CNBP Is the Major Purine Strand Binding Activity

Two cycles of purine strand affinity chromatography, each requiring elution with denaturants followed by renaturation, yielded the zinc-dependent purine strand binding activity copurifying with a doublet of polypeptides at approximately 19 kDa (Fig. 6, lanes 4 and 5); this doublet comprised the vast majority of the protein in this sample. To identify and characterize the polypeptides binding to the affinity column, the eluted fractions were digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Six unambiguous sequences were obtained (), all identical to predicted tryptic fragments of CNBP, a zinc finger protein previously shown to bind the purine-rich single strand of the sterol response element (SRE) and postulated to play a role in sterol metabolism (16) . Several possibilities could account for the appearance of a doublet, including proteolysis, posttranslational modification, or forms of CNBP specified by alternately spliced mRNAs. The existence of another protein comigrating with, but unrelated to CNBP, was also considered. In order to help distinguish between these possibilities, reverse transcription PCR was performed using primers derived from extreme amino- and carboxyl-terminal CNBP sequence; this yielded a doublet at 550 base pairs (data not shown). Cloning and sequencing of these PCR products revealed DNA encoding the previously described CNBP (referred to hereafter as CNBP-L), as well as an alternately spliced cDNA in which 7 amino acids (residues 36-42) were deleted (CNBP-S). These alternately spliced RNAs most likely encode the purified protein doublet.

Both CNBP-L and CNBP-S, expressed as recombinant glutathione S-transferase-fusion proteins, bound specifically to the CT element purine strand (Fig. 7); complex formation was eliminated by preincubation of the recombinant protein with EGTA (data not shown). Removal of the gst portion of the recombinant protein with thrombin generated a purine strand binding activity with an EMSA mobility identical to that of the major purine strand binding activity in nuclear extract (Fig. 8, lanes 5 and 6). As further proof of identity, EMSA purine strand complexes generated using either HeLa nuclear extract, were supershifted with affinity-purified antibodies to recombinant CNBP (Fig. 8, lane 2), but not with antibodies to either hnRNP K ( lane 3), or glutathione S-transferase ( lane 4). Although the purine strand was the preferred ligand for recombinant as well as HeLa CNBP, double strand CT element nevertheless competed effectively for binding to the CNBPs at 5-fold greater concentrations (data not shown).


Figure 7: Recombinant CNBP-L and CNBP-S account for the major purine strand activity. 1, 5, 25, or 125 ng of either specific ( lanes 2-5) or nonspecific oligonucleotides ( lanes 6-9 and 10-13) were incubated with 30 ng of recombinant gst-CNBP-L ( top), or gst-CNBP-S ( bottom) using the purine single strand as probe. Complexes were resolved by nondenaturing polyacrylamide electrophoresis.




Figure 8: CNBP comprises the major purine single strand mobility shift. Left, affinity-purified antibodies to either CNBP ( lane 2), hnRNP K ( lane 3), or glutathione S-transferase ( lane 4), were added to a mixture of P-labeled purine single-stranded oligonucleotide and 3 µg of HeLa cell nuclear extract. Complexes were resolved by EMSA. Arrows indicate the two complexes that contain CNBP. Asterisk to the left indicates supershifted CNBP complex. Right, purified recombinant CNBP-L ( lane 5) and recombinant CNBP-S ( lane 6) were digested with thrombin according to manufacturer's (Sigma) specifications and incubated with labeled purine single-stranded oligonucleotide. The upper shift most likely contains multiple CNBP molecules/DNA strand (probe contains 3 CT repeats) and is quantitatively variable (compare lanes 1 and 7). Free probe (probe alone, lane 8) is not shown in lanes 1-4.



CNBP-L and CNBP-S Can Be Distinguished by DNA Binding

The alternately spliced mRNA encoding CNBP-S disrupts the sequence GRGGF found in CNBP-L. As GRGGF constitutes a motif found most frequently in a variety of proteins that interact with single-stranded nucleic acid (21) , it seemed possible that CNBP-L and -S might not interact with all nucleic acids equivalently. CNBP-L was originally identified by its binding to the purine-rich single strand of the sterol response element upstream of the low density lipoprotein receptor gene. Therefore, recombinant CNBP-L and -S were compared quantitatively for their ability to bind to the SRE and the CT element purine-rich strands. Although the 7-amino acid difference between the two spliced forms did not significantly affect binding to the CT oligonucleotide (Fig. 9 a), it caused CNBP-S to have an approximately 27-fold higher affinity for the SRE oligonucleotide relative to CNBP-L (Fig. 9 b, compare lanes 2 and 13).


