Nucleosome structure and dynamics
Our cumulative analysis of chromatin structure and thermodynamics challenges the long-held notion that in vivo nucleosomes exclusively adopt the conformation observed by x-ray crystallography. Nucleosome conformation and stability are major determinants of DNA accessibility, which ultimately impacts transcription, replication and repair. We have compelling evidence that nucleosomes exist in several inter-convertible states under physiological conditions. In addition, we have developed quantitative assays to measure nucleosome stability, and have used these assays to examine what affects nucleosome thermodynamics, as well as the equilibria between different structural states.
Interaction of Chromatin with Nuclear Proteins
Many nuclear proteins bind nucleosomes in vivo, with profound effects on chromatin architecture, leading to varied biological outcomes. We use a combination of approaches to investigate the thermodynamics and architecture of such complexes, and to monitor the structural changes in chromatin and interacting proteins. Several of the factors under investigation are of clinical importance, either as targets for anticancer drugs (e.g. Poly-(ADP-ribose) polymerase (PARP-1, 2, and 3), or because mutations in the corresponding gene are correlated with disease states (e.g. MeCP2).
Histone Chaperones and Nucleosome Assembly Factors
Histone chaperones are structurally diverse proteins that bind histones with high affinity and assist in the various steps of nucleosome assembly and disassembly. We study the structure of histone chaperone complexes with histones and nucleosome assembly intermediates, and have developed assays to dissect their mechanism in vitro and in vivo. We find that different histone chaperones promote different steps of nucleosome assembly and disassembly. Therefore, we are investigating synergies between histone chaperones, and between chaperones and ATP-dependent chromatin remodeling factors. The function of histone chaperones in transcription is also analyzed (see below). Mechanisms by which various histone chaperones are targeted to their respective sites of action are also investigated.
Transcription in a chromatin context
Nucleosomes present formidable barriers for the transcription machinery. We are using a recombinant in vitro system to study how RNA polymerase II navigates through nucleosomes, and determine the role of various nuclear factors in displacing nucleosomes in front of the polymerase, and in reassembling them in its wake (Fig. 3). This system also allows us to dissect the role of epigenetic modifications on RNA polymerase progression.