Uncovering the Cross-Talk and Dynamics of the Human SAGA HAT Module with Designer Nucleosomes

Abstract

Chromatin structure and gene expression are regulated by a vast network of post-translational modifications (PTMs) on histone proteins. These modifications rarely act in isolation; they engage in combinatorial cross-talk in which one mark influences the writing, erasing, or reading of another. The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is a ~1.5 MDa transcriptional coactivator that catalyzes histone H3 acetylation through its histone acetyltransferase (HAT) module, comprising GCN5, ADA2B, ADA3, and SGF29. While SAGA function has been extensively studied in yeast and much of the complex has been visualized by X-ray crystallography and cryo-electron microscopy, the kinetic behavior of the human SAGA HAT module on nucleosomal substrates and the structural basis for its engagement within the full complex or with chromatin remain incompletely understood. In this dissertation, we present a quantitative biochemical and structural characterization of the human SAGA HAT module on nucleosomal substrates. Using a chemically defined nucleosome platform generated by sortase-mediated ligation of synthetic H3 tail peptides onto recombinant tailless nucleosomes, we systematically tested how individual histone PTMs and their combinations modulate hSAGA HAT activity. We further leveraged this platform to generate asymmetric nucleosomes in which the two H3 tails carry distinct modifications, enabling direct dissection of cis versus trans cross-talk effects within a single nucleosome. We find that hSAGA HAT acetylates H3K9 and H3K14 on 185 bp nucleosomes with V/[E] of 1.2 ± 0.3 min⁻¹ on unmodified substrates, and that DNA linker length is a major determinant of activity, with 185 bp nucleosomes acetylated substantially more efficiently than 147 bp nucleosomes. H3K14 acetylation and tetra-acetylation of H3 each modestly stimulate K9 acetylation (1.4- and 1.5-fold respectively), while H3K4 trimethylation produces robust activation (~4-fold). Most strikingly, asymmetric nucleosome experiments reveal that K4me3 activates hSAGA HAT in cis but inhibits in trans, providing direct evidence that the SGF29 Tudor domain and GCN5 catalytic domain engage the same H3 tail during productive acetylation. To gain structural insight, we prepared bisubstrate analog nucleosomes bearing symmetric and asymmetric H3K4me3 and H3K14-CoA modifications and characterized HAT module-nucleosome complexes by AlphaFold3 modeling, hydrogen-deuterium exchange mass spectrometry, and cryo-electron microscopy. HDX-MS identified two GCN5 regions, a disordered segment in the PCAF homology domain and the bromodomain, that engage the nucleosome and contact linker DNA, while cryo-EM data demonstrated that the HAT module-nucleosome complex remains too conformationally dynamic for high-resolution reconstruction even with the bisubstrate trap. Together, this work establishes a quantitative framework for hSAGA HAT enzymology on nucleosomes, provides direct biochemical evidence for cis-directed reader-writer coupling through SGF29, and reveals that hSAGA HAT-nucleosome engagement is fundamentally driven by flexible histone tail and linker DNA contacts. These findings inform our understanding of how SAGA selectively acetylates chromatin at active promoters and provide a foundation for therapeutic targeting of the SAGA HAT module in cancer and other diseases.