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The methyltransferase SETD3 is an enzyme essential for catalyzing histidine-73 methylation on β-Actin, thereby promoting its polymerization and regulating muscle contraction. Although increasing evidence suggests that SETD3 is involved in multiple physiological or pathological events, its biological functions remain incompletely understood. In this study, we utilize in situ proximity labeling combined with mass spectrometry analysis to detect potential interacting partners of SETD3. Unexpectedly, we find that many splicing factors are associated with SETD3. Genome-wide RNA sequencing reveals that SETD3 regulates pre-mRNA splicing events, predominantly influencing exon skipping. Biochemical and bioinformatic analyses suggest that SETD3 interacts with hnRNPK, and they collaboratively regulate exon skipping in a common subset of genes. Functionally, we demonstrate that SETD3 and hnRNPK are required for retention of exon 7 skipping in the FNIP1 gene. This promotes FNIP1-mediated nuclear translocation of the transcription factor TFEB and the subsequent induction of lysosomal and mitochondrial biogenesis. Overall, this study uncovers a novel function of SETD3 in modulating mRNA exon splicing.

Abstract Phosphorylation of Ser10 of histone H3 (H3S10p), together with the adjacent methylation of Lys9 (H3K9me), has been proposed to function as a ‘phospho‐methyl switch’ to regulate mitotic chromatin architecture. Despite of immense understanding of the roles of H3S10 phosphorylation, how H3K9me2 are dynamically regulated during mitosis is poorly understood. Here, it is identified that Plk1 kinase phosphorylates the H3K9me1/2 methyltransferase G9a/EHMT2 at Thr1045 (pT1045) during early mitosis, which attenuates its catalytic activity toward H3K9me2. Cells bearing Thr1045 phosphomimic mutant of G9a (T1045E) show decreased H3K9me2 levels, increased chromatin accessibility, and delayed mitotic progression. By contrast, dephosphorylation of pT1045 during late mitosis by the protein phosphatase PPP2CB reactivates G9a activity and upregulates H3K9me2 levels, correlated with decreased levels of H3S10p. Therefore, the results provide a mechanistic explanation of the essential of a ‘phospho‐methyl switch’ and highlight the importance of Plk1 and PPP2CB‐mediated dynamic regulation of G9a activity in chromatin organization and mitotic progression.

Methyltransferase Set2‐mediated methylation of histone H3 lysine 36 (H3K36), which involves the addition of up to three methyl groups at this site, has been demonstrated to function in many chromatin‐coupled events. The methylation of H3K36 is known to recruit different chromatin effector proteins, affecting transcription, mRNA splicing and DNA repair. In this study, we engineered two yeast set2 mutants that lack H3K36 mono/dimethylation (H3K36me1/2) and trimethylation (H3K36me3), respectively, and characterized their roles in the production of antisense transcripts under nutrient‐rich conditions. Using our new bioinformatics identification pipeline analysis, we are able to identify a larger number of antisense transcripts in set2∆ cells than has been published previously. We further show that H3K36me1/2 or H3K36me3 redundantly repressed the production of antisense transcripts. Moreover, gene ontology (GO) analysis implies that H3K36me3‐mediated antisense transcription might play a role in DNA replication and DNA damage repair, which is independent of regulation of the corresponding sense gene expression. Overall, our results validate a coregulatory mechanism of different H3K36 methylation states, particularly in the repression of antisense transcription.

BackgroundThe development of a new strategy to overcome chemoresistance to hepatocellular carcinoma (HCC) treatment is a long-standing issue. We have previously found that upregulated SETD3 levels are closely correlated with HCC. This study aims to explore the mechanism underlying how upregulation of SETD3 promotes liver carcinogenesis.MethodsRNA-Sequencing analysis was used to explore the correlation of SETD3 with regulatory targets. In vitro assays including cell proliferation and migration were performed to study the oncogenic roles of SETD3 and PLK1. Western blotting, immunohistochemical staining, and blood biochemical assays were performed to examine protein expression or pathological index in tumor tissues and mice liver tissues. Luciferase reporter system and chromatin immunoprecipitation assays were used to explore the mechanism.ResultsWe revealed that SETD3 regulates gene expression in subgroups, including cell division, cell proliferation, and cell cycle, in hepatocellular tumor cells. We found that SETD3 upregulation is associated with elevated PLK1 level in both hepatic tumor cells and clinical liver tissues. We further showed that overexpression of SETD3 promoted tumor cell proliferation and migration, whereas inhibition of PLK1 activity attenuated these phenotypes caused by SETD3. By taking advantage of the Sleep Beauty transposase system, we confirmed that upregulated mouse Setd3 promoted hepatic carcinogenesis in situ, but knockdown of mouse Plk1 mitigated Setd3-promoted tumorigenesis in mice. Mechanistically, we showed that SETD3 could be recruited to the promoter of PLK1 gene to facilitate PLK1 transcription.ConclusionsOur data demonstrate that elevated SETD3 may promote HCC by enhancing PLK1 expression, which suggests that SETD3 may act as a potential drug target combined with PLK1 inhibition to treat HCC.

