Establish novel protocols to profile the binding of transcription factors or histone complexes

DNA adenine methylation identification (DamID) has been developed in 2000 (van Steensel et al., 2000; van Steensel et al., 2000) and has garnered much attention as a robust and simple method to profile DNA-binding sites of virtually any DNA- or chromatin-interacting protein, including transcription factors, RNA Polymerase II or even histone modifier complexes (Southall et al., 2013; Cheetham et al., 2018; Marshall et al., 2017). In this fashion, DamID can replace many of the standard methods to characterize gene expression and regulation such as RNAseq or ChIPseq. However, despite its great potential, high reproducibility and flexible set-up, only few groups and laboratories have actually implemented the technology. This is partly due to the length of the protocol and the need for establishing the technique in the laboratory as commercial DamID-assays are not available. In parallel, the available software to analyze DamID data is outdated or insufficient to cope with standard replicate numbers (Marshall et al., 2015; Gutierrez-Triana et al., 2016; Vázquez-Marín et al, 2019). Hence, while a parallel project will develop state-of-the-art analytical software for DamID data, the here outlined project will address the length of the DamID protocol to provide a short, streamlined version reliant on exclusively commercially available reagents. By relying on biochemistry otherwise employed in ATACseq protcols, DamID will simultaneously become more sensitive promising a higher signal-to-noise ratio and thus detection of even rare binding events.

Together these endeavors will make DamID a completely open-access, easily implemented, sensitive and affordable technique that with high likelihood will be the alternative of choice for commercially available assays.

For this, the project will cover a wide range of techniques starting from 1) molecular biology (transformation and propagation of bacteria, plasmid extraction), 2) human cell culture (cell propagation, plasmid electroporation), 3) next-generation sequencing library preparation and 4) in house, hands-on sequencing (ATACseq, DamID) culminating in the 5) detailed analysis of the acquired data. No particular skills are required as everything can be learned during the project. The only requirement is excitement.

While we will initially use plasmids encoding for ATF4, a transcription factor involved in integrated stress responses, the project can also be expanded to give space to creativity by the respective Master student to study other factors involved in gene regulation. Similarly, the amount of computational, statistical and analytical work can be modified dependent on the students interest and preferences.

The project will take place in the lab of Associate Professor Robin Andersson with me as supervisor. The lab is part of the Section for Computational and RNA Biology at the Department for Biology, University of Copenhagen. Collaborations with the lab of Jelle van den Ameele at the University of Cambridge will strengthen the project further.