The Epigenetics Revolution

One morning, after the cells had been cultured with 5-azacytidine for several weeks, the researchers found that there was a strange lumpy bit in one of the culture flasks. To the naked eye, this initially looked like a mould infection. Most people would just discard the flask and make a silent promise to be a bit more careful when culturing their cells in future, to stop this happening again. But Peter Jones did something else. He looked at the lump more closely and discovered it wasn’t a stray bit of mould at all. It was a big mass of cells, which had fused to form giant cells containing lots of nuclei. These were little muscle fibres, the syncytial tissue we met in the discussion of X inactivation. Sometimes the little muscle fibres would even twitch[168].

This was very odd indeed. Although the cell line had originally been derived from a mouse embryo, it never usually formed anything like a muscle cell. It tended instead to form epithelial cells – the cell type that lines the surfaces of most of our organs. Peter Jones’ work showed that 5-azacytidine could change the potential of these embryonic cells, and force them to become muscle cells, instead of epithelial cells. But why would a compound that killed cancer cells, presumably by disrupting production of DNA and mRNA, have an effect like this?

Peter Jones carried on working on this when he moved from South Africa to the University of Southern California. Two years later, he and his PhD student Shirley Taylor showed that cell lines treated with 5-azacytidine didn’t only form muscle. They could also form other cell types. These included fat cells (adipocytes) and cells called chondrocytes. These produce cartilage proteins, such as those that line the surfaces of joints so that the two planes can glide smoothly over each other.

These data showed that 5-azacytidine wasn’t a special muscle-specifying factor. Very presciently, Professor Jones made the suggestion in his paper reporting this work that, ‘5-azacytidine … causes a reversion to a more pluripotent state’[169]. In other words, this compound was pushing the ball a little way back up Waddington’s epigenetic landscape. The ball was then rolling back down the valleys between the hills, into a different final resting place.

But there was still no theory as to why 5-azacytidine had this unusual effect. Peter Jones himself tells a lovely self-deprecating story about the turning point in our understanding. His original appointment at the University of Southern California was in the Department of Paediatrics, but he wanted a joint appointment with the Department of Biochemistry. Part of the procedure for obtaining this joint appointment included an extra interview, which he considered quite pointless. Peter Jones described his work with 5-azacytidine in this interview and explained that no-one knew why the compound affected cell pluripotency. Robert Stellwagen, another scientist at the same university who was taking part in the interview asked, ‘Have you thought of DNA methylation?’. Our candidate admitted he not only hadn’t thought of it, he hadn’t even heard of it[170].

Peter Jones and Shirley Taylor immediately began to focus on DNA methylation and in a very short time showed that this was indeed key to the effects of 5-azacytidine. 5-azacytidine inhibited DNA methylation. Peter Jones and Shirley Taylor created a number of related compounds and tested them for their effects in cell culture. The ones that inhibited DNA methylation also caused the changes in phenotype originally observed for 5-azacytidine. Compounds that didn’t inhibit DNA methylation had no effect on phenotype[171].

The methylation cul-de-sac

Cytidine (base C) and 5-azacytidine are very similar in chemical structure. They are shown in Figure 11.1, which for simplicity only shows the most relevant parts of the structure (called cytosine and 5-azacytosine, respectively).

^{Figure 11.1 5-azacytosine can be incorporated into DNA during the DNA replication which takes place prior to cell division. 5-azacytosine takes the place of a C base, but because it contains a nitrogen atom where there is usually a carbon atom, the foreign base cannot be methylated by DNMT1 in the way that was described in Figure 4.2.}

The top half of the diagram is very similar to Figure 4.1, showing that cytosine can be methylated by a DNA methyltransferase (DNMT1, DNMT3A or DNMT3B) to create 5-methylcytosine. In 5-azacytosine, a nitrogen atom (N) replaces the key carbon atom (C) that normally gets methylated. The DNA methyltransferases can’t add a methyl group to this nitrogen atom.