An origin of bidirectional DNA replication is located within a CpG island at the 3′ end of the chicken lysozyme gene

Loc Phi-van 1 Wolf H. Strtling 0 1 0 Institut fr Physiologische Chemie, Universitt Hamburg , Martinistrasse 52, 20246 Hamburg, Germany 1 Institut fr Tierzucht und Tierverhalten , Bundesforschungsanstalt fr Landwirtschaft, Drnbergstrasse 25-27, 29223 Celle , Germany We previously identified a broad initiation zone of DNA replication at the chicken lysozyme gene locus. However, the existence of a highly preferred origin of bidirectional replication (OBR), often found in initiation zones, remained elusive. In order to re-examine this issue we used a competitive PCR assay to determine the abundance of closely spaced genomic segments in a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand abundance occurred at the 3' end of the gene, where initiation events were 17 times more frequent than upstream of the gene. This primary initiation site, active in lysozyme expressing myelomonocytic HD11 cells and non-expressing hepatic DU249 cells, was found to reside within an unusually located CpG island. While most CpG islands are found at the 5' end of genes, the lysozyme gene island extends from the 3' end of the second intron and includes ~1.2 kb of 3' flanking DNA. As diagnosed by methylation-sensitive restriction enzymes, the island is largely non-methylated in HD11 cells, DU249 cells and inactive chicken erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is composed of two subsites separated by ~100 bp, was localised very close to the segment with the highest initiation activity. Our results suggest that the non-methylated CpG island and the HS provide an accessible chromatin structure for the lysozyme gene origin of replication. - Mapping initiation sites for DNA replication in the chromosomes of flies, frogs and mammals has revealed at least 22 replication origins (reviewed in 1 and references cited below). While these studies agree that DNA synthesis initiates at specific sites but not randomly throughout cellular chromosomes, an important feature of replication origins appears paradoxical. Several studies using labelling of early replication intermediates with nucleotide precursors conclude that initiation occurs from narrow zones encompassing as little as 0.5 kb (24). In contrast, when replication intermediates were fractionated by two-dimensional gel electrophoresis and then identified by hybridisation with sequence-specific probes, initiation appears to occur at many sites distributed throughout much larger regions. In several cases this paradox could be clarified by use of techniques that allow quantitative determination of the relative frequency of initiation at densely spaced sites (4,5). The chicken lysozyme gene locus is one of the best studied chromatin domains in vertebrates. The gene is flanked by well characterised regulatory sites (enhancers, silencer, HRE) (6) and nuclear matrix attachment regions (MARs) (7). To fill a gap in our knowledge, we started to study replication of the lysozyme locus, and as a first step, previously identified a broad initiation zone of DNA replication (8). Yet unfortunately, our previous studies failed to reveal a preferred origin of bidirectional replication (OBR) in the lysozyme initiation zone. We therefore decided to re-examine this issue and used the nascent-strand abundance assay to quantitatively determine by PCR the relative abundance of more densely spaced genomic sites within the 1.0 kb size fraction of nascent DNA from chicken myelomonocytic HD11 cells and hepatic DU249 cells. Furthermore, we concentrated our efforts on the 3' half of the gene and its 3' flanking DNA, as our previous studies seemed to indicate that the frequency of initiation is greatest within the 3' located sub-zone (8). Presently very little is known about the distribution of origins of DNA replication in relationship to other genomic landmarks. Origins were found to occur near promoters (3,9), within transcriptional units (10), at the 3' end of genes (3) and in intergenic regions (2,4,11). A close correlation of DNA replication with transcription is demonstrated by the fact that almost all constitutively expressed (housekeeping) genes replicate within the first half of S phase in many cell types, while many tissue specific genes when active replicate in early S phase, but when inactive in late S phase (12,13). A very prominent landmark within the vertebrate genome is 12 kb regions of densely spaced CpG dinucleotides, named CpG islands. They are associated with the 5' end of ~50% of all mammalian genes, where they cover the promoter and one or more of the 5' exons (14,15). While the majority of the methylated CpGs are found in retrotransposons and centromeric satellite DNA, CpG islands are free of methylation in all tissues, including those in which the associated genes are silent, with the exception of those on the inactive X chromosome (16) and those associated with the inactive allele of imprinted genes (17,18). The chromatin encompassing CpG islands is free of histone H1, highly acetylated at histones H3 and H4 N-termini, and preferentially accessible to restriction enzymes (19). These features provide an open chromatin configuration. In a recent nascent-strand abundance analysis of three hamster genes and one human gene, that all contain CpG islands at their 5' ends, CpG island DNA but not flanking DNA was found to be present in a short nascent strand size fraction (20). Furthermore, CpG island-like fragments were enriched in the short nascent DNA fraction from human erythroleukaemic cells, suggesting that initiation occurs in CpG islands at a significant frequency. In order to evaluate the generality of this connection, it would be interesting to determine the origin of replication in genomic loci that contain CpG islands at non-5' end regions. Besides by DNA sequences, origins of replication are determined by chromatin structure. Initiation of DNA replication is strongly inhibited by packaging of DNA into chromatin. This effect can be abolished by interaction of specific proteins, e.g. initiation proteins and some transcription factors, with replication origins (21,22). The origin of simian virus 40 minichromosome and origins of Saccharomyces cerevisiae have been shown to be maintained in an open chromatin structure highly accessible to DNA endonucleases (2325). Furthermore, the chromatin structure at origins in yeasts and origins in human cells is modulated during the cell cycle (23,26,27). Additionally, it has been shown that attachment to the nuclear matrix mediates cell cycle-specific alterations in chromatin structure at the hamster DHFR ori-b and ori-g (28). Our present results localise a primary initiation site (OBR) to a 650 bp region at the 3' end of the lysozyme gene. This OBR resides within an ~1.8 kb CpG island, which extends from the 3' end of the second intron and includes ~1.2 kb of 3' flanking DNA. Furthermore, the OBR is coincident with two closely spaced DNase I hypersensitive sites. These r (...truncated)