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Abnormality of enhancer regulation has emerged as one of the critical features for cancer cells. KDM5C is a histone H3K4 demethylase and frequently mutated in several types of cancer. It is critical for H3K4me3 and activity of enhancers, but its regulatory mechanisms remain elusive. Here, we identify TRIM11 as one ubiquitin E3 ligase for KDM5C. TRIM11 interacts with KDM5C, catalyzes K48-linked ubiquitin chain on KDM5C, and promotes KDM5C degradation through proteasome. TRIM11 deficiency in an animal model represses the growth of breast tumor and stabilizes KDM5C. In breast cancer patient tissues, TRIM11 is highly expressed and KDM5C is lower expressed, and their expression is negatively correlated. Mechanistically, TRIM11 regulates the enhancer activity of genes involved in cell migration and immune response by targeting KDM5C. TRIM11 and KDM5C regulate MCAM expression and cell migration through targeting H3K4me3 on MCAM enhancer. Taken together, our study reveals novel mechanisms for enhancer regulation during breast cancer tumorigenesis and development.

MLL3 is a histone H3K4 methyltransferase that is frequently mutated in cancer, but the underlying molecular mechanisms remain elusive. Here, we found that MLL3 depletion by CRISPR/sgRNA significantly enhanced cell migration, but did not elevate the proliferation rate of cancer cells. Through RNA-Seq and ChIP-Seq approaches, we identified TNS3 as the potential target gene for MLL3. MLL3 depletion caused downregulation of H3K4me1 and H3K27ac on an enhancer ~ 7 kb ahead of TNS3. 3C assay indicated the identified enhancer interacts with TNS3 promoter and repression of enhancer activity by dCas9-KRAB system impaired TNS3 expression. Exogenous expression of TNS3 in MLL3 deficient cells completely blocked the enhanced cell migration phenotype. Taken together, our study revealed a novel mechanism for MLL3 in suppressing cancer, which may provide novel targets for diagnosis or drug development.

Gene transcription is coordinately regulated by multiple transcription factors. However, a systematic approach is still lacking to identify co-regulators for transcription factors. Here, we performed ChIP-Seq analysis and predicted the regulators for p53-mediated transcription process, from which we confirmed the roles of GLIS2, MAZ and MEF2A in regulating p53 target genes. We revealed that GLIS2 selectively regulates the transcription of PUMA but not p21. GLIS2 deficiency caused the elevation of H3K27ac and p53 binding on the PUMA enhancer, and promoted PUMA expression. It increased the rate of apoptosis, but not cell cycle. Moreover, GLIS2 represses H3K27ac level on enhancers, regulates the gene expression related with focal adhesion and promotes cell migration, through inhibiting p300. Big data analysis supports GLIS2 as an oncogene in colon cancer, and perhaps other cancers. Taken together, we have predicted candidates for p53 transcriptional regulators, and provided evidence for GLIS2 as an oncogene through repressing enhancer activation.

In eukaryote cells, core components of chromatin, such as histones and DNA, are packaged in nucleus. Leakage of nuclear materials into cytosol will induce pathological effects. However, the underlying mechanisms remain elusive. Here, cytoplasmic localization of nuclear materials induced by chromatin dysregulation (CLIC) in mammalian cells is reported. H3K9me3 inhibition by small chemicals, HP1α knockdown, or knockout of H3K9 methylase SETDB1, induces formation of cytoplasmic puncta containing histones H3.1, H4 and cytosolic DNA, which in turn activates inflammatory genes and autophagic degradation. Autophagy deficiency rescues H3 degradation, and enhances the activation of inflammatory genes. MRE11, a subunit of MRN complex, enters cytoplasm after heterochromatin dysregulation. Deficiency of MRE11 or NBS1, but not RAD50, inhibits CLIC puncta in cytosol. MRE11 depletion represses tumor growth enhanced by HP1α deficiency, suggesting a connection between CLIC and tumorigenesis. This study reveals a novel pathway that heterochromatin dysregulation induces translocation of nuclear materials into cytoplasm, which is important for inflammatory diseases and cancer., The current study reveals a novel pathway leading to cytoplasmic localization of nuclear material induced by chromatin dysregulation (CLIC). HP1α knockdown, SETDB1 knockout or H3K9me3 inhibition by drugs, induces cytoplasmic puncta containing histones H3.1, H4 and DNA, which is regulated by MRE11 and NBS1. CLIC activates inflammatory gene expression, γH2AX formation, and autophagy, and is related with inflammation and tumorigenesis.

