In eukaryotic cells, genomic DNA is organized into small units called nucleosomes, each of which generally consists of a histone octamer and DNA wrapping around this histone core, so that the whole genome can be efficiently packed into a small nucleus. On the other hand, the presence of nucleosome remodelers ensures the genetic information still remains accessible to regulatory factors. Although it was known for a long time that these remodelers can weaken the interaction between the DNA and histones in an ATP-dependent manner to expose part of the DNA sequence, the underlying mechanisms have not been fully explored, especially for the CHD remodeler family.
In the project, we will assemble recombinant nucleosomes and will use them as a substrate to test the remodeling ability of CHD4 and also NuRD (Nucleosome Remodeling Deacetylase) complex, using single molecule FRET (fluorescence resonance energy transfer) experiments. The FRET experiment involves fluorescent labelling of one of the histones and also of the DNA, which allows the monitoring of the translocation of the nucleosome in real time.
Other experimental approaches, including measurement of the ATP hydrolysis rate of CHD4 during remodeling (using a fluorescent phosphate-sensing protein), and gel-based remodeling assays will also be carried out. Together with structural studies, these experiments will be used to delineate the biochemical mechanism of remodelling by CHD4-family chromatin remodelling proteins.