Investigating the Role of Lysine Methylation in Neuronal Differentiation

Abstract

Neuronal differentiation is a critical process during brain development, and aberration in neuronal differentiation has emerged as a major point of convergence for neurodevelopmental disorders (NDDs). Thus, there is a critical need to understand the molecular mechanisms that regulate neuronal cell differentiation. The reversible post-translational modification lysine methylation has reported regulatory roles in neuronal differentiation. While histone lysine methylation is well-studied, insights into the role of non-histone lysine methylation in differentiation remain limited, partly due to the lack of high-throughput profiling in neuronal models. The enzymes that mediate lysine methylation – lysine methyltransferases (KMTs) and demethylases (KDMs) – are critical for brain development. Over a third of KMTs/KDMs have been associated with NDDs, and haploinsufficiency of a number of these enzymes, including the lysine methyltransferase ASH1L, results in aberrations in neuronal differentiation. The overall objective of this work was to gain mechanistic insight into the regulation of neuronal differentiation by lysine methylation of histone and non-histone proteins. Toward this end, we employed a quantitative proteomics approach (tandem mass tag LC-MS/MS) to profile global changes in lysine methylation across differentiation of human neural progenitor cells into post-mitotic, dopaminergic-like neurons using the Lund human mesencephalic (LUHMES) cell model. We quantified hundreds of lysine methylation events on a range of diverse non-histone proteins of biological and clinical interest. To our knowledge, this is the first report of global profiling of lysine methylation across neuronal differentiation by quantitative mass spectrometry. We also sought to determine the contribution of the lysine methyltransferase activity of the NDD-associated enzyme ASH1L toward regulation of LUHMES differentiation. We found that treatment with AS-99, a small molecule inhibitor against ASH1L KMT activity, resulted in deficiencies in neurite length and branching, supporting a critical role for ASH1L KMT activity in LUHMES differentiation regulation. Using biochemical approaches, we confirmed histone H3K36 as a lysine methylation substrate of ASH1L in vitro, and we elucidated the substrate selectivity of ASH1L. Future work will determine the impact of ASH1L-mediated substrate methylation toward regulation of LUHMES differentiation. Taken together, this work supports a critical role for lysine methylation in the regulation of neuronal differentiation.