Transient receptor potential vanilloid 2 (TRPV2) is a multimodal ion channel implicated in diverse physiopathological processes. Its important involvement in immune responses has been suggested such as in the macrophages’ phagocytosis process. However, the endogenous signaling cascades controlling the gating of TRPV2 remain to be understood. Here, we report that enhancing tyrosine phosphorylation remarkably alters the chemical and thermal sensitivities of TRPV2 endogenously expressed in rat bone marrow-derived macrophages and dorsal root ganglia (DRG) neurons. We identify that the protein tyrosine kinase JAK1 mediates TRPV2 phosphorylation at the molecular sites Tyr(335), Tyr(471), and Tyr(525). JAK1 phosphorylation is required for maintaining TRPV2 activity and the phagocytic ability of macrophages. We further show that TRPV2 phosphorylation is dynamically balanced by protein tyrosine phosphatase non-receptor type 1 (PTPN1). PTPN1 inhibition increases TRPV2 phosphorylation, further reducing the activation temperature threshold. Our data thus unveil an intrinsic mechanism where the phosphorylation/dephosphorylation dynamic balance sets the basal chemical and thermal sensitivity of TRPV2. Targeting this pathway will aid therapeutic interventions in physiopathological contexts.

Summary: TGF-β has been demonstrated to promote tumor metastasis, and the regulatory mechanisms are poorly understood. Here, we report the role of USP2a in promoting metastasis by facilitating TGF-β-triggered signaling. USP2a interacts with TGFBR1 and TGFBR2 upon TGF-β stimulation and removes K33-linked polyubiquitin chains from Lys502 of TGFBR1, promoting the recruitment of SMAD2/3. Simultaneously, TGFBR2 phosphorylates Ser207/Ser225 of USP2a, leading to the disassociation of SMAD2/3 from TGFBR1. The phosphorylation of USP2a and SMAD2 is positively correlated in human tumor biopsies, and USP2a is hyper-phosphorylated in lung adenocarcinomas with lymph node invasion. Depletion or pharmacologic inhibition of USP2a dampens TGF-β-triggered signaling and metastasis. Our findings have characterized an essential role of USP2a as a potential target for treatment of metastatic cancers. : Zhao et al. find that USP2a deconjugates K33-linked ubiquitination of TGFBR1 at Lys502 and is phosphorylated at Ser207/Ser225 by TGFBR2 after TGF-β stimulation. This allows R-SMAD recruitment to and subsequent disassociation from the TGFBR1/2 receptor complex. Keywords: USP2a, TGF-β, epithelial-to-mesenchymal transition, metastasis, ubiquitination, phosphorylation, SMAD2/3, TGFBR1/2, signaling transduction

Epigenetic alterations are a fundamental pathological hallmark of Alzheimer's disease (AD). Herein, we show the upregulation of G9a and H3K9me2 in the brains of AD patients. Interestingly, treatment with a G9a inhibitor (G9ai) in SAMP8 mice reversed the high levels of H3K9me2 and rescued cognitive decline. A transcriptional profile analysis after G9ai treatment revealed increased gene expression of glia maturation factor ß (GMFB) in SAMP8 mice. Besides, a H3K9me2 ChIP-seq analysis after G9a inhibition treatment showed the enrichment of gene promoters associated with neural functions. We observed the induction of neuronal plasticity and a reduction of neuroinflammation after G9ai treatment, and more strikingly, these neuroprotective effects were reverted by the pharmacological inhibition of GMFB in mice and cell cultures; this was also validated by the RNAi approach generating the knockdown of GMFB/Y507A.10 in Caenorhabditis elegans. Importantly, we present evidence that GMFB activity is controlled by G9a-mediated lysine methylation as well as we identified that G9a directly bound GMFB and catalyzed the methylation at lysine (K) 20 and K25 in vitro. Furthermore, we found that the neurodegenerative role of G9a as a GMFB suppressor would mainly rely on methylation of the K25 position of GMFB, and thus G9a pharmacological inhibition removes this methylation promoting neuroprotective effects. Then, our findings confirm an undescribed mechanism by which G9a inhibition acts at two levels, increasing GMFB and regulating its function to promote neuroprotective effects in age-related cognitive decline. [ABSTRACT FROM AUTHOR]

Research has been conducted on fibrilization and amyloid-like aggregation of alpha-synuclein. The authors discuss the importance of nine-residue peptide motif in fibrilization and aggregation of human alpha Syn.

