THE ROLE OF LYSINE-SPECIFIC DEMETHYLASE 1 IN GLUCOCORTICOID RECEPTOR-MEDIATED GENE EXPRESSION

Abstract

In order to regulate gene expression, nuclear receptors interact with multiple coregulatory complexes which contain various enzymatic activities. The glucocorticoid receptor (GR), upon glucocorticoid (GC) binding, has been shown require the activity of lysine deacetylases (KDACs) in order to regulate transcription. We have previously demonstrated that KDACs can function as either transcriptional coactivators or corepressors for GR-target genes, depending on gene context. Chromatin Immunoprecipitation assays reveal a loss of di-methylated H3K4 at the transcription start sites of repressed genes when KDACs 1 and 2 are inhibited using Valproic Acid (VPA). Because no concurrent change in tri-methylated H3K4 was observed, KDACs are likely to regulate a demethylase to reduce H3K4 di-methylation. Lysine-Specific Demethylase 1 (LSD1) was selected as the candidate, because it is a component of CoREST and NuRD complexes, which also contain KDACs 1 and 2, and it only demethylates mono- and dimethylated H3K4. Because LSD1 additionally plays a role in the expression of androgen and estrogen receptor target genes, the aim of this investigation was to determine the role of Lysine- Specific Demethylase 1 with respect to GR-mediated gene expression. Through Co- Immunoprecipitation assays, we confirmed that LSD1 associates with both KDACs 1 and 2. By monitoring global levels of LSD1 over time, we demonstrated that VPA does not cause changes in LSD1 expression, nor global changes in mono- and tri-methylated H3K4. Using siRNAmediated depletion of LSD1, we determined that LSD1 activity is necessary for transcriptional repression of GR target genes, but plays little role in activation. However, pharmacological inhibition of LSD1 using Pargyline, OG-L002, and Bizine did not have any appreciable effects on the expression of GR activated and repressed genes, though additional assays are needed to adequately determine the efficacy of the drugs for in vitro studies.