IN-VIVO PROTEIN-DNA INTERACTIONS AT A HUMAN DNA-REPLICATION ORIGIN

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream, DNase I treatment of nuclei isolated from both exponential ly growing and nonproliferating HL-60 cells showed that this region ha s an altered, highly accessible, chromatin structure, High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction, In growing HL-60 cel ls, footprints at sequences homologous to binding sites for known tran scription factors (members of the basic-helix-loop-helix family, nucle ar respiratory factor 1, transcription factor Sp1, and upstream bindin g factor) were detected in the region corresponding to the promoter of the downstream gene, Upon conversion of cells to a nonproliferative s tate, a reduction in the intensity of these footprints was observed th at paralleled the diminished transcriptional activity of the genomic a rea. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that e xtended over 70 nucleotides on one strand only, This footprint was abs ent from nonproliferating HL-60 cells, indicating that this specific p rotein-DNA interaction might be involved in the process of origin acti vation.