Ankle1, a LEM protein involved in genomic rearrangement

`LEM-domain` proteins are characterized by the presence of a conserved structural motif first identified in the nuclear envelope proteins Lamina associated polypeptide 2 (LAP2), Emerin and MAN1. The LEM motif mediates the interaction with the DNA-bridging protein, Barrier-to-Autointegration Factor (BAF). LEM proteins serve important functions in the maintenance of nuclear architecture, chromatin organization and cell signaling. While most characterized LEM proteins are type II integral inner nuclear membrane proteins and interact with the nuclear lamina, database searches revealed novel, non-membrane-bound LEM proteins with so far unknown molecular functions. Mutations in the LEM proteins Emerin, LAP2 and MAN1 cause heritable human diseases. The molecular mechanisms underlying these diseases are poorly understood. The long-term goal of our research is to understand how LEM proteins regulate chromatin organization and determine nuclear architecture, and which of these functions are impaired in human disease conditions. In this project we will focus on the novel, so far uncharacterized non-membrane LEM protein `Ankyrin and LEM- domain containing 1` (Ankle1). Ankle1 primary sequence is highly conserved between multicellular animals. Our preliminary data on mammalian Ankle1 and data from our collaborators on the C. elegans Ankle1 ortholog point to a function of the protein in DNA repair and genomic rearrangement. Intriguingly, we found evidence that mammalian Ankle1 may be part of an endonuclease complex predominantly expressed in lymphoid tissues. Aim 1 of this study will characterize Ankle1`s potential endonuclease activity and test the hypothesis that Ankle1 is a developmentally regulated and tissue-specific endonuclease in humans and mice. Aim 2 will identify Ankle1- interacting proteins in order to determine Ankle1-associated DNA repair and recombination complexes and pathways. Furthermore, the involvement of Ankle1-BAF interaction in Ankle1 activities will be tested. Aim 3 will test Ankle1 functions in vivo, focusing on genomic rearrangement processes in wild-type and Ankle1-deficient mice. We will study the potential role of Ankle1 in mammalian lymphocyte development and in genomic stability. Sensitivity of (cancer) cells towards DNA-damaging agents will be tested upon knock-down and overexpression of Ankle1. Altogether, this study will provide a comprehensive biochemical and functional characterization of the novel LEM protein Ankle1.

In this project we identified and characterized a novel component in the cell nucleus that contributes to the repair of damaged genomic DNA. Understanding DNA damage repair and components involved therein at molecular detail is important and highly relevant for health, since defects in these repair pathways can lead to cancer. The total DNA, called the human genome, which encodes the building plan of the cells, organs and the whole organism, is packed into chromatin fibers and highly organized and regulated in the cell nucleus. The genome has to be maintained stably throughout the lifetime of cells and faithfully transmitted to daughter cells. Only in a few cell types, such as the antibody-producing immune cells and in sperm cells and oocytes, genomic DNA has to be rearranged during cell development and maturation in a highly regulated pathway. In addition, in all cells of our body, the DNA is exposed to damaging conditions in the environment (e.g. UV radiation, chemicals) and within the cell (oxygen radicals). These factors cause stochastic DNA damage that has to be faithfully and efficiently repaired, as changes in the genome can lead to diseases. Numerous DNA-repair components exist in the cell nucleus that are activated during the physiological genome rearrangements and upon unwanted DNA damage, but a large number of these components are not yet known or their mode of action is not understood. In our project we studied a group of nuclear proteins that associate with DNA. We identified a hitherto uncharacterized DNA-binding protein, termed Ankyrin repeat and LEM domain containing protein 1 (Ankle1) that showed features of DNA damage repair components. Our studies revealed that Ankle1 is primarily found in cells of the immune system in bone marrow, spleen and blood, suggesting a function of Ankle1 in the physiological genomic rearrangements during maturation of immune cells. We generated a transgenic mouse model completely lacking Ankle1 and tested maturation of immune cells. Our data unexpectedly showed that Ankle1 is not essential for immune cell maturation but may be important for the repair of unwanted DNA damage. Our findings are in line with another recent study showing that women containing a slightly different form of Ankle1 have a significantly higher risk to develop breast and ovarian cancer. Such genes, which do not cause cancer by themselves but increase the susceptibility for the development of cancer through known cancer-inducing factors and genes, are called modifier genes. Detailed knowledge of the properties and function of these sparsely studied modifier genes is important for diagnosis and therapy outcome prognosis. Altogether, our project identified a potential modifier gene contributing to the development of cancer and our data set the basis for further analyses of its contribution in cancer formation in patients.