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Structure and Dynamics of Chromatin
Summary: Karolin Luger is investigating the structural biology of chromatin. Luger hopes to refine the overall view of chromatin's architecture by understanding how the nucleosome interfaces with the cellular machinery through sequence variations and post-translational modifications in its own proteins or interactions with outside molecules.
Our long-term goal is to investigate the structural properties of the nucleosome and of chromatin higher-order structures, 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 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, conventional biochemistry, molecular biology, and yeast genetics, to investigate fundamental questions of chromatin structure control.
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. Nucleosomal DNA is highly distorted and partially occluded from the solvent because of its tight interaction with the histone octamer. Thus, nucleosome architecture greatly affects the accessibility of nucleosomal DNA for global and specific regulators. Linker histones and other nonhistone proteins promote or stabilize the folding of nucleosomal arrays into superstructures of increasing complexity and largely unknown architecture. We are investigating the fundamental question of how chromatin "switches" between varying levels of packaging through the concerted action of post-translational modifications of histones and histone variants, histone chaperones, and chromatin architectural proteins.
Interaction of Nucleosomes with Transcription Factors and Other Nuclear Proteins
Because the vast majority of eukaryotic DNA exists in complex with histones, many sequence-specific DNA-binding proteins must recognize their binding site in the structural context of the nucleosome. Other regulatory factors may recognize the specific topography of nucleosomal DNA in a sequence-independent manner, but possibly with contributions from the histone octamer. We use a combination of methods to investigate the structural determinants and the structural changes that are inflicted on the nucleosome and the interacting protein upon binding to nucleosomal DNA. We also study the effects on higher-order structure with analytical ultracentrifugation and atomic force microscopy. 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.
Nucleosome Assembly and Histone Exchange
Eukaryotic chromatin is highly dynamic. Acidic histone chaperones such as nucleosome assembly protein 1 (NAP1) are implicated in histone turnover during transcription and replication. Homologs of NAP1 have been identified in all eukaryotes. Although initially identified as histone chaperones and chromatin assembly factors, these homologs have additional functions—including roles in tissue-specific transcription regulation, apoptosis, histone shuttling, and cell cycle regulation—that extend beyond those of a simple chaperone and assembly factor. Several have been characterized as oncoproteins, and many have been found in complex with enzymes that post-translationally modify histones or are implicated in ATP-dependent chromatin remodeling.
The crystal structure of yeast NAP1 reveals a novel fold with implications for histone transport and exchange. The structure suggests several hypotheses regarding the function of the NAP1 protein family that are now being tested by a variety of methods, including small-angle x-ray scattering, FRET, and in vivo studies. We are also investigating the intricate interplay between histone chaperones and histone acetyl transferases.
Post-translational Modifications of Histones and Their Effects on Chromatin Structure and Stability
We have developed an assay to measure the thermodynamic stability of the nucleosome and of chromatin fibers under physiological conditions. We are now testing the hypothesis that histone modifications affect factor-mediated chromatin assembly/disassembly by altering the affinity of histones for the assembly factor, by altering nucleosome stability (that is, histone-histone and histone-DNA interactions), or by a combination of the two. Similarly, the post-translational modification of assembly factors also affects this delicate equilibrium between assembly and disassembly of chromatin. Our studies have already resulted in surprising insights into the mechanisms of assembly factors and their roles in nucleosome positioning.
We have determined the structures of nucleosomes carrying a variety of post-translational modifications, and we have studied their folding behavior in an in vitro system. The emerging picture is that post-translational modifications primarily affect the interaction of histone tails with the nucleosome surface, thus promoting or disfavoring the formation—and perhaps also the stability—of chromatin higher-order structure.
Histone Variants
The replacement of canonical histones with histone variants is an important pathway to alter the biochemical makeup of chromatin locally, with the potential to exert considerable influence on the structure and function of chromatin. We have made considerable progress in elucidating the structure and function of nucleosomes and chromatin containing several histone variants. One emerging theme arising from our studies is that the overall structure of the nucleosome is by and large maintained upon incorporation of histone variants, but subtle differences in the surface and stability of the nucleosome or in how the ends of the DNA are organized by the histone octamer differ between major-type and variant histones. For example, in H2A.Z, surface changes result in changes in higher-order structure formation that are ultimately important for development. In other instances (e.g., macroH2A), extranucleosomal nonhistone domains change local chromatin structure by recruiting histone-modifying enzymes and other nonhistone chromatin-associated proteins.
These studies were supported in part by the Human Frontier Science Program, the March of Dimes Birth Defects Foundation, the National Institutes of Health, and the International Rett Syndrome Foundation.
Last updated May 15, 2009
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