DNase-sensitive chromatin structure near a chromosomal origin of bidirectional replication of the avian alpha-globin...

We used an in vitro nuclear run-off replication assay to analyze the direction of replication in a 13-kb region 5' to the avian alpha-globin genes. Previous work from this laboratory suggested that the alpha-globin genes of 5-day chick red blood cells and avian MSB cells replicate in vivo in the transcriptional direction, possibly from a chromosomal origin near the alpha pi-globin gene. Here we extend those studies by showing that replication forks move divergently from an 8-kb region of alpha-globin 5'-flanking DNA. One potential zone for the initiation of bidirectional replication was located approximately 2.5 kb 5' to the alpha pi-globin gene in both of these cell types. Additionally, a barrier to replication fork movement, which may be located in a second origin zone, was found approximately 5 kb farther upstream. Both in 5-day RBCs, where the alpha-globin genes are expressed, and in MSB cells where they are not expressed, DNase I hypersensitive structures were found approximately 5 kb 5' to the alpha pi-globin gene, in the putative replication initiation domain. Another DNase I hypersensitive site was confirmed to exist in 5-day RBC nuclei upstream of the transcribed alpha pi-globin gene. These results suggest that replication of the alpha-globin genes initiates in the nearby 5'-flanking DNA of this locus in a region marked by distinct chromatin structures.