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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 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 and structural aspects of these questions. We are using multipronged approaches, including x-ray crystallography, small-angle x-ray scattering, fluorescence resonance energy transfer, 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. Although we now have detailed knowledge of the structure of nucleosomes from a variety of species and from examples of mutants and histone variants, molecular details of the multiple levels of chromatin higher-order structure have remained elusive. Nor do we know how global and specific regulators (for example, linker histone H1 or transcription factors) access DNA in the various structural contexts. Finally, we are investigating the fundamental question of how chromatin structure "switches" between varying degrees of complexity.
Interaction of Nucleosomes with Transcription Factors and Other Cellular 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, most notably x-ray crystallography and fluorescence resonance energy transfer, 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 protein LANA (latency-associated nuclear antigen) 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 via a tight hairpin motif. This study (which is the result of an ongoing collaboration with Kenneth Kaye [Harvard Medical School]) unequivocally demonstrates how a highly structured nucleosomal surface acts as an interaction platform for molecular recognition. These 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. Structural and biochemical investigations of nucleosomes bearing post-translational modifications in the structured regions of the histones are ongoing.
Nucleosome Assembly and Histone Exchange
Eukaryotic chromatin is highly dynamic and turns over rapidly in the absence of DNA replication and transcription. Acidic histone chaperones such as nucleosome assembly protein 1 (NAP-1) are implicated in this process. Homologs of NAP-1 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. Some family members are essential in mammals because of a still uncharacterized role in neuronal development. 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.
In vitro, NAP-1 reversibly removes and replaces H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange. Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position. Our results suggest novel roles for NAP-1 (and perhaps for other histone chaperones) in mediating chromatin fluidity by incorporating histone variants and assisting nucleosome sliding.
The crystal structure of yeast NAP-1 is the first for a NAP- family member and reveals a novel fold with implications for histone transport and exchange. The structure suggests several hypotheses regarding the function of the NAP-1 protein family that are now being tested by a variety of methods, including small-angle x-ray scattering, FRET, and in vivo studies. (These studies are supported by the National Institutes of Health.)
Histone Variants
The replacement of canonical histones with histone variants has emerged as 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. Histone variants are distinct nonallelic forms of conventional, major-type histones that form the bulk of nucleosomes during replication and whose synthesis is tightly coupled to S phase. They are found in most eukaryotic organisms and are expressed in all tissue types.
We have made considerable progress in elucidating the structure and function of nucleosomes and chromatin containing the histone variants H2A.Z, H2A.Bbd, and macroH2A and the centromeric H3 histone variant CENP-A. 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 by the Human Frontier Science Program, the March of Dimes Birth Defects Foundation, and the National Institutes of Health.)
Last updated: September 15, 2008
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