Functions, functional determinants and mechanisms of intrinsically disordered regions in yeast general regulatory factors

• Applicant: Professor Dr. Philipp Korber
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 568768619

Eukaryotic genomes are regulated by the interplay of sequence-specific DNA binders, collectively called transcription factors (TFs), and chromatin. Both TFs and chromatin factors are highly enriched in intrinsically disordered regions (IDRs). Understanding the roles of these IDRs is key to understanding genome regulation. For example, IDRs in TFs, which are transactivators, function as activation domains, receive signalling input, drive condensate formation and modulate DNA binding. However, much less clear are the roles of IDRs in TFs that lack activation domains, but, e.g., organize nucleosomes or insulate chromatin domains. General regulatory factors (GRFs) from yeast are a model for such non-transactivator TFs. GRFs are mostly essential for viability, bind thousands of sites in the genome, are nucleosome positioning barriers, broadly affect genomic processes, like transcription and replication, and consist only of DNA binding domains (DBDs) and long IDRs. We explore the IDR-mediated functions and mechanisms as well as the IDR properties required for function (functional determinants) of GRFs. Our approach is to fuse IDR variants to GRF DBDs and assay these constructs for various functions. The yeast model is ideal to generate many IDR variants and to test them rapidly in well-established functional assays. Starting with the GRF Abf1, we already demonstrated that the correlation of GRF-IDR variants with growth phenotypes successfully delineated functional determinants of the IDR. This led to the identification of a short linear “Essential Motif” in combination with a mostly acidic IDR context as basis for viability conferred by Abf1. Surprisingly, this “context + motif” IDR-mode could be replaced by a “context only” IDR-mode. While we learned a lot regarding IDR properties, we still do not know the function(s) and mechanism(s) mediated by the IDRs of Abf1 or other GRFs. To find out, we will now assay our Abf1-IDR variants, which encompass a range of growth phenotypes and two IDR-modes, for known GRF functions (DNA site selection, nucleosome positioning, effects on transcription/replication) and potential new functions (3D chromatin folding, condensate formation). For other GRFs, analogous IDR variants will be generated. IDRs likely mediate protein interactions. Previous co-immunoprecipitation/mass spectrometry studies failed to identify interactions of GRF IDRs, probably as these are too transient. We will employ a proximity labeling approach optimized for yeast. The comparison of interactors for functionally stratified IDR variants is a powerful approach to inform on functions and mechanisms. Together, our project will provide a detailed understanding of functions, functional determinants and mechanisms of IDRs in non-transactivator TFs that broadly orchestrate genomic processes. As in the past, such fundamental genome regulation mechanisms elucidated in yeast are very likely instructive for multicellular species and dysregulated in cancer.

• DFG Programme: Research Grants