Fuzhuan Tea (FZT) protects against obesity and obesity-related disorders. However, little is known about its effects on high-sugar toxicity in Caenorhabditis elegans. Here, we find that FZT limits the life-shortening effects of a high-sugar (glucose, sucrose, galactose, and fructose) diet, but does not extend lifespan on a normal diet or protect against oxidative stress. FZT also can prolong healthspan and alleviate the detrimental effects of a glucose-rich diet. Furthermore, we report that effects of FZT on glucose toxicity and advanced glycation end products (AGEs) damage can be dissociated. Notably, FZT is dependent on DAF-16/FOXO and the insulin/IGF-1 signaling (IIS) pathway to promote lifespan and decrease fat content on a glucose-rich diet; concomitantly, FZT reduces AGEs levels by regulating SKN-1/Nrf and p38/mitogen-activated protein kinase (MAPK) pathway, independent of DAF-16/FOXO and IIS pathway. Our findings indicate that FZT protects against high glucose toxicity by a mechanism other than protection against AGEs damage.

Hippo pathway is involved in tumorigenesis, and its regulation in cytosol has been extensively studied, but its regulatory mechanisms in the nuclear are not clear. In the current study, using a FBS-inducing model following serum starvation, we identified KDM3A, a demethylase of histone H3K9me1/2, as a positive regulator for hippo target genes. KDM3A promotes gene expression through two mechanisms, one is to upregulate YAP1 expression, and the other is to facilitate H3K27ac on the enhancers of hippo target genes. H3K27ac upregulation is more relevant with gene activation, but not H3K4me3; and KDM3A depletion caused H3K9me2 upregulation mainly on TEAD1-binding enhancers rather than gene bodies, further resulting in H3K27ac decrease, less TEAD1 binding on enhancers and impaired transcription. Moreover, KDM3A is associated with p300 and required for p300 recruitment to enhancers. KDM3A deficiency delayed cancer cell growth and migration, which was rescued by YAP1 expression. KDM3A expression is correlated with YAP1 and hippo target genes in colorectal cancer patient tissues, and may serve as a potential prognosis mark. Taken together, our study reveals novel mechanisms for hippo signaling and enhancer activation, which is critical for tumorigenesis of colorectal cancer.

SETDB1, a histone H3K9 methyltransferase, has been reported to be upregulated in a variety of tumors and promotes cancer development. However, the exact pathogenesis of SETDB1 in human colorectal cancer (CRC) is hitherto unknown. Here, we showed that SETDB1 expression was highly amplified in CRC. Functionally, SETDB1 downregulation in SW480 and HCT116 cells reduced cell proliferation, migration, invasion, and increased CRC cells apoptosis. In contrast, SETDB1 overexpression promoted CRC cells proliferation, migration, and invasion. High expression of SETDB1 was associated with a more aggressive phenotype in vitro. Flow cytometry showed that cell cycle was arrested in G1 phase after SETDB1 silencing. Furthermore, depletion of SETDB1 in vivo suppressed CRC cells proliferation. Mechanistically, p21 was identified as the target of SETDB1. After transfected with siSETDB1, expression of p21 was distinctly increased. In contrast, expression of p21 was significantly decreased after overexpression SETDB1. We also showed that SETDB1 could be involved in the regulation of epithelial–mesenchymal transition (EMT) in HCT116 cells. Moreover, we confirmed that SETDB1 could regulate the activity of p21 promoter by dual-luciferase repoter assay, and proved that SETDB1 could bind to the promoter of p21 and regulate its H3K9me3 enrichment level by ChIP-PCR experiment. Finally, we verified that silencing of SETDB1 inhibited CRC tumorigenesis in vivo. In conclusion, our results indicate that SETDB1 is a major driver of CRC development and might provide a new therapeutic target for the clinical treatment of CRC.

