Saturday, 13 September 2014

Epigenetics: all fingers and thumbs: Molecule of the Month for September

I was trying to find a context for this month's choice for molecule of the month, to avoid accusations of self indulgence (I work on these enzymes)! My choice is the family of enzymes responsible for modifying the genome in many organisms that forms a major part of the Biological phenomenon called "epigenetics" or increasingly, "epigenomics". Epi simply means on top of, and so epigenetics is something that modifies the phenotype of a gene. Think of it like seasoning your food: chips without salt and vinegar, or coffee without milk! For some, like me, black coffee is fine as it is, but some need milk or even cream, before they can enjoy it. The study of epigenetics really came to prominence around 25 years ago when despite considerable experimental challenges, scientists including Timothy Bestor and Adrian Bird began to develop a molecular framework for understanding DNA methylation in higher organisms. The methylation of DNA is found in most organisms (but not all!), sometimes the adenine is modified and in other organisms the cytosine. In some organisms, both nucleotides can be modified. In bacteria, methylation of the genome is part of a mechanism that protects the DNA from attack by restriction endonucleases, enzymes that cleave double stranded DNA in two. These enzymes formed the basis for the development of molecular cloning in the 1970s and '80s in particular (enter Rich Roberts in the search field for more on this). 

The methylation of cytosine bases in DNA not only provides bacteria with a primitive immune system (I should emphasis that the role of methylation and its evolutionary emergence is not as simple as this, but a discussion of this would be a distraction here), it is also at the heart of gene regulation and genomic regulation in vertebrates. After Tim Bestor identified the first vertebrate DNA methylase (DNA MT1), several more have been discovered, but it is clear from work in his lab and that of "transgenic" scientists like Rudolf (Rudi) Jaenisch in the USA, that methylation drives a number of fundamental processes during development. What is fascinating about the enzymes (such as DNA MT1) is that the methylation chemistry is carried out by a structure that is very similar in organisms as evolutionary distinct as microbes and humans. The conservation of mechanism is also preserved in the genome and can be visualised by simple BLAST searching. Therefore, when Rich Roberts and Xiaodong Cheng (and colleagues) determined the 3D structure of the cytosine-specific DNAMTase from Haemophilus haemolyticus (M.HhaI) around 20 years ago, it proved to be an excellent model for understanding the molecular basis of DNA methylation in all Life. 

The structure I want to discuss is the complex of M.HhaI with its DNA duplex,
caught in the act of methylation, before the enzyme leaves the product. This was achieved by the combination of chemical synthesis and Molecular Biology to facilitate X-ray crystallography. If you look closely you will see that the enzyme wraps around most of the DNA double helix, but remarkably, the target Cytosine base is flipped out. This means that the catayltic (active) site can now readily transfer the methyl group from the cofactor S-adenosyl-L-methionine (one of two methyl donors in Nature: what is the other?), to the target base. The reaction over, the enzyme pops the modified base back and goes on its way. 

At first glance this seems like a very elaborate mechanism, but without giving you chapter and verse, what has emerged since this landmark study, is that many enzymes that repair or modify DNA, have been shown to use base flipping as a means of reaching the parts that they would otherwise find difficult to reach. (It is possible to unwind the strands to access the bases, but this leaves the DNA vulnerable to degradation and requires greater molecular sophistication). It seems that Nature has found ways of capturing flipped bases, during a natural "breathing" phenomenon, or proteins have developed that readily push out the target base, suggesting that this is a primitive molecular function. Enzymes that repair the damage of sunlight (where Thymine bases that lie next to each other can spontaneously fuse) utilise base flipping as do enzymes that repair mismatches in the otherwise faithfully replicated DNA helix.

The crystal structure of the flipped base was made possible by the use of a fluorinated version of the cytosine, incorporated into the short synthetic DNA molecule, mimicing a drug that is used to treat a number of diseases: 5-fluorouracil. This combination of synthetic chemistry, X-ray crystallography and molecular biology (to engineer recombinant protein expression) is a wonderful example of multi-disciplinary Science. Moreover, while the catalytic questions can be analysed using the model of M.HhaI, Bestor and many others have now developed ways of tracking DNA MTase by fusing it with GFP and establishing its role with other genome modifiers in modulating the genome in a diverse number of ways (see top RHS, the fluorescence of PCNA, a marker of human DNA replication, colocalizes with DNMT1).

Returning to the fingerprints of the title, one Y13 student is asking the question, why do we have different fingerprints, and why indeed do identical twins also have non-identical prints? This is at the heart of epigenetics, when presumably differences in local concentrations of small molecules re-programme the genotype to produce a new phenotype. It was actually over 60 years ago that the famous mathematician, Alan Turing, proposed a simplified model, referred to as the reaction-diffusion system in which he proposed that morphogenesis resulted from the coming together of environmental chemistry and genetics (often referred to as chemical biology). Simple physical laws underpin complex biological events (see top LHS). The field of epigenetics is emerging as an important direction for anti cancer drug discovery and for me, illustrates how serendipity (searching for a molecular understanding of an interesting aspect of nucleic acid biochemistry) can have a major impact on medicine. 

Finally, if you think this is interesting, one of the recent acquisitions in the Institute of Integrative Biology's Centre for Genomic Research in our sponsoring institute, the University of Liverpool, is a new generation gene sequencer manufactured by Pacific Biotechnology. Already Rich Robert's group (see Rich talking recently about the work, RHS) at New England Biolabs have shown that this methodology can not only read the As, Cs, Gs and Ts of DNA, but the MeCs and MeAs, and with it will be a new era of methylome analysis as we unlock the link between our genes and our environment across entire genomes. Exciting times for us all I think ! 

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