NM23-H1 and NM23-H2 Repress Transcriptional Activities of Nuclease-hypersensitive Elements in the Platelet-derived Growth Factor-A Promoter

Abstract

The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3-portions of both 5-SHS strands in either singlestranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5-and 3-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (82 to 8). Activity of the negative regulatory region (1853 to 883), which contains the 5-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.