Hypoxia-inducible factor-1α (HIF-1α) mediates hypoxic responses and regulates gene expression involved in angiogenesis, invasion and metabolism. Among the various HIF-1α posttranslational modifications, HIF-1α methylation and its physiological role have not yet been elucidated. Here we show that HIF-1α is methylated by SET7/9 methyltransferase, and that lysine-specific demethylase 1 reverses its methylation. The functional consequence of HIF-1α methylation is the modulation of HIF-1α stability primarily in the nucleus, independent of its proline hydroxylation, during long-term hypoxic and normoxic conditions. Knock-in mice bearing a methylation-defective Hif1aKA/KA allele exhibit enhanced retinal angiogenesis and tumour vascularization via HIF-1α stabilization. Importantly, S28Y and R30Q mutations of HIF-1α, found in human cancers, are involved in the altered HIF-1α stability. Together, these results demonstrate a role for HIF-1α methylation in regulating protein stability, thereby modulating biological output including retinal and tumour angiogenesis, with therapeutic implications in human cancer.
Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Although Pontin is frequently overexpressed in human cancers of various types and implicated in oncogenic functions, the upstream signaling network leading to the regulation of Pontin that in turn affects transcription of downstream target genes has not been extensively studied. Here, we identify Pontin is methylated by G9a/GLP methyltransferases in hypoxic condition and potentiates HIF-1α-mediated activation by increasing the recruitment of p300 coactivator to a subset of HIF-1α target promoters. Intriguingly, Pontin methylation results in the increased invasive and migratory properties by activating downstream target gene, Ets1. In contrast, inhibition of Pontin methylation results in the suppression of tumorigenic and metastatic properties. Together, our data provide new approaches by targeting Pontin methylation and its downstream targets for the development of therapeutic agents for human cancers.epigenetics | transcriptional regulation | covalent nonhistone modification
Adeno-associated virus (AAV) vectors can deliver transgenes to diverse cell types and are therefore useful for basic research and gene therapy. Although AAV has many advantages over other viral vectors, its relatively small packaging capacity limits its use for delivering large genes. The available transgene size is further limited by the existence of additional elements in the expression cassette without which the gene expression level becomes much lower. By using alternative combinations of shorter elements, we generated a series of AAV expression cassettes and systematically evaluated their expression efficiency in neurons to maximize the transgene size available within the AAV packaging capacity while not compromising the transgene expression. We found that the newly developed smaller expression cassette shows comparable expression efficiency with an efficient vector generally used for strong gene expression. This new expression cassette will allow us to package larger transgenes without compromising expression efficiency.
The inflammatory response mediated by nuclear factor κB (NF-κB) signaling is essential for host defense against pathogens. Although the regulatory mechanism of NF-κB signaling has been well studied, the molecular basis for epigenetic regulation of the inflammatory response is poorly understood. Here we identify a new signaling axis of PKCα-LSD1-NF-κB, which is critical for activation and amplification of the inflammatory response. In response to excessive inflammatory stimuli, PKCα translocates to the nucleus and phosphorylates LSD1. LSD1 phosphorylation is required for p65 binding and facilitates p65 demethylation, leading to enhanced stability. In vivo genetic analysis using Lsd1 mice with ablation of LSD1 phosphorylation and chemical approaches in wild-type mice with inhibition of PKCα or LSD1 activity show attenuated sepsis-induced inflammatory lung injury and mortality. Together, we demonstrate that the PKCα-LSD1-NF-κB signaling cascade is crucial for epigenetic control of the inflammatory response, and targeting this signaling could be a powerful therapeutic strategy for systemic inflammatory diseases, including sepsis.
The circadian clock is a self-sustaining oscillator that controls daily rhythms. For the proper circadian gene expression, dynamic changes in chromatin structure are important. Although chromatin modifiers have been shown to play a role in circadian gene expression, the in vivo role of circadian signal-modulated chromatin modifiers at an organism level remains to be elucidated. Here, we provide evidence that the lysine-specific demethylase 1 (LSD1) is phosphorylated by protein kinase Cα (PKCα) in a circadian manner and the phosphorylated LSD1 forms a complex with CLOCK:BMAL1 to facilitate E-box-mediated transcriptional activation. Knockin mice bearing phosphorylation-defective Lsd1(SA/SA) alleles exhibited altered circadian rhythms in locomotor behavior with attenuation of rhythmic expression of core clock genes and impaired phase resetting of circadian clock. These data demonstrate that LSD1 is a key component of the molecular circadian oscillator, which plays a pivotal role in rhythmicity and phase resetting of the circadian clock.
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