Active Mammalian Replication Origins Are Associated with a High-Density Cluster of ^mCpG Dinucleotides

Abstract

ori-␤ is a well-characterized origin of bidirectional replication (OBR) located ϳ17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The ϳ2-kb region of ori-␤ that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine ( m C). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of m CpGs that was ϳ10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5 end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of m CpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins.At least 16 initiation sites for DNA replication have now been mapped in the chromosomes of mammals (21,32,70). These studies have shown that DNA synthesis is not initiated randomly throughout cellular chromosomes but at specific DNA sites. However, these sites appear more complex than those found in the simpler genomes of bacteria, bacteriophages, yeasts, animal viruses, and mitochondria. Most DNA synthesis is initiated at specific genomic loci that are contained within 0.5 to 2 kb of DNA. These loci represent the functional origins of bidirectional replication (OBR). Replication bubbles also are detected throughout a larger initiation zone, ranging in size from 6 kb to greater than 55 kb, that encompasses the OBR and contains alternative initiation sites. Thus, metazoan cells appear to designate specific chromosomal loci as regions where initiation can occur and then to select from the many potential initiation sites within this region one site (or a small cluster of sites) that acts as a primary origin of DNA replication. What are the parameters that determine where DNA replication begins?Metazoan replication origins are determined at least in part by specific DNA sequences. The same origins that are utilized in cells containing single copies of a unique genetic locus are also utilized in cells containing hundreds of copies of the same locus (12,18,39,72). Moreover, origins can be translocated to other chromosomal sites and still retain their activity (33, 56) and origin activity can be eliminated by deletion of specific sequences (2, 41). Several reports of autonomo...

Differential Patterns of Methylation of the IFN-γ Promoter at CpG and Non-CpG Sites Underlie Differences in IFN-γ Gene Expression Between Human Neonatal and Adult CD45RO− T Cells

“…There are few precedents for this possibility in the current literature, and reports of non-CpG methylation in mammalian gene sequences have been sporadic and largely restricted to repetitive DNA elements. Densely methylated CpG islands occurring at the ori S14 and ori-␤ origins of replication in Chinese hamster ovary cells are bilaterally methylated at CpN dinucleotides (28) and are conserved in humans (36,37). Bisulfite sequencing of two regions of pBluescript II SK ϩ plasmid that had been transfected into mouse F9 embryonal carcinoma cells and mouse NIH-3T3 fibroblasts revealed methylated CpNpG sites at different cytosines along both DNA strands for each of the two regions of the plasmid (25).…”

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis

“…According to the protocol by Rein et al (1997), 1 mg of DNA was incubated with 0.6 N NaOH in a ®nal volume of 20 ml for 15 min at 378C. One hundred and twenty microliters of a solution containing 3.6 M sodium bisul®te (freshly prepared, Sigma) and 0.6 mM hydroquinone (freshly prepared, Sigma) was added, and the sample underwent 20 cycles of denaturation of 958C for 30 s and incubation at 508C for 15 min.…”

DNA Methylation at Mammalian Replication Origins

“…The bisulfite method was used to detect methylated cytosines at specific DNA sequences as described previously (24). For analysis of nascent DNA, 1 g of ⌽X174 DNA was added as carrier prior to bisulfite conversion.…”

“…In Escherichia coli, DNA methylation plays a direct role in regulating the efficiency of origin usage and the timing of origin activation (see "Discussion"). In mammals, two replication origins in hamster cells (24) and one in human cells (32) are associated with an unusually dense cluster of methylated CpG dinucleotides (CpGs) while other origins contain sufficient CpGs to generate a similar pattern of DNA methylation (24). In one case (24), these CpGs were shown to be methylated in active origins as well as in the total cell population.…”

“…In one case (24), these CpGs were shown to be methylated in active origins as well as in the total cell population. Earlier reports that mammalian replication origins were associated with an unusual "densely methylated island" consisting of ϳ100 to ϳ500 base pairs in which all cytosines were methylated, regardless of their dinucleotide composition (25,26), apparently resulted from incomplete DNA cleavage by methylation-sensitive restriction endonucleases and incomplete reaction of DNA with bisulfite followed by selective amplification of unreacted DNA segments (24,27).…”