Nuclear Structure and Function Research Group

The DNA molecules that run the length of each human chromosome are arguably the longest and most important biomolecules known. But although we now know the sequence of the billions of bases in the DNA of each of the 46 chromosomes, we still know little about how those bases are folded in 3-D space within the nucleus of a living cell. Common sense suggests that there must be some underlying order within the apparent tangle, and our current research is concerned with elucidating what that order might be, and how it affects gene function. Our ultimate goal is to generate structural and functional maps of nuclei using a multi-disciplinary approach (e.g., combining molecular analysis, high-resolution imaging, and computer modelling).

We are faced with a considerable problem. The human genome is made up of 23 pairs of DNA molecules that are incredibly thin and long, with a width of ~2 nm and a combined length of ~2 m. All 46 molecules must be packed into a nucleus only ~10 μm wide, a packing problem analogous to folding a kite string that stretches from New York to Chicago into a sphere 10 metres across. Real-sized DNA strings are probably packed into real-sized nuclei by a combination of coiling into higher-order spirals, looping by attachment to underlying structures, and random folding (like pasta in a bowl). Yet this tangle must still allow individual genes to perform their functions; for example, inheritance requires that each DNA molecule must be copied exactly and sorted precisely so that each daughter cell receives one copy.

We are currently analyzing where the processes of "replication" (copying DNA into DNA) and "transcription" (copying DNA into the RNA so that the genetic information can be "translated" into protein) take place within the nuclear tangle. To do so, we gently permeabilize cells, and allow them to make DNA and RNA in the presence of tagged building blocks (i.e., phospho-sugars carrying modified bases); these are incorporated by the cellular machinery into newly-made DNA and RNA. Then, we bind antibodies to the tags, and attach fluorescent labels to those antibodies so that the newly-made DNA and RNA appear in our high-powered microscopes as green and red spots respectively. To our surprise, it turns out that the cellular machines involved in replication and transcription do not act alone. Instead, tens – and sometimes hundreds – are housed in enormous "factories" (diameters 0.05 – 1 μm) where each reels in one of the loops as they copy each base as it passes by (illustrative movie). These results beg several questions. How and when are the factories built? What is the relationship between the different factories involved in replication and transcription? What happens to the factories as cells grow and differentiate, or become malignant? Do some transcription factories specialize in the transcription of particular genes? We are currently trying to characterize the properties of a transcription factory in detail.

We would like to study these processes in living cells, and recently it has become possible to do so using a protein found in a jellyfish that lives in the Pacific ocean. This protein is responsible for the bioluminescence seen in a ship's wake. In response to an attack (e.g., by a ship!), the protein is induced to emit a green flash, presumably blinding the attacker. Other scientists have found that the fluorescence of the jellyfish protein is retained even when fused to other proteins, so giving them a fluorescent tag. We have constructed a hybrid gene encoding the jellyfish protein fused to a human protein that lies at the core of the machine that copies DNA into RNA. Then, we introduced the hybrid gene into a living (Chinese hamster) cell where it was copied into RNA, and the RNA translated into a hybrid protein. In turn, this hybrid protein is incorporated into the copying machine. We are now using our microscopes to see exactly where the now-fluorescent copying machines are in the living cell, and measuring how long they take to copy a gene.

As outlined above, genetic information is copied from DNA into RNA, and then translated into protein. All the textbooks tell us that the process of translation does not take place close to the gene in the nucleus; rather, the RNA moves out to the cytoplasm and is translated there. However, we have recently found that some translation occurs in the nucleus using a similar approach; permeabilized cells were allowed to make proteins in the presence of fluorescently-tagged building blocks (in this case, amino acids). We saw newly-made (fluorescent) proteins in the cytoplasm (as expected), as well as in the nucleus – in the transcription factories. We are now studying the uses that Nature has found for this nuclear protein synthesis.

We are also collaborating with physicists and mathematicians in an ambitious project – our ultimate goal is to write equations that will enable us to predict both the way genomes are folded in 3-D space, and how folding determines function. One approach of the theoreticians is to treat the chromosome as a long polymer, and allow it to "diffuse" in their powerful computers so that it samples many different configurations (this is a so-called " Monte Carlo " simulation as it is based upon chance events). We imagine that a chromosome in a living cell samples similar configurations over time, subject to the limitations imposed by tight packing. We hope to check experimentally whether the configurations seen in the computer are actually found in the living cell, and we will tune our theoretical models so that they fit the data obtained experimentally. As we gain experience, we would then try to model more and more chromosomes until we build up a complete picture.

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