An integrated view of the essential eukaryotic chaperone FACT in complex with histones H2A-H2B

Summary: Structure of the FACT chaperone domain in complex with
histones H2A-H2B, and a model for FACT-mediated nucleosome
reorganization Nucleosomes are the smalles unit of chromatin: two
coils of DNA are wrapped around a histone octamer core, which
neutralizes its charge and `packs' the lengthy molecule.
Nucleosomes confer a barrier to processes that require access to
the eukaryotic genome such as transcription, DNA replication and
repair. A variety of nucleosome remodeling machines and histone
chaperones facilitate nucleosome dynamics by depositing or evicting
histones and unwrapping the DNA. The eukaryotic FACT complex
(composed of the subunits Spt16 and Pob3) is an essential and
highly conserved chaperone. It assists the progression of DNA and
RNA polymerases, for example by facilitating transcriptional
initiation and elongation. Further, it promotes the genome-wide
integrity of chromatin structure, including the suppression of
cryptic transcription. Genetic and biochemical assays have shown
that FACT's chaperone activity is crucially mediated by a direct
interaction with histones H2A-H2B. However, the structural basis
for how H2A-H2B are recognized and how this integrates with FACT’s
other functions, including the recognition of histones H3-H4 and of
other nuclear factors, is unknown. In my PhD research project, I
was able to reveal the structure of the yeast chaperone domain in
complex with the H2A-H2B heterodimer and show that the Spt16M
module in FACT’s Spt16 subunit establishes the evolutionarily
conserved H2A-H2B binding and chaperoning function. The structure
shows how an alpha-helical `U-turn' motif in Spt16M interacts with
the alpha-1-helix of H2B. The U-turn motif scaffolds onto a tandem
pleckstrin-homology-like (PHL) module, which is structurally and
functionally related to the H3-H4 chaperone Rtt106 and the Pob3M
domain of FACT. Biochemical and in vivo assays validate the crystal
structure and dissect the contribution of histone tails and H3-H4
toward FACT binding. My results show that Spt16M makes multiple
interactions with histones, which I suggest allow the module to
gradually invade the nucleosome and ultimately block the strongest
interaction surface of H2B with nucleosomal DNA by binding the H2B
alpha-1-helix. Together, these multiple contact points establish an
extended surface that could reorganize the first 30 base-pairs of
nucleosomal histone–DNA contacts. Further, I report a brief
biochemical analysis of FACT’s heterodimerization domain. Its PHL
fold indicates shared evolutionary origin with the H3-H4-binding
Spt16M, Pob3M and Rtt106 tandem PHL modules. However, the
Spt16D–Pob3N heterodimer does not bind histones, rather it connects
FACT to replicative DNA polymerases. The snapshots of FACT’s
engagement with H2A-H2B and structure-function analysis of all its
domains lay the foundation for the systematic analysis of FACT’s
vital chaperoning functions and how the complex promotes the
activity of enzymes that require nucleosome reorganization.