Nitric Oxide Regulates Protein Methylation during Stress Responses in Plants.

Methylation and nitric oxide (NO)-based S-nitrosylation are highly conserved protein posttranslational modifications that regulate diverse biological processes. In higher eukaryotes, PRMT5 catalyzes Arg symmetric dimethylation, including key components of the spliceosome. The Arabidopsis prmt5 mutant shows severe developmental defects and impaired stress responses. However, little is known about the mechanisms regulating the PRMT5 activity. Here, we report that NO positively regulates the PRMT5 activity through S-nitrosylation at Cys-125 during stress responses. In prmt5-1 plants, a PRMT5C125S transgene, carrying a non-nitrosylatable mutation at Cys-125, fully rescues the developmental defects, but not the stress hypersensitive phenotype and the responsiveness to NO during stress responses. Moreover, the salt-induced Arg symmetric dimethylation is abolished in PRMT5C125S/prmt5-1 plants, correlated to aberrant splicing of pre-mRNA derived from a stress-related gene. These findings define a mechanism by which plants transduce stress-triggered NO signal to protein methylation machinery through S-nitrosylation of PRMT5 in response to environmental alterations.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells.

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

Prmt5 promotes ciliated cell specification of airway epithelial progenitors via transcriptional inhibition of Tp63.

The epithelium of the pulmonary airway is composed of several distinct cell types that differentiate from common progenitor cells to provide defense against environmental insults. Epigenetic mechanisms regulating lineage differentiation of airway epithelial progenitors remain poorly understood. Protein arginine methyltransferase 5 (Prmt5) is a predominant type II arginine methyltransferase that methylates >85% of symmetric arginine residues. Here, we provide evidence for the function of Prmt5 in promoting ciliated cell fate specification of airway epithelial progenitors. We show that lung epithelial-specific deletion of Prmt5 resulted in a complete loss of ciliated cells, an increased number of basal cells, and ecotopic-expressed Tp63-Krt5+ putative cells in the proximal airway. We further identified that transcription factor Tp63 is a direct target of Prmt5, and Prmt5 inhibited Tp63 transcription expression through H4R3 symmetric dimethylation (H4R3sme2). Moreover, inhibition of Tp63 expression in Prmt5-deficient tracheal progenitors could partially restore the ciliated cell deficient phenotype. Together, our data support a model where Prmt5-mediated H4R3sme2 represses Tp63 expression to promote ciliated cell fate specification of airway progenitors.

Theanine transporters identified in tea plants (Camellia sinensis L.).

Theanine, a unique non-proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H+ -dependent. The tissue-specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root-to-bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root-to-shoot delivery in the tea plant.

Histone arginine methylation by Prmt5 is required for lung branching morphogenesis through repression of BMP signaling.

Branching morphogenesis is essential for the successful development of a functional lung to accomplish its gas exchange function. Although many studies have highlighted requirements for the bone morphogenetic protein (BMP) signaling pathway during branching morphogenesis, little is known about how BMP signaling is regulated. Here, we report that the protein arginine methyltransferase 5 (Prmt5) and symmetric dimethylation at histone H4 arginine 3 (H4R3sme2) directly associate with chromatin of Bmp4 to suppress its transcription. Inactivation of Prmt5 in the lung epithelium results in halted branching morphogenesis, altered epithelial cell differentiation and neonatal lethality. These defects are accompanied by increased apoptosis and reduced proliferation of lung epithelium, as a consequence of elevated canonical BMP-Smad1/5/9 signaling. Inhibition of BMP signaling by Noggin rescues the lung branching defects of Prmt5 mutant in vitro Taken together, our results identify a novel mechanism through which Prmt5-mediated histone arginine methylation represses canonical BMP signaling to regulate lung branching morphogenesis.

PRMT7 is involved in regulation of germ cell proliferation during embryonic stage.

Arginine methylation is one of the most important post-translational modifications which is catalyzed by protein arginine methyltransferases (PRMTs). Previous studies have demonstrated that Prmt5 plays important role in germ cell development. Prmt7 is the only family member responsible for mono-methylation of arginine residue. However, whether Prmt7 is also involved in germ cell development remains unclear. In this study, we find that PRMT7 is abundantly expressed in the male germ cells during embryonic stage (from E10.5). Depletion of Prmt7 results in the defect of germ cell proliferation during embryonic stage and the number of primordial germ cells is significantly reduced in Prmt7-/- mice at E11.5. We also find that the size of testes is reduced in Prmt7-/- mice at P5 with reduced germ cell number and the diameter of seminiferous tubules. Further study reveals that the expression of BMPs and TGF-ß singling pathway is significantly changed in germ cells of Prmt7-/- mice at E12.5. However, no defect of testes development is observed in adult Prmt7-/flox; Mvh-Cre mice. Collectively, this study demonstrates that Prmt7 plays roles in male germ cell proliferation during embryonic stages and it is not required for germ cell development postnatally.

