Gang Ouyang

Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation

Hypoxia-inducible factor (HIF)-1α and HIF-2α are the main regulators of cellular responses to hypoxia. Post-translational modifications of HIF-1α and 2α are necessary to modulate their functions. The methylation of non-histone proteins by Set7, an SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. In this study, we show that Set7 methylates HIF-1α at lysine 32 and HIF-2α at lysine K29; this methylation inhibits the expression of HIF-1α/2α targets by impairing the occupancy of HIF-α on hypoxia response element of HIF target gene promoter. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 exhibit upregulated HIF target genes. Set7 inhibitor blocks HIF-1α/2α methylation to enhance HIF target gene expression. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 or inhibition of Set7 by the inhibitor subjected to hypoxia display an increased glucose uptake and intracellular adenosine triphosphate levels. These findings define a novel modification of HIF-1α/2α and demonstrate that Set7-medited lysine methylation negatively regulates HIF-α transcriptional activity and HIF-1α-mediated glucose homeostasis.

Eaf1 and Eaf2 negatively regulate canonical Wnt/β-catenin signaling.

Eaf factors play a crucial role in tumor suppression and embryogenesis. To investigate the potential mechanism of Eaf activity, we performed loss- and gain-of-function assays in zebrafish using morpholino and mRNA injections, respectively. We found that eaf1 and eaf2 inhibit Wnt/β-catenin signaling, thereby modulating mesodermal and neural patterning in the embryo. Moreover, ectopic expression of eaf1 and eaf2 in embryos and cultured cells blocked β-catenin reporter activity. By immunoprecipitation, we also observed that Eaf1 and Eaf2 bound to the Armadillo repeat region and C-terminus of β-catenin, as well as to other β-catenin transcription complex proteins, such as c-Jun, Tcf and Axin, suggesting the formation of a novel complex. In addition, the N-terminus of Eaf1 and Eaf2 bound to β-catenin and exhibited dominant-negative activity, whereas the C-terminus appeared to either harbor a suppression domain or to recruit a repressor. Both the N- and C-terminus must be intact for Eaf1 and Eaf2 suppressive activity. Lastly, we demonstrate a conservation of biological activities for Eaf family proteins across species. In summary, our evidence points to a novel role for Eaf1 and Eaf2 in inhibiting canonical Wnt/β-catenin signaling, which might form the mechanistic basis for Eaf1 and Eaf2 tumor suppressor activity.

ELL targets c-Myc for proteasomal degradation and suppresses tumour growth

Increasing evidence supports that ELL (eleven–nineteen lysine-rich leukaemia) is a key regulator of transcriptional elongation, but the physiological function of Ell in mammals remains elusive. Here we show that ELL functions as an E3 ubiquitin ligase and targets c-Myc for proteasomal degradation. In addition, we identify that UbcH8 serves as a ubiquitin-conjugating enzyme in this pathway. Cysteine 595 of ELL is an active site of the enzyme; its mutation to alanine (C595A) renders the protein unable to promote the ubiquitination and degradation of c-Myc. ELL-mediated c-Myc degradation inhibits c-Myc-dependent transcriptional activity and cell proliferation, and also suppresses c-Myc-dependent xenograft tumour growth. In contrast, the ELL(C595A) mutant not only loses the ability to inhibit cell proliferation and xenograft tumour growth, but also promotes tumour metastasis. Thus, our work reveals a previously unrecognized function for ELL as an E3 ubiquitin ligase for c-Myc and a potential tumour suppressor.

pVHL Negatively Regulates Antiviral Signaling by Targeting MAVS for Proteasomal Degradation

The von Hippel–Lindau (VHL) gene is a well-defined tumor suppressor linked to human heredity cancer syndromes. As a component of the VHL-elongin B/C E3 ligase complex, pVHL performs its tumor function by targeting proteins for proteasomal degradation. It is largely unknown whether pVHL functions in antiviral immunity. In this article, we identify that pVHL negatively regulates innate antiviral immunity, which acts mainly by inducing degradation of mitochondrial antiviral-signaling protein (MAVS, also known as Cardif, IPS-1, or VISA). Overexpression of pVHL abrogated the cellular response to viral infection, whereas knockdown of pVHL exerted the opposite effect. pVHL targeted the K420 residue of MAVS to catalyze the formation of K48-linked polyubiquitin chains, leading to proteasomal degradation of MAVS. After viral infection, Mavs levels remained low in wild type zebrafish embryos but became much higher in vhl-deficient (vhl−/−) zebrafish embryos. Higher MAVS levels correlated with a greatly exaggerated antiviral response. In this work, we demonstrate that pVHL exhibits a previously unknown role in innate antiviral immunity.

