On the molecular mechanisms of chromatin modification

Andrea Mattevi
Department of Biology and Biotechnology,
University of Pavia, Italy

Human LSD1, Lysine-specific demethylase working on histone tail as chromatin remodeler, is typically found in association with CoREST1 and HDACs, forming a stable sub-complex module which acts in concert removing the epigenetic marks for gene activation. LSD1 is involved in a large number of physiological and pathological processes: from gene silencing to cell-growth and differentiation, from genetic syndromes to cancer. Despite many biochemical data are available, the molecular bases for the substrate recognition by LSD1-CoREST1 complex are not known.
The core aim of our research is to reveal the molecular details – mostly in terms of protein/protein and protein/DNA interactions – of the assembly and recognition of LSD1-CoREST1 in complex with the nucleosome. While H3 tail and DNA-binding properties of the demethylase complex have been analyzed in depths, one of the main challenges of this work is the stabilization of the complex since it is physiologically transient. To address this question, we employed cutting-edge techniques for the study the reconstituted LSD1-CoREST1/Nucleosome complex. In this regard, the LSD1-CoREST1 heterodimer and several surface mutants have been purified in recombinant form, with the goal of mapping the enzyme-nucleosome interactions. At the same time, fully recombinant nucleosomal particles have been purified following published protocols, and the whole LSD1-CoREST/Nucleosome complex reconstituted in vitro. We devised a methodology to covalently bind the complex components and stabilize the assembly using nucleosomal particles carrying a chemical moiety for the stable trapping of the H3 tail inside LSD1 active site. The data obtained from analytical chromatography, SPR and FP techniques were combined with the structural analysis of the complex using SAXS. Altogether, data led us to develop a validated model for the recognition that involves two steps: the nucleosome is initially recognized by CoREST1 through its SANT2 domain, which probes DNA to engage the nucleosome in a productive mode. Only at this point LSD1 is able to recognize the histone tail for demethylation, thereby producing hydrogen peroxide.
A few questions still remain unsolved: are the other histones involved in the recognition process? Are there domains other than the active site interacting with the nucleosome? If yes, how? Are LSD1-produced rOS enganged in signaling processes? The full characterization – now being further investigated using AFM and EM – would be key to understand in depth the mechanisms that, once altered, trigger pathological events. This, also thanks to the studies on new inhibitor molecules, can represent a milestone not only in epigenetics, but also in applied medical research.

1. Pilotto, S., Speranzini, V., Tortorici, M., Durand, D., Fish, A., Valente, S., Forneris, F. , Mai, A., Sixma T.K., Vachette, P., Mattevi, A (2015)
Interplay between nucleosomal DNA, histone tails and CoREST underlies LSD1-mediated H3 demethylation Proc. Natl. Acad. Sci. USA 112:2752

2. Speranzini, V., Pilotto, S., Sixma, T.K., Mattevi A. (2016)
Touch, act and go: landing andoperating on nucleosomes. EMBO J. 35:376