Enhancers are cis-acting elements with many sites bound by transcription factors and activate transcription over long distance. Histone modifications are critical for enhancer activity and utilized as hallmarks for the identification of putative enhancers. Monomethylation of histone H3 lysine 4 (H3K4me1) is the mark for enhancer priming; acetylation of histone H3 lysine 27 (H3K27ac) for active enhancers and trimethylation of histone H3 lysine 27 (H3K27me3) for silent enhancers. Recent studies from multiple groups have provided evidence that enhancer reprogramming, especially gain of enhancer activity, is closely related to tumorigenesis and cancer development. In this review, we will summarize the recent discoveries about enhancer regulation and the mechanisms of enhancer reprogramming in tumorigenesis, and discuss the potential application of enhancer manipulation in precision medicine.

SPOP is one of the important subunits for CUL3/SPOP/RBX1 complex tightly connected with tumorigenesis. However, its exact roles in different cancers remain debatable. Here, we identify CYCLIN E1, as a novel substrate for SPOP. SPOP directly interacts with CYCLIN E1 and specific regulates its stability in prostate cancer cell lines. SPOP/CUL3/RBX1 complex regulates CYCLIN E1 stability through poly-ubiquitination. CDK2 competes with SPOP for CYCLIN E1 interaction, suggesting that SPOP probably regulates the stability of CDK2-free CYCLIN E1. CYCLIN E1 expression rescued proliferation, migration, and tumor formation of prostate cancer cell suppressed by SPOP. Furthermore, we found SPOP selectively regulates the substrates’ stability and signaling pathways in prostate cancer and CCRC cell lines, suggesting that complicated mechanisms exist for SPOP to regulate substrate specificity. Altogether, we have revealed a novel mechanism for SPOP in suppressing prostate cancer and provided evidence to show SPOP has dual functions in prostate cancer and CCRC.

Although the exact etiology of inflammatory bowel disease (IBD) remains unclear, exaggerated immune response in genetically predisposed individuals has been reported. Th1 and Th17 cells mediate IBD development. Macrophages produce IL-12 and IL-23 that share p40 subunit encoded by IL12B gene as heteromer partner to drive Th1 and Th17 differentiation. The available animal and human data strongly support the pathogenic role of IL-12/IL-23 in IBD development and suggest that blocking p40 might be the potential strategy for IBD treatment. Furthermore, aberrant alteration of some cytokines expression via epigenetic mechanisms is involved in pathogenesis of IBD. In this study, we analyzed core promoter region of IL12B gene and investigated whether IL12B expression could be regulated through targeted epigenetic modification with gene editing technology. Transcription activator-like effectors (TALEs) are widely used in the field of genome editing and can specifically target DNA sequence in the host genome. We synthesized the TALE DNA-binding domains that target the promoter of human IL12B gene and fused it with the functional catalytic domains of epigenetic enzymes. Transient expression of these engineered enzymes demonstrated that the TALE-DNMT3A targeted the selected IL12B promoter region, induced loci-specific DNA methylation, and down-regulated IL-12B expression in various human cell lines. Collectively, our data suggested that epigenetic editing of IL12B through methylating DNA on its promoter might be developed as a potential therapeutic strategy for IBD treatment.

ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the misfolded proteins are dislocated from ER membrane into cytosol, which requires the AAA ATPase p97/VCP and its cofactor, the NPL4-UFD1 dimer. Here, we performed a CRISPR-based screen and identify many candidates for ERAD regulation. We further confirmed four proteins, FBOX2, TRIM6, UFL1 and WDR20, are novel regulators for ERAD. Then the molecular mechanism for WDR20 in ERAD is further characterized. Depletion of WDR20 inhibits the degradation of TCRα, a typical ERAD substrate, while WDR20 overexpression reduces TCRα protein level. WDR20 associates with TCRα and central regulators of the ERAD system, p97, GP78 and HRD1. A portion of WDR20 localizes to the ER-containing microsomal membrane. WDR20 expression increases TCRα ubiquitination, and HRD1 E3 ligase is essential for the process. WDR20 seems to serve as an adaptor protein to mediate the interaction between p97 and TCRα. Our study provides novel candidates and reveals an unexpected role of WDR20 in ERAD regulation.