Set2-mediated H3 Lys36 methylation is a histone modification that has been demonstrated to function in transcriptional elongation by recruiting the Rpd3S histone deacetylase complex to repress intragenic cryptic transcription. Recently, we identified a trans-histone pathway in which the interaction between the N terminus of Set2 and histone H4 Lys44 is needed to mediate trans-histone H3 Lys36 diand trimethylation. In the current study, we demonstrate that mutation of the lysine 44 residue in histone H4 or the Set2 mutant lacking the histone H4 interaction motif leads to intragenic cryptic transcripts, indicating that the Set2 and histone H4 interaction is important to repress intragenic cryptic transcription. We also determine that histone H2A residues (Leu116 and Leu117), which are in close proximity to histone H4 Lys44, are needed for proper trans-histone H3 Lys36 methylation. Similar to H4 Lys44 mutants, histone H2A Leu116 and Leu117 mutations exhibited decreased H3 Lys36 diand trimethylation, increased histone H4 acetylation, increased resistance to 6-azauracil, and cryptic transcription. Interestingly, the combined histone H4 Lys44 and H2A mutations have more severe methylation defects and increased H4 acetylation levels. Furthermore, we identify that additional histone H2A and H3 core residues are also needed for H3 Lys36 diand tnmethylation. Overall, our results show and suggest that multiple H4, H2A, and H3 residues contribute to and form a Set2 docking/recognition site on the nucleosomal surface so that proper Set2-mediated H3 Lys36 diand trimethylation, histone acetylation, and transcriptional elongation can occur. [ABSTRACT FROM AUTHOR]

This work examines the role of the Arabidopsis thaliana RING E3 ligase, HISTONE MONOUBIQUlTINATION1 (HUB1) disease resistance. Loss-of-function alleles of HUB1 show increased susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola, whereas HUB1 overexpression conferred resistance to B. cinerea. By contrast, responses to the bacterial pathogen Pseudomonas syringae are unaltered in hub1 plants, hub1 mutants have thinner cell walls but increased callose around an infection site. HUB1 acts independently of jasmonate, but ethylene (ET) responses and salicylate modulate the resistance of hub1 mutants to necrotrophic fungi. The ET response factor ETHYLENE INSENSITIVE2 is epistatic to HUB1 for A. brassicicola resistance but additive to HUB1 for B. cinerea resistance. HUB1 interacts with MED21, a subunit of the Arabidopsis Mediator, a conserved complex that regulates RNA polymerase II. RNA interference lines with reduced MED21 expression are highly susceptible to A. brassicicola and B. cinerea, whereas T-DNA insertion alleles are embryonic lethal, suggesting an essential role for MED21. However, HUB1-mediated histone H2B modification is independent of histone H3 and DNA methylation. In sum, histone H2B monoubiquitination is an important chromatin modification with regulatory roles in plant defense against necrotrophic fungi most likely through modulation of gene expression. [ABSTRACT FROM AUTHOR]

Fibrillization of α-synuclein (α-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkinson's disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit α-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of α-Syn chain, generating α-Syn–quinone adducts. It is likely that the amino groups of α-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked α-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact α-Syn, suggesting that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of α-Syn with DA is relevant to the selective dopaminergic loss in PD. [ABSTRACT FROM AUTHOR]

Amyloid-like aggregation or fibrillization of α-synuclein (α-Syn) and the filamentous deposits in Lewy bodies are believed to be closely associated with several fatal neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Here, we report the importance of a nine-residue peptide motif, [sup 66]VGGAVVTGV[sup 74], in the fibrillization and cytotoxicity of human α-Syn. Mutagenesis combined with thioflavin T fluorescence detection, atomic force microscopic imaging, and cytotoxicity assays reveal that deletion of this sequence completely eliminates α-Syn fibrillization and cell toxicity. However, deletion of the [sup 71]VTGV[sup 74] sequence decreases the fibrillization rate while the cytotoxicity remains unchanged. Incorporation of charged residues within this region slows aggregation and even impedes filament formation. In addition, substitution of Gly68 with Ala or C-terminal truncations of α-Syn accelerate the fibrillization processes. Circular dichroism studies suggest that β-sheet formation is often concomitant with filament formation. Thus, this segment, namely, the GAV motif, is responsible for aggregation or fibrillization of α-Syn and perhaps other amyloidogenic proteins. The oligomers formed during fibrillogenesis might be associated with the cytotoxicities of various α-Syn species. This finding may provide further insight into the understanding of the molecular mechanism underlying the fibrillogenesis implicated in neurodegeneration as well as aid in drug design and development of transgenic models. [ABSTRACT FROM AUTHOR]

