Structural insights into dynamic histone post-translational modification regulation

Abstract

Nucleosome is the basic unit of eukaryotic chromatin, consisting of histone proteins and a segment of DNA. Transcription activities are relatively repressed due to repeated units of nucleosomes package DNA into higher ordered and condensed form. However, histone post-translational modifications (PTMs) reveal new insights to gene transcription regulation. Dynamic changes of histone PTMs can provide transient opportunities for nucleosome-DNA unwrapping and rewrapping, exposing particular DNA section for protein binding and resulting in repression or activation of gene transcription. The PTM players, “writer”, “eraser” and “reader” participate in histone PTM regulation by adding, removing, and reading various conjugated modifications on histones. Structural biology has been regarded as a strong tool to identify and characterize PTM players to particular histone PTMs, providing the deeper understanding of binding mechanisms and insights to drug design. Here, in this thesis, two cases of histone PTM players were investigated. The first case is about lysine lactylation (Kla), a novel histone PTM discovered in late 2019. It can directly stimulate gene transcriptions, resulting in a series of physiological changes, such as macrophage polarization. In this thesis, the human NAD+-dependent deacylases, Sirtuin family proteins have been screened to test whether they are the eraser of Kla. After a series of biochemical experiments including NAD+ consumption/cycling assay, isothermal titration calorimetry and HPLC/MS analysis, SIRT3 was found to present the highest eraser activity against H4K16la site. The SIRT3-H3K23la and SIRT3-H4K16la complex structures were solved, revealing the binding mechanism of SIRT3-Kla. Preconfigured hydrophobic pocket of SIRT3 accommodated the hydrocarbon portion of Kla, and the hydroxyl-group of the Kla was stabilized by Q228 of SIRT3 via water bridges. The western blot demonstrated that knock down of SIRT3 caused increased level of H3K18la and H4K16la. More ChIP- seq studies are required to investigate the role of SIRT3 in H4K16la related gene transcription.

The second case is about the glioma amplified sequence 41 (GAS41), one of the YEATS domain-containing reader family. GAS41 is highly expressed in several glioblastoma multiforme cell lines and promotes oncogene transcription via recognition of particular histone PTMs. Here, a crystal structure of apo decamer GAS41 was solved, which contains 10 monomers in each asymmetric unit. And these monomers aligned as a double-layered ring. Each ring is constituted of 5 monomers and the ring-to-ring interaction is mainly facilitated by hydrophobic interaction. Notably, in the single layer, the protruding N-terminal of each monomer interacts into the reader pocket of the adjacent monomer mainly by hydrogen bonds. Moreover, the N-terminal was well superimposed with acyl-lysine in other published GAS41 structures. The decamer structure reveals a new insight for developing peptide-based inhibitors of GAS41. Together, these structures provide molecular basis for the functions of PTM players and shed light on the dynamics of histone PTM regulation.