Background/Aims: Angiotensin II (Ang II)-mediated hypertension is a major risk factor for cardiovascular diseases. Ang II induces changes in vessel structure and function through the activation of genes related to signaling pathways. However, the changes in the gene expression profiles of blood vessels in response to Ang II remain unclear. Methods: Wild-type C57BL/6 mice were infused with Ang II (1500 ng/kg/min) using an osmotic pump for 1, 3, and 7 days. Vascular wall inflammation and remodeling were evaluated by pathological examination. Time-series microarray and quantitative PCR analyses were performed. Bioinformatics analyses were conducted to identify key genes, pathways, and biological processes. Results: After Ang II infusion, blood pressure and aortic remodeling were increased over time. Microarray analysis identified a totally of 3631 differentially expressed genes in aortas at days 1, 3, and 7 of Ang II infusion. These genes were involved in multiple biological processes, including cell adhesion, angiogenesis, cell migration, protein phosphorylation, immune system, and cell cycle, which may play important roles in regulating Ang II-induced arterial injury during hypertension. The genes were classified into 50 profiles by hierarchical cluster analysis, and finally, 14 significant profiles were identified. Among these genes, protein kinase cAMP-activated catalytic subunit alpha (Prkaca), a gene that directly regulated 137 neighboring genes, was located at the center of the gene network in Ang II-infused aortas. Further, Prkaca protein expression and cAMP level were downregulated in a time-dependent manner in Ang II-infused aortas. Conclusions: The combined use of DNA microarrays and cluster and gene network analyses identified Prkaca as a key Ang II-responsive gene that may mediate early vascular injury and hypertension.

Under RNA virus infection, retinoic acid-inducible gene I (RIG-I) in host cells recognizes viral RNA and activates the expression of type I IFN. To investigate the roles of protein methyltransferases and demethylases in RIG-I antiviral signaling pathway, we screened all the known related enzymes with a siRNA library and identified LSD1 as a positive regulator for RIG-I signaling. Exogenous expression of LSD1 enhances RIG-I signaling activated by virus stimulation, whereas its deficiency restricts it. LSD1 interacts with RIG-I, promotes its K63-linked polyubiquitination and interaction with VISA/MAVS. Interestingly, LSD1 exerts its function in antiviral response not dependent on its demethylase activity but through enhancing the interaction between RIG-I with E3 ligases, especially TRIM25. Furthermore, we provide in vivo evidence that LSD1 increases antiviral gene expression and inhibits viral replication. Taken together, our findings demonstrate that LSD1 is a positive regulator of signaling pathway triggered by RNA-virus through mediating RIG-I polyubiquitination., Author summary RIG-I signaling pathway is critical for human cells to defend from RNA virus infection, such as SARS-CoV-2, influenza virus, and Vesicular Stomatitis Virus (VSV). LSD1 is a histone demethylase regulating transcription. The current study reveals a novel function of LSD1 in regulating the activation of RIG-I signaling pathway. LSD1 interacts with RIG-I and promotes RIG-I poly-ubiquitination independent of its demethylase activity. LSD1 facilitates the interaction between RIG-I and its ubiquitin E3 ligase TRIM25, which is crucial for recruitment of downstream proteins. The mice with LSD1 deficiency are susceptible to virus infection and have lower survival rate. Taken together, our findings demonstrate a novel molecular mechanism for regulating the anti-viral RIG-I signaling pathway.

