Substrate Trapping To Discover The Role Of Histone Deacetylase Proteins Beyond Epigenetics

Gene expression is regulated by chromatin remodeling factors and histone modifications, such as phosphorylation, methylation and acetylation. Acetylation is regulated by histone acetyltransferases and histone deacetylases. Histone deacetylases remove acetyl groups from ε-N-acetyl lysine amino acids, which allows DNA to wrap around the histones more tightly. The discovery of a wide variety of acetylated proteins suggests that HDAC proteins likely deacetylate other substrates in addition to histones. By identifying non-histone substrates, HDAC proteins have been linked to multiple cellular processes in addition to gene expression. However, with only few known substrates, the full role of HDAC proteins in cellular events was unclear. In prior HDAC-related research, substrate identification has been largely serendipitous. This thesis work focused on the development of substrate trapping mutants of HDAC proteins, namely HDAC1 and HDAC6, to discover new substrates. Discovery of novel substrates for HDAC1 and HDAC6 using trapping mutants may lead to a better understanding of the role of the proteins in diseases. Given the high sequence similarity between HDAC family members, substrate trapping was applied to identify novel substrates of HDAC6. Due to the presence of two catalytic domains in HDAC6, four mutants (two from each domain) were screened to assess the relative trapping ability towards a known substrate, tubulin. H611A, a second catalytic site mutant, was identified as the optimal substrate trapping mutant for HDAC6. Proteomics-based substrate trapping using H611A identified a profile of potential HDAC6 substrates. Many of the previously known HDAC6 substrates were identified, validating the method. Protein arginine methyltransferase 5 (PRMT5) was identified as a new substrate of HDAC6, which further revealed a crosstalk between acetylation and methyltransferase activity. Another HDAC6 substrate, Serine/threonine kinase 38 like protein (STK38L), was shown to have a novel role in ciliogenesis through HDAC6-mediated deacetylation. To optimize substrate trapping for HDAC1, the relative trapping abilities of 17 inactive HDAC1 mutants were assessed using two known substrates of HDAC1. HDAC1 H141A, F150A, and C151A showed strong binding to the protein substrates LSD1 and p53. Interestingly, each mutant preferentially trapped a different substrate. By combining several inactive mutants, the trapping strategy facilitates discovery of new HDAC1 substrates and shed light on the variety of HDAC1-related functions in cell biology. In summary, this thesis work established a systematic method to identify novel substrates of HDAC proteins to uncover new HDAC-related functions