Role of SUMOylation within the vitamin D response

Abstract

Vitamin D is a pleiotropic nutrient that elicits various physiological responses in a cell type- or tissue-specific manner. Cellular vitamin D signalling is mediated by a member of nuclear receptor superfamily, vitamin D receptor (VDR), which transactivates vitamin D target genes, forming heterodimer with retinoid X receptor (RXR). Given the wide spectrum of vitamin D response, those receptors are considered to be regulated through a mechanism that is closely related to cell type- or tissue-specific
environment, and as one of the potential mechanisms, recent studies have revealed that both VDR and RXR are post-translationally modified by small ubiquitin-like modifier (SUMO), with the level of SUMO modification (SUMOylation) modulated by PIAS4 (protein inhibitor of activated STAT 4) and SENPs (sentrin/SUMO-specific proteases), enzymes that enhance and reverse SUMOylation, respectively. Corresponding to the level of SUMOylation, VDR transactivity has been shown to be repressed and enhanced by PIAS4 and SENPs, respectively, suggesting that SUMOylation has a negative impact upon vitamin D signalling. To better understand the molecular mechanism by which SUMOylation and its enzymes regulate vitamin D signalling,
this study was aimed to obtain clues to how PIAS4 and SENPs impact upon functions of VDR and RXR, such as intracellular trafficking, transcriptional coregulator association and transactivity. When VDR was overexpressed in cells, PIAS4 showed
its ability to sequester VDR into a specific subnuclear compartment that closely associated with nuclear matrix, while SENP2 showed its potential to counteract it. At the endogenous level, however, such a sequestration was not observed. As another
mechanism, PIAS4 was found to alter the pattern of VDR-coregulator interaction in a manner dependent upon its enzymatic activity, suggesting an involvement of PIAS4-mediated SUMOylation of VDR and/or coregulators in this process. The transactivity of RXR, on the other hand, was found to be modulated by PIAS4 and SENPs in an analogous fashion to the regulation of VDR transactivity, but unlike VDR, PIAS4 changed neither intracellular trafficking nor coregulator association of RXR. However, the experiments provided an implication that SUMOylation on RXR alters the efficiency of heterodimerisation with specific dimeric partners including VDR. Finally, to accelerate future studies on the role of SUMOylation in the regulation of vitamin D signalling, a mass spectrometry-based, proteome-wide analytical method to identify SUMOylation sites on VDR was designed, and the functionality of SUMO mutant constructs that were required for the analysis was evaluated. These results provide multifaceted evidence that helps illustrating an intricate regulatory mechanism for vitamin D signalling that involves SUMOylation and its modulation by SUMOylation enzymes, PIAS4 and SENPs. Such an insight provided by this study will serve as a
foundation in the future efforts to further detail the complex molecular mechanism that regulates vitamin D signalling, leading to clearer mechanistic definition of the diversity and specificity of vitamin D response.

Date of Award	Jul 2020
Original language	English
Supervisor	Tara Moore (Supervisor) & Paul Thompson (Supervisor)