Histone positioning and modifications on viral genomes are important factors regulating virus replication. To investigate the dynamics of modified histones on the viral genome and their potential roles in antiviral response, we studied the dynamic changes of histone modifications across the HSV-1 genome in THP-1 cells. Histone modifications were detected on the HSV-1 genome soon after infection, including H3K9me3, H3K27me3, H3K4me3 and H3K27ac. These modifications emerged on the viral genome soon after infection and changed rapidly along with virus life cycle progression. The transcription repression marks, H3K9me3 and H3K27me3, decreased on the viral genome during the infection process; the transcription activation mark H3K27ac increased. Treatment with C646, an inhibitor of H3K27ac transferase p300, significantly repressed virus replication and viral gene expression. Our study reveals the relationship between histone modifications and viral gene expression and provides potential novel strategies for antiviral treatment.

Endometrial carcinoma (EC) is a female-specific malignant tumor. Although current treatments can achieve good outcomes and improve patient survival, there remains a high incidence of treatment-induced infertility, a serious side effect that is unacceptable to those of childbearing age. Studies have demonstrated that micro ribonucleic acids (microRNAs or miRNAs) such as miR-544a regulate tumor-related gene expression. However, whether miR-544a is involved in the progression of EC is unknown. This study aimed to investigate the biological functions and underlying mechanisms of miR-544a in EC in vivo and in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed miR-544a overexpression in EC tissue and cell lines, which was associated with a decreased in overall survival as revealed by Kaplan–Meier analysis. Functionally, the miR-544a inhibitor restricted the proliferation [detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay], invasion, and migration (detected by transwell assay) of human endometrial adenocarcinoma cells (HEC-1B and Ishikawa) and facilitated cell apoptosis (detected by flow cytometry assay). Western blotting analysis revealed that the miR-544a inhibitor decreased the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 and elevated the levels of cleaved caspase3 and cleaved poly (ADP-ribose) polymerase. Furthermore, animal experiments indicated that the miR-544a antagonist (antagomir-544a) suppressed tumor growth significantly in a mouse xenograft model. The mechanistic, qRT-PCR, and immunohistochemical indications were that a reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and miR-544a had inverse expression changes in EC. Bioinformatics analysis revealed RECK as a potential target for miR-544a, and this was verified by the dual-luciferase reporter assay. Subsequently, in vitro experiments, including transwell assay, MTT assay, flow cytometry assay, and Western blotting analysis, demonstrated that RECK exerted antitumor effects on EC, which were negatively regulated by miR-544a. Taken together, our study findings suggested miR-544a as a valuable target in EC therapy.

MicroRNAs (miRNAs) exhibit aberrant expression in the initiation and progression of a variety of human cancers, including colorectal cancer (CRC). However, the exact mechanisms are not well defined. miRNA expression profiles were characterized by microarrays in CRC samples, and miRNA 18b (miR-18b) was increased significantly in tumor tissues. The expression of miR-18b was confirmed in the CRC cell lines SW480 and HCT116 and 44 clinical specimens by quantitative real-time PCR (qRT-PCR). Multiple linear regression analysis showed a strong correlation of miR-18b expression with lymph node and distant metastasis. Overexpression of miR-18b promoted cell proliferation by facilitating cell cycle progression, and knockdown of miR-18b significantly suppressed migration in CRC cells. CDKN2B was identified as a target of miR-18b by high-throughput RNA sequencing and bioinformatics. After transfection with a miR-18b mimic, expression of CDKN2B was reduced significantly in CRC cells, and the effect was restored when a miR-18b inhibitor was transfected. A luciferase assay indicated miR-18b directly binds to the 3′ untranslated region (UTR) of CDKN2B. Expression of CDKN2B was downregulated in patient cancer tissues and negatively correlated with miR-18b. In a model of ectopic expression of miR-18b and CDKN2B, CDKN2B overexpression antagonized the effects of miR-18b in vitro and in vivo. The data show that miR-18b is involved in CRC carcinogenesis through targeting CDKN2B.

