Characterisation of CHRAC14 and CHRAC16, the two Histone Fold Subunits of the Chromatin Accessibility Complex

In eukaryotic nuclei, the DNA double helix is wound up and
condensed into chromatin through the interaction with histones and
further proteins. Several factors regulate the chromatin structure,
allow unfolding or condensation of the chromatin fibre and permit
or restrict access to DNA. One prominent class of chromosomal
regulators is represented by ATP-dependent chromatin remodelling
complexes, which use the energy derived from ATPhydrolysis to break
or alter histone-DNA contacts. The ATP-utilising Chromatin Assembly
and Remodelling Factor (ACF) and the Chromatin Accessibility
Complex (CHRAC) are two closely related ATP-dependent chromatin
remodelling factors. ACF consists of the ATPase ISWI and ACF1, a
large protein that influences both the quality and efficiency of
ISWI activity. CHRAC contains ISWI and ACF1 as well, but in
addition the two small histone fold proteins CHRAC14 and CHRAC16.
In this work, the CHRAC14 and CHRAC16 subunits are characterised
both structurally and functionally. The generation of a bicistronic
expression plasmid allowed the expression and purification of
highly pure recombinant CHRAC14-CHRAC16 in stoichiometric amounts.
The crystal structure of the CHRAC14-CHRAC16 complex was solved at
a resolution of 2.4 Å and demonstrates that the two proteins
interact with each other via their histone fold motifs, thereby
closely resembling the structure of histones H2A-H2B and NFYB-NFYC,
the histone fold subunits of nuclear factor Y (NF-Y). Rat
monoclonal antibodies against CHRAC14 and CHRAC16 were raised and
characterised, but due to their poor affinity, they turned out to
be only of limited use for the analysis of the two proteins.
CHRAC14-CHRAC16 interact with the N-terminus of ACF1, including the
conserved WAC motif. They have a weak affinity for DNA, and studies
with CHRAC14-CHRAC16 deletion variants revealed that their
C-termini play important but distinct roles in DNA binding.
Finally, CHRAC14-CHRAC16 facilitate ACF-dependent nucleosome
mobilisation, and their ability to enhance ACF activity depends on
both the interaction with the ACF1 N-terminus and the dynamic
binding to DNA. In the light of profound similarities to the
effects of HMGB1 (high mobility group box protein 1) on nucleosome
sliding, these data imply that the CHRAC14-CHRAC16 subcomplex
operates as a ‘DNA chaperone’ and assists ACF1 and ISWI during
ATPdependent nucleosome remodelling by providing a transient DNA
binding surface. This work provides the basis for further
experiments to gain more insights into the mechanistic details of
CHRAC-dependent nucleosome remodelling and to explore the roles of
CHRAC in the living cell.