Svend Petersen-Mahrt
Svend Petersen-Mahrt
affiliation: IFOM-FIRC Institute of Molecular Oncology
research area(s): Molecular Biology, Immunity And Infection
Course: Molecular Medicine: Molecular Oncology and Computational Biology
University/Istitution: Università di Milano, UNIMI-SEMM
Dr. Svend Petersen-Mahrt is the head of the DNA Editing in Immunity and Epigenetics lab at IFOM - IEO. The work in his laboratory focuses on the notion that DNA instability, usually associated with cancer, can be advantageous for an organism. The bulk of the laboratories' work focuses on analysing a novel family of enzymes - DNA deaminases. While a research fellow with Prof. Michael Neuberger at the MRC-LMB in Cambridge, UK, they discovered a truly breakthrough process, which - for the first time - identified a class of proteins that mutate our genomes over a billion times a day. This work provided the backbone for a whole new area of research that has spawned over a hundred laboratories world wide (and is continuing to grow). He then applied his previous research background in virology and antibody engineering to analyse this novel phenomena in various biological systems. This allowed him to provide the epigenetics field with a new direction on how to approach a long standing issue - How are DNA methylation marks erased? Most importantly, his recent work may have provided the answer to one of the important question in oncology, which was already posed over 125 years ago - How does the hormone estrogen cause cancer? With his work and approaches, Dr. Petersen-Mahrt has been able to provide both fundamental insight into biological processes as well as have a direct impact on patient care.

The Editing laboratory is working on: how AID is Activated in terms of transcriptional regulation, and how AID is Targeted and gains access to the specific loci. At the ssDNA target site, we are interested in what types of enzymatic Kinetics are regulating the activity. Once the deaminated dU (or dT) is present in the reformed dsDNA as a DNA mismatch, we are studying how this lesion is processed to induce immune diversity in Acquired Immunity and protects our cells via Innate Immunity. Our discovery that AID also functions outside the immune-system lead us to characterise the molecular aspects of Epigenetic Reprogramming and Meiotic Recombination. As an enzyme capable of inducing mutations and recombination it is self-evident that we also investigate how AID can induce Oncogenesis.
The DNA in our cells provides the blue-print of our body temporally as well as spatially. Evolution has placed a premium on keeping this beautiful molecule stable, with DNA instability leading to mutations and possibly oncogenesis. Opposition this dogma, our laboratory is studying a group of enzymes called DNA deaminases, which induce DNA instability via deaminating dC residues to dU. Isolated as necessary factors for the development of our immune system, they have now been implicated in a number of fundamental aspects of cellular and organismal physiology, thereby providing the outline of our working hypothesis: DNA instability is an evolutionary desirable phenomena and will help in survival of the organism.

DNA deaminases are a family of proteins that deaminate cytosine bases in single stranded DNA (ssDNA). Originally thought to function as genome guardian to protect against foreign DNA (e.g. retroviruses, transposons etc.), the ancestral member, AID, has co-evolved with the immunoglobulin loci, and is essential for the formation of a functional humoral immune response. While our recent efforts, as well as those of other laboratories, identified AID to deaminate 5-methyl-cytosine (5meC), and with it playing a role in epigenetic reprogramming. Because the physiological function of DNA deaminases is to induce DNA damage, they are very powerful mutagens and may be involved in all stages of oncogenesis.

There are numerous pathways of how AID could become activated, including transcription, translation, protein complex formation, etc. We have recently been able to show that AID can be directly activated by the hormone estrogen. This discovery has far reaching consequences, as it may have answered an over 100 year old question: How can estrogen induce cancer? As estrogen itself is non-mutagenic, directly activating AID transcription, estrogen is able to induce a DNA mutator in a variety of hormonally responsive cells.

Having a DNA mutator roam freely in the nucleus would be very ‘dangerous’ as it could mutate off-target loci and induce genome instability. Rather than using a hypothesis driven approach, we used classical biochemistry to understand which proteins are associated with AID on chromatin. This unbiased approach identified the RNA pol II elongation complex as the centre of a targeting mechanism. Although it may be counterintuitive, as RNA pol II is widely distributed throughout the genome, this discovery in combination with our work on AID’s involvement in epigenetics (see below) and its direct link to DNA repair (also below), provides a framework for a novel hypothesis.

