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Introduction to our Research

 

   

The long-term goal of our research is to investigate the structural properties of the nucleosome and of chromatin higher order structures, and to understand in molecular detail the fundamental question of how transcription, replication, recombination, and repair take place within the context of highly compacted chromatin. We are using multi-pronged approaches including x-ray crystallography, small angle  x-ray scattering, fluorescence resonance energy transfer and anisotropy, analytical ultracentrifugation, atomic force microscopy, as well as biochemistry and molecular biology, and yest genetics, to elucidate structural and mechanistic aspects of these processes.

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 and chromatin structure (and thus DNA accessibility) may be altered by DNA sequence incorporation of histone variants, by the posttransitional modification of histone tails (intrinsic modulators of chromatin structure).  Extrinsic modulators are distinct from the nucleosome and include nucleosome-binding proteins, histone-modifying enzymes and histone chaperones. Key proteins of interest are chromatin architectural proteins H1, MeCP2 and PARP-1, viral protein LANA, and chaperones Nap1, Vps75, Scm3 and FACT. We characterize their activities using approaches ranging from crystallography and small-angle x-ray scattering to fluorescence resonance energy transfer and in vivo yeast genetic assays. Our studies indicate that intrinsic and extrinsic modulators of chromatin dynamics are functionally connected, and this concept is currently the focus of our investigations.

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 the hypothesis that intrinsic and/or extrinsic modulators affect nucleosome thermodynamics, as well as the equilibria between different structural states.

Selected Reviews (opens in a new window)

Image of Nucleosome in four orientations

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Individual areas of research:
  1. Intrinsic modulators of chromatin structure......................................................................
    • Histone variants
    • Posttransitional modifications of histones
    • DNA Sequence

  2. Extrinsic modulators of chromatin structure.....................................................................
    • Histone chaperones
    • Nucleosome binding proteins

  3. Assay Development............................................................................................................
     
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