Loss of the golgin GM130 causes Golgi disruption, Purkinje neuron loss, and ataxia in mice.

The Golgi apparatus lies at the heart of the secretory pathway where it is required for secretory trafficking and cargo modification. Disruption of Golgi architecture and function has been widely observed in neurodegenerative disease, but whether Golgi dysfunction is causal with regard to the neurodegenerative process, or is simply a manifestation of neuronal death, remains unclear. Here we report that targeted loss of the golgin GM130 leads to a profound neurological phenotype in mice. Global KO of mouse GM130 results in developmental delay, severe ataxia, and postnatal death. We further show that selective deletion of GM130 in neurons causes fragmentation and defective positioning of the Golgi apparatus, impaired secretory trafficking, and dendritic atrophy in Purkinje cells. These cellular defects manifest as reduced cerebellar size and Purkinje cell number, leading to ataxia. Purkinje cell loss and ataxia first appear during postnatal development but progressively worsen with age. Our data therefore indicate that targeted disruption of the mammalian Golgi apparatus and secretory traffic results in neuronal degeneration in vivo, supporting the view that Golgi dysfunction can play a causative role in neurodegeneration.

Phosphorylation of serine/arginine-rich splicing factor 1 at tyrosine 19 promotes cell proliferation in pediatric acute lymphoblastic leukemia.

Serine/arginine-rich splicing factor 1 (SRSF1) has been linked to various human cancers including pediatric acute lymphoblastic leukemia (ALL). Our previous study has shown that SRSF1 potentially contributes to leukemogenesis; however, its underlying mechanism remains unclear. In this study, leukemic cells were isolated from pediatric ALL bone marrow samples, followed by immunoprecipitation assays and mass spectrometry analysis specific to SRSF1. Subcellular localization of the SRSF1 protein and its mutants were analyzed by immunofluorescence staining. Cell growth, colony formation, cell apoptosis, and the cell cycle were investigated using stable leukemic cell lines generated with lentivirus-mediated overexpressed WT or mutant plasmids. Cytotoxicity of the Tie2 kinase inhibitor was also evaluated. Our results showed the phosphorylation of SRSF1 at tyrosine 19 (Tyr-19) was identified in newly diagnosed ALL samples, but not in complete remission or normal control samples. Compared to the SRSF1 WT cells, the missense mutants of the Tyr-19 phosphorylation affected the subcellular localization of SRSF1. In addition, the Tyr-19 phosphorylation of SRSF1 also led to increased cell proliferation and enhanced colony-forming properties by promoting the cell cycle. Remarkably, we further identified the kinase Tie2 as a potential therapeutic target in leukemia cells. In conclusion, we identify for the first time that the phosphorylation state of SRSF1 is linked to different phases in pediatric ALL. The Tyr-19 phosphorylation of SRSF1 disrupts its subcellular localization and promotes proliferation in leukemia cells by driving cell-cycle progression. Inhibitors targeting Tie2 kinase that could catalyze Tyr-19 phosphorylation of SRSF1 offer a promising therapeutic target for treatment of pediatric ALL.

Histone Arginine Methylation by PRMT7 Controls Germinal Center Formation via Regulating Bcl6 Transcription.

B cells are the center of humoral immunity and produce Abs to protect against foreign Ags. B cell defects lead to diseases such as leukemia and lymphomas. Histone arginine methylation is important for regulating gene activation and silencing in cells. Although the process commonly exists in mammalian cells, its roles in B cells are unknown. To explore the effects of aberrant histone arginine methylation on B cells, we generated mice with a B cell-specific knockout of PRMT7, a member of the methyltransferases that mediate arginine methylation of histones. In this article, we showed that the loss of PRMT7 led to decreased mature marginal zone B cells and increased follicular B cells and promoted germinal center formation after immunization. Furthermore, mice lacking PRMT7 expression in B cells secreted low levels of IgG1 and IgA. Abnormal expression of germinal center genes (i.e., Bcl6, Prdm1, and Irf4) was detected in conditional knockout mice. By overexpressing PRMT7 in the Raji and A20 cell lines derived from B cell lymphomas, we validated the fact that PRMT7 negatively regulated Bcl6 expression. Using chromatin immunoprecipitation-PCR, we found that PRMT7 could recruit H4R3me1 and symmetric H4R3me2 to the Bcl6 promoter. These results provide evidence for the important roles played by PRMT7 in germinal center formation.

Akt Phosphorylates Wnt Coactivator and Chromatin Effector Pygo2 at Serine 48 to Antagonize Its Ubiquitin/Proteasome-mediated Degradation.