The von hippel-lindau protein suppresses androgen receptor activity.

The androgen receptor (AR) plays a pivotal role in prostate homeostasis and prostate cancer development. To understand the mechanism underlying the regulation of the AR holds a promise for developing novel therapeutic approaches for prostate cancer. Here, we show that the Von Hippel-Lindau gene product, pVHL, physically interacts with AR and inhibits AR transcription activity but does not induce AR turnover. Moreover, pVHL also suppresses androgen-induced cell proliferation, implicating a physiological role of pVHL in androgen-induced signaling pathway. In addition, we provide evidence to show that pVHL actually enhanced AR de-ubiquitination instead of inducing AR ubiquitination, uncovering a noncanonical role of pVHL in the ubiquitin proteasome pathway. Our data reveal a novel function of pVHL in the regulation of AR transcription activity, which may expand the scope of pVHL in tumor suppression and provide mechanistic insight into prostate cancer initiation and progression.

ELL Inhibits E2F1 Transcriptional Activity by Enhancing E2F1 Deacetylation via Recruitment of Histone Deacetylase 1

ABSTRACT ELL (eleven-nineteen lysine-rich leukemia protein) was first identified as a translocation partner of MLL in acute myeloid leukemia; however, the exact mechanism of its action has remained elusive. In this study, we identified ELL as a direct downstream target gene of E2F1. Coimmunoprecipitation assays showed that ELL interacted with E2F1 in vitro and in vivo, leading to inhibition of E2F1 transcriptional activity. In addition, ELL enhanced E2F1 deacetylation via recruitment of histone deacetylase 1 (HDAC1). Notably, the MLL-ELL fusion protein lost the inhibitory role of ELL in E2F1 transcriptional activity. Furthermore, DNA damage induced ELL in an E2F1-dependent manner and ELL protected cells against E2F1-dependent apoptosis. Our findings not only connect ELL to E2F1 function and uncover a novel role of ELL in response to DNA damage but also provide an insight into the mechanism for MLL-ELL-associated leukemogenesis.

Forkhead Transcription Factor 3a (FOXO3a) Modulates Hypoxia Signaling via Up-regulation of the von Hippel-Lindau Gene (VHL)

FOXO3a, a member of the forkhead homeobox type O (FOXO) family of transcriptional factors, regulates cell survival in response to DNA damage, caloric restriction, and oxidative stress. The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of the E3 ubiquitin ligase complex that mediates hypoxia-inducible factor α degradation under aerobic conditions, thus acting as one of the key regulators of hypoxia signaling. However, whether FOXO3a impacts cellular hypoxia stress remains unknown. Here we show that FOXO3a directly binds to the VHL promoter and up-regulates VHL expression. Using a zebrafish model, we confirmed the up-regulation of vhl by foxo3b, an ortholog of mammalian FOXO3a. Furthermore, by employing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) technology, we deleted foxo3b in zebrafish and determined that expression of hypoxia-inducible genes was affected under hypoxia. Moreover, foxo3b-null zebrafish exhibited impaired acute hypoxic tolerance, resulting in death. In conclusion, our findings suggest that, by modulating hypoxia-inducible factor activity via up-regulation of VHL, FOXO3a (foxo3b) plays an important role in survival in response to hypoxic stress.

ELL Protein-associated Factor 2 (EAF2) Inhibits Transforming Growth Factor β Signaling through a Direct Interaction with Smad3.

