Structural and functional characterization of the INO80 chromatin remodelling complex

Schall, Kevin (2022): Structural and functional characterization of the INO80 chromatin remodelling complex. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

DNA is the carrier of the genetic information in all kingdoms of life. Cells face the challenge to pack DNA and ensure its integrity on the one hand while enabling access to the genetic code on the other hand. This holds in particular true for eukaryotes, whose genomes are typically larger than those of prokaryotes and organized in multiple linear DNA molecules, termed chromosomes, within the nuclear envelope. Their genetic information is stored as a nucleoprotein complex referred to as chromatin, in which DNA is associated with histone proteins. It compacts DNA and at the same time provides an elaborate platform to regulate access to the genetic code. Various fundamental cellular processes depend on this access and thus are regulated by the organization of chromatin, such as transcription, cell division, cell differentiation and DNA repair. The fundamental unit of chromatin is the nucleosome core particle, in which 147 bp of DNA are wrapped around an octamer of the four core histone proteins (H2A, H2B, H3 and H4) or variants thereof, giving rise to a disk-shaped particle. The nucleosome core particle originated from archaea. These possess one or two histone proteins, which are orthologous to eukaryotic histones and assemble with DNA in an overall similar fashion. Being the fundamental unit of chromatin, the formation, disassembly, localization and composition of the individual nucleosome core particles directly impacts the chromatin landscape and therefore gene regulation. These actions are carried out by chromatin remodelling complexes (‘remodellers’). The catalytic core of all remodellers is a Snf2-type ATPase, which converts the energy of ATP hydrolysis in DNA translocation. Based on flanking domains and additional subunits, remodellers can be grouped into four families: ISWI, CHD, SWI/SNF and INO80. While ISWI and CHD carry out their function as small complexes or even as single subunits, remodellers of the SWI/SNF and INO80 families form multi-subunit complexes in the megadalton range. In the past twenty years, several hallmark studies characterized the biological functions of these multi-subunit complexes and analyzed their composition and architecture. However, insights on a detailed structural level into how the individual subunits cooperate remained elusive, mainly due to technical limitations. These could partly be overcome in the past years, especially by the advent of high-resolution cryogenic electron microscopy. This thesis analyzes the INO80 chromatin remodelling complex (INO80), the founding member of the INO80 family, from a structural and functional perspective with an emphasis on its action on the nucleosome core particle. INO80 translocates DNA around the nucleosome core particle and spaces nucleosomes to form genic arrays. The presented results reveal, how the evolutionarily conserved subunits of INO80 interact with the nucleosome and catalyze DNA translocation in a coordinated fashion. A cryo-EM structure of the core module of INO80 bound to the nucleosome core particle demonstrates that the ATPase domain and the actin fold of Arp5 bind nucleosomal DNA at SHL -6 and SHL -3, respectively, while the insert domain of Arp5 contacts the acidic patch. The Rvb1/2 heterohexamer connects these subunits without forming major nucleosome contacts. This arrangement provides valuable information about the mechano-chemical catalysis cycle of INO80, in which the ATPase domain acts as a motor, Arp5 as a counter grip and the Rvb1/2 ring as a stator element. The ATPase pumps DNA inside the nucleosome core particle against Arp5, which leads to a DNA strain. Once sufficient force is generated, the counter grip is released and DNA translocation occurs. Thus, these results explain the biochemically and biophysically observed step size of 10 – 20 bp of DNA translocation catalyzed by INO80. The X-ray structure of the Arp8 module in combination with biochemical data shows that the module binds outside the nucleosome core particle to extranucleosomal DNA. Arp8, actin and Arp4 organize the HSA domain of Ino80 in a way that a number of conserved and positively charged lysine and arginine residues interact with entry DNA ahead of the ATPase domain. This interaction is crucial for the catalysis of DNA translocation by INO80. The combination of these structures leads to a composite model of the evolutionarily conserved subunits of INO80, which is supported by a more recent cryo-EM structure. It suggests that the Arp8 module prevents DNA residing in a transition state between the ATPase and Arp5 from slipping back. Moreover, the Arp8 module could also act as a molecular ruler as its footprint matches the distance between two nucleosome core particles in genic arrays formed by INO80. Small molecule analysis reveals that histone tails regulate nucleosome invasion by INO80. They constitute a regulatory barrier and constrain conformations of nucleosome-bound INO80.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• 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: 20. Dezember 2022
• 1. Bericht­erstatter:in: Hopfner, Karl-Peter