Odontoblasts derive from neural crest–derived odontogenic mesenchymal cells, and they are an important barrier of defense for the host. Survival and immunity of odontoblasts play important roles in protecting the dentin-pulp structure. Autophagy can eliminate damaged organelles and recycle cellular components to facilitate cellular homeostasis. Autophagy can be activated with external stressors, such as starvation, hypoxia, and infection. In this study, the role of autophagy in inflamed odontoblasts was explored, and its possible mechanism was investigated. Cell viability was not affected by mild lipopolysaccharide (LPS) stimulation, and autophagy was activated during this process. Immunofluorescence of light chain 3 confirmed that autophagy was induced with LPS treatment. Early-stage autophagy inhibition resulted in down-regulated cell viability, contrary to the up-regulated cell viability at late-stage autophagy inhibition. Western blot suggested that p-Akt and survivin were not activated in the early stage, and they gradually increased and peaked in the late stage. Meanwhile, autophagy was down-regulated through the Akt/mTOR/survivin pathway in the late stage. Thus, autophagy has a dual role in inflamed odontoblasts, which indicates its importance in maintaining the microenvironment homeostasis of odontoblasts. Autophagy was induced as a survival mechanism in the early stage, and it decreased through the Akt/mTOR/survivin signaling pathway in the late stage.

Epigenetic factors and related small molecules have emerged to be strongly involved in autophagy process. Here we report that 2-PCPA and GSK-LSD1, two inhibitors of histone H3K4 demethylase KDM1A/LSD1, induce autophagy in multiple mammalian cell lines. The two small molecules induce accumulation of LC3II, formation of autophagosome and autolysosome, and SQSTM1/p62 degradation. 2-PCPA treatment inhibits cell proliferation through cell cycle arrest but does not inducing cell death. Exogenous expression of KDM1A/LSD1 impaired the autophagic phenotypes triggered by 2-PCPA. The autophagy induced by 2-PCPA requires LC3-II processing machinery. But depletion of BECN1 and ULK1 with siRNA did not affect the LC3-II accumulation triggered by 2-PCPA. 2-PCPA treatment induces the change of global gene expression program, including a series of autophagy-related genes, such as SQSTM1/p62. Taken together, our data indicate that KDM1A/LSD1 inhibitors induce autophagy through affecting the expression of autophagy-related genes and in a BECN1-independent manner.

Unlike other members of the polycomb group protein family, EZH1 has been shown to positively associate with active transcription on a genome-wide scale. However, the underlying mechanism for this behavior still remains elusive. Here, we report that EZH1 physically interacts with UXT, a small chaperon-like transcription co-activator. UXT specifically interacts with EZH1 and SUZ12, but not EED. Similar to upon knockdown of UXT, knockdown of EZH1 or SUZ12 through RNA interference in the cell impairs the transcriptional activation of nuclear factor (NF)-κB target genes induced by TNFα. EZH1 deficiency also increases TNFα-induced cell death. Interestingly, chromatin immunoprecipitation and the following next-generation sequencing analysis show that H3K27 mono-, di- and tri-methylation on NF-κB target genes are not affected in EZH1- or UXT-deficient cells. EZH1 also does not affect the translocation of the p65 subunit of NF-κB (also known as RELA) from the cytosol to the nucleus. Instead, EZH1 and SUZ12 regulate the recruitment of p65 and RNA Pol II to target genes. Taken together, our study shows that EZH1 and SUZ12 act as positive regulators for NF-κB signaling and demonstrates that EZH1, SUZ12 and UXT work synergistically to regulate pathway activation in the nucleus.

Endoplasmic reticulum (ER)-associated degradation (ERAD) of unassembled T-cell receptor alpha-chain (TCRalpha) is reliant on the presence of two basic residues in the transmembrane (TM) segment of TCRalpha. The precise role of these residues in ER quality control is unclear. Here, we show that a TCRalpha mutant lacking these intramembrane charged residues has a tendency to form homooligomers through an interchain disulfide bond that involves a specific pair of cysteine residues. Covalent oligomerization of TCRalpha appears to stabilize it at the ER membrane. The presence of a single lysine residue at specific positions within the TCRalpha TM domain abolishes its oligomerization and causes its rapid degradation. Conversely, when TCRalpha oligomerization is induced by a bivalent compound, the degradation of TCRalpha is inhibited. Together, these results suggest that the intramembrane charged residues in TCRalpha do not function as a signal for substrate recognition in ERAD. Instead, their primary role is to reduce TCRalpha oligomerization, maintaining it in a retrotranslocation-competent state. Our results also suggest that the ERAD machinery is inefficient when coping with oligomerized substrates, indicating a requirement for chaperone-mediated protein disassembly in the ER lumen prior to retrotranslocation.

