Structural basis for histone H3 acetylation by Rtt109 in complex with histone chaperones Asf1 and Vps75

Abstract

In eukaryotes, genetic material is stored as chromatin, a DNA-protein complex whose structure is tightly regulated. Histones are the main protein components of chromatin and their post-translational modifications (PTMs) influence several cellular processes, from gene expression to epigenetic regulation and inheritance. Histone acetyltransferases carry out the acetylation of histones, an abundant modification, the function of which is determined by the position of the modified residue. Histone chaperones can influence the specificity of the histone acetyltransferases, however, the underlying mechanism of this process is not fully understood. Rtt109 is a fungal histone acetyltransferase which is essential for the acetylation of newly synthesized histone H3. Two distinct histone chaperones, Asf1 and Vps75, have been reported to alter its activity and specificity: while Asf1 is necessary for H3 K56 acetylation, Vps75 promotes acetylation of H3 K9, K23 and K27, which are located in a long disordered N-terminal tail of H3. Despite the availability of structures of Rtt109 in complex with Vps75, the mechanism of regulation of Rtt109 activity by Asf1 and Vps75 remains elusive. In order to understand how Asf1 and Vps75 stimulate the Rtt109 activity towards specific substrates, I reconstituted in vitro the complex containing Rtt109, histones H3:H4 and both chaperones. With multi angle light scattering and nuclear magnetic resonance (NMR), I could show that the Vps75 dimer assembles a non-symmetric complex with one copy of Rtt109 and Asf1-bound histones. Using an integrative structural biology approach combining distance restraint information from NMR and low-resolution shape information from small-angle neutron scattering (SANS) data, I could obtain a structural model of this complex. The structure revealed that the chaperones form a bagel-shaped complex with Rtt109 and the histones, bringing the enzyme and the substrate together and positioning H3 K56 next to the Rtt109 active center. A combination of NMR data with biochemical experiments and computational studies, revealed that the flexible H3 tail is chaperoned by Asf1 and is guided towards the catalytic pocket of Rtt109 by both folded and unfolded structural elements of Vps75. These results, taken together with existing literature and further mutational studies, allowed me to propose a mechanism by which the histone chaperones promote acetylation of the disordered H3 N-terminal tail.