In order to accommodate almost 2 meters of the genomic DNA into the confines of the cell’s nucleus with a diameter of only few microns, genomic DNA has to be tightly packaged and highly organized into chromatin structure. Simultaneously, chromatin must remain dynamic to allow access of its genetic information for gene expression, DNA
repair, and DNA replication. It is clear that nucleosomal impedance has to be dealt with when it comes to transcriptional activity and DNA binding sites. The controlled access to the genomic material is gained through local or global relaxation of chromatin structure
and/or changes in its dynamics. One of the major regulators responsible for chromatin modulation are histone Post-Translational Modifications (PTMs), where regulation occurs either by directly altering DNA-histone interactions or by altering the binding properties of modification dependent chromatin remodellers.
Here we utilize biophysical methods of Single-Molecule (SM) Magnetic Tweezers (MT), SM Atomic Force Microscopy (AFM), restriction enzyme kinetics, and Fluorescence Resonance Energy Transfer (FRET) analysis to investigate the impact of PTMs, positioned throughout the distinct structured regions of the nucleosome, on nucleosome stability, structure, and dynamics. Namely, acetylation of histone H3 at lysine 56 in the entry/exit region (H3 (K56ac)), double acetylation of histone H4 at lysines 77 and 79 within the loss of rDNA Silencing (LRS) domain (H4 (K77ac, K79ac)), and double acetylation of histone H3 at lysines 115 and 122 in the nucleosome dyad (H3 (K115ac, K122ac)). We find that modifications in separate structural regions of the nucleosome control distinct dynamic events: (1) acetylations of lysines when close to the dyad increase nucleosome disassociation and thus control disassembly, and (2) acetylations of lysines near the entry/exit region regulate DNA unwrapping and transcription factor binding. These results
show that dyad region controls for disassembly, while the entry-exit region controls partial unwrapping.
Phosphorylation of histone H3 at threonine 118 (H3 (T118ph)), a different type of histone PTM located near the nucleosome dyad, alters nucleosome structure by promoting ’dimerization’. SM particle analysis from AFM micrographs and also mass content analysis from Electrohoretic Mobility-Shift Assay (EMSA) reveal formation of
hexadecasomes containing one or two DNA molecules. We also show that nucleosomes containing phosphorylated H3 (T118) promote higher-order chromatin compaction when integrated into chromatin fibers.
We further provide mechanical and biological studies examining the role of Lens Epithelium-Derived Growth Factor (LEDGF) in catalyzing retroviral integration with Integrase (IN) proteins from Prototype Foamy Virus (PFV) and Human Immunodeficiency Virus (HIV). Although the current state of the study is preliminary, our results point
towards the conclusion that LEDGF has a multi-functional role during the integration step of retroviral replication cycle: (1) it helps stabilize the IN - Complementary DNA (cDNA) assembly, (2) it serves as molecular tether helping with targeting the integration sites, and (3) it catalyzes the strand exchange by causing DNA to bend.