Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80

Niebauer, Vanessa (2020): Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Knowing the explicit locations of nucleosomes in a genome is a pre-requisite for understanding the regulation of genes. Predominantly at regulatory active promoter sites, regular spaced arrays phased at reference points shape the chromatin landscape. In eukaryotic cells ATP-dependent chromatin remodeler align nucleosomes at reference points and are pivotal in the formation of the stereotyped promoter pattern. Chromatin remodeler of the ISWI, CHD, SWI/SNF and INO80 family convert energy derived from ATP hydrolysis to operate on their nucleosomal substrates to accomplish nucleosome spacing, eviction and editing reactions. Recent structural elucidations provided mechanistic insights into how chromatin remodelers engage their nucleosomal substrates (Eustermann et al., 2018, Aramayo et al., 2018, Willhoft et al., 2018, Ayala et al., 2018, Farnung et al., 2017, Wagner et al., 2020, Yan et al., 2019, He et al., 2020, Han et al., 2020) and brought about a unifying DNA wave mechanism underpinning ATP-dependent DNA translocation by chromatin remodeling complexes (Yan and Chen, 2020). Understanding how phased arrays of equally spaced nucleosomes are generated by chromatin remodelers represents an ultimate long-term goal in chromatin biology. What remains unclear is the underlying mechanism that directs nucleosome positioning by chromatin remodelers in absolute terms. How do ATP-dependent chromatin remodelers generate phased arrays of regularly spaced nucleosomes? How are the distances between nucleosomes and phasing sites and between adjacent nucleosomes set? Is DNA shape read-out part of nucleosome positioning driven by chromatin remodelers? Do remodelers have intrinsic ruler-like elements that set spacing and phasing distances? The aim of this thesis was to delineate whether, and if so, what type of genomic information is read by a remodeler in the stereotypic placement of nucleosomes at physiological sites, and how the remodeler activities fit into the unifying framework of ATP-dependent DNA translocation mechanism of chromatin remodelers. To gain an insight into nucleosome positioning driven by Saccharomyces cerevisiae (S.c.) ATP-dependent chromatin remodelers, a combination of a minimalistic genome-wide in vitro reconstitution system, biochemical analysis, high-resolution structures and structure-guided mutagenesis of the S.c. INO80 model system was applied. Findings of this work would have an impact on the mechanistic understanding of nucleosome positioning driven by ATP dependent chromatin remodelers based on the ruler concept that has been described earlier for the ISW1a chromatin remodeler (Yamada et al., 2011). The ISW1a, Chd1 and ISW2 remodelers demonstrated “clamping” activity and used ruler elements to set 1 Abstract distances with a defined linker length (21-26 bp at all densities, 12-13bp at all densities, 54-58 bp at low/medium densities, respectively). Mutagenesis of the INO80 model system identified and tuned the INO80 ruler element, which is comprised of the Ino80_HSA domain of the ARP module, the NHP10 module and Ino80 N-terminal residues. Regularly spaced symmetrical arrays were generated at the Reb1 reference point sites as well as at BamHI-introduced dsDNA break sites. Nucleosome positioning on the genomic sequences of S. c., Schizosaccharomyces pombe (S.p.) as well as Escherichia coli (E.coli) showed no significant differences. Mutagenesis of residues located within the Ino80_HSA domain established a causal link between nucleosome positioning by INO80 and DNA shape read-out by the INO80_HSA domain. The spacing and phasing distances generated by ATP-dependent chromatin remodelers point towards a remodeler-intrinsic ruler activity that is independent of underlying DNA sequences and can be sensitive to nucleosome density. This study measured linker lengths set by remodeler-intrinsic ruler-like functionalities in absolute terms, which will be instrumental to dissect contributions from individual remodelers in nucleosome positioning in vivo. This provides the starting point to understand how remodeler-driven nucleosome dynamics direct stable steady-state nucleosome positions relative to DNA bound factors, DNA ends and DNA sequence elements. Sequence-dependent DNA shape features have been mainly associated with binding of transcription factors as well as general regulatory factors and more static DNA binding events. This study augments the general description of nucleosome positioning sequences for chromatin remodelers by establishing nucleosome positioning motifs based on DNA shape analysis. This study provides an intriguing framework to implement DNA shape read-out in the tracking mechanism of DNA-translocating machineries.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Chromatin Structure, ATP-dependent Chromatin Remodeler, Nucleosome Positioning, INO80
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 29. Juli 2020
• 1. Bericht­erstatter:in: Hopfner, Karl-Peter