本研究旨在探討讀者劇場對技術型高中餐飲科學生英語學習動機及學習成就之影響，並透過實驗組與控制組之比較，來檢視其差異。研究對象為臺北市某技術型高中餐飲科二年級學生共72 人，採用準實驗研究設計，隨機分派成實驗組與控制組，兩組課程內容均 以餐旅英文與會話課程之飲料服務與用餐服務兩單元為主題進行設計。實驗組採用讀者劇場方式進行教學，控制組則維持傳統教學模式。教學實驗歷經八週共十六節課，於教學前後施予測驗，再以單因子共變異分析探討英語學習動機及學習成就的差異，並佐以 小組討論紀錄與回饋評量。結果顯示實驗組學生在英語學習動機及學習成就上的表現優於控制組學生。實施讀者劇場讓學生透過小組練習和團隊合作，以學生為中心的教與學模式，提供學習者能有更多嘗試機會與一個練習舞台，不僅可有效增進學生的學習興趣、 降低口說焦慮並培養人際互動的社會能力，也能顯著提升學生的學習成就。 This study examined the influence of Readers Theatre strategies integrated into a hospitality English and conversation class on the English learning motivation and learning performance of vocational high school students. Using a quasi-experimental design, this study involved 72 fourteen-graders who were randomly assigned to either the experimental group or the control group. The former adopted Readers theatre teaching, a performance strategy of a play or a show, while the latter followed traditional teaching methods. Before and after the experiment, all participants filled out the English Learning Motivation and Achievement Test. Through data collected from the students’ responses and records, the results of one-way ANCOVA indicated that the experimental group outperformed the control group for the dependent variables. The advantages of Readers theatre over traditional models are that it utilizes a student-centered teaching and learning model. It includes more group practice and teamwork and allows students to try and exercise, and in this study it showed positive effects on the students’ English learning motivation and learning performance. Finally, several recommendations and suggestions were provided for further implementation as well as for forthcoming studies.

The article presents a study on methylation-phosphorylation switch. It outlines its identification of polo-like kinase 1 (Plk1) activity in repairing damaged DNA. It outlines the antagonization of phosphorylation by methyltransferase G9a monomethylates. An overview of the significant role of the kinase mutant K209me1 in the machinery of recovery is also provided.

Histone modifications, including lysine methylation, are epigenetic marks that influence many biological pathways. Accordingly, many methyltransferases have critical roles in various biological processes, and their dysregulation is often associated with cancer. However, the biological functions and regulation of many methyltransferases are unclear. Here, we report that a human homolog of the methyltransferase SET (SU(var), enhancer of zeste, and trithorax) domain containing 3 (SETD3) is cell cycle-regulated; SETD3 protein levels peaked in S phase and were lowest in M phase. We found that the-isoform of the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7β) specifically mediates SETD3 degradation. Aligning the SETD3 sequence with those of well known FBXW7 substrates, we identified six potential non-canonical Cdc4 phosphodegrons (CPDs), and one of them, CPD1, is primarily phosphorylated by the kinase glycogen synthase kinase 3 (GSK3β), which is required for FBXW7β-mediated recognition and degradation. Moreover, depletion or inhibition of GSK3β or FBXW7β resulted in elevated SETD3 levels. Mutations of the phosphorylated residues in CPD1 of SETD3 abolished the interaction between FBXW7β and SETD3 and prevented SETD3 degradation. Our data further indicated that SETD3 levels positively correlated with cell proliferation of liver cancer cells and liver tumorigenesis in a xenograft mouse model, and that overexpression of FBXW7β counteracts the SETD3's tumorigenic role. We also show that SETD3 levels correlate with cancer malignancy, indicated by SETD3 levels that the 54 liver tumors are 2-fold higher than those in the relevant adjacent tissues. Collectively, these data elucidated that a GSK3β-FBXW7β-dependent mechanism controls SETD3 protein levels during the cell cycle and attenuates its oncogenic role in liver tumorigenesis. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis. [ABSTRACT FROM AUTHOR]