Posted on behalf of Retread
An incredible article appeared last month in the journal Science. If it can be verified and if it applies generally, our conception of just how genes coding for protein are turned on will be radically changed (yes, there are many other kinds of genes other than those coding for proteins). If DNA compaction, nucleosomes, histones, lysine methylation and demethylation, the histone code, nuclear hormone receptors (particularly the estrogen receptor), DNA glycosylase and topoisomerase aren’t old friends have a look at the first comment on this post for the background you need. Don’t worry, there is plenty of chemistry to follow.
Some histone code modifications are reversible, particularly acetylation of the epsilon amino group of lysine. Enzymes acetylating histone lysines are called histone acetylases, those removing it are called histone deacetylatases (HDACs). However, lysine methylation was thought to be permanent until ’04 when several enzymes able to demethylate lysine were found. One such enzyme is called LSD1 (it has nothing to do with the hallucinogen). It removes the two methyl groups from lysine #9 of histone #3 (H3K9me2). If this modification is present on a nucleosome near a gene, the gene is silenced, so the methyls must be removed so the protein it codes for can be made.
The estrogen receptor + estrogen complex bound to the ERE (the estrogen response element – a 15 nucleotide DNA sequence) triggers H3K9me2 removal. The process of demethylation is oxidative (how else would you split a nitrogen to hydrocarbon bond?). Hydrogen peroxide is produced, a loose cannon which oxidizes the juicy electron-rich bases of DNA nearby, forming in particular 8 oxo-guanine, as guanine is the most easily oxidized DNA base. Since 21% of the DNA bases in our genome are guanine, H2O2 doesn’t have far to look. This calls in some fairly heavy artillery (DNA glycosylase to remove the 8 oxo-guanine, topoisomerase IIbeta to unwind the DNA so it can be repaired, the repair enzymes, etc, etc…). Naturally this opens up the compacted DNA structure around the gene allowing RNA polymerase II to do its work transcribing the estrogen responsive gene into mRNA (once the damage is repaired).
So according to this paper, estrogen turns on gene transcription by damaging DNA. This is fantastic (if true). There’s more. The estrogen receptor is but one member of a group of proteins called nuclear hormone receptors. The name comes from the fact that other hormones (progesterone, androgen, thyroid, glucocorticoids, mineralocorticoids) have their own proteins that turn on (or turn off) genes the same way. Subsequently it was found that some vitamin metabolites (vitamin D3, vitamin A) have similar receptors even though they aren’t hormones. The human genome contains 48 such proteins. Less than half of them have known ligands. Those with known ligands have their finger in just about every metabolic pie in the cell.
One final point. It has been estimated that 8-oxoguanine is formed 100,000 times each day in every cell. Perhaps its formation is physiologic rather than pathologic. Where does that leave antioxidant therapy, which has been touted to do everything but cure hemorrhoids? Well, one such trial was done on 29,000 Finnish men at high risk for lung cancer (they were smokers) [New England J. Med. vol. 330 pp. 1029-1035 (1994)] Alpha tocopherol (one antioxidant used in the study) didn’t decrease the incidence of lung cancer, and there was an 18% higher incidence of lung cancer among the men receiving beta carotene (another antioxidant). In medicine, theory is great but data trumps it every time.
Retread
Chemists think in Angstroms. Relative sizes are easier to think of that way. No one thinks of the carbon-carbon bond as .154 nanometers long. Nanometers do have their uses however. The pi electron cloud of an aromatic ring is .34 or so nanometers thick and we have about 3.2 billion nucleotides in our genome. This comes out nicely to one meter if all the aromatic rings of the nucleotides in one cellular genome are stacked on top each other (as most pictures of DNA place them in biochemistry books). Somehow all this has to fit inside a nucleus of the order of 10 microns in diameter, a 100,000 fold compaction at a minimum.
We know the first order of compaction. About 150 nucleotides of DNA are nearly twice wrapped around a collection of 8 histone proteins (the nucleosome) like thread around a spool. The diameter of the nucleosome with its DNA wrapping is about 10 nanometers, so this only shrinks the length of DNA by a factor of 5. Another 20,000 fold further compaction is needed. People have seen a 30 nanometer chromatin fiber, but its actual structure is still being debated. Beyond that it’s mostly speculation.
Somehow, specialized proteins are able to recognize different sequences of DNA bases despite the compaction which must be occurring (something I regard as nearly miraculous). One well studied example is the estrogen receptor. Estrogen diffuses inside the cell (it is lipid soluble), binds to the receptor (a protein) which changes its conformation allowing it to bind to a particular 15 nucleotide DNA sequence called the estrogen response element (ERE). Since there 2^30 different possible 15 nucleotide sequences, this is plenty of specificity. Once bound to the ERE, the estrogen receptor + estrogen complex recruits the megadalton (yes megadalton) copying machine (RNA polymerase II) which makes an RNA copy (mRNA) of a gene near the ERE.
There is far more to the story than just DNA and histones. Many other proteins associate with histones. The association is quite specific because the parts of the histones sticking out from the nucleosomal spool (the histone tails) are chemically modified. The hydroxyl groups of serine and threonine can be phosphorylated or not. The epsilon amino group of lysine can be acetylated, mono, di or trimethylated (or not). Arginine can be singly or multiply methylated on the guanidino nitrogens. At least four other covalent histone tail modifications are known. Some 17 different methylated lysines on various histones are known. Proteins binding modified histones are quite specific, binding to trimethylated lysine #4 on histone #3, but not unmodified lysine at this position or lysines elsewhere. This is the histone code – modifications which determine just which protein can bind to a given nucleosome. Depending on the proteins bound, whole stretches of DNA can be shut down (heterochromatin) so its genes aren’t expressed, or a compacted structure can be opened up so its genes are transcribed. It depends on just what histone modifications are present and which proteins bind to them.
The double helix of DNA makes a complete turn every 10 nucleotides, so there are 320,000,000 such turns in our genome. To copy DNA into mRNA, the two strands of the helix must be locally separated (so that one can be read and copied). Separation means unwinding the turns of the helix which causes overcoiling of the two DNA strands on either side of the separation. Since it takes 3 nucleotides to code for one amino acid the DNA for even a 100 amino acid protein (not particularly large) has to be unwound 30 times. That’s where the topoisomerases come in. Some of them break the double helix, attach the ends to themselves, and pass through the gap another part of the DNA helix relieving the torsion produced by the unwinding. Other topoisomerases actually add torsion but that’s another story. Pardon the gee whiz, but I find this sort of thing amazing and again rather miraculous.
Lastly, recall that nucleotide means DNA base + sugar + phosphate and that the DNA backbone is sugar–phosphate–sugar–phosphate with the DNA bases hanging off the sugar. DNA glycosylases just remove the DNA base from the sugar. They are part of the repair process for modified DNA bases (such 8 oxo-guanine). The way in which the DNA glycosylase for 8-oxoguanine actually finds the damaged needle in the 3.2 gigabase haystack is a great story. Have a look at Proc. Natl. Acad. Sci. USA vol. 103 pp. 15020-15025 (2006) if you have the time.
Now back to the main event.
Retread
For what it’s worth department: [ J. Am. Med. Association vol. 297 pp. 842 – 857 ‘07 ] Is a meta-analysis or 68 randomized trials of antioxidant dietary supplements. I don’t trust meta-analyses — they have been spectacularly wrong in the past most notably about the ‘benefits’ of hormonal supplementation after menopause. There was no evidence that the supplements had any beneficial effects of mortality, in fact suggesting that some of them (you’ll have to read the article to find out which ones) actually INCREASED the risk of death.