One of the key factors in formulating an energy strategy is environmental information such as nutritional availability. As many metabolism-associated genes are epigenetically regulated40
, nutrient-driven epigenetic factors may have important roles in forming metabolic phenotypes3
. LSD1 is a unique demethylase that does not contain the jumonji
domain but as a flavoenzyme does have the FAD-dependent amine oxidase domain1
. Our present study clearly indicates that LSD1 negatively regulates energy expenditure that can be reversed by inhibiting LSD1 function and FAD biosynthesis (). Cellular FAD potentiates LSD1 to repress energy-expenditure genes such as PGC-1
α through H3K4 demethylation in adipocytes where excess energy is stored as triglycerides. Moreover, our experiments using mature adipocytes and isolated adipose tissues revealed the metabolic state-dependent effects of LSD1 inhibition. Thus, the transcriptional and epigenetic regulation by FAD-dependent LSD1 may be central in nutrient-driven metabolic adaptation.
Schematic model of LSD1 function in the metabolic gene regulation.
In this study, we identified a set of energy expenditure-associated genes as direct targets of LSD1-mediated repression. We focused on the genes that were commonly induced by LSD1-KD, BHC80-KD and TC treatment. This criterion increased the likelihood of picking up genes that were directly regulated through H3K4 demethylation by LSD1, because BHC80 is reportedly the LSD1 partner required for H3K4 demethylation-dependent repression, and TC irreversibly inhibits the catalytic activity of LSD1 (refs 16
). This finding was further confirmed by the use of SLIs with minimized nonspecific effects. Our microarray results also revealed the non-overlapping effects of LSD1-KD and BHC80-KD on the genome-wide expression profile. This suggests that in many cases, LSD1 and BHC80 regulate gene expression independently, and that BHC80 is dispensable for LSD1 function other than H3K4 demethylation. In fact, it is reported that BHC80 recognizes H3K4 to facilitate the demethylation by LSD1 (ref. 16
), and only a fraction of LSD1 was associated with BHC80 in our experiment (Supplementary Fig. S1c
). It is also noteworthy that reported phenotypes of LSD1
- and BHC80
-KO mice are distinctively different, indicating the non-overlapping biological function of these genes11
Mechanistically, LSD1 disruption induced moderate enrichment of methylated H3K4 on actively transcribed promoters. Histone methylation status can be determined by the equilibrium of methylating and demethylating forces, and, in many cases, LSD1 demethylation activity is counteracted by the lysine methyltransferase Set7/9 (refs 9
). Interestingly, a previous report has shown that glucose-dependent induction of NF-κB gene expression was coincident with Set7/9-dependent H3K4 methylation, and was counteracted by LSD1, implying the possible involvement of Set7/9 in the metabolic gene regulation42
. In our study, Set7/9-KD resulted in the partial reduction of H3K4 demethylation at LSD1-target promoters (). Thus, it is possible that LSD1 fine-tunes the expression of metabolic genes so that the cellular energy balance can be properly maintained.
We found in this study that the restriction of cellular FAD availability weakened the LSD1-dependent transcriptional repression of energy-expenditure genes. LSD1, like other flavoenzymes, requires FAD for its catalytic activity and converts it into the reduced form, FADH2
). The catalytic activity of LSD1 may be directly connected to the cellular metabolic state via the fluctuation of the FAD/FADH2
ratio depending on the FAD oxidation processes such as fatty acid β-oxidation and the TCA cycle. Another intriguing possibility is that a physical association between the LSD1 and FAD production machinery determines the LSD1 activities. Indeed, a recent study identified the existence of RFK in the protein complex containing TNFα receptor-1, in which RFK seemed to directly provide NADPH oxidase with FAD, facilitating the TNFα signalling44
. Such a mechanism might explain why even a small reduction in FAD content in RFK-KD and lumiflavin-treated cells was sufficient for the LSD1 inhibition. In mitochondria, where the majority of FAD production occurs, flavoenzymes may stably associate with FAD even under FAD-reduced conditions33
. As the FAD reserve for nuclear flavoenzymes is relatively small, their enzymatic activity could be highly sensitive to FAD restriction.
It is of great interest that FAD-dependent LSD1-mediated demethylation may be analogous to, but clearly distinct from, NAD+
-dependent Sirtuin 1 (Sirt1)-mediated deacetylation. Sirt1 is the orthologue of yeast Sir2 histone deacetylase, and promotes mitochondrial activation that contributes to calorie restriction-induced metabolic adaptation45
. Regarding the regulation of the PGC-1α function, Sirt1 activates PGC-1α protein by deacetylating its lysine residue46
, whereas LSD1 negatively regulates the expression of the PGC-1
α gene. Such opposing functions of LSD1 and Sirt1 suggest the existence of reciprocal switches for energy homeostasis in which FAD and NAD+
serve as coenzymatic sensors.
Aberration of cellular energy metabolism is associated with a wide range of multifactor and/or polygenic diseases including obesity-associated diseases, neurological disorders and cancer47
. As epigenetic mechanisms are often linked to the pathogenesis of these diseases, the epigenetic factors responsible would be attractive as pharmacological therapeutic targets5
. Our study depicts a novel mechanism of the crosstalk between energy metabolism and epigenetic gene regulation in which the FAD-dependent LSD1 activity regulates energy-expenditure genes. Thus, LSD1 inhibitors may be a new class of epigenetic drugs that can therapeutically benefit a wide range of metabolic disorders.