Computational, biochemical and NMR-driven structural studies on nucleosomal DNA

Abstract

Chromatin provides the required structural compaction of DNA to fit in the nucleus and plays crucial roles in controlling cell fate and protecting genome integrity. Chromatin function ultimately depends on the structural and dynamical properties of the nucleosome, its fundamental repeating unit. The nucleosome acts as a gate keeper by regulating the binding of proteins that carry out DNA-templated processes like transcription, replication and repair. The studies described in this thesis aimed to contribute to our basic understanding of chromatin structure and function, involving chromatin remodeling and pioneer factor binding in particular. We specifically focused on the contribution of the nucleosomal DNA in these processes. In Chapter 2, we used computational modeling to study newly discovered archaeal histones that were only identified at the sequence level. We analyzed their propensity to form a continuous histone-DNA complex, or hypernucleosome, analogous to what was recently reported for archaeal histones HMfA and HMfB. As eukaryotes are classified as being part of the archaeal evolutionary branch, this complex might represent the evolutionary ancestor of chromatin organization into nucleosomes in eukaryotes. In Chapter 3, we introduced the method of ramified rolling circle amplification (rRCA) for the efficient large-scale production of a genomic nucleosomal DNA sequence from the human LIN28B locus. We showed that rRCA can be used to produce milligram amounts of nucleosomal DNA in an isothermal, one-pot reaction overnight that is easily scalable. Importantly, rRCA offers flexibility in choice of sequence. This may facilitate future studies on native, genomic nucleosomes, which might have structural and dynamic properties different from nucleosomes reconstituted on the common artificial strong-positioning sequences. Chromatin remodelers are ATP-dependent molecular motors that are essential for proper control of gene expression and protection of genome integrity. In Chapter 4, we applied Fluorescence Anisotropy (FA) and Nuclear Magnetic Resonance (NMR) to identify and characterize conformational changes of the ISWI remodeler upon interaction with its activating nucleosomal epitopes. To prevent spurious consumption of ATP, these lobes are locked in an inactive conformation in the free state of the remodeler, and this auto-inhibition is only relieved by interaction with the substrate, in particular the nucleosomal DNA and histone H4 tail. We showed that the ISWI ATPase domain is only able to bind the H4 tail in presence of dsDNA and that this ISWI-DNA- H4 complex requires presence of ATP-mimic ADP-BeFx for stability. We proposed that the first step of autoinhibition release is the interaction of ISWI with dsDNA. As the nucleosomal DNA plays a crucial role in nucleosome interactions with chromatin remodelers, pioneer transcription factors and other proteins, in Chapter 5, we explored the possibility of directly observing the nucleosomal DNA in NMR studies. We developed a method for segmental one-strand isotope-labeling of six thymine residues in the nucleosomal DNA-601 sequence, which creates the opportunity of using MeTROSY NMR experiments to improve signal to noise ratios in large complexes like the nucleosome.