Eukaryotic genomes are organized into active and inactive domains referred to euchromatin and heterochromatin. This functional organization plays an important role in chromosome segregation, telomere maintenance and genome stability. A key component of heterochromatin are the HP-1 family of proteins, which bind to a histone 3 lysine-9 methyl mark and act as a platform for diverse regulators. A biochemical activity thought to be important for the spread of heterochromatin is the ability of HP-1 proteins to polymerize. Recent work on the fission yeast HP-1 protein, Swi6, reveals that polymerization is regulated by autoinhibition. A critical and poorly understood question is the extent of the conformational transition between closed, inactive and open, active forms of Swi6 that drive heterochromatin spread. This gap in knowledge derives from the fact that HP-1 proteins and their complexes with nucleosomes are conformationally dynamic in solution and difficult to crystallize. Here we will define the structural dynamics of the Swi6-chromatin complex and link the structural states to function.
In Aim 1 we will determine the structure of the autoinhibited form of Swi6 using an integrated modeling approach that employs restraints NMR spectroscopy and small-angle x-ray scattering in solution (SAXS). The biological significance of the structural models will be tested in gene silencing assays in the fission yeast S. pombe.
In Aim 2, we will determine the degree of conformational rearrangements of chromatin when Swi6 engages the nucleosome to form a spreading competent state. The structure and dynamics of the nucleosome-Swi6 complex will be interrogated by a combination of biophysical methods, such as Methyl-TROSY NMR and HD-exchange mass-spectrometry (MS), that can provide residue specific structural information in solution. Cross-linking mass-spectrometry in conjunction with cryoEM will be employed to obtain models of the spreading competent form if Swi6 bound to nucleosomes. This approach will shed insights into conformational control of heterochromatin formation by Swi6 in fission yeast and provide a conceptual foundation for how heterochromatin is regulated in human cells.
Heterochromatin and its key component HP1 proteins are essential for gene silencing and genome stability. When dysregulated, pathologies such as blood cancers can arise. The project combines NMR, biochemical and in vivo assays to study the HP1-chromatin architecture in the model organism fission yeast. The overall goal is to structurally dissect how HP1 proteins influence chromatin structure in order to advance our understanding of heterochromatin formation and HP1 function in both in normal and cancer cells.
