Roger L. P. Adams

Inactive chromatin spreads from a focus of methylation

The detailed mechanisms of inhibition of transcription by DNA methylation are still unknown, but it has become obvious that the formation of chromatin plays an important role in this process. Using an approach enabling us to methylate, in vitro, chosen regions in a plasmid, we now show that specific methylation of nonpromoter sequences results in transcriptional inhibition of a reporter gene construct and that this inhibition is independent of the position of the methylated region within the plasmid. In plasmid minichromosomes containing a short region of methylated DNA, both methylated and unmethylated sequences are protected from limited MspI digestion. Our results show that inactive chromatin is present at unmethylated regions in partially methylated minichromosomes and can thereby inhibit gene expression. Spreading of the inactive chromatin is not inhibited by the presence of active promoters, nor is it a consequence of transcriptional inactivity.

Deoxyribonucleoside triphosphate pools in synchronized and drug-inhibited L929 cells

Abstract 1. The method of Solter and Handschumacher ( A. W. Solter and R. E. Handschumacher , Biochim. Biophys. Acta, 174 (1969) 585) has been applied to an analysis of the deoxyribonucleoside triphosphate pools of L929 mouse cells. The average size of these pools is between 15 and 20 pmoles/106 cells. 2. Aminopterin depresses the pool of dTTP but incubation with 5 mM thymidine increases the dTTP pool up to 790 pmoles/106 cells while reducing the sizes of the pools of dATP, dGTP and dCTP. 3. Synchronised cells show larger pool sizes after the end of the S-phase than during DNA synthesis. 4. Hydroxyurea reduces the pool sizes of purine deoxyribonucleoside triphosphates but evidence is presented that hydroxyurea may also exert an effect on DNA synthesis by interfering with the localization of the deoxyribonucleotide pool within the cell nucleus.

The in vivo methylation of DNA in mouse fibroblasts

Abstract Studies on the in vivo methylation of DNA occurring in cultured mouse fibroblasts (Strain L929) treated with hydroxyurea have suggested that the only DNA base to be methylated, namely cytosine, is methylated after the initial synthetic stage of DNA replication and is a process which proceeds efficiently only in the presence of a continual supply of nascent DNA. Experiments with synchronised cell cultures demonstrated that cytosine methylation occurred within an hour of the synthesis of DNA in the S phase of the cell cycle. Additional ‘chase’ experiments showed that the 5-methylcytosine of DNA so formed is stable, no evidence being found for its demethylation, deamination or excision from the polymer.

Methylation of mosquito DNA

Cells of the mosquito Aedes albopictus have 5.6 pg DNA/cell. This DNA is 58 mol% (A + T) and has about 0.03 mol% each of 5-methylcytosine and 6-methylaminopurine. The DNA is largely degraded by the restriction enzyme HpaII showing the virtual absence of the sequence CMeCGG. Small amounts of enzymic activity recovered largely in non-nuclear fractions transfer methyl groups from S-adenosylmethionine to cytosine and adenine.

The relationship between synthesis and methylation of DNA in mouse fibroblasts.

Abstract Studies on the in vivo methylation of DNA in cultured mouse L cells confirm the presence of a distinct lag between synthesis of DNA and its methylation. This lag is most pronounced for the DNA made in late S phase and the DNA is methylated to only 50 % the extent of that DNA made in early S phase. Methylation of DNA does not occur in stationary L cell cultures nor prior to DNA synthesis when such cultures are stimulated to grow. Neither does methylation occur on the DNA of phytohaemagglutinin stimulated horse lymphocytes prior to DNA synthesis.

Newly synthesised DNA is not methylated

Abstract In cultured mouse L929 cells DNA synthesis has been shown to occur by initial synthesis of short DNA chains which are subsequently joined together to form higher molecular weight DNA. The short chains are readily denatured on extraction of the DNA, as is DNA containing cytosine arabinoside. As measured by a two 2-min pulse at 30 °C with deoxy[14C]cytidine only 15 % of the DNA is readily denatured, yet neither it, nor the stable, high molecular weight DNA are significantly methylated. This demonstrates that methylation is not required for stabilisation of DNA.

Eukaryotic DNA methylation

Abstract The evidence for a role of 5-methylcytosine in the control of transcription is examined and some possible additional functions are considered.

Inhibition of poly(ADP-ribosyl)ation induces DNA hypermethylation: a possible molecular mechanism

The pattern of DNA methylation established during embryonic development is necessary for the control of gene expression and is preserved during the replicative process. DNA regions of about 1–2 kb in size, termed CpG islands and located mostly in the promoter regions of housekeeping genes, are protected from methylation, despite being about 6–10 times richer in the dinucleotide CpG than the rest of DNA. Their unmethylated state guarantees the expression of the corresponding housekeeping genes. At present, the mechanism by which CpG islands remain protected from methylation is not clear. However, some results suggest that poly(ADPribosyl)ation, an enzymatic process that introduces a postsynthetic modification onto chromatin proteins, might be involved. Here we show in L929 mouse fibroblast cells that inhibition of poly(ADP‐ribose) polymerase(s) at different cell‐cycle phases increases the mRNA and protein levels of the major maintenance DNA methyltransferase (DNMT1) in G1/S border. Increase of DNMT1 results in a premature PCNA‐DNMT1 complex formation, which facilitates robust maintenance, as well as de novo DNA methylation processes during the G1/S border, which leads to abnormal hypermethylation.

The effect of 5-azadeoxycytidine on cell growth and DNA methylation.

By growing cells in the presence of 3 mM thymidine and 5-azadeoxycytidine up to 20% of DNA cytosines have been substituted with azacytosine. No substitution was obtained on incubating with 5-methyldeoxycytidine. Azacytosine-substituted DNA has a very low level of 5-methylcytosine and cells, which survive azadeoxycytidine treatments maintain this low level of methylation in the absence of the drug. The DNA of such cells is undermethylated fairly evenly in all classes of DNA e.g., satellite and unique DNA. Incubation of cells in azadeoxycytidine leads to high cell mortality which is not related to the lack of DNA methylation but may be linked to the altered interactions of proteins with the substituted DNA. This effect, rather than reduced DNA methylation, may be the cause of differentiative changes observed on treatment of cells with 5-azacytidine.

Stimulation of de novo methylation following limited proteolysis of mouse ascites DNA methylase

The ability of mouse Krebs II ascites cell DNA methylase to add methyl groups to native, unmethylated DNA (de novo activity) is stimulated by limited proteolysis. The affinity of the enzyme for DNA is not altered by this treatment but the rate of reaction is increased so that 40% or more of methylatable sites are methylated within 4.5 h. The activation is associated with a decrease in size of the enzyme to 6.2 S.