2 PhD projects offered in the IPP summer call 2020
Modified DNA bases play an important epigenetic role in the regulation of gene expression in development and disease. In mammals there are four different cytidine modifications in genomic DNA known to have an epigenetic function, most prominently 5-hydroxymethylcytosine. There are many more known DNA and RNA modifications, whose function remains unknown and awaits characterization, including the cellular enzymes that affect these modifications, which will be the aim of the projects.
PhD project 1: RNA Epitranscriptomics & Embryonic Stem Cells
Some 170 RNA modifications have been described on different types of RNA, most of which remain poorly understood. Some RNA modifications are present on messenger RNA where they can play critical role in post-transcriptional gene regulation. Similar to epigenetic marks on the DNA and Histones, these modifications are often highly conserved and can be dynamically regulated in development and disease. This new layer of gene expression regulation has been coined as “epitranscriptomics”.
Identifying novel effectors of the mammalian RNA methylome would result in new insights into transcription, translation, splicing, and various other RNA-mediated cellular processes. Using liquid chromatography-coupled mass spectrometry (LC-MS/MS), we devised a method to screen for effectors of ~50 RNA modifications. Our screens several hits for modifying enzymes and the corresponding RNA modifications.
In this project, you will characterize these RNA modifying enzymes and the RNA modifications. You will monitor the modifications by LC-MS/MS. You will study their role in embryonic stem cell self-renewal and pluripotency, as well as in differentiation. You will create mutants using CRISPR/Cas9 mediated genome editing and characterize them. You will use Next generation sequencing methods and carry out bioinformatics analyses to learn about affected genes and regulatory networks.
PhD project 2: Mass spectrometry of novel DNA & RNA modifications
Not only RNA but also DNA is modified in cells to regulate gene activity epigenetically. For example 5’-methylcytosine (5mC) serves as an epigenetic modification, which provides a memory state for silencing gene expression. We utilize highly sensitive mass spectrometry (Triple Quadrupole Mass Spec) and stable isotope metabolic labelling to profile epigenetic and epitranscriptomic changes, including 5mC, 5hmC, 5fC, 5caC, m6A, and others.
The successful candidate will have a strong background in Organic Chemistry or Biochemistry to study nucleic acid modifying enzymes to unravel how chemical modifications in genomic DNA and DNA:RNA hybrids (“R-loops”) regulate pluripotency and differentiation of mammalian embryonic stem cells and embryos. You will use LC-MS/MS, genome-edited CRISPR/Cas9 stem cell mutants, as well as various NGS protocols to carry out a multi- dimensional analysis to address: Which modifications can be identified and how do they change with different cellular states? What enzymes mediate them? Where in the genome do the modifications occur? What are molecular determinants involved in targeting of modifications to specific sites? Critical methods you will use are LC-MS/MS, embryonic stem cell self-renewal and pluripotency, CRISPR/Cas9 mediated genome editing and Next generation sequencing (NGS) methods. The project will involve strong interaction with computational biologists.
Publications relevant to the projects
Arab, K., Karaulanov, E., Musheev, M., Trnka P., Schäfer A., Grummt, I. and Niehrs, C. (2019). GADD45A binds R-loops and recruits TET1 to CpG island promoters.Nat. Genet. 51:217-223 133.
Schüle KM, Leichsenring M, Andreani T, Vastolo V, Mallick M, Musheev MU, Karaulanov E, Niehrs C. GADD45 promotes locus-specific DNA demethylation and 2C cycling in embryonic stem cells. (2019). Genes Dev. 33:782-798.
Han D, Schomacher L, Schüle KM, Mallick M, Musheev MU, Karaulanov E, Krebs L, von Seggern A, Niehrs C. (2019). NEIL1 and NEIL2 DNA glycosylases protect neural crest development against mitochondrial oxidative stress.eLIFE. 49044.
Musheev MU, Baumgärtner A, Krebs L, Niehrs C. (2020). The origin of genomic N6-methyl-deoxyadenosine in mammalian cells.Nat. Chem Biol. doi: 10.1038/s41589-020-0504-2.
Leismann, J., et al (2020) The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior in Drosophila.EMBO Rep. (in press)