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 ␤-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 ␤-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 (5HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5HS2-5 in the normal chromosomal context of the human ␤-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5HS2-5 in the classically defined LCR do not control replication in the human ␤-globin locus. Recent studies also show that targeted deletion of 5HS2-5 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.The eukaryotic genome is divided into independently regulated domains that initiate DNA replication from defined sequences at specific times during S phase. Control mechanisms exist to specify both the sites of replication initiation (origins) and the temporal order of replication throughout the genome. Although great progress has been made in understanding the control of DNA replication in viruses, bacteria, and yeasts, we are only beginning to understand the control of DNA replication in higher eukaryotes. Over a dozen origins have been mapped in metazoans (reviewed in reference 10), and it appears that DNA replication in these organisms, in contrast to prokaryotes and lower eukaryotes, does not depend only on origin-proximal sequences, but is subject to long-distance regulation as well. The human ␤-globin locus is one of several loci in which this long-distance regulation has so far been demonstrated; sequences over 50 kb from the origin of replication are necessary for proper replication initiation and replication timing of the locus. Because of the tools available with which to make genetic modifications and perform functional analyses, the human ␤-globin locus offers an excellent opportunity to dissect possible replication control mechanisms.A single bidirectional origin, located in the vicinity of the ␤-globin gene, is used to replicate the human ␤-globin locus ( Fig. 1) (1, 35). Although this origin is used in both erythroid and nonerythroid cells, replication timing follows patterns of gene activity and chromatin structure: in erythroid cells, the globin genes are transcribed, the locus is gen...

Identification of New Human Origins of DNA Replication by an Origin-Trapping Assay

“…The importance of such distal elements has been demonstrated for origins located near the human ␤-globin and the hamster DHFR loci (12,27,31). In embryonic extracts from frogs, flies, or fish, many genomic sites can be used as replication origins.…”

Orchestration of chromatin-based processes: mind the TRRAP

“…However, the role of acetylation in replication timing may not be limited to yeast. In human cells, histone acetylation has been correlated with replication timing (Cimbora et al, 2000), and HBO1 HAT (Iizuka and Stillman, 1999), associates with components of the replication machinery (Burke et al, 2001). This was confirmed by a study showing that histones are hyperacetylated at the active origins of replication and that this coincides with binding of the origin recognition complex (Aggarwal and Calvi, 2004).…”

Replication Timing Profile Reflects the Distinct Functional and Genomic Features of the MHC Class II Region

“…This is consistent with a report that replication timing in the human b-globin locus correlates with an open chromatin architecture rather than expression per se in certain cell types. 29 The classical class II region contains clusters of coordinately controlled, immune-related genes that are likely to have arisen during evolution by segmental duplications. 12 It thus behaves as a distinct genomic region in terms of its expression.…”