Transcription regulation emerged to be one of the key mechanisms in regulating autophagy. Inhibitors of H3K9 methylation activates the expression of LC3B, as well as other autophagy-related genes, and promotes autophagy process. However, the detailed mechanisms of autophagy regulated by nuclear factors remain elusive. In this study, we performed a drug screen of SMYD2-/- cells and discovered that SMYD2 deficiency enhanced the cell death induced by BIX01294, an inhibitor of histone H3K9 methylation. BIX-01294 induces accumulation of LC3 II and autophagy-related cell death, but not caspase-dependent apoptosis. We profiled the global gene expression pattern after treatment with BIX-01294, in comparison with rapamycin. BIX-01294 selectively activates the downstream genes of p53 signaling, such as p21 and DOR, but not PUMA, a typical p53 target gene inducing apoptosis. BIX-01294 also induces other autophagy-related genes, such as ATG4A and ATG9A. SMYD2 is a methyltransferase for p53 and regulates its transcription activity. Its deficiency enhances the BIX-01294-induced autophagy-related cell death through transcriptionally promoting the expression of p53 target genes. Taken together, our data suggest BIX-01294 induces autophagy-related cell death and selectively activates p53 target genes, which is repressed by SMYD2 methyltransferase.

Toll-like receptor 3 (TLR3) recognizes dsRNA generated during viral infection and activation of TLR3 results in induction of type I interferons (IFNs) and cellular anti-viral response. TLR3 is associated with a TIR domain-containing adapter protein TRIF, which activates distinct downstream pathways leading to activation of NF-kappaB and ISRE sites in the promoters of type I IFNs. We show here that A20, a NF-kappaB-inducible zinc finger protein that has been demonstrated to be an inhibitor of TNF-induced NF-kappaB activation and a physiological suppressor of inflammatory response, potently inhibited TLR3- and Sendai virus-mediated activation of ISRE and NF-kappaB and IFN-beta promoter in reporter gene assays. A20 also inhibited TRIF-, but not its downstream signaling components TBK1-, IKKbeta-, and IKKepsilon-mediated activation of ISRE and NF-kappaB and IFN-beta promoter. Moreover, A20 interacted with TRIF in co-immunoprecipitation experiments. Finally, expression of A20 could be induced at protein level by Sendai virus infection. These data suggest that A20 targets TRIF to inhibit TLR3-mediated induction of IFN-beta transcription and functions as a feedback negative regulator for TLR3 signaling and cellular anti-viral response.

The tumor necrosis factor receptor-associated factor (TRAF) protein family members are critically involved in activation of NF-kappaB, JNK, and p38 activation triggered by tumor necrosis factor (TNF) receptor family members and toll/interleukin-1 receptor (TIR)-containing receptors. TRAF proteins (except for TRAF1) contain an N-terminal RING finger domain that is essential for their functions. In this report, we identified a protein designated as TRAF7, which contains a RING finger domain and a zinc finger domain that are mostly conserved with those of TRAFs. TRAF7 also contains seven WD40 repeats at its C terminus. TRAF7 specifically interacted with MEKK3 and potentiated MEKK3-mediated AP1 and CHOP activation. Depletion of TRAF7 by antisense RNA inhibited MEKK3-mediated AP1 and CHOP activation. Moreover, overexpression of TRAF7 induced caspase-dependent apoptosis. Domain mapping experiments indicated that TRAF7 potentiated MEKK3-mediated AP1 and CHOP activation and induced apoptosis through distinct domains. Our studies identified a novel TRAF family member that is involved in MEKK3 signaling and apoptosis.

