SAH ratio

In figure 4 they looked at several of the enzymes and other proteins that might be involved with changes in the SAM : SAH ratio. They looked at the expression level by qPCR of SAHH of two methyl transferases dNMT3a and dNMT3b these are the de-novo methylases not dNMT1 which is maintenance. Then they looked at a methyl CpG binding protein, the expression of one of those genes NBD2. When they looked at SAHH they see that adding levels of homocysteine cause a reduction in the levels of this enzyme and this in a sense may be a compensation where less SAHH is needed because we know that the enzyme SAHH converts SAH to homocysteine, so high levels of homocysteine could cause the cell to not want this enzyme any more in order to reduce homocysteine levels. When we look at the level of dNMT3a and dNMT3b, the authors were surprised to see an increase in expression of these genes because the ratio for methylation goes against methylating the genome and it doesn’t makes sense that more of this enzyme would be useful, but one of their discussion points is based on compensation; it would make sense that if the ability to do DNA methylation is becoming harder and harder to do because the ratio is being thrown off, that the cell might be trying to compensate by producing more enzyme to push through the biochemistry and to favor the forward reaction of methylating the genome. Finally, the expression of NBD2 is reduced possibly because the number of CpGs it would see are not around so the cell does not need a lot of protein to bind to methylated substrate if the methylated substrate were going away. They do a bit of hand waving when they discuss these last two proteins, but they were trying to tell a story of how homocysteine might affect the overall level of DNA methylation in the cell.

When they look at these ALU and LINE-1 elements, and they do PCR using their different probes with homocysteine treatment, they see that they show reduced or hypo-methylation associated with treatment of homocysteine treatment. And this is consistent with the fact that one the SAM : SAH ratio is off which would affect the cell’s ability to methylate the genome. Even though the cell might be compensating by making more enzyme, dNMT3a and dNMT3b, making more of that enzyme isn’t going to get the job done because that enzyme is still going to be inhibited by SAH. The increase in dNMT3a and 3b might explain why there is a reduction in SAM. Initially, you might think that if SAH levels are increasing they would inhibit the methyl transferase and thus preserve the reactant SAM, but they noted that SAM levels went down a bit when they treated it with homocysteine. The authors in the discussion talked about that the slight reduction in SAM was because there were more enzymes dNMT3a and 3b being made to compensate. If that’s the case that means that those enzymes are actually methylating. If they’re methylating the genome as a way to compensate clearly they weren’t methylating the ALU or LINE-1 elements for compensation because they saw a reduction there. Overall their conclusions were that the homocysteine levels could throw off the balance of this folate and SAM cycle and they noted some compensation mechanisms that could occur within in the cell. However, overall they saw that despite these compensation reactions, that still it produced, at the genomic scale as assayed by these two regions, a level of hypo-methylation that might result in a diseased condition because certain portions of the genome, now being hypo-methylated would produce genes and gene products that are not according to normal biology.