Our long-term goals are to visualize the structure of the nucleosome and of chromatin higher-order assemblies and to understand in molecular detail how transcription, replication, recombination, and repair take place within the context of highly compacted chromatin. We are particularly interested in mechanistic, structural, and thermodynamic aspects of these fundamental questions. We are using multipronged approaches, including x-ray crystallography, small-angle x-ray scattering, fluorescence resonance energy transfer (FRET), and analytical ultracentrifugation, as well as atomic force microscopy, biochemistry, molecular biology, and yeast genetics, to investigate chromatin structure regulation.
The nucleosome is the elemental repeating unit in 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. Nucleosome structure and dynamics (and thus DNA accessibility) may be altered by DNA sequence, by the incorporation of histone variants or by the post-translational modification of histones (intrinsic modulators of chromatin structure). Extrinsic modulators are distinct from the nucleosome and include nucleosome-binding proteins and histone chaperones. Key extrinsic factors of interest in my lab are chromatin architectural proteins H1, MeCP2, and PARP-1; the viral protein LANA; and histone chaperones Nap1, Vps75, Scm3, and FACT.
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 interconvertible states under physiological conditions. We have developed quantitative assays to measure nucleosome stability and the thermodynamics of nucleosome assembly and disassembly, and we have used these assays to examine the hypothesis that intrinsic and/or extrinsic modulators affect nucleosome thermodynamics, as well as the equilibria between different structural states.
Post-translational Modifications and Histone Variants
We have determined the structures of nucleosomes carrying a variety of post-translational modifications and studied their folding behavior in an in vitro system. We have also investigated the structure and function of nucleosomes and chromatin containing histone variants H2A.Z, H2A.Bbd, macroH2A, and CenH3. The emerging picture is that post-translational modifications and histone variants have only minor effects on the crystal structures, but affect the interaction between nucleosomes, thus promoting or disfavoring the formation—and perhaps also the stability—of chromatin higher-order structure.
Complementary to x-ray crystallography, we use small-angle x-ray scattering (SAXS) and single-molecule methods to investigate nucleosome structure and dynamics in solution. These techniques allow us to realize functional aspects of nucleosome structure and dynamics hidden by crystal lattice constraints, including "breathing" of terminal DNA segments and alternative conformational states. SAXS reveals that DNA sequence and certain histone variants promote a more extended nucleosome conformation. We have also developed an assay to measure nucleosome stability and the thermodynamics of nucleosome assembly under physiological conditions. We are now engaged in a comprehensive evaluation of the effects of intrinsic modulators on chromatin structure and dynamics.
Interaction of Nucleosomes with Nuclear Proteins
Many abundant nuclear proteins bind nucleosomes in vivo with profound effects on chromatin architecture. The molecular and thermodynamic characterization of their interactions with the nucleosome has been hampered for decades by technical difficulties inherent to multicomponent, high-affinity, low-specificity systems. This has severely limited our understanding of their biological function. We use a combination of novel approaches to investigate the thermodynamics of the interactions, as well as the structural changes in the nucleosome and interacting proteins. Several of the factors under investigation are of clinical importance, either as targets for anticancer drugs or because mutations in the corresponding gene are correlated with certain disease states.
For example, by studying how Kaposi's sarcoma herpesvirus LANA protein enables the viral genome to tether onto chromosomes so that virus is not lost from cells, we found that LANA engages histones H2A and H2B to dock onto chromosomes by binding to the nucleosomal surface. Our experiments have also provided unanticipated insight into the mechanisms by which the nucleosomal surface contributes to chromatin higher-order structure formation. We have found that subtle changes in the charge and shape of the nucleosomal surface profoundly alter the propensity of nucleosomal arrays to form condensed chromatin. Some post-translational modifications of the histones have similar effects.
Eukaryotic chromatin is highly dynamic. Histone chaperones are a structurally diverse class of proteins that bind histones and assist in nucleosome assembly and disassembly. We investigate the structure of histone chaperones and have developed assays to study the mechanisms by which they assemble and disassemble nucleosomes in vitro and in vivo.
For example, thermodynamic and in vivo yeast studies reveal that a member of the nucleosome assembly protein family, yeast Nap1, assembles chromatin by disfavoring noncanonical histone-DNA interactions, perhaps by stabilizing a folded state. This activity requires the acidic, disordered C-terminal tail of Nap1. We are now studying several Nap1 paralogs as well as other, unrelated histone chaperones from mammals to elucidate functional differences.
These studies were supported in part by the National Institutes of Health and the International Rett Syndrome Foundation.
As of May 30, 2012