Absence of an Unusual “Densely Methylated Island” at the Hamster dhfr ori-β

Abstract

An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-␤ locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G 1 , S, or G 2 ؉ M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines ( m C) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of m C residues that complements the more commonly used hydrazine method. These methods were capable of detecting m C in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori-␤ DMI was not detected in any of these cell populations using any of these methods.DNA methylation at CpG dinucleotides in mammalian cells has been implicated as an important component of such pivotal processes as transcription (1), imprinting (2), recombination (3), carcinogenesis (4), development (5), and replication timing (6). CpG methylation is carried out by a methyltransferase that is associated with replication foci in the nucleus (7). In general, this enzyme methylates specifically hemimethylated CpG dinucleotides, although de novo methylation of unmethylated CpG dinucleotides can occur but at a low rate (8). In addition, this enzyme occasionally methylates cytosines in sequences other than CpG (9, 10). However, given the specificity of the mammalian methyltransferase, this non-target methylation should be of little significance in vivo because the pattern of non-CpG methylation will not be maintained during the subsequent round of DNA replication.Recently, an unusual form of DNA methylation has been implicated as an important component of mammalian origins of DNA replication. A densely methylated island (DMI) 1 has been reported at three different mammalian origins of DNA replication (11, 12). These DMIs were found only in proliferating cell populations and consisted of a 127-, 258-, or 516-bp stretch of DNA in which all cytosines were methylated, regardless of their sequence context (12). The discovery of DMIs has several important implications. First, this unprecedented methylation pattern suggested that a second (or modified) methylation enzyme exists in mammalian cells. Interestingly, limited proteolysis of the mamm...

Differential Reactivity of the RatS100A4(p9Ka) Gene to Sodium Bisulfite Is Associated with Differential Levels of the S100A4 (p9Ka) mRNA in Rat Mammary Epithelial Cells

“…Similar, diverse, non-symmetric sites of methylation have been described previously as "densely methylated islands" at origins of replication associated with ribosomal protein S14 and the dhfr locus in Chinese hamster ovary cells (33). However, it has been suggested that these densely methylated islands of methylated cytosine residues might be artifactual, arising from incomplete denaturation of the DNA prior to treatment with sodium bisulfite, at least at the dhfr locus (34). In the present experiments, it is unlikely that the unusual pattern of methylation arises from incomplete denaturation of the DNA.…”

Direct detection of double-stranded DNA: molecular methods and applications for DNA diagnostics

“…The bisulfite method requires denaturing double-stranded DNA and bisulfite treatment for 4-18 h, followed by sequencing, microarray detection, or methylation-specific PCR 71 and their variations. 72 Some problems arising from these approaches are: (a) DNA occasionally partially degrades; 73 (b) incomplete bisulfite reactions result in false positives; 74 (c) resulting singlestranded DNA adopts alternate folded conformations 75 that prevent PCR amplifications; (d) primer design becomes problematic in methylation-specific PCR; and (e) microarray detection technologies are expensive. 76 Many of the above artifacts can be solved 77 with appropriate changes in experimental conditions, however the total time for this reaction and analysis is long and remains constant.…”

DNA Methylation at Mammalian Replication Origins

“…Chromosomal DNA was extracted and cleaved with restriction enzymes to eliminate the possibility that subsequent purification of nascent Br-DNA retrieved c-myc sequences whose origin may not have fired but were connected to an active, BrdUrd-labeled origin located some distance away. Nascent Br-DNA was affinity-purified and analyzed for m CpGs using the bisulfite method as described previously (27). An example of these data is shown in Fig.…”

“…A strong signal from methylated DNA (indicated by cytosine residues in the C lane at the positions of CpG dinucleotides) was evident within 1 min of pulse labeling, whereas the signal from Closed lollipops denote methylated cytosines at CpG dinucleotides that were determined using four independent methods, including the bisulfite method (24,27). Nine of these m CpGs are clustered within a 356-bp region (shaded box) adjacent to the OBR that was defined by the transition between discontinuous and continuous DNA synthesis (55).…”

“…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).…”