Development of auxiliary-mediated protein semisynthesis methods toward the study of histone SUMOylation

Abstract

Eukaryotic DNA is packaged into chromatin, which consists of a fundamental repeating unit, the nucleosome, and its associated proteins. Nucleosomes are made up of histone protein octamers, containing two each of histones H2A, H2B, H3, and H4, around which ~147 bp of DNA is wound. The tails of histones are disordered and extend beyond the DNA where they may be accessed by reader, writer, and eraser proteins. Post-translational modifications (PTMs) on histone tails are crucial to regulating chromatin structure and its interacting proteins, which collectively dictate the transcriptional state of the overlying DNA. The dysfunction of DNA-templated processes underlies a variety of human pathologies, including neurodegenerative and autoimmune diseases, and multiple human cancers. Importantly, these conditions are all associated with the misregulation of various histone PTMs. We focused on the poorly-understood effects of histone modification with the small ubiquitin-like modifier (SUMO). The SUMOylation of histone H4 is conserved between yeast and humans and is implicated in transcriptional silencing and DNA repair. However, a mechanistic understanding of how H4 SUMOylation leads to these outcomes has remained elusive for well over a decade, and insight into its precise roles in gene regulation may lead to the identification of new therapeutic targets. We were interested in the dynamic interplay, or cross-talk, between SUMOylated H4 (suH4) and other histone PTMs, potentially mediated by transcriptional repressive complexes. To generate suH4 for in vitro assays, we utilized protein semisynthesis. Toward this goal, we developed chemical methodology for facile, site-specific native SUMOylation of protein substrates, including suH4, based on the small molecule native chemical ligation auxiliary (2-aminooxy)ethanethiol. We further utilized this auxiliary to accomplish protein SUMOylation under non-denaturing conditions, which greatly expanded the scope of substrates accessible to this methodology. Over the course of developing non-denaturing ligation, we discovered that the thiol additive, 4-mercaptophenylacetic acid (MPAA), promoted N-O bond reductive cleavage and auxiliary removal without the low pH and harsh denaturants previously required. Extensive characterization of the reaction revealed that it is promoted by the spontaneous formation of MPAA thiyl radicals in solution. Finally, these methodologies enabled us to generate multi-milligram quantities of homogenous suH4 to test the hypothesis that SUMO recruits the transcriptional repressor histone deacetylase 1 (HDAC1) to chromatin. In vitro deacetylation assays with SUMOylated mononucleosomes (MN) demonstrated that a repressive complex of HDAC1 and the corepressor protein CoREST is preferentially recruited to SUMOylated MN, which enhances their deacetylation. Thus, we have identified a novel cross-talk relationship between histone SUMOylation and acetylation, which represents one possible pathway by which histone SUMOylation can mediate transcriptional repression.