Figure 9: Differential binding of CNBP alternately spliced forms to the sterol response elements. Crude extract (3 µg, lane 1), or decreasing concentrations of gst-CNBP-L ( lanes 2-9) or gst-CNBP-S ( lanes 10-17), were added to 1 ng of P-labeled CT ( panel a) or SRE ( panel b) purine-rich oligonucleotides and subjected to EMSA. Recombinant CNBP shifts are indicated by the arrows at the right. Recombinant protein amounts are as follows: lanes 2 and 10, 750 ng; lanes 3 and 11, 250 ng; lanes 4 and 12, 80 ng; lanes 5 and 13, 27 ng; lanes 6 and 14, 9 ng; lanes 7 and 15, 3 ng; lanes 8 and 16, 0.9 ng; and lanes 9 and 17, 0.3 ng. Probe alone is shown in lane 18.



CNBP Can Stimulate CT Activation in Vivo

Does CNBP participate directly in the regulation of CT-mediated expression? To address this question, a genomic CNBP expression vector encoding both the S and L forms was cotransfected with either a CT- or AP1-dependent reporter construct into HeLa cells. CT-dependent expression was stimulated 5-fold by the CNBP expression vector (Fig. 10). In contrast, AP1-driven chloramphenicol acetyltransferase activity was not altered by CNBP expression. Thus CNBP behaves as a transactivator of the CT element in vivo.


DISCUSSION

CNBP was found to be the second single strand-specific DNA binding protein interacting with the CT element, a segment of DNA that up-regulates expression of the c- myc gene. CNBP binds to the purine-rich strand of the CT element, opposite to hnRNP K, which can bind to pyrimidine-rich strand. Both proteins up-regulate CT element-driven chloramphenicol acetyltransferase expression in transient transfection assays. These properties define a system whereby formation of an open complex at the CT element composed of unpaired strands, CNBP, hnRNP K, and any additional factors, could be favored by several mechanisms. Modulation of the concentrations of either active CNBP or active hnRNP K, in vivo, could influence the ability of the other to find the CT element by exposing its binding site. Because five CT elements reside in the c- myc promoter, open complex formation would have an exponential dependence on the concentrations of individual components, rendering the complex sensitive to even modest fluctuation in the levels of its components. Thus physiological conditions that alter either CNBP or hnRNP K levels, or that sequester one or the other protein, would be expected to modulate CT element activity. Importantly, hnRNP K has been shown to interact with several regulators of cell growth or gene expression, including the oncoproteins c-Src (23, 24) and Vav (25) , thus highlighting potential links between the CT element and important cellular processes. The protein neighbors of CNBP remain to be explored. Because Sp1 can bind to the double-stranded CT element and activate transcription, at least in vitro, a complex equilibrium exists at this site. Cellular conditions such as transcription driven supercoiling of upstream sequence or replication could favor single strand CT formation with concomitant CNBP and hnRNP K action, whereas activation of a quiescent c- myc gene might require Sp1 binding to the duplex CT element.

If hnRNP K and CNBP were to bind the CT element in vivo, then a complex pattern of sensitivity to, and protection from, agents that cleave single strand DNA might be expected. Indeed, the in vivo profile of the reactivity of the CT region with the single strand-specific oxidizing agent KMnOreveals hypersensitivity on each strand both at the extreme boundaries of the element and between the two most 3` repeats (data not shown).

Several features of CNBP as a transcription factor are noteworthy. The protein exists in two forms with different nucleic acid recognition properties. The form of CNBP first identified, CNBP-L, was devoid of an effect on the sterol response element in cotransfection experiments (16) . However, CNBP-L binds particularly poorly to the G-rich single strand of the SRE as compared with the CT element or to CNBP-S binding to the SRE. Two recently cloned proteins, sterol regulatory element binding proteins 1 and 2, have emerged as important up-regulators of genes in the cholesterol biosynthetic pathway, thus suggesting that any role for CNBP in the regulation of these genes may be a negative one (26, 27, 28) . This would be consistent with the increase in CNBP levels under sterol-repressed conditions (16) . It is noteworthy that both of the elements recognized by CNBP in single-stranded form are also bound by a transcription factor in double-stranded form; Sp1 has a positive effect on transcription from the CT element,and SREBP-1 and -2 have been shown to stimulate transcription through the SRE (26, 27) . Whether the binding of these factors is regulated by modulation of the single-stranded character of the loci will require additional experiments. The array of regulatory factors displayed along the c- myc gene, the complicated interweaving of DNA topology, and protein-protein interaction, and the control of transcription by promoter utilization, initiation, and elongation, all serve to indicate that the inputs of many pathways are integrated to control transcription of this important protooncogene.

  
Table: Amino acid sequences of CNBr fragments obtained from purine strand affinity purified protein



FOOTNOTES

*
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.

§
To whom correspondence should be addressed: Laboratory of Pathology, National Cancer Inst., NIH, Bethesda, MD 20892. Tel.: 301-496-2176; Fax: 301-402-0043.

The abbreviations used are: hnRNP, heterogeneous ribonucleoprotein; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; SRE, sterol response element; CNBP, cellular nucleic acid binding protein.

E. F. Michelotti and D. Levens, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Lance Liotta and Lou Staudt for critical evaluation of the manuscript. We thank Kevin Gardner for helpful discussions during the course of the work and Suzanne Sanford for superb technical assistance.


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