Periodic Reporting for period 3 - INO3D (Mechanism of ATP Dependent Chromatin Modelling and Editing by INO80 Remodellers)
Reporting period: 2022-10-01 to 2024-03-31
Our chromosomal DNA is packaged and organized in the cells nuclei in the form of chromatin, the collective assembly of nuclear DNA and associated proteins. The most basic unit of chromatin are nucleosomes, a protein – DNA complex, where 146 basepairs of genomic DNA are wrapped ~1.7 times around a proteinaceous core of eight histone proteins. Nucleosomal packaging compacts and protects the DNA from nucleases, regulate access of DNA binding proteins to DNA and are carriers of epigenetic information. Besides the canonical core histones H2A,H2B,H3 and H4, each present in two copies in nucleosomes, eukaryotic cells have a variety of histone variants that can replace their canonical counterpart in the histone octamer. The position, epigenetic state and histone composition nucleosomes are critical regulators of gene expression, chromatin state and DNA repair and are central players in defining cellular identity, differentiation and formation of aberrant cells.
To define, alter and dynamically regulate the position and composition of nucleosomes, eukaryotic cells possess a variety of ATP dependent molecular machines, denoted chromatin remodeler. The remodeler INO80 is a 15-subunit protein complex with a molecular mass exceeding 1 megadalton. Its structure, mechanism and function is studied in this proposal in order to understand how INO80 shapes chromatin and helps regulate gene expression and DNA repair. INO80 emerges as the remodeler that is responsible for organizing nucleosome positions around promoter DNA and can both de novo position nucleosomes as well as generate regularly spaced nucleosomal arrays. We use a combination of cryo-electron microscopy the structures and mechanisms of human and fungal INO80 to unravel their mode of action at high resolution. Our insights will not only help understand fundamental mechanisms of shaping nuclear chromatin; our results will also aid the development of specific drugs aimed at inhibiting INO80 for new routes of therapeutic intervention, since altered chromatin due to mis-regulation of chromatin remodelers is an emerging feature of many cancer cells.
The overall objectives are:
1) provide structural framework for architecture of INO80 remodellers
2) reveal the ATP dependent mechanism how INO80 moves nucleosomes
3) investigate how INO80 reads out genomic information to position nucleosomes at promoter regions
To define, alter and dynamically regulate the position and composition of nucleosomes, eukaryotic cells possess a variety of ATP dependent molecular machines, denoted chromatin remodeler. The remodeler INO80 is a 15-subunit protein complex with a molecular mass exceeding 1 megadalton. Its structure, mechanism and function is studied in this proposal in order to understand how INO80 shapes chromatin and helps regulate gene expression and DNA repair. INO80 emerges as the remodeler that is responsible for organizing nucleosome positions around promoter DNA and can both de novo position nucleosomes as well as generate regularly spaced nucleosomal arrays. We use a combination of cryo-electron microscopy the structures and mechanisms of human and fungal INO80 to unravel their mode of action at high resolution. Our insights will not only help understand fundamental mechanisms of shaping nuclear chromatin; our results will also aid the development of specific drugs aimed at inhibiting INO80 for new routes of therapeutic intervention, since altered chromatin due to mis-regulation of chromatin remodelers is an emerging feature of many cancer cells.
The overall objectives are:
1) provide structural framework for architecture of INO80 remodellers
2) reveal the ATP dependent mechanism how INO80 moves nucleosomes
3) investigate how INO80 reads out genomic information to position nucleosomes at promoter regions
INO80 reads out DNA shape features: One of the key questions in the field of remodelers is how they are regulated. The structural mechanism derived in my laboratory suggests that INO80 pumps DNA into the nucleosome core, whereby a regulatory module contacts entry DNA. The structure of INO80 raised the question whether entry DNA features or sequences could regulate INO80. For instance, particular DNA features present in promoter DNA might inhibit further sliding of nucleosomes into the nucleosome-free region and by this mechanism help place the +1 and -1 nucleosomes. We setup a recombinant system for INO80 that enabled us to not only study its architecture by cryo-EM and chemical crosslinking mass spectrometry, but we could introduce point mutations and assemble partial complexes for structure-function analysis and biochemical studies.
In collaboration with colleagues (Ha laboratory), we investigated whether INO80 is biochemically modulated by DNA bendability. It turned out that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibited nucleosome sliding into the linker by INO80. The mechanism can explain how INO80 might organise ab initio promoter chromatin and flanking nucleosomal arrays in the absence of other factors.
(Basu et al, Nature 2021, doi: 10.1038/s41586-020-03052-3)
We also investigated which elements of INO80 are critical for nucleosome positioning and whether there are correlations between INO80's nucleosome positioning activity and DNA sequence in a yeast genome wide scale. Here, we engaged in a collaboration with the Korber lab at LMU, who pioneered genome wide chromatin reconstitution assays using purified factors and DNA. The recombinant IN080 has the capability, together with purified DNA and recombinant histones, to assemble proper promoter nucleosomes and generate flanking arrays. In this study, we also identified specific ruler elements in INO80, which are critical for the regular spacing of nucleosomes in arrays. A particular structural module containing nuclear actin and actin related proteins Arp4 and Arp8 of IN080 is an important regulatory element in this mechanism.