Acquired Immunity
Inducing dU lesions in the immunoglobulin locus initiates DNA repair pathways that do not result in repair, but mutation and recombination. We have begun to dissect the molecular mechanisms of AID induced lesion resolution. Using an in vitro biochemical approach in cellular extracts (the first such system available), we have been able to show that post-lesion generation, AID recruits DNA repair proteins to the deaminated dC and alters repair functions - a novel activity of AID. We are currently analysing an AID interacting factor part of the DNA damage response pathway. The system also provides an insight into how single AID-induced events can lead to complex processive like alterations in the DNA.

Epigenetic Reprogramming
From the outset, we considered DNA deamination to be part of a general genome guardian mechanism. Our discovery that AID is expressed in oocytes and other important developmentally regulated tissues, was hinting towards a second physiological function of AID. Combining this finding with our biochemical analysis showing that AID can deaminate 5meC in the context of a CpG, indicated that AID may play a role in epigenetic reprogramming. Our current work is centred around the precise molecular mechanisms that are needed to initiate AID induced de-methylation. Our in vitro system has again provided novel insight into this process and may provide a paradigm shift on how DNA demethylation is initiated - via AID’s role in recruiting DNA repair proteins and inducing processive demethylation.

Understanding the fundamental principles of any biological process is vital for science and society, yet at the same time one should never forget that more immediate concerns have to be addressed as well. DNA deaminases are the first proteins identified to have mutagenic activity as their physiological function. This changes the outlook of how to analyse any disease with genetic instability, as it is no longer the mutant protein that is causing the abnormality, but how the cell controls and regulates a mutator. Our work on hormones has shown that stimulating normal cells with physiological amounts of estrogen leads to oncogenic mutations and translocations. Moreover, our kinetic analysis showed that enzyme activity can be used to control potential oncogenic activity.

1) Siim Pauklin and Svend K. Petersen-Mahrt. 2009. Progesterone Inhibits AID by Binding to the Promoter. Journal of Immunology: 189(2):1238-44. (ISI 6.1).

2) Siim Pauklin, Julia S. Burkert, Julie Martin, Fekret Osman, Sandra Weller, Simon J. Boulton, Matthew C. Whitby and Svend K. Petersen-Mahrt. 2009. Alternative Induction of Meiotic Recombination from Single Base Lesions of DNA Deaminases. Genetics: 182(1):41-54. (ISI 4.0)
Highlighted by Editor of Genetics 182:xx.

3) Siim Pauklin, Isora V. Sernández, Gudrun Bachmann, Almudena R. Ramiro and Svend K. Petersen-Mahrt. 2009. Estrogen directly activates AID transcription and function. Journal of Experimental Medicine 206:99-111. (ISI 15.6)
a) Women, autoimmunity, and cancer: a dangerous liaison between estrogen and activation-induced deaminase? Robert W. Maul and Patricia J. Gearhart. J. Exp. Med. 2009 206: 11-13.
b) Hormone wrecks DNA. Research Highlights. Nature Medicine 15: 148 - 149 (2009).
c) Public Press: Daily Mail, Evening Standard, Mail on Sunday (Full Page coverage and Interview), Medical News Today, Oncology Times, Lab Times, Faculty 1000. Blogs: WellSphere Cancer Community, Medical Research News.

4) Heather A. Coker and Svend K. Petersen-Mahrt. 2006. The nuclear DNA deaminase AID functions distributively whereas cytoplasmic APOBEC3G has a processive mode of action. DNA Repair 6:235-243. (7)
Commentary: DNA Repair (Amst). 2007 Jun 1;6(6):689-92.
Response: DNA Repair (Amst). 2007 Jun 1;6(6):693-4.

5) Hugh D. Morgan, Wendy Dean, Heather A. Coker, Wolf Reik and Svend K. Petersen-Mahrt. 2004. Aid Deaminates 5-methylcytosine in DNA and is Expressed in Pluripotent Tissues. Journal of Biological Chemistry 279:52353-523560. (72)
a) Editors' Choice. Science: 22 October 2004; 306 (5696).
b) Public Press: BBC News, Faculty 1000, Medical News Today, Genetics Society News, various Blogs.

