UCPH

Palaeogenomics is the nascent discipline concerned with sequencing and analysis of genome-scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR-based Sanger sequencing, following the dawn of the second-generation sequencing revolution, it has rapidly become a reality. Indeed, so much so, that popular perception has moved away from if extinct species’ genomes can be sequenced, to when it will happen - and even, when will the first extinct animals be regenerated. Unfortunately this view is naïve, and does not account for the financial and technical challenges that face such attempts. I propose an exploration of exactly what the limits on genome reconstruction from extinct or otherwise historic/ancient material are. This will be achieved through new laboratory and bioinformatic developments aimed at decreasing the cost, while concomitantly increasing the quality of genome reconstruction from poor quality materials. In doing so I aim to build a scientifically-grounded framework against which the possibilities and limitations of extinct genome reconstruction can be assessed. Subsequently genomic information will be generated from a range of extinct and near-extinct avian and mammalian species, in order to showcase the potential of reconstructed genomes across research questions spanning at least three different streams of research: De-extinction, Evolutionary Genomics, and Conservation Genomics. Ultimately, achievement of these goals requires formation of a dedicated, closely knit team, focusing on both the methodological challenges as well as their bigger picture application to high-risk high-gain ventures. With ERC funding this can become a reality, and enable palaeogenomics to be pushed to the limits possible under modern technology.

TET2 (Tet Methylcytosine Dioxygenase 2) is a DNA demethylase frequently mutated in patients with Acute Myeloid Leukemia (AML). DNA methylation is an epigenetic modification with key roles in the specification of cellular identity, and, when deregulated, in cancer. DNA methylation is a reversible process and, only recently, it was discovered that TET (Ten-eleven Translocation) proteins mediate DNA demethylation. Mammals have three TET homologues (TET1-3). While TET1 and TET3 share an N-terminal DNA-binding domain (CXXC motif), TET2 has lost its CXXC motif during evolution. Therefore, it is not understood how TET2 binds DNA. Due to the lack of a known DNA-binding domain in TET2, I hypothesize that TET2 may require to interact with proteins to be recruited to DNA, which is essential for its function. Hence, the main goal of my proposal is to understand how TET2 binds to DNA and to identify interacting proteins that participate in this recruitment. I also aim to unveil whether cancer-associated mutations affect TET2 recruitment. To achieve these goals, I will perform a structural-functional analysis of TET2. First, I will perform Chromatin Immunoprecipitation (ChIP) followed by whole genome sequencing of wildtype and truncated TET2. Thus, I will identify the region(s) required for the binding of TET2 to DNA. Next, I will seek proteins that interact with TET2 through the previously identified region by Mass Spectrometry and I will test their involvement in TET2 recruitment to DNA. Furthermore, I will perform similar experiments to inspect the impact of TET2 missense mutations found in human cancer on TET2 recruitment to DNA. Altogether, this project will yield a better understanding of how TET2 is recruited to DNA. The results obtained will significantly impact the epigenetic field and allow for a better understanding of the mechanisms by which TET2 exerts its function in normal cells and how TET2 mutations contribute to cancer.

Signal transduction via post-translational modifications (PTMs) of proteins maintains essential cellular processes in all eukaryotes. Similar to protein phosphorylation, O-GlcNAcylation is a vital signaling mechanism that involves the dynamic cycling of sugar molecules on proteins and these PTMs exhibit extensive crosstalk for regulation of core cellular processes. The only eukaryotic cell type that lacks both signaling mechanisms is yeast and it has been difficult to understand how yeast survive without the essential functions of O-GlcNAcylation. This proposal is based on our discovery demonstrating that baker’s yeast has an O-linked mannose (O-Man) glycosylation system that operates in nuclear, cytoplasmic and mitochondrial compartments. The localization of these O-Man modifications on yeast proteins mirrors that of O-GlcNAcylation found in higher eukaryotes and this discovery demonstrates that yeast possess a hitherto unknown signaling mechanism involved a myriad of cellular processes. This research project aims to explore when and where yeast utilize the nucleocytoplasmic O-Man signaling system and to understand the functional consequences of this novel modification. In addition, the project aims to identify and characterize the enzymes responsible for the attachment and removal of nucleocytoplasmic O-Man modifications in order to enable manipulation of the system for improvements in yeast-based bioproduction and bioprocessing platforms. The project will open an entirely new area of research by bringing novel knowledge on how yeast orchestrate cellular signaling and advance our understanding on how essential cellular processes are controlled in eukaryotes. This holds promise to bring unique opportunities to manipulate yeast for improvements and open for wide applications in industry and biotechnology.

The development of healthy organisms requires the formation of different cellular systems, such as a blood, bone and muscle. All these different cell types arise during embryonic life from specific pools of mesodermal progenitors (MPs). A central question is how distinct MPs are specified and ultimately why different cells respond to signalling pathways in different ways? Critical insights here are directly relevant to conceive strategies for increasing the efficiency of mesodermal differentiation aimed for treating muscular degenerative diseases. To date, in vitro, somitic mesoderm differentiation for regenerative purpose has had limited success. Wnt signalling promotes Embryonic Stem Cell (ESC) differentiation of all the MPs, including skeletal muscles. The activity of specific pioneer transcription factors (TFs) may be the key for converting Wnt signalling pathways into a specific transcriptional program. Pioneer TFs shape the chromatin landscape by opening the chromatin and allowing the recruitment of lineage specific TFs, and thus ultimately control the TF binding dynamics and the acquisition of a specific cell fate. Pbx proteins are pioneer TFs, which are specifically expressed in the primitive streak, the region of the embryo that will produce all mesoderm, and are critical for promoting mesodermal specification. Here, I will establish how Pbx proteins specify MPs and determine the competence of early mesoderm to respond to Wnt signalling. To this end, I will use mouse embryos and murine epiblast stem cells, which are analogous to human ESC (hESCs). My approach combines the strength of an in vitro ESC differentiation method, routinely used at DanStem, with chromatin immunoprecipitation and transcriptional regulation assays, which I have extensively mastered during my postdoctoral training. I expect that my findings will provide novel tools for rational design of strategies to combat genetic and degenerative muscular diseases, such as muscular dystrophies.

The Large Hadron Collider and particle physics as a whole is entering a high precision era. A powerful tool for studying new physics in precision experiments where new particles may not be produced directly, but their influence on lower energy physics may be seen indirectly, is that of Effective Field Theories. I propose to use the Background Field Method, a useful formulation in perturbative quantum field theory, to aid in the calculation of one-loop amplitudes in the Standard Model Effective Field Theory. After completing basic calculations allowing for the one-loop program to proceed, I will focus on calculations relevant to Z boson physics first as this is the area most in need of precision calculation and will open the door for important studies of next generation collider design. Then I will finish by calculating the Higgs boson width to one loop order with up to 3 final state particles in the Standard Model Effective Field Theory. This will serve as an update to the tree level Higgs width package currently being produced by myself and my supervisor. A core element of this proposal is the production of another package, tentatively named "SMEFT-ONE," in which I will release the results of all one loop calculations produced as a result of this proposal. The purpose of this package will be the dissemination of the work to the particle phenomenology community as a whole with a focus on the ability to directly use the results in independent studies.