Mechanisms of LSD1 regulation

Abstract

The precise spatiotemporal regulation of gene expression is essential for all aspects of cellular function. Dysregulation of gene transcription can lead to serious pathologies, including severe malignancies and ultimately cell death. Consequently, complex regulatory mechanisms are essential to maintain and adapt gene expression in response to internal or external cues. Among the key enzymes of transcriptional regulation is the Lysine Specific Demethylase 1 (LSD1), a chromatin-modifying enzyme present in numerous multiprotein complexes. LSD1 is critical for proper cellular differentiation and its overexpression is associated with poor prognosis in various cancers, highlighting the need for a comprehensive understanding of its function and regulation. Previous work in the Rathert lab established a fluorescent reporter system that allows to investigate the gene repressive function of LSD1 or other factors in a biological context. In combination with this reporter system, a multiplexed RNAi screen identified both novel and known coregulators of LSD1. One of the top candidate hits, the NIMA-related kinase NEK6, previously not reported to impact LSD1, was validated and further characterized in this thesis. A combination of in vitro and cell-based approaches demonstrated that NEK6 directly modulates LSD1 via phosphorylation at serine 126 (S126). This residue is located within the unstructured N-terminus, a region lacking any secondary or tertiary structure, which is therefore defined as intrinsically disordered region (IDR). Extensive laboratory work demonstrated biomolecular condensation of the IDR of LSD1, which was impacted by phosphorylation of S126. Further, the IDR was directly linked to the cellular function of LSD1. Up to now, the impact of the unstructured region towards the functionality of LSD1 was neglected in the scientific field. Therefore, a detailed structural and functional analysis was conducted, which revealed a previously uncharacterized acidic patch within the IDR. Building on this observation, detailed characterization was performed to explain the impact of the respective acidic patch towards LSD1 functionality. Although absent in current structural models, this acidic patch is located in close proximity to the catalytic center and impacts substrate binding in vitro. It further plays a role in the biomolecular condensation behaviour of LSD1, suggesting a dual role in modulating both structural organization and enzymatic function. Together, the findings presented in this dissertation illustrate how integrative cellular and biochemical approaches revealed novel regulatory mechanisms of LSD1. These insights expand the understanding of LSD1 function and its modulation by both, intrinsic structural elements and external signalling stimuli. These findings also underscore the disregarded functional relevance of IDRs, highlighting the need for broader investigation into their specific functions.