Labtimes 2017-06

page 62 Lab Times 6-2017 Methods A naive view of the genome is: it is a string of symbols bearing the mes- sage that on its own, is enough to build an organism. Some bits of the string are comparatively easy to understand, namely the protein-coding parts, where each codon triplet stands for an amino acid. Some parts are a little more subtle, such as the special sequences that mark the start of a protein-coding gene, or mark where se- quences are to be spliced. No doubt there are yet higher-orders of meaning in the message, too, which remain to be discov- ered. But the basic idea is that the genome is a kind of Turing machine. In other words, it works just like a computer programme, where one string encodes not only the data, but also the instructions for manipulating those data. But perhaps we have been too influ- enced by this metaphor of the “genome-as- programme”. That is certainly one point made by the many biologists, who have looked at not only the sequence of the ge- nome but also at the chromosomes as a whole, with their own three-dimensional structure. A chromosome's job is not an easy one. Unpack all the DNA in just one of your cells, line it up end to end and it will be some twenty metres in length. All of that has to be packed into a spherical container less than ten μm in diameter. Oh, and by the way, you can’t just pack it any old way – you’d have to allow access to various sites in the sequence at various times: when you want to transcribe a gene or replicate the DNA, for instance. In other words, the formation of chro- mosomes is little short of an engineering miracle. More than a packing problem And if you don’t think those design specifications are tough enough, hang on, because it gets worse. It is emerging that the 3D structure of chromosomes is not just the solution to a difficult packing problem but also plays a role in controlling how genes are expressed. Right, so we need to under- stand the 3D structure of chromosomes. But how are we to determine that structure? One solution is microscopy but its resolu- tion is far too low to answer the questions being asked today. The biggest advance in mapping the structure of chromosomes comes from a field of related approaches called Chro- mosome Conformational Capture (3C as it is called). The basic idea behind all these techniques is to measure the distance be- tween pairs of sites on the DNA sequence. Imagine you want a map of a city, showing the location of all the key buildings. Imag- ine further that a clever cartographer de- cides she wants to save bandwidth by send- ing you not a map but a spreadsheet, listing the distances between every pair of build- ings. Irritating, to say the least, but being an even cleverer biologist, you realise that it is indeed possible, if perhaps not easy, to work out the relative position of each building in space just from the distance data alone. How is this done in Chromosome Con- formation Capture? There are three key steps: ‘freezing’ the DNA into place using formaldehyde cross-linking, joining the cross-linked DNA fragments together into single DNA strands, then using DNA chem- istry on those strands, to figure out who was sitting next to whom. To understand the latest incarnation of 3C, we need to go over its history. You can sum up that history as being ‘one-to-one’, ‘one-to-many’, ‘many-to-many’ and finally ‘all-to-all’. One-to-one 3C was the first type to be described and is the starting point for all the others, and for the rest of this arti- cle, I will use the term 3C to refer to this particular version. Latest 3C incarnation The first step is to cross-link the DNA strands together with formaldehyde. The assumption is that the closer two loci are together and the more frequently they are close together, the more likely they are to be cross-linked. That cross-linking can oc- cur between loci on the same chromosome or loci on different chromosomes. It is important to get the concentration of formaldehyde right but, as with so many techniques, there is no easy way of telling what that ideal concentration is and how long the fixation should take. The usual range is between one and four percent for between five and ten minutes, but no sys- Bench philosophy (71): Chromosome conformation capture methods Mapping Chromatin Interactions The three-dimensional structure of the chromosomes has a major impact, on which genes get expressed and when . For the last ten years, a growing number of scientists has been working on better ways to look at this 3D structure. Photo: Chi (Alice) Lu & Seongjun Park Photo: Peter Jonas Chromosomes fold into complex three-dimensional structures that affect, e.g. gene function. Photo: NIH

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