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

Hartlepp, Klaus F. (2006): Characterisation of CHRAC14 and CHRAC16, the two Histone Fold Subunits of the Chromatin Accessibility Complex. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

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.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: chromatin, histone, ATPase, remodelling, CHRAC
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 22. März 2006
• 1. Bericht­erstatter:in: Becker, Peter