3D organisation of the mammalian genome and its function

Speaker: Wouter de Laat
Department: Bionanoscience
Subject: 3D organisation of the mammalian genome and its function
Location: Delft University of Technology
Date: 2016-02-11
Author: Romano van Genderen

The seminar started by debunking a common myth, namely that 97% of all DNA, which is often considered “junk DNA” actually consists of all sorts of regulative elements and switches, which are turned off and on in specific tissues, causing the morphological changes. But not only the genetic information on this non-coding plays a regulative role, also the role it plays in changing the 3D organisation of the coding DNA plays a role.

The first experiment he presented that showed evidence for this was done by the Spitz lab. They showed that by moving a so-called transposon cassette, a piece of DNA that can be inserted in the genome, across the genome, the expression of specific proteins can be influenced. Sometimes a small step with the cassette leads to a large change in expression, while a large step does not make a huge difference. This was used to show that these two sites in the large step are separate on the genome, but topologically very close.

Another experimental method he presented was the 3C (chromosome conformation capture) method and other derived methods. 3C is based on formaldehyde linking the proteins on the DNA together into a sort of knot, then digesting them to cut the knot out. This is ligated and read to show which parts of the DNA are close together in space. He showed an example where this method was used to study the haemoglobin protein. Also he showed some derivative methods like 4C and Hi-C.

Afterwards he explained the basis of higher-scale genome folding in mammals. The main point he wanted to introduce were the so-called topologically active domains, also called TAD’s. These are loops in the DNA, separated from one another by a protein called CTCF. These domains also act as functional domains with promotors and enhancers relatively close by. A next step in folding is made by sorting the genome on the basis of activity. The active domains bind one another and so do the inactive domains. Afterwards, the inactive domains move towards the nuclear periphery. He showed a practical application of this by explaining how leukaemia originates.

Next he elaborated on the CTCF protein, which forms the anchor of a chromatin loop. The most important property of the protein is that it has a direction. In order for it to function properly and form a loop, the two proteins must not be in the same direction. This is better explained in the following image:

fx1.jpg

Image 1: Image explaining the directionality of CTCF. Source: E. de Wit et al. CTCF binding polarity determines chromatin looping, Mol Cell, 60 (2015), pp. 676–684

Finally, he showed how 3C methods could be used for haplotyping, so to differentiate the maternal, paternal and foetal genome. This is because the more advanced 4C method can detect translocations in the genome. Because these C methods are based on location and not on sequence, a small difference in genome, like a few more SNP’s, are ignored. Using this haplotyping, a non-invasive form of prenatal diagnosis can be done using the fragments of foetal DNA found in the mother’s blood.

The thing I found most striking about this presentation is the situation that occurred during the questions. There was one person who was more of a proponent of the topological supercoiling model than the TAD model. Their discussions showed that sometimes one model is not sufficient to explain all the experiments.

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