Investigating Ioc4 and its unique PWWP domain in chromatin organization and Isw1b remodeler function

• Applicant: Professor Andreas Gerhard Ladurner, Ph.D., since 2/2023
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry
• Term: from 2018 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 410995368

DNA in eukaryotic cells is packaged into chromatin. To access DNA during gene transcription and other nuclear processes requires reshaping and modulation of chromatin organization by chromatin remodelers and histone modifying enzymes. Remodelers, including the ISWI family are highly conserved molecular machines that utilize the energy generated by ATP hydrolysis to slide, evict or exchange histone proteins. The yeast Saccharomyces cerevisiae contains two ISWI homologs, Isw1 and Isw2. Isw1 further forms two different complexes, Isw1a and Isw1b. In a previous study we have shown that Isw1b is required to retain and recycle existing histones in the wake of RNA polymerase II transcription in vivo. Thereby, Isw1b maintains chromatin organization over genes in a non-permissive, hypo-acetylated state and limits transcription of non-coding (nc) RNAs. Understanding the cellular mechanisms in place to suppress non-coding transcription is vital, especially since a growing number of ncRNAs has been implicated in the regulation of gene expression. In contrast, mis-expression of ncRNAs is associated with numerous processes, from human disease to ageing in yeast.Isw1b localization to gene bodies is dependent on its Ioc4 subunit and histone H3K36 methylation. The remodeler ensures retention of the modified histones and simultaneously prevents the incorporation of new, soluble histone proteins. This system breaks down in the absence of either the Isw1b chromatin remodeler or H3K36 methylated histones, resulting in the deposition of new, highly acetylated histones, which leads to a more open chromatin organization and increased transcription of ncRNAs. We currently do not understand how the remodeler recycles existing histones. We focus our research on deciphering the molecular mechanisms underpinning the newly proposed role for Ioc4/Isw1b as a factor able to stabilize nucleosomes and act as a histone chaperone. Also, we will dissect the contributions of newly identified Ioc4 features such as its unique subdomain and dependence on DNA interactions for remodeler recruitment and function, using a combination of in vitro biochemistry and genomic approaches. Together, these efforts will provide profound mechanistic insights regarding chromatin organization by the Isw1b remodeler. Further, our results can be extrapolated to understand and manipulate the association and/or function of other factors in a chromatin context.Our previous work and track record on chromatin regulation and experience performing detailed in vitro binding and remodeling assays using purified components as well as genome-wide in vivo analyses of remodeler chromatin localization and non-coding transcription put us in an ideal position to pursue these goals (Hepp et al, Biochim Biophys Acta 2017; Dutta et al, Genes Dev 2014; Huang et al, Genes Dev 2014; Mosley et al, Mol Cell Proteomics 2013; Smolle et al Nat Struct Mol Biol 2012; Venkatesh et al, Nature 2012; Gkikopoulos et al, Science 2011).

• DFG Programme: Research Grants
• Ehemalige Antragstellerin: Professorin Dr. Michaela Smolle, until 1/2023