Our long-term goals are to understand in molecular detail how transcription, replication, recombination, and repair take place within the context of chromatin. Our approaches include x-ray crystallography, spectroscopy, analytical ultracentrifugation, mass spectrometry (HDX-MS), atomic force microscopy, as well as biochemistry, molecular biology, whole-genome approaches and yeast genetics.
Nucleosome Structure and Dynamics
The nucleosome is the fundamental repeating unit of chromatin, consisting of two copies each of the four histone proteins (the histone octamer) around which 146 base pairs of DNA are wrapped in nearly two turns of a tight superhelix (Fig. 1). Nucleosomes are not static structures, rather, they exist in several interconvertible states under physiological conditions (Fig. 2). Similarly, the packing of nucleosomes in supramolecular complexes, previously thought to be highly ordered, can occur in a number of interchangeable arrangements. These structural transitions are key regulators of DNA accessibility, and thus influence all genome transactions.
Nucleosome structure and dynamics, as well as the arrangement of nucleosomes and intervening linker DNA in higher order structures, are affected by DNA sequence, by the incorporation of histone variants, or by the post-translational modification of histones (intrinsic modulators of chromatin structure). Extrinsic modulators include nucleosome-binding proteins, histone chaperones, chromatin assembly and disassembly factors, ATP-dependent chromatin remodelers, and the RNA polymerase itself. We are investigating how intrinsic and/or extrinsic modulators affect nucleosome thermodynamics, and the equilibria between different structural states (Fig. 2).
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 and assist in the various steps of nucleosome assembly and disassembly. We study the structure of several histone chaperone complexes with histones and nucleosome assembly intermediates, and have developed assays to study their mechanism in vitro and in vivo. We find that different histone chaperones promote different steps of nucleosome assembly and disassembly. Therefore, we have started to investigate synergies between histone chaperones, and between chaperones and ATP-dependent chromatin remodeling factors. 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.
These studies were supported in part by the National Institutes of Health.
As of January 15, 2016