title: Structural and biochemical studies of  ATP-dependent chromatin remodelers from  the INO80/SWR1 family
creator: Jungblut, Anna
subject: ddc-570
subject: 570 Life sciences
description: The genome of a eukaryotic cell is packaged by histone proteins into nucleosomes. ATP-dependent  chromatin remodeling enzymes shape this first level of chromatin organisation and play a fundamental  role in regulating access of nuclear factors to DNA. Eukaryotes possess four evolutionary conserved  families of chromatin remodelers. The INO80/SWR1 family members assemble into mega-Dalton sized, multi-subunit complexes and configure the nucleosome landscape at gene promoters, origins of replications and sites of DNA damage. In Saccharomyces cerevisisae (S.c.), the 15-subunit  INO80 complex translocates DNA through ATP hydrolysis along the histone core leading to positioning  of +1 nucleosomes downstream of promoter DNA. Subsequently, the 14-subunit S.c. SWR1  complex exchanges the canonical histone H2A/H2B dimers with the variant H2A.Z/H2B through  ATP hydrolysis. H2A.Z containing +1 nucleosomes are proposed to serve as a gateway for gene  transcription. Despite of this importance, the precise molecular mechanisms underlying these nucleosome  remodeling reactions are poorly understood. In addition, it remains unclear what defines  the substrate specificity and the different reaction outcomes of INO80 and SWR1 remodelers which  in principle share a common subunit composition including nuclear actin and Actin-related proteins  (Arps). To address these questions, I characterized the structural basis of nucleosome remodeling  by INO80 and SWR1 complexes by using a combination of cryo-electron microscopy (cryoEM) and  biochemistry.  In the first part of this thesis, I present the mechanistic basis of hexasome remodeling by the INO80  complex. Hexasomes are non-canonical nucleosomes lacking one copy of H2A/H2B and are a hallmark  of actively transcribed genes. My findings demonstrate how INO80 identifies non-canonical  DNA and histone marks of hexasomes that result from the absence of H2A/H2B. INO80 is activated  by directly sensing an exposed H3/H4 histone interface, operating independently of the H2A/H2B  acidic patch. This is enabled through a spin-rotated binding mode of the Ino80 motor domain while  its Arp module remains tethered to the now unwrapped linker DNA. These findings shed light on  how the absence of H2A/H2B creates altered substrates for chromatin remodeling, thereby creating  a different, however unexplored layer of energy-mediated chromatin regulation.  In the second part, I present the first structural and biochemical study of SWR1 from Chaetomium  thermophilum (C.t.). I identified the subunit composition of C. t. SWR1 and implemented a recombinant  expression and purification strategy for both S.c. and C.t. SWR1 that enabled subsequent  cryoEM studies. In addition, I investigated the structure of the Arp module by using an integrative  structural biology approach building on cryoEM, AI-powered AlphaFold structure predictions and  crosslinking mass spectrometry. Thereby, I determined an almost complete model of the Apo SWR1  complex in which the Arp module is flexibly tethered to the core of SWR1. A kink in the Arp module  enables histone tail recognition of the Yaf9 subunit, while recognition of linker DNA might occur by  switching SWR1 into an open conformation. Histone tail recognition and tethering to linker DNA  may specify SWR1 activity at +1 nucleosomes.  INO80 and SWR1 share similar architectures, yet drive distinct functional outcomes. My thesis provides  a structural framework for understanding the underlying molecular mechanisms.
date: 2025
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/35220/1/Thesis_Jungblut2024.pdf
identifier: DOI:10.11588/heidok.00035220
identifier: urn:nbn:de:bsz:16-heidok-352205
identifier:   Jungblut, Anna  (2025) Structural and biochemical studies of ATP-dependent chromatin remodelers from the INO80/SWR1 family.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/35220/
rights: info:eu-repo/semantics/openAccess
rights: Please see front page of the work (Sorry, Dublin Core plugin does not recognise license id)
language: eng