The c-myc proto-oncogene encodes a nuclear phosphoprotein with leucine zipper and helix-loop-helix structural motifs which appears to be important in the molecular biology of normal and abnormal cellular proliferation. Myc is implicated in the control of both differentiation and replication (Cole, Annu. Rev. Genet. 20:361-384 (1986)), and recent reports link myc to apoptotic cell death (Askew et al., Oncogene 6:1915-1922 (1991), Evan et al., Cell, 69:119-125 (1992), and Neiman et al., Proc. Natl. Acad. Sci. USA 88:5857-5861 (1991), each of which is incorporated herein by reference). Myc and its dimerization partner Max form stable heterodimers through their helix-loop-helix and leucine zipper domains and bind specifically to a core "E box" CACGTG DNA sequence (Blackwood et al., Science 251:1211-1217 (1991), incorporated herein by reference). Max homodimers may serve as transcriptional repressors, whereas myc/max heterodimers can activate transcription (Kretzner et al., Nature 359:426-429 (1992), incorporated herein by reference). Certain of the biological functions of myc may be mediated by transcriptional regulation of putative target genes.
Despite recent progress in defining the mechanism of myc action on "down stream" events, less progress has been made in defining the proteins regulating the expression of c-myc itself. Both transcriptional and post-transcriptional mechanisms appear to play a role in regulation of c-myc gene expression (Cole, Annu. Rev. Genet. 20:361-384 (1986), Spencer et al., Cancer Res. 56:1-48 (1991), and Marcu et al., Annual Rev. Biochem. 61:809-860 (1992), each of which is incorporated herein by reference). Maintenance of the level of the c-myc MRNA is achieved by regulation of both transcriptional initiation and elongation. Both initiation, and elongation of the c-myc mRNA, depend upon promoter elements which interact specifically with particular nuclear factors (Spencer, Oncogene 5:777-785 (1990) and Spencer et al., Cancer Res. 56:1-48 (1991), each of which is incorporated herein by reference). A general map of mouse and human c-myc transcription elements has been suggested and nuclear factors which bind to these elements have been reported. In certain cases novel cDNA's encoding such factors have been isolated and sequenced including: ZF87 (also called MAZ), a proline-rich six Zn-finger protein binding to ME1a1/ME1a2 elements within P2 promoter of the murine c-myc gene (Pyrc et al., Biochem. 31:4102-4110 (1992) and Bossone et al., Proc. Natl. Acad. Sci. USA, 89:7452-7456 (1992), each of which is incorporated herein by reference); a 37-kDa protein, MBP-1, which appears to be a negative regulator of the human c-myc promoter (Ray et al., Mol. Cell. Biol. 11:2154-2161 (1991), incorporated herein by reference); and nuclease sensitive element protein-1 (NSEP-1) which binds to a region necessary for efficient P2 initiation (Kolluri and Kinniburgh, Nucl. Acids Res. 17:4771 (1991), incorporated herein by reference). In addition, an Rb binding protein E2F which recognizes an E1A-transactivation site in the human c-myc promoter (Thalmeier et al., Genes Dev. 3:527-536 (1989), incorporated herein by reference) has also been cloned (Helin et al., Cell 70:337-350 (1992), incorporated herein by reference).
The chicken c-myc 5'-flanking region is at least 10-fold enriched in CpG-pairs compared with total chicken DNA and is presently thought to be a member of the family of CpG-rich islands involved in regulating certain house keeping genes (Bird et al., Nature 321:209-213 (1986), incorporated herein by reference). Overall high GC content (.about.80%) of the 5'-flanking region predicts that most of the potential regulatory DNA elements will be GC-rich. Analysis of DNA-protein interactions within the 5'-flanking region of the chicken c-myc gene revealed multiple GC-rich sequences which specifically interact with nuclear proteins (Lobanenkov et al., Eur. J. Biochem. 159:181-188 (1986), incorporated herein by reference). Proteins binding to one specific region within a hypersensitive site approximately 200 base pairs upstream of the start of transcription have reportedly been analyzed (Lobanenkov et al., Oncogene 5:1743-1753 (1990) and Lobanenkov et al., Gene Reg. and AIDS, Portfolio Publishing Corp., Texas, p. 45-68 (1989), incorporated herein by reference). Three nuclear factors were found that bind to several overlapping sequences within 180-230 bp upstream of the start of transcription. Two of the proteins appear to resemble the transcription factor Sp1, the other is a factor which seems to bind to a GC-rich sequence containing three regularly spaced repeats of the core sequence CCCTC. The CCCTC-binding factor was termed CTCF (Lobanenkov et al., Oncogene 5:1743-1753 (1990) and Lobanenkov et al., Gene Reg. and AIDS, Portfolio Publishing Corp., Texas, p. 45-68 (1989), incorporated herein by reference).
Studies suggest that during embryonic development the regulatory state of c-myc transcription can determine whether a cell continues to proliferate, or stops, and enters a pathway to terminal differentiation. Failure to properly regulate myc may be one pathway to malignancy. Thus, identifying the suppressor mechanisms by which myc is regulated would provide important reagents and assays useful in the detection of mutants that are indicative of a disease state such as cancer and the development of candidate therapeutic agents can that regulate cell proliferation, for example, inhibiting cell proliferation in cancer on the one hand, or stimulating cell proliferation in a damaged tissue on the other hand. Quite surprisingly, the present invention fulfills these and other related needs.