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 the 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(se abrirá en una nueva ventana))
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(se abrirá en una nueva ventana)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).
We could also show that the human INO80 core complex also has the ability to mobilize hexasomes. We observed again a spin rotation of INO80 on the hexasome, but also found spin rotation capacities even on nucleosomes, depending on the degree of unwrapping. Thus INO80, at least in the human system, has a topological mode of nucleosome binding. The defining features for recognition are the location where entry DNA enters the nucleosome core. Depending on the degree of unwrapping, INO80 can flexibly adapt to mobilise different nucleosome variants. (Aggarwal et al., BioRxiv doi:
https://doi.org/10.1101/2025.08.25.671740(se abrirá en una nueva ventana)).
In summary, our work uncovered fundamental mechanisms by which the INO80 chromatin remodeler senses and responds to DNA and nucleosomal shape to organize chromatin architecture.
