Histone demethylase JMJD1C is phosphorilated by mTOR to activate de novo lipogenesis

da | Giu 26, 2020 | Biologia Molecolare

Abstract

The synthesis of fatty acids and triglycerides increases considerably in response to food and insulin. JMJD-1C is a specific histone demethylase, which is phosphorylated by mTOR, assisted with his cofactor (α-KG) promotes lipogenesis in liver. This mechanism contributes to the development of hepatosteatosis.

The paradox of selective hepatic insulin resistance, wherein the insulin-resistant liver fails to suppress glucose production but continues to produce lipids, has been central to the pathophysiology of hepatosteatosis and hyperglycaemia (Heng Wu, 2016). Non-alcoholic fatty liver disease (NAFLD) is a major type of metabolic disorder, which has become a severe public health problem due to its high association with metabolic syndrome and progression of liver diseases, including non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma (Bin Liu, 2018).

Epigenetic modifications, such as acetylations or methylations, have an important impact on the development of this pathology; generally, demethylases act on Lysine and require particular cofactors and cosubstrates (Berger, 2007). The article presented by Viscarra and collaborators (2020) focuses the attention at the correlation established between JMJD-1C’s phosphorylation by the mTOR complex and de novo lipogenesis in the hepatoteatosis desease. JMJD-1c is a Histone demethylase of Jumonnji family, which performs its function on histone H3K9me2.The researchers identified it as a critical epigenetic factor for lipogenesis.

VIscarra and collaborators first decided to inquire the interaction between USF-1, a trasnscription factor, and JMJD-1C. USF-1 is a central player as a molecular switch for lipogenesis during fasting/feeding by recruiting distinct TFs and signalling molecules. The scientists, using GST pull down and co-IP demonstrated the direct interaction between USF-1 and JMJD-1C.  Moreover, through Fas luciferase they noticed a synergistic action of the two proteins.

The researchers conducted a treatment with Methylstat, JMJD-1C domain inhibitor containing demethylases, demonstrating the necessity for the demethylase’s action. Next, they examined the expression of lipogenic genes in response to insulin treatment by gaining and losing function of JMJD-1C through RT-qPCR in HepG2 cells, underling the functional role of JMJD-1C. In vivo experiments were conducted to investigate the significance of JMJD-1C for the activation of lipogenic genes in liver of previously fed mice, finding respectively an increase and decrease in presence of overexpression or  silenced JMJD-1C.

From literature emerges the role of alpha-ketoglutarate as an obliged cofactor for Jumonji family. For this reason, the researchers examined the alterations of alpha-ketoglutarate levels in response to the presence or absence of insulin in HepG2 cells and in the presence or absence of food in mice. The study revealed an increased in level of alpha-ketoglutarate in the presence of insulin and or food. Moreover, HepG2 cells treated with dimethyl-ketoglutarate (DMKG), a cell-permeable analog of α-KG, observed a further FAS increase and the expression of other lipogenic genes in a dose-dependent manner, which underlined the importance of the alpha-ketoglutarate as a cofactor.

Scientists also examined H3K9 methylation status of lipogenic promoters by ChIP following insulin/DMKG treatment. Insulin-treated cells had lower enrichment of H3K9me3 and H3K9me2, and greater enrichment of H3K9me1. Cells in which insulin and DMKG were added had even lower enrichment of H3K9me2, and greater accumulation of H3K9me1, at lipogenic promoters compared to the insulin-treated cells.

To examine whether changes in α-KG levels can fully explain the JMJD-1C-mediated activation of lipogenic genes in response to insulin/feeding treatment, they next assayed the demethylase activity of JMJD-1C purified from HepG2 cells in the excess of all of the required co-factors and substrates, including α-KG.  Notably, they detected significantly higher demethylase activity of JMJD-1C purified from insulin-treated HepG2 cells. Similarly, they detected higher demethylase activity with the immuno-purified JMJD-1C from livers of fed mice. Higher demethylase activity of JMJD-1C from insulin/feeding treatment, suggests that JMJD-1C could be post-translationally modified in response to insulin / feeding.

Through the use of antibodies capable of selectively binding phosphorylated Serines or Threonines, they found a main phosphorylation (T505). It emerged that the phosphorylation of Threonine in position 505 is a post-translational modification that occurs exclusively in the presence of insulin.

By using of site-specific point mutations in correspondence of Serines and Threonines phosphorylation they noticed an increased activity of the T505D mutant and a decreased  activity of the T505A mutant. The results obtained from the article therefore support the hypothesis that phosphorylation at the level of Threonine 505 (T505) plays a key role in the activation of the FAS promoter by means of JMJD-1C. From the literature it emerged that Threonine could be phosphorylated by mTOR (Masahiro Morita, 2015).

In the article was also discovered that mTOR also has an impact on the expression of lipogenic genes because, through silencing of this protein or use of selective inhibitors, it can cause a decrease in the expression of them. The study supports the hypothesis that in the presence of food (Minassian C., 1994) or insulin the alpha ketoglutarate’s levels increase, thus confirm the role of the aforementioned molecule as a co-substrate of JMJD-1C for the demethylation of H3K9 (which involves the activation of the genes lipogenic). An increase of alpha-ketoglutarate’s level was also found in presence of hepatostatosis, classifying  as a possible diagnostic marker for this disease.

Figure 1

The article is well structured, and all the experiments are complete and explained in a clear way, but researchers have not clarified why the mutation with Aspartic Acid may lead to an increase in mRNA levels of lipogenic genes. Furthermore, although the article suggests a possible application of α-KG as a possible marker, the scientists do not pay due attention to this molecule, not including it in the molecular pathway that leads lipogenesis and the metabolic disorders related to it.

References

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Laura Leonardi

Master Industrial Biotechnology student

Oriana Lupo

Master Industrial Biotechnology student