No abstract

Numerous genes contain promoter elements that are nuclease hypersensitive. These elements frequently possess polypurine/polypyrimidine stretches and are usually associated with altered chromatin structure. We have previously isolated a clone that binds a class of CT-rich promoter elements. We have further characterized this clone, termed the nuclease-sensitive element protein-1, or NSEP-1. NSEP-1 binds both duplex CT elements and the CT-rich strand of these elements in a 'generic' sequence specific manner and has overlapping but distinct single-and double-strand DNA binding domains. The minimal peptide region sufficient for both duplex and single-strand DNA binding includes two regions rich in basic amino acids flanking an RNP-CS-1 like octapeptide motif. Deletion analysis shows that the single-strand DNA binding activity is mediated by the RNP-CS-1 like octapeptide motif and is the key peptide region necessary for single-strand binding. NSEP-1's affinity for CT rich promoter elements with strand asymmetry in addition to its double- and single-strand DNA binding properties suggests that it may be a member of a class of DNA binding proteins that modulate gene expression by their ability to recognize DNA with unusual secondary structure.

We have located a positive, cis-acting DNA sequence element within the 5' flanking DNA of the c-myc gene (-125 base pairs). This DNA sequence element has a large purine-pyrimidine strand asymmetry and can assume the H-DNA conformation. A factor with the properties of a ribonucleoprotein (RNP) interacts with this DNA region. The interaction of the c-myc DNA sequence element and the RNP involves an RNase H-sensitive mechanism and, therefore, may involve an RNA-DNA hybrid. In addition, a protein factor(s) binds to this DNA sequence element. DNA footprinting and mutant oligonucleotide binding/competition assays implicate a punctate, poly(G-C) recognition/binding sequence for the RNP factor, whereas the major protein factor requires two ACCCT sequence motifs for maximal binding. These results suggest that RNP and protein factors act as positive transcriptional regulators of the c-myc gene, perhaps by altering DNA topology.Several genes have been identified that regulate growth and differentiation. One such gene is the protooncogene c-myc. Enforced expression of c-myc in cultured mouse erythroleukemia cells disrupts differentiation and allows continued growth (1-3). When the c-myc gene is linked to the immunoglobulin A enhancer, and made a transgene in mice, a pre-B cell hyperplasia results. A large percentage of these transgenics progress to a B-cell lymphoma (4, 5). c-myc antisense oligonucleotides inhibit cellular growth and induce differentiation in the human promyelocytic leukemia cell line HL-60 (6). These results demonstrate a direct role for c-myc in the regulation of cellular growth and an indirect role in cellular differentiation.Sequence-specific DNA binding proteins regulate eukaryotic transcription (7)(8)(9)(10)(11)(12). Although several protein factors have been shown to bind to the c-myc gene, the binding of these factors has not shown a correlation with the activity of the c-myc gene (13-15).We, and others, have observed non-B DNA structures in the 5' flanking region of the c-myc gene (ref. 16; T.L.D. and A.J.K., unpublished results). One of the single-strand nuclease-sensitive sites in the c-myc gene maps near and may correspond to a DNase I-hypersensitive site termed III1 (17). DNase I sensitivity at the I1I1 site disappears when cells become committed to terminal differentiation, a time when c-myc transcription is turned off (18). These data imply that the II1 nuclease-hypersensitive site is a cis-acting regulatory element of the c-myc gene. Therefore, we examined the role of this DNA region in c-myc expression. MATERIALS AND METHODSc-myc Fusion Gene Constructs, Gene Transfection, and Chloramphenicol Acetyltransferase (CAT) Assays. Deletions were made in a subclone of the 866-base-pair (bp) Pvu II fragment containing exon I and 353 bp of 5' flanking DNA.This subclone was digested with Sma I, and deletions were created with BAL-31 exonuclease and were sequenced by the dideoxy method (19). CAT constructs were transfected into HeLa cells as described (20). CAT assays were performed as described...

Transcripts of the proto-oncogene c-myc are composed of a rapidly degraded polyadenylated RNA species and an apparently much more stable, nonadenylated RNA species. In this report, the extended kinetics of c-myc RNA turnover have been examined in rapidly growing cells and in cells induced to differentiate. When transcription was blocked with actinomycin D in rapidly growing cells, poly(A)+ c-myc was rapidly degraded (t1/2 = 12 min). c-myc RNA lacking poly(A) initially remained at or near control levels; however, after 80 to 90 min it was degraded with kinetics similar to those of poly(A)+ c-myc RNA. These bizarre kinetics are due to the deadenylation of poly(A)+ c-myc RNA to form poly(A)- c-myc, thereby initially maintaining the poly(A)- c-myc RNA pool when transcription is blocked. In contrast to growing cells, cells induced to differentiate degraded both poly(A)+ and poly(A)- c-myc RNA rapidly. The rapid disappearance of both RNA species in differentiating cells suggests that a large proportion of the poly(A)+ c-myc RNA was directly degraded without first being converted to poly(A)- c-myc RNA. Others have shown that transcriptional elongation of the c-myc gene is rapidly blocked in differentiating cells. We therefore hypothesize that in differentiating cells a direct, rapid degradation of poly(A)+ c-myc RNA may act as a backup or fail-safe system to ensure that c-myc protein is not synthesized. This tandem system of c-myc turnoff may also make cells more refractory to mutations which activate constitutive c-myc expression.

I have used chemical probes and an oligonucleotide-association assay to determine the structure of a nuclease-sensitive, c-myc DNA region. I find that this DNA region can form a triplex-single stranded conformer in vitro--the H-DNA conformer. This DNA region has been shown previously to be a positive, cis-acting transcription element of the c-myc gene and to bind nuclear factors, including a base-paired ribonucleoprotein. Therefore, H-DNA may be a functionally important in vivo topoisomer where the H-DNA and B-DNA conformers have different transcriptional activities.