Pygopus 2 (Pygo2/PYGO2) is an evolutionarily conserved coactivator and chromatin effector in the Wnt/ß-catenin signaling pathway that regulates cell growth and differentiation in various normal and malignant tissues. Although PYGO2 is highly overexpressed in a number of human cancers, the molecular mechanism underlying its deregulation is largely unknown. Here we report that Pygo2 protein is degraded through the ubiquitin/proteasome pathway and is posttranslationally stabilized through phosphorylation by activated phosphatidylinositol 3-kinase/Akt signaling. Specifically, Pygo2 is stabilized upon inhibition of the proteasome, and its intracellular level is regulated by Cullin 4 (Cul4) and DNA damage-binding protein 1 (DDB1), components of the Cul4-DDB1 E3 ubiquitin ligase complex. Furthermore, Pygo2 is phosphorylated at multiple residues, and Akt-mediated phosphorylation at serine 48 leads to its decreased ubiquitylation and increased stability. Finally, we provide evidence that Akt and its upstream growth factors act in parallel with Wnt to stabilize Pygo2. Taken together, our findings highlight chromatin regulator Pygo2 as a common node downstream of oncogenic Wnt and Akt signaling pathways and underscore posttranslational modification, particularly phosphorylation and ubiquitylation, as a significant mode of regulation of Pygo2 protein expression.

SKB1/PRMT5-mediated histone H4R3 dimethylation of Ib subgroup bHLH genes negatively regulates iron homeostasis in Arabidopsis thaliana.

Histone modifications play critical roles in the perception of environmental cues by plants. Here, we report that Shk1 binding protein 1 (SKB1/AtPRMT5), which catalyzes the symmetric dimethylation of histone H4R3 (H4R3sme2), is involved in iron homeostasis in Arabidopsis. The SKB1 lesion mutant exhibited higher iron accumulation in shoots and greater tolerance to iron deficiency than the wild type. The expression of SKB1 was not affected by iron, but the level of H4R3sme2 mediated by SKB1 was related to iron status in plants. We showed by chromatin immunoprecipitation (ChIP) and genome-wide ChIP-seq that SKB1 associated with the chromatin of the Ib subgroup bHLH genes (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101), and symmetrically dimethylated histone H4R3. The quantity of SKB1 that associated with chromatin of the Ib subgroup bHLH genes and the level of H4R3sme2 corresponded to the iron status of plants (higher with increased iron supply and lower when iron was removed). We conclude that SKB1-mediated H4R3sme2 regulates iron homeostasis in Arabidopsis in the context of increasing or decreasing expression of Ib subgroup bHLH genes. Iron deficiency may cause an increase in the disassociation of SKB1 from chromatin of the bHLH genes and a decrease in the level of H4R3sme2, thereby elevating their transcription and enhancing iron uptake. Our findings provide new insight into the molecular mechanisms of iron homeostasis in strategy I plants.

Protein arginine methyltransferase 5 (Prmt5) is required for germ cell survival during mouse embryonic development.

In mammals, germ cells undergo massive epigenetic remodeling during fetal development. However, the physiological functions of epigenetic modification in germ cell development remain unclear. In this study, we found that protein arginine methyltransferase 5 (Prmt5) was abundantly expressed in the germ cells of both male and female gonads. Deletion of Prmt5 by crossing with Tnap-Cre mice resulted in germ cell depletion in adult mice. Germ cell loss was first observed between Embryonic Days 12.5 and 13.5 (E12.5 and E13.5), and very few of these cells remained at birth. Oct4, Sox2, and Nanog were abundantly expressed in Prmt5-deficient germ cells at E13.5 and E15.5, whereas the expression of these genes was dramatically decreased in control germ cells. Interestingly, the expression of meiosis-associated genes was virtually absent in Prmt5-deficient female germ cells at E13.5, whereas the expression of other germ cell-specific genes was not changed. Further study revealed that H4R3me2s was completely absent after Prmt5 inactivation, whereas the level of H3R2me2s was not changed in Prmt5-deficient germ cells. Collectively, this study demonstrated that Prmt5 plays critical roles in germ cell development that are required for germ cell survival during embryonic stages.

NF-kappa B mediated up-regulation of CCCTC-binding factor in pediatric acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most frequently occurring malignant neoplasm in children. Despite advances in treatment and outcomes for ALL patients, the pathogenesis of the disease remains unclear. Microarray analysis of samples from 100 Chinese children with ALL revealed the up-regulation of CTCF (CCCTC binding factor). CTCF is a highly conserved 11-zinc finger protein that is involved in many human cancers; however, the biological function of CTCF in pediatric ALL is unknown. METHODS: The expression patterns of CTCF were evaluated in matched newly diagnosed (ND), complete remission (CR), and relapsed (RE) bone marrow samples from 28 patients. The potential oncogenic mechanism of CTCF and related pathways in leukemogenesis were investigated in leukemia cