Most of dual-specificity protein phosphatases (DSPs) play an important role in the regulation of mitogenic signal transduction and controlling the cell cycle in response to extracellular stimuli. In this study, a novel human dual-specificity protein phosphatases gene named dual-specificity phosphatase 23 (DUSP23) was isolated by large-scale sequencing analysis of a human fetal brain cDNA library. Its cDNA was 726 bp in length, encoding a 150-amino acid polypeptide which contained a dual-specificity phosphatase catalytic (DSPc) domain but not a CDC25 homology (CH2) domain. Reverse transcription-PCR (RT-PCR) revealed that the DUSP23 was expressed in most fetal tissues and two adult tissues: testis and colon. Transient transfection experiment suggested that DUSP23 was localized in the cytoplasm of HEK293 cells. DUSP23 showed distinctive phosphatase activity toward p-nitrophenyl phosphate (pNPP), as well as oligopeptides containing phospho-tyrosine and phospho-threonine residues. Furthermore, DUSP23 could dephosphorylate p44ERK1 but not p38 and p54SAPKbeta in vitro. All the results indicated that DUSP23 was a novel protein phosphatase with dual substrate specificity.

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.

As a core component of the mitotic checkpoint complex, BubR1 has a modular organization of molecular functions, with KEN box and other motifs at the N terminus inhibiting the anaphase-promoting complex/cyclosome, and a kinase domain at the C terminus, whose function remains unsettled, especially at organismal levels. We generate knock-in BubR1 mutations in the Drosophila genome to separately disrupt the KEN box and the kinase domain. All of the mutants are homozygously viable and fertile and show no defects in mitotic progression. The mutants without kinase activity have an increased lifespan and phenotypic changes associated with attenuated insulin signaling, including reduced InR on the cell membrane, weakened PI3K and AKT activity, and elevated expression of dFoxO targets. The BubR1 kinase-dead mutants have a reduced cap cell number in female germaria, which can be rescued by expressing a constitutively active InR. We conclude that one major physiological role of BubR1 kinase in Drosophila is to modulate insulin signaling.

Lysine-specific demethylase 1 (LSD1) is well-known for its role in decommissioning enhancers during mouse embryonic stem cell (ESC) differentiation. Its role in gene promoters remains poorly understood despite its widespread presence at these sites. Here, we report that LSD1 promotes RNA polymerase II (RNAPII) pausing, a rate-limiting step in transcription regulation, in ESCs. We found the knockdown of LSD1 preferentially affects genes with higher RNAPII pausing. Next, we demonstrate that the co-localization sites of LSD1 and MYC, a factor known to regulate pause-release, are enriched for other RNAPII pausing factors. We show that LSD1 and MYC directly interact and MYC recruitment to genes co-regulated with LSD1 is dependent on LSD1 but not vice versa. The co-regulated gene set is significantly enriched for housekeeping processes and depleted of transcription factors compared to those bound by LSD1 alone. Collectively, our integrative analysis reveals a pleiotropic role of LSD1 in promoting RNAPII pausing.

RAP1 interacting factor 1 (Rif1) is highly expressed in mice embryos and mouse embryonic stem cells (mESCs). It plays critical roles in telomere length homeostasis, DNA damage, DNA replication timing and ERV silencing. However, whether Rif1 regulates early differentiation of mESC is still unclear.

Transposable elements, including endogenous retroviruses (ERVs), constitute a large fraction of the mammalian genome. They are transcriptionally silenced during early development to protect genome integrity and aberrant transcription. However, the mechanisms that control their repression are not fully understood. To systematically study ERV repression, we carried out an RNAi screen in mouse embryonic stem cells (ESCs) and identified a list of novel regulators. Among them, Rif1 displays the strongest effect. Rif1 depletion by RNAi or gene deletion led to increased transcription and increased chromatin accessibility at ERV regions and their neighboring genes. This transcriptional de-repression becomes more severe when DNA methylation is lost. On the mechanistic level, Rif1 directly occupies ERVs and is required for repressive histone mark H3K9me3 and H3K27me3 assembly and DNA methylation. It interacts with histone methyltransferases and facilitates their recruitment to ERV regions. Importantly, Rif1 represses ERVs in human ESCs as well, and the evolutionally-conserved HEAT-like domain is essential for its function. Finally, Rif1 acts as a barrier during somatic cell reprogramming, and its depletion significantly enhances reprogramming efficiency. Together, our study uncovered many previously uncharacterized repressors of ERVs, and defined an essential role of Rif1 in the epigenetic defense against ERV activation.

Ticks, blood-feeding arthropods, and secrete immunosuppressive molecules that inhibit host immune responses and provide survival advantages to pathogens. In this study, we characterized the immunosuppressive function of a novel tick salivary protein, DsCystatin, from Dermacentor silvarum of China. DsCystatin directly interacted with human Cathepsins L and B and inhibited their enzymatic activities. DsCystatin impaired the expression of inflammatory cytokines such as IL1β, IFNγ, TNFα, and IL6 from mouse bone marrow-derived macrophages (BMDMs) that had been stimulated with LPS or Borrelia burgdorferi. Consistently, DsCystatin inhibited the activation of mouse BMDMs and bone marrow-derived dendritic cells by downregulating the surface expression of CD80 and CD86. Mechanically, DsCystatin inhibited LPS- or B. burgdorferi-induced NFκB activation. For the first time, we identified that DsCystatin-attenuated TLR4 signaling by targeting TRAF6. DsCystatin enhanced LPS-induced autophagy, mediated TRAF6 degradation via an autophagy dependent manner, thereby impeded the downstream phosphorylation of IκBα and the nuclear transport of NFκB. Finally, DsCystatin relieved the joint inflammation in B. burgdorferi or complete Freund's adjuvant induced mouse arthritis models. These data suggested that DsCystatin is a novel immunosuppressive protein and can potentially be used in the treatment of inflammatory diseases.

Previous studies have implicated an essential role for UHRF1-mediated histone H3 ubiquitination in recruiting DNMT1 to replication sites for DNA maintenance methylation during S phase of the cell cycle. However, the regulatory mechanism on UHRF1-mediated histone ubiquitination is not clear. Here we present evidence that UHRF1 and USP7 oppositely control ubiquitination of histones H3 and H2B in S phase of the cell cycle and that DNMT1 binds both ubiquitinated H3 and H2B. USP7 knockout markedly increased the levels of ubiquitinated H3 and H2B in S phase, the association of DNMT1 with replication sites and importantly, led to a progressive increase of global DNA methylation shown with increased cell passages. Using DNMT3A/DNMT3B/USP7 triple knockout cells and various DNA methylation analyses, we demonstrated that USP7 knockout led to an overall elevation of DNA methylation levels. Mechanistic study demonstrated that USP7 suppresses DNMT1 recruitment and DNA methylation through its deubiquitinase activity and the interaction with DNMT1. Altogether our study provides evidence that USP7 is a negative regulator of global DNA methylation and that USP7 protects the genome from excessive DNA methylation by attenuating histone ubiquitination-dependent DNMT1 recruitment.

Transposable elements (TEs) through evolutionary exaptation have become an integral part of the human genome, offering ample regulatory sequences and shaping chromatin 3D architecture. While the functional impacts of TE-derived sequences on early embryogenesis have been recognized, their roles in malignancy are only starting to emerge. Here we show that many TEs, especially the pluripotency-related human endogenous retrovirus H (HERVH), are abnormally activated in colorectal cancer (CRC) samples. Transcriptional upregulation of HERVH is associated with mutations of several tumor suppressors, particularly ARID1A. Knockout of ARID1A in CRC cells leads to increased transcription at several HERVH loci, which involves compensatory contribution by ARID1B. Suppression of HERVH in CRC cells and patient-derived organoids impairs tumor growth. Mechanistically, HERVH transcripts colocalize with nuclear BRD4 foci, modulating their dynamics and co-regulating many target genes. Altogether, we uncover a critical role for ARID1A in restraining HERVH, whose abnormal activation can promote tumorigenesis by stimulating BRD4-dependent transcription.

Global DNA hypomethylation is a most common epigenetic alteration in cancer, but the mechanism remains elusive. Previous studies demonstrate that UHRF1 but not UHRF2 is required for mediating DNA maintenance methylation by DNMT1. Here we report unexpectedly a conserved function for UHRF1 and UHRF2: inhibiting de novo DNA methylation by functioning as E3 ligases promoting DNMT3A degradation. UHRF1/2 are frequently overexpressed in cancers and we present evidence that UHRF1/2 overexpression downregulates DNMT3A proteins and consequently leads to DNA hypomethylation. Abrogating this negative regulation on DNMT3A or overexpression of DNMT3A leads to increased DNA methylation and impaired tumor growth. We propose a working model that UHRF1/2 safeguards the fidelity of DNA methylation and suggests that UHRF1/2 overexpression is likely a causal factor for widespread DNA hypomethylation in cancer via suppressing DNMT3A.

In mammals it is unclear if UHRF1-mediated DNA maintenance methylation by DNMT1 is strictly dependent on histone H3K9 methylation. Here we have generated an Uhrf1 knockin (KI) mouse model that specifically abolishes the H3K9me2/3-binding activity of Uhrf1. The homozygous Uhrf1 KI mice are viable and fertile, and exhibit ∼10% reduction of DNA methylation in various tissues. The reduced DNA methylation occurs globally in the genome and does not restrict only to the H3K9me2/3 enriched repetitive sequences. In vitro UHRF1 binds with higher affinity to reconstituted nucleosome with hemi-methylated CpGs than that with H3K9me2/3, although it binds cooperatively to nucleosome with both modifications. We also show that the nucleosome positioning affects the binding of methylated DNA by UHRF1. Thus, while our study supports a role for H3K9 methylation in promoting DNA methylation, it demonstrates for the first time that DNA maintenance methylation in mammals is largely independent of H3K9 methylation.

Global DNA hypomethylation is a most common epigenetic alteration in human neoplasia. However, accumulative evidence shows that global DNA hypomethylation impacts tumorigenesis in a tissue-specific manner, promoting tumorigenesis in some but suppressing tumorigenesis in others including colorectal cancer. The underlying mechanisms, especially how DNA hypomethylation suppresses tumorigenesis, remain largely unknown. Here, we investigate how DNA hypomethylation affects intestinal tumorigenesis by using an Uhrf1 tandem tudor domain knockin mutant mouse model (Uhrf1ki/ki) that exhibits a moderate ~10% reduction of global DNA methylation. We found that both chemical-induced colorectal carcinogenesis and Apc loss of heterozygosity (LOH)-induced intestinal tumorigenesis are substantially suppressed in the Uhrf1 mutant mice. Furthermore, unlike Dnmt1 hypomorphic mice in which DNA hypomethylation suppresses the incidence of macroscopic intestinal tumors but promotes the formation of microadenoma in ApcMin/+ background, Uhrf1ki/ki/ApcMin/+ mice have markedly reduced incidence of both microadenoma and macroadenoma. DNA hypomethylation does not appear to affect Apc LOH, activation of the Wnt or Hippo pathway, or tumor cell proliferation, but acts cooperatively with activated Wnt pathway to enhance the caspase-3 gene expression, activation, and apoptosis. Furthermore, increased caspase-3 expression correlates with DNA hypomethylation within the caspase-3 enhancer regions. Taken together, we present a new mouse model for investigating the role of and the molecular mechanisms by which DNA hypomethylation suppresses intestinal tumorigenesis. Our finding that a moderate DNA hypomethylation is sufficient to suppress intestinal tumorigenesis by promoting caspase-3 expression and apoptosis sheds new light on DNA-methylation inhibitor-based colorectal cancer therapeutics.

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remain largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. PRMT5 was identified as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. PRMT5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that PRMT5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, mice were treated with middle cerebral artery occlusion with the PRMT5 inhibitor EPZ015666 and found that PRMT5 inhibition sustains protection against neuronal death in vivo. Together, these findings revealed a novel epigenetic mechanism of PRMT5 in cerebral ischemia and uncovered a potential target for neuroprotection.

Dispositional envy is distinguished by definition and neurally from episodic envy. While the neural correlates of episodic envy have been evaluated by specific tasks in previous studies, little is known about the structural neural basis of dispositional envy. In this study, we investigated the structural neural basis of dispositional envy underlying individual differences across two independent samples comprising a total of 100 young healthy adults. Firstly, 73 subjects' data (sample 1) was analyzed, and we assessed the association between regional gray matter volume (rGMV) and dispositional envy using voxel-based morphometry (VBM). Furthermore, we explored the role of emotional intelligence in the association between GMV and dispositional envy. VBM indicated that dispositional envy was positively correlated with GMV in the left dorsolateral prefrontal cortex (DLPFC) and superior temporal gyrus (STG). We also found that emotional intelligence partially mediated the association between DLPFC volume and dispositional envy. These results were replicated in another independent sample (Sample 2, n = 27). These results provide the first evidence that dispositional envy exhibits a structural neural correlation with the DLPFC and STG, and give a neutral explanation for why individuals with high emotional intelligence exhibit less envy.

Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment.

Mouse embryonic stem cells (mESCs) cycle in and out of a transient 2-cell (2C)-like totipotent state, driven by a complex genetic circuit involves both the coding and repetitive sections of the genome. While a vast array of regulators, including the multi-functional protein Rif1, has been reported to influence the switch of fate potential, how they act in concert to achieve this cellular plasticity remains elusive. Here, by modularizing the known totipotency regulatory factors, we identify an unprecedented functional connection between Rif1 and the non-canonical polycomb repressive complex PRC1.6. Downregulation of the expression of either Rif1 or PRC1.6 subunits imposes similar impacts on the transcriptome of mESCs. The LacO-LacI induced ectopic colocalization assay detects a specific interaction between Rif1 and Pcgf6, bolstering the intactness of the PRC1.6 complex. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis further reveals that Rif1 is required for the accurate targeting of Pcgf6 to a group of genomic loci encompassing many genes involved in the regulation of the 2C-like state. Depletion of Rif1 or Pcgf6 not only activates 2C genes such as Zscan4 and Zfp352, but also derepresses a group of the endogenous retroviral element MERVL, a key marker for totipotency. Collectively, our findings discover that Rif1 can serve as a novel auxiliary component in the PRC1.6 complex to restrain the genetic circuit underlying totipotent fate potential, shedding new mechanistic insights into its function in regulating the cellular plasticity of embryonic stem cells.

Bile acids (BAs) are closely related to nutrient supply and modified by gut microbiota. Gut microbiota perturbations shape BA composition, which further affects host metabolism.

Dengue virus (DENV) is the most important vector-borne virus globally. The safe and effective vaccines are still under development and there are no antiviral drugs for DENV induced diseases. In this study, we obtained five DENV1 isolates (DENV1 A to E) from the outbreak of dengue fever in 2014 of Guangzhou, China, and analyzed their replication efficiency and virulence in vitro and in vivo. The results suggested that among the five DENV1 strains, DENV1 B has the highest replication efficiency in both human and mosquito cells in vitro, also causes the highest mortality to suckling mice. Further study suggested that nonstructural proteins from DENV1B have higher capacity to suppress host interferon signaling. In addition, the NS2B3 protease from DENV1B has higher enzymatic activity compared with that from DENV1 E. Finally, we identified that the 64th amino acid of NS2A and the 55th amino acid of NS2B were two virulence determining sites for DENV1. This study provided new evidences of the molecular mechanisms of DENV virulence.

Viral infection activates innate immune pathways and interferons (IFNs) play a pivotal role in the outcome of a viral infection. Ubiquitin modifications of host and viral proteins significantly influence the progress of virus infection. Ubiquitin-conjugating enzyme E2s (UBE2) have the capacity to determine ubiquitin chain topology and emerge as key mediators of chain assembly.

In order to eliminate viral infections, hundreds of interferon-stimulated genes (ISGs) are induced via type I interferons (IFNs). However, the functions and mechanisms of most ISGs are largely unclear. A tripartite motif (TRIM) protein encoding gene TRIM69 is induced by dengue virus (DENV) infection as an ISG. TRIM69 restricts DENV replication, and its RING domain, which has the E3 ubiquitin ligase activity, is critical for its antiviral activity. An in vivo study further confirmed that TRIM69 contributes to the control of DENV infection in immunocompetent mice. Unlike many other TRIM family members, TRIM69 is not involved in modulation of IFN signaling. Instead, TRIM69 interacts with DENV Nonstructural Protein 3 (NS3) directly and mediates its polyubiquitination and degradation. Finally, Lys104 of NS3 is identified as the target of TRIM69-mediated ubiquitination. Our study demonstrates that TRIM69 restricts DENV replication by specifically ubiquitinating a viral nonstructural protein.