(Oberbeckmann, Niebauer et al., doi: https://doi.org/10.1101/2020.02.28.969618(opens in new window))
In a parallel study employing recombinant INO80 and the chromatin reconstitution/shaping assays we addressed protein modules as well as DNA shape features that are enriched in INO80 positioned nucleosomes. In this study, we established a first mechanism how INO80 processes and integrates genomic information to organize the first level of chromatin.
Oberbeckmann et al, doi: https://doi.org/10.1101/2020.11.03.366690(opens in new window)
DNA bending and ATPase cycle by SNF2 ATPases :A central goals of the project is to understand the basis of ATP driven alterations to DNA structure to govern remodelling reactions. The SNF2 ATPase Modifier of Transcription 1 (Mot1), regulates gene experssion by moving the TATA box-binding protein (TBP) from promoter DNA. To understand how Mot1 does this, we use cryo-EM to observe different stages of Mot1 at work, which allowed us to derive a "molecular video" of how Mot1 displaces TBP. The results derive from Mot1 revealed a new principle for remodelling, showed interesting parallels to certain SF2 family immune sensors and help us to understand and study the more complex nucleosome remodellers as well (Woike et al: Nature Struct & Mol Biol 10.1038/s41594-023-00966-0)
Remodeling of non-canonical nucleosomes: Gene transcription is linked to formation of non-canonical nucleosomes, in particular hexasomes contain six instead of the usual eight histones. In this paper, our collaboration partners and us describe how the INO80 complex binds to hexasomes through spin rotation that is markedly different from its interaction with nucleosomes. DNA, but not histone interactions appear to dictate nucleosome recognition. (Zhang et al. Science 2023 doi: 10.1126/science.adf62)
In collaboration with colleagues (Ha laboratory), we investigated whether INO80 is biochemically modulated by DNA bendability. It turned out that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibited nucleosome sliding into the linker by INO80. The mechanism can explain how INO80 might organise ab initio promoter chromatin and flanking nucleosomal arrays in the absence of other factors.
(Basu et al, Nature 2021, doi: 10.1038/s41586-020-03052-3)
We also investigated which elements of INO80 are critical for nucleosome positioning and whether there are correlations between INO80's nucleosome positioning activity and DNA sequence in a yeast genome wide scale. Here, we engaged in a collaboration with the Korber lab at LMU, who pioneered genome wide chromatin reconstitution assays using purified factors and DNA. The recombinant IN080 has the capability, together with purified DNA and recombinant histones, to assemble proper promoter nucleosomes and generate flanking arrays. In this study, we also identified specific ruler elements in INO80, which are critical for the regular spacing of nucleosomes in arrays. A particular structural module containing nuclear actin and actin related proteins Arp4 and Arp8 of IN080 is an important regulatory element in this mechanism.
(Oberbeckmann, Niebauer et al., doi: https://doi.org/10.1101/2020.02.28.969618(opens in new window))
In a parallel study employing recombinant INO80 and the chromatin reconstitution/shaping assays we addressed protein modules as well as DNA shape features that are enriched in INO80 positioned nucleosomes. In this study, we established a first mechanism how INO80 processes and integrates genomic information to organize the first level of chromatin.
Oberbeckmann et al, doi: https://doi.org/10.1101/2020.11.03.366690(opens in new window)
DNA bending and ATPase cycle by SNF2 ATPases :A central goals of the project is to understand the basis of ATP driven alterations to DNA structure to govern remodelling reactions. The SNF2 ATPase Modifier of Transcription 1 (Mot1), regulates gene experssion by moving the TATA box-binding protein (TBP) from promoter DNA. To understand how Mot1 does this, we use cryo-EM to observe different stages of Mot1 at work, which allowed us to derive a "molecular video" of how Mot1 displaces TBP. The results derive from Mot1 revealed a new principle for remodelling, showed interesting parallels to certain SF2 family immune sensors and help us to understand and study the more complex nucleosome remodellers as well (Woike et al: Nature Struct & Mol Biol 10.1038/s41594-023-00966-0)
Remodeling of non-canonical nucleosomes: Gene transcription is linked to formation of non-canonical nucleosomes, in particular hexasomes contain six instead of the usual eight histones. In this paper, our collaboration partners and us describe how the INO80 complex binds to hexasomes through spin rotation that is markedly different from its interaction with nucleosomes. DNA, but not histone interactions appear to dictate nucleosome recognition. (Zhang et al. Science 2023 doi: 10.1126/science.adf62)
NO80 chromatin remodelers are highly complex molecular machines, consisting of a least 15 subunits in a >1 Megadalton complex. Due to their architectural complexity, flexibile and modular structure, as well as their complex nucleosomal substrates, remodelers such as IN080 are a formidable challenge for structural biology as well as a structure-function analyses. Techniques developed in this project with respect to structural analyses and the biochemistry of recombinant remodelers enable us to reveal how these molecular machines read and integrate genomic information to shape chromatin. A more detailed and resolved view of functional states will not only help understand one of the fundamental aspects of genome biology but also facilitate development of epigenetic drugs aimed at modulating remodeler activity.