Summary The transcriptional coactivator p300 is a histone lysine acetyltransferase that is typically recruited to transcriptional enhancers and regulates gene expression by acetylating chromatin. Here we show that p300 activation directly depends on the activation and oligomerisation status of transcription factor (TF) ligands. Using two model TFs, IRF3 and STAT1, we demonstrate that TF dimerization enables trans-autoacetylation of p300 in a highly conserved and intrinsically disordered autoinhibitory lysine-rich loop (AIL), resulting in HAT activation. We describe a p300 crystal structure in which the AIL invades the active site of a neighbouring HAT domain thus revealing a snap-shot of a trans-autoacetylation reaction intermediate. Substrate access to the active site involves rearrangement of an autoinhibitory RING domain. Our data explain how cellular signalling, TF activation and dimerization controls p300 activation thus explaining why gene transcription is associated with chromatin acetylation.

Nuclear protein in testis (Nut) is a universal oncogenic driver in the highly aggressive NUT midline carcinoma, whose physiological function in male germ cells has been unclear. Here we show that expression of Nut is normally restricted to post-meiotic spermatogenic cells, where its presence triggers p300-dependent genome-wide histone H4 hyperacetylation, which is essential for the completion of histone-to-protamine exchange. Accordingly, the inactivation of Nut induces male sterility with spermatogenesis arrest at the histone-removal stage. Nut uses p300 and/or CBP to enhance acetylation of H4 at both K5 and K8, providing binding sites for the first bromodomain of Brdt, the testis-specific member of the BET family, which subsequently mediates genome-wide histone removal. Altogether, our data reveal the detailed molecular basis of the global histone hyperacetylation wave, which occurs before the final compaction of the male genome.

We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale.