AID Recruitment · Targeting of DNA deaminase AID via transcription and the RNA pol II elongation complex

Targeting of DNA deaminase AID via transcription and the RNA pol II elongation complex

As much as society (especially the western industrialised society) would like to eradicate 'cancer' as a disease, it is not a disease as defined by the Koch Postulates, as the agent provocateur is a mixture of environmental factors, genetic predisposition, and random events. Complex diseases such as cancer require understanding of not only a diseased cell, but also the fundamentals in human biology. The discoveries from this work has and will provide insight into the fundamental understanding of how the human immune system functions, while I was also able to draw conclusions on how genetic changes (a predisposition for cancer formation) such as mutations in our genome can arise over time. The genome (the blueprint of our cells and therefore our body) has at its core DNA, and it is the stability of DNA that impacts on all aspects of life. The survival of the organism requires the DNA in somatic (most normal) tissue to be stable, avoiding genetic pathologies such as cancer or Huntington's. While on the other hand in the germ cells (those needed for reproductions) a diverse genetic pool from DNA recombination and mutation provides the specie a chance of adaptation. In humans, most genetic instability in somatic tissue is associated with cancer, importantly though the adaptive immune system - without which we could not survive - requires DNA instability to provide us with active immune cells. Over a decade ago, a family of proteins was discovered - the DNA deaminases - that are actively recruited to DNA and deaminate one of the building blocks of DNA - cytosine (dC) - to uracil (dU). This change, if not repaired by the cells, would lead to a mutation in the genome. In the immune system the mutations are required to provide us with a pleiotropy of antibodies to fight all invading pathogens, with the DNA deaminase AID being the inducing agent. Work from the Petersen-Mahrt laboratory had implicated AID to also play an important role outside the immune system: during epigenetic reprogramming AID can deaminate methylated cytosines - a classical DNA epigenetic mark. Epigenetic changes are alterations in the function of the genome that do not require changes in the DNA. It is a major determinant for spacial and temporal regulation of protein expression. More importantly, the epigenetic status of a genome is usually altered in cancer cells. AID induced deamination of this mark (methylated cytosine) leads to the re-introduction of cytosine, thereby changing the epigenetic status of that locus. The outcome of the DNA deaminase induced lesion (dU), repair, demethylation, or mutation/recombination is dependent on AID itself, the configuration of the target region, as well as the physiological state of the cell. As AID's natural activity - mutating DNA for a better immune system and epigenetic control - is also the organisms Achilles heel, as unregulated DNA deaminase activity will lead to non-specific DNA instability in other somatic tissues and lead to oncogenesis. Clearly understanding how AID is functioning and regulated during normal growth and development, would give us a direct insight into how DNA mutations can arise in pathology. Discovery I: Although AID is a relative small protein, to date it has not been possible to determine its 3D structure. This has been a real limitation in understanding its precise enzymatic activity, as well as developing drugs to inhibit its function during oncogenesis. To circumvent this limitation we used chemical probing of AID, where synthetic molecules were tested in their ability to change AID activity. We discovered that there is a size limitation within the AID molecule and the deaminase can only accept methyl-cytosine modifications; see our publication Rangam, Schmitz, and Petersen-Mahrt 2012. This discovery also provided a novel fundamental insight in that AID was not able to alter other DNA epigenetic marks - such as hydroxy-methyl cytosine. In recent years a number of laboratories speculated that AID could act on this mark, but did not provide proofs. Our discovery ensured that the epigenetic field is separating the various reprogramming systems. Discovery II: Our past work as well as the key finding in Discovery I provided a platform to revisit a number of theories and discoveries in the field of epigenetics. We therefore wrote a review article for Annual Reviews of Genetics concerning the topic 'DNA demethylation' (Franchini, Schmitz, and Petersen-Mahrt 2012). Because of our expertise in AID and DNA repair, we were able to outline for the first time the various pathways and mechanisms that would provide the epigenetics field with a comprehensive overview of DNA demethylation. Discovery III: We re-analysed publicly available cancer patient databases that contained mutations in key regulatory genes - the tumour suppressors. Using bioinformatic analysis, we identified 'foot-prints' surrounding cytosine mutations in p53 and APC genes. Those 'foot-prints' were compared with the 'foot-prints' of the DNA deaminases (including AID) and we were able to reveal a strong correlations of putative mutator (DNA deaminases) and cancer type (colon, skin, liver, etc); see our publication Schmitz and Petersen-Mahrt, 2012. Discovery IV: One of the key questions in understanding the function of DNA deaminases is, why are certain cytosines more likely to be targeted by DNA deaminases than others in our genomes. We discovered that AID would associate itself with the gene expressing machinery - RNA polymerase II - in order to reach its target. I joint this ongoing research work, and was able to provide key findings that AID is present at the target site during the pausing of the expression, and not before. Furthermore, AID itself was able to influence the RNA polymerase II activity at target sites (Willmann et al. 2013). This work was a key finding as it provided a missing link from other laboratories discoveries in the cascade of events leading to targeted DNA deamination. Importantly, this work was only possible, due to our intensive collaboration with previous lab members, with the labs of Dr. Bernardo Reina-San Martin (Strasbourg, France) and Dr. Silvo Conticello (Turin, Italy). Discovery V: While revealing that AID is a potential regulatory factor, I was interested in studying how AID was regulated. I used this opportunity, to write a comprehensive review paper on this topic with 2 PhD students. We focused on how AID was activated by hormones such as oestrogen, and how this could lead to a link between oestrogen and cancer or autoimmunity (Incorvaia et al., 2013). As the senior author on this work, under my supervision/guidance, we prepared the manuscript 'Hormones and AID - balancing immunity and autoimmunity'.

Data: CORDIS, © European Union