Background: EAF2 plays an important role in affecting cellular processes, but the mechanisms underlying its effects are poorly understood. Results: EAF2 was identified as a novel repressor of TGF-β signaling through a direct interaction with Smad3. Conclusion: EAF2 specifically interacts with Smad3 to prevent Smad3 nuclear translocation, Smad4-Smad3 complex formation, and p300-Smad3 complex formation. Significance: EAF2 may function by inhibiting TGF-β signaling. A series of in vitro and in vivo studies has shown that EAF2 can affect multiple signaling pathways involved in cellular processes. However, the molecular mechanisms underlying its effects have remained elusive. Here we report the discovery of a new functional link between EAF2 and TGF-β signaling. Promoter reporter assays indicated that EAF2 suppresses Smad3 transcriptional activity, resulting in inhibition of TGF-β signaling. Coimmunoprecipitation assays showed that EAF2 specifically interacts with Smad3 in vitro and in vivo but not with other Smad proteins. In addition, we observed that EAF2 binding does not alter Smad3 phosphorylation but causes Smad3 cytoplasmic retention, competes with Smad4 for binding to Smad3, and prevents p300-Smad3 complex formation. Furthermore, we demonstrated that EAF2 suppresses both TGF-β-induced G1 cell cycle arrest and TGF-β-induced cell migration. This study identifies and characterizes a novel repressor of TGF-β signaling.

pVHL acts as a downstream target of E2F1 to suppress E2F1 activity

The VHL (von Hippel-Lindau) gene is a well-defined tumour suppressor linked to hereditary cancer syndromes. Although it is well documented that pVHL (von Hippel-Lindau protein) mediates HIF (hypoxia-inducible factor)-1/2α degradation under conditions of normoxia, accounting for a major mechanism of pVHL in tumour suppression, it remains elusive whether other HIF-independent functions contribute to the pVHL tumour suppressive function. In the present study, we found that pVHL is a downstream target of E2F1, which harbours an E2F1-binding site in its promoter. Moreover, pVHL binds to E2F1 in vitro and in vivo, resulting in inhibition of E2F1 transcriptional activity. Mechanistic studies showed that pVHL binding enhances E2F1 deacetylation. Further immunoprecipitation assays indicated that the pVHL interaction diminishes P/CAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] and p300 association with E2F1, but enhances Sirt1 (sirtuin 1) binding to E2F1. In addition, upon DNA damage, pVHL is induced. Knockdown of pVHL sensitizes cells to DNA-damage-induced apoptosis dependent on E2F1, uncovering a role for pVHL in the response to DNA damage. The findings of the present study reveal a novel function of pVHL and demonstrate a negative-feedback loop between pVHL and E2F1, which may shed new light on the explanation of the role of pVHL in tumour suppression.

Tet1 facilitates hypoxia tolerance by stabilizing the HIF-alpha proteins independent of its methylcytosine dioxygenase activity

Abstract Because of the requirement of oxygen (O2) to produce energy, aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen in both acute status and chronic status. To date, how organisms tolerate acute hypoxia and the underlying mechanisms remain largely unknown. Here, we identify that Tet1, one member of the ten-eleven translocation (TET) family of methylcytosine dioxygenases, is required for hypoxia tolerance in zebrafish and mice. Tet1-null zebrafish and mice are more sensitive to hypoxic conditions compared with their wild-type siblings. We demonstrate that Tet1 stabilizes hypoxia-inducible factor α (HIF-α) and enhances HIF-α transcription activity independent of its enzymatic activity. In addition, we show that Tet1 modulates HIF-2α and HIF-1α through different mechanisms. Tet1 competes with prolyl hydroxylase protein 2 (PHD2) to bind to HIF-2α, resulting in a reduction of HIF-2α hydroxylation by PHD2. For HIF-1α, however, Tet1 has no effect on HIF-1α hydroxylation, but rather it appears to stabilize the C-terminus of HIF-1α by affecting lysine site modification. Furthermore, we found that Tet1 enhances rather than prevents poly-ubiquitination on HIF-α. Our results reveal a previously unrecognized function of Tet1 independent of its methylcytosine dioxygenase activity in hypoxia signaling.