The Role of Protein Condensation in Gene Activation

2 PhD projects offered in the IPP summer call Molecular Mechanisms in Genome Stability & Gene Regulation

Scientific Background

Transcription is one of the most fundamental processes of all cellular life. Precise regulation in time and space is needed for proper development and to adequately react to environmental changes. Misregulation, on the other hand, can lead to the establishment of serious diseases most notably cancer. The regulation of gene activation is achieved via a complex, multi-step process that starts by the recognition of DNA enhancer elements through gene-specific transcription factors which recruit downstream factors that help position RNA polymerase II correctly at the transcription start site for RNA synthesis to begin. Recently, it was discovered that transcription initiation sites constitute protein condensates with liquid-like properties. This exciting new finding has attracted much attention in the transcription community, as it can potentially explain cellular observations that were puzzling previously. This includes the simultaneous firing of different genes or the bursting behaviour of transcription. While we recently succeeded in reconstituting the formation of transcription factor condensates in vitro, little is known about their function or regulation inside cells. Furthermore, cellular condensates contain thousands of different proteins but how their composition is regulated is not fully understood.
Our newly developed reconstitution systems (see Morin et al. and Quail et al.) provide us with a tool to build initiation condensates in the test tube and to increase their complexity in a stepwise manner. To achieve this, we will use different biochemical methods including protein purification and characterization, reconstitution of condensates on DNA, and DNA binding assays. The insights obtained in this way will later on be tested in the complex environment of the cell to investigate the effect of condensation on gene expression more directly. Here, mammalian tissue culture will be utilized and a variety of genome-wide methods (e.g. ChIP-Seq, RNA-Seq, ATAC-Seq) will be performed. Different collaborations inside and outside Mainz will ensure the interdisciplinarity of our investigations. 

We are a new research group that has started at the IMB in September 2023 and are looking for motivated PhD students to decipher how initiation condensates regulate gene expression. 

PhD Project 1: Investigating the role of transcription factor condensation during chromatin opening

During de novo gene activation, chromatin has to transition from a closed to an open state that allows binding of the transcription machinery. This project will address important open questions about this transition: Can condensates form on nucleosomes and with which epigenetic modifications? Is transcription factor condensation important for histone modification? And finally, what is the role of condensation in chromatin opening (during gene activation)?

Here, we aim to reconstitute nucleosomes and recreate some of the early steps of chromatin opening in the test tube. Using the methods detailed above, the initial experiments will be performed in vitro using our reconstitution system to simplify the problem to its most relevant components. The biochemical results will be used to formulate testable hypotheses which we will investigate inside cells. This strategy will enable us to gain physiologically relevant insight into the interaction between transcription factor condensation and chromatin modification. My previous research focused on the famous Yamanaka pluripotency factor Klf4 which will be utilized here, too, together with other pioneer and non-pioneer transcription factors. 

If you are interested in this project, please select Wittmann (TFC) as your group preference in the IPP application platform.

PhD Project 2: Formation and function of Ngn3 condensates during pancreas development

In the pancreas, different cell types develop from pancreas progenitor cells. One of them constitutes the hormone-producing endocrine beta-cells. Interestingly, the master transcription factor Neurogenin-3 (Ngn3) is both necessary and sufficient for the differentiation into endocrine cells. Using this system as a tool, we will be able to investigate the physiological role of condensation in pancreas development. 

In this project, we will use the in vitro methods described above to test different Ngn3 variants that affect the protein’s condensation behaviour. After identifying interesting variants in this way, we will introduce them into pancreas progenitor cells for differentiation into beta cells. This approach will allow us to study the effect of Ngn3 condensation on cell differentiation. The entire project will be done in collaboration with a pancreas expert and will give insights into the biological role of transcription factor condensation.

If you are interested in this project, please select Wittmann (NGN) as your group preference in the IPP application platform.

 

Publications relevant to the projects

Morin JA, Wittmann S, Choubey S, Klosin A, Golfier S, Hyman AA, Jülicher F, Grill SW. (2022) Surface condensation of a pioneer transcription factor on DNA. Nat Physics, 18: 271–276 Link

Wittmann S, Alberti S. (2019) ERα condensates: chronic stimulation is hard to ignore. Nat Struct Mol Biol 26(3): 153-154 Link

Wittmann S, Renner M, Watts BR, Adams O, Huseyin M, Baejen C, El Omari K, Kilchert C, Heo DH, Kecman T, Cramer P, Grimes JM, Vasiljeva L. (2017) The conserved termination factor Seb1 bridges RNA polymerase II and nascent RNA. Nat Commun 8: 14861 Link

Quail T, Golfier S, Elsner M, Ishihara K, Murugesan V, Renger R, Jülicher F, Brugués J. (2021) Force generation by protein–DNA co-condensation. Nat. Phys. 17, 1007–1012 Link

Sabari BR, Dall'Agnese A, Young RA. (2020) Biomolecular Condensates in the Nucleus. Trends Biochem Sci. 45(11):961-977. Link

Zhang X, McGrath P, Salomone J, Rahal M, McCauley H, Schweitzer J, Kovall R, Gebelein B, Wells J. (2019) A Comprehensive Structure-Function Study of Neurogenin3 Disease-Causing Alleles during Human Pancreas and Intestinal Organoid Development. Dev Cell. 50(3):367-380e7 Link

 

Contact Details

Dr Sina Wittmann
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