Molecular Characterization of the HIRA Histone Chaperone Complex from Chaetomium thermophilum

Abstract

The HIRA histone chaperone complex is an essential epigenetic regulator which deposits the histone variant H3.3 onto DNA to condense it into chromatin and to regulate the epigenetic landscape of the cell. Because the HIRA complex and H3.3 deposition is associated with areas of active transcription in the genome, HIRA dysregulation is associated with disordered development, aging, and maintenance of terminally differentiated cell types. The goal of our studies is to understand the molecular basis of H3.3‐specific histone deposition by the HIRA complex. Toward this goal, we recombinantly express orthologs of the four HIRA complex components‐ HIRA, UBN1, CABIN1, and ASF1a‐ from the thermophilic fungus Chaetomium thermophilum for biochemical and structural characterization. We show efficient recombinant expression of each subunit, determine complex stoichiometry using analytical ultracentrifugation, and begin structural analysis using a combination of X‐ray crystallography and cryo‐electron microscopy of the subunits alone, in complex, and with histones. Support or Funding Information Structural Biology and Molecular Biophysics Training Program, NIH 5‐T32‐GM‐008275‐29; Epigenetics of Aging and Age‐Associated Diseases P01, NIH 5P01AG031862‐12