Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.     

Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.     

Regulation of activator/dissociation transposition by replication and DNA methylation

In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids ("chromatid selectivity"). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.     

Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro

CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.     

In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.     

Hypomethylation of HeLa cell DNA and the absence of 5-methylcytosine in SV40 and adenovirus (type 2) DNA: analysis by HPLC

Methylation of the purified virion DNA of both SV40 and adenovirus (type 2) was measured by high performance liquid chromatography (HPLC) and compared to their hosts, African green monkey kidney cells and Hela cells, respectively. Essentially, no 5-methylcytosine was detected in either viral DNA. Implications for viral gene regulation by methylation are discussed. In comparison with normal human cell DNA methylation levels, HeLa cell DNA methylation is reduced significantly.     

SINE retroposons can be used in vivo as nucleation centers for de novo methylation

SINEs (short interspersed elements) are an abundant class of transposable elements found in a wide variety of eukaryotes. Using the genomic sequencing technique, we observed that plant S1 SINE retroposons mainly integrate in hypomethylated DNA regions and are targeted by methylases. Methylation can then spread from the SINE into flanking genomic sequences, creating distal epigenetic modifications. This methylation spreading is vectorially directed upstream or downstream of the S1 element, suggesting that it could be facilitated when a potentially good methylatable sequence is single stranded during DNA replication, particularly when located on the lagging strand. Replication of a short methylated DNA region could thus lead to the de novo methylation of upstream or downstream adjacent sequences.     

Methylation in eucaryotes influences the repair of G/T and A/C DNA basepair mismatches

Although methylation of DNA at some sites regulates gene expression, 5mC at many sites does not appear to have any effect. We present evidence that hemimethylation at many different sites can act as a discrimination signal in mismatch repair. Deamination of 5mC in a symmetrically methylated doublet CpG yields the mismatched base pair T/G in a hemi-methylated doublet pair. Because both bases in the mismatched pair are normal constituents of DNA, identifying the incorrect base is problematic. The only apparent distinction of the two is the methylation on the strand opposite the deamination event. Using available methylases we have produced hemi-methylated SV40 DNAs that are mismatched at a single T/G or A/C basepair in a sequence that mimics the lesion resulting from the deamination of a 5mCpG. Methylation at the adjacent cytosine results in the replacement of the T much more frequently than when no methylation is present in the heteroduplex. Cytosine methylation at sites farther removed from the mismatch is equally effective in replacing the incorrect T at the mismatch. Although methylation in vertebrates is almost exclusively on cytosine in the doublet CpG, methylation of cytosines in other doublets, as well as methylation of adenosine, also act as strand discrimination signals. Perhaps some of the excess methylation in vertebrate DNAs may serve to direct mismatch repair.     

Investigating the effects of the presence of foreign DNA on DNA methylation and DNA repair events in cultured eukaryotic cells

Methylation of DNA in eukaryotic cells, global as well as gene-specific, is affected by endogenous and endogenous factors. In this paper, it is reported that deviations in DNA methylation and expression of genes involved in DNA repair and the cell cycle are affected in 143B cultured cells containing an expression vector. Global DNA methylation analysis with cytosine-extension assay revealed a decreased global DNA methylation in the presence of the expression vector. Less promoter-specific methylation, as measured by bisulfite-MS PCR, was observed for MGMT and p16INK4a in vector-containing cells. Comet assay investigations revealed a negative effect on the DNA repair capacity of both BER and NER in Complex III compromised cells. This was reflected in the down-regulation of hOGG1 and ERCC1 expression. The results presented in this paper support the existence of a strong relationship between impaired mitochondrial function and deviations in DNA methylation and extend this relationship to impaired DNA repair.     

Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women

Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle, and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1, and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy, and methylation score for several CpG sites in CDH1, GSTP1, SFRP1, and RBP1. Methylation of RASSF1 was positively correlated with women’s age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.     

Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women

Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1 and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy and methylation score, for several CpG sites in CDH1, GSTP1, SFRP1 and RBP1. Methylation of RASS F1 was positively correlated with women''s age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.Key words: biomarker, pyrosequencing, promoter methylation, breast epithelial cells, breast milk, breast cancer risk, parity, age-related promoter methylation, pregnancy-associated protection from breast cancer     

Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line

We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the 'immediate' methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the 'delayed' methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.     

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.     

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.     

Tissue specific methylation of c-myc in adult chickens

Methylation of cytosine in DNA has long been correlated with modulation of specific gene expression in eukaryotes. Methylation of the c-myc locus was examined in six tissues from adult Leghorn chickens. The c-myc locus was found to be variably methylated in all examined tissues, except blood, where erythrocytic DNA showed no evidence of significant methylation of c-myc. This is contrasted with the observed sever methylation of the beta actin locus and the generally high methylation patterns found in avian erythrocytic DNA.     

Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites

Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown. In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction. Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair. We prepared a set of A·C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions. However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias. By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect. In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.     

Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line

We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the ‘immediate’ methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the ‘delayed’ methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.