Long-Distance Control of Origin Choice and Replication Timing in the Human β-Globin Locus Are Independent of the Locus Control Region

Abstract

DNA replication in the human β-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the β-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5′HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5′HS2-5 in the normal chromosomal context of the human β-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5′HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5′HS2-5 in the classically defined LCR do not control replication in the human β-globin locus. Recent studies also show that targeted deletion of 5′HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

ACKNOWLEDGMENTS

This work was supported by a fellowship from the American Cancer Society to D.M.C., a fellowship from the Deutsche Forschungsgemeinschaft to D.S., a fellowship from the Leukemia Research Foundation to A.R., and NCI grant CA54337 and NIH grants HL57620 and DK44746 to M.G.

We thank Jim Roberts and Ross Hardison for critical reading of the manuscript; R. Scott Hansen and Mirit Aladjem for helpful discussions; and Urszula Maliszewski, Agnes Telling, and the FHCRC Flow Cytometry and Image Analysis Facilities for technical assistance.