6) Silvestro G. Conticello, Cornelia J.F. Thomas, Svend Petersen-Mahrt and Michael S. Neuberger. 2005. Evolution of the AID/APOBEC family of (deoxy)cytidine deaminases. Journal Molecular Evolution 22:367-377. (104)

7) Rupert C. L. Beale*, Svend K. Petersen-Mahrt*, Ian N. Watt, Reuben S. Harris, Cristina Rada, and Michael S. Neuberger. 2004. Differential context-dependence of DNA deamination by APOBEC enzymes: comparison with mutation spectra in vivo. Journal of Molecular Biology. 337:585-596. (120)

8) Svend Petersen-Mahrt and Michael S. Neuberger. 2003. In vitro deamination of cytosine to uracil in single-stranded DNA by APOBEC1. Journal of Biological Chemistry 278:19583-19586. (53)

9) Reuben S. Harris*, Svend Petersen-Mahrt* and Michael S. Neuberger. 2002. RNA editing enzyme APOBEC1 and some of its homologues can act as DNA mutators. Molecular Cell 10:1247-1253. (189)

10) Svend Petersen-Mahrt*, Reuben S. Harris* and Michael S. Neuberger. 2002. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418:99-104. (421)
Commentary: In Nature, Science, The Scientist, and Cell.

11) Reuben S. Harris, Kate N. Bishop, Ann M. Sheehy, Heather M. Craig, Svend K. Petersen-Mahrt, Ian N. Watt, Michael S. Neuberger, and Michael H. Malim. 2003. DNA deamination mediates innate immunity to retroviral infection. Cell 113:803-809. (518)

12) Reuben S. Harris, Julian E. Sale, Svend Petersen-Mahrt, and Michael S. Neuberger. 2002. Immunoglobulin V gene conversion in a cultured B cell line is dependent upon AID. Current Biology 12:435-438. (124)
Project Title:
DNA lesion resolution in immunity and epigenetics
How DNA instability leads to mutations and recombination is a fundamental aspect of cancer research. Our work on AID and cancer poses a related important question: How are AID induced lesions being resolved - leading either to repair, mutation, or recombination? Classical genetics has provided some insight into the mechanism and identified key players, but a complete understanding has been missing. This is predominantly due to a lack of a sufficiently robust in vitro biochemical assay. Our proposal is to utilise our past experience on DNA deaminase and combine it with a novel in vitro assay, leading to new in vivo understanding and new targets. This system is the first to analyse a singly defined biological created DNA base modification, which is unlike other systems where DNA base modifications are synthetically generated (chemically synthesised or chemically induced).
DNA demethylation has long been hypothesised about, but only recently has genetic and preliminary biochemical evidence been able to identify key players. To date, there is no in vitro system available to dissect the molecular pathways that lead from 5meC to dC. AID has been implicated genetically as well as biochemically in active DNA demethylation. We therefore want to utilise our IVR system similar to AIM 1 to begin to understand this important genetic and epigenetic phenomena.

Project Title:
Molecular Mechanisms and Novel Targets in Hyper−Immunoglobulin M Syndrome II
Hyper-IgM syndrome 2 (HIGM2) is a hereditary immuno-deficiency disease, that is genetically identified through recessive or dominant mutations in the activation induced deaminase (AID) gene. AID is required for the normal development of a healthy immune system, inducing antibody diversification through class switch recombination and somatic hypermutation. AID catalyses the hydrolytic deamination of cytosine residues in DNA, with the resulting uracil inducing DNA instability leading to immunoglobulin (Ig) diversification. This targeted DNA instability is precisely regulated, but not well understood. Although RNA pol II transcription had been implicated, no direct evidence existed linking AID activity and transcription.
The aim of this application is the analysis of our recently identified AID containing transcription complexes and the role of RNA pol II modification during Ig diversification and immune pathologies. To understand the physiological relevance of AID interacting with RNA pol II complexes, biochemical analysis in vitro and in cell lines will identify direct interactions and binding domains of AID. This will allow us to create new AID mutations in cell lines and mice, that can be tested for their role in immune diversification. Genetic analysis of identified candidates will delineate the molecular pathways of how transcription alters the efficiency of AID and immune diversification. While newly discovered AID mutations and candidate genes could provide new pharmacological targets. Complementing the experimental approaches, sequence analysis of AID and candidate genes in DNA from HIGM patients will provide a clinical insight into disease mechanism.