The transcription factor NFκB plays important roles in immune regulation, inflammatory responses, and anti-apoptosis. Activation of NFκB requires the activity of IκB kinase, a kinase complex that contains two catalytic subunits, IKKα and IKKβ, and a non-enzymatic regulatory subunit, IKKγ. To understand how NFκB activation is regulated at the IKKγ level, we searched for IKKγ-interacting proteins by the yeast two-hybrid system. This search identified ZNF216, a zinc finger protein with unknown biological functions. ZNF216 contains an A20-like zinc finger domain (ZnF-A20) at its N terminus and an AN1-like domain (ZnF-AN1) at its C terminus. Similar to A20, ZNF216 interacted with IKKγ, RIP, and TRAF6 in co-immunoprecipitation experiments. Domain mapping experiments indicated that the ZnF-A20 domain was responsible for interacting with IKKγ and RIP, whereas the ZnF-AN1 domain interacted with TRAF6. ZNF216 inhibited NFκB activation triggered by overexpression of RIP and TRAF6 but not of p65. ZNF216 also inhibited tumor necrosis factor (TNF)-, interleukin-1-, and Toll-like receptor 4-induced NFκB activation in a dose-dependent manner. The ZnF-A20 domain was essential for ZNF216-mediated inhibition of NFκB activation. The ZnF-A20 and ZnF-AN1 domains of ZNF216 could interact with each other, whereas ZNF216 could form homo-oligomers or hetero-oligomers with A20. Unlike A20, which inhibits TNF-induced apoptosis, overexpression of ZNF216 sensitized cells to TNF-induced apoptosis. Our findings suggest that ZNF216 and A20 have redundant and distinct roles in regulating NFκB activation and apoptosis.

Disrupting protein glycosylation induces ER (endoplasmic reticulum) stress, resulting in the activation of UPR (unfolded protein response) pathways. A key function of the UPR is to restore ER homeostasis, but prolonged or unsolved ER stress can lead to apoptosis. MLL1 (Mixed Lineage Leukemia 1, also named ALL-1 or HRX), a histone H3K4 methyltransferase in mammals, plays important roles in leukemogenesis, transcriptional regulation, cell cycle and development. Here, we find that Mll1 deficiency enhances UPR and apoptosis induced by the glycosylation inhibitor TM (tunicamycin). The abnormal regulation of the UPR appears to be caused by a defect in protein glycosylation. Furthermore, Mll1 directly binds to the promoters of H6pd , Galnt12 and Ugp2 , which regulates H3K4 trimethylation and the subsequent expression of these genes. The knockdown of H6pd , Galnt12 or Ugp2 enhances TM-induced apoptosis in Mll1 +/+ MEF cells, whereas the ectopic expression of these proteins inhibits TM-induced apoptosis in Mll1 −/− MEF cells. Together, our data suggest that the maturation of glycoproteins in the ER is subject to regulation at the epigenetic level by a histone methyltransferase whose abnormality can lead to cancer and developmental defects.

Genes of the mixed lineage leukemia (MLL) family regulate transcription by methylating histone H3K4. Six members of the MLL family exist in humans, including SETD1A, SETD1B and MLL1-MLL4. Each of them plays non-redundant roles in development and disease genesis. MLL1 regulates the cell cycle and the oscillation of circadian gene expression. Its fusion proteins are involved in leukemogenesis. Here, we studied the role of MLL1 in innate immunity and found it selectively regulates the activation of genes downstream of NF-κB mediated by tumor necrosis factor (TNFα) and lipopolysaccharide (LPS). Real-time PCR and genome-wide gene expression profile analysis proved that the deficiency of MLL1 reduced the expression of a group of genes downstream of nuclear factor κB (NF-κB). However, the activation of NF-κB itself was not affected. The MLL1 complex is found both in the nucleus and cytoplasm and is associated with NF-κB. CHIP assays proved that the translocation of MLL1 to chromatin was dependent on NF-κB. Our results suggest that MLL1 is recruited to its target genes by activated NF-κB and regulates their transcription.

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. Our previous observations demonstrate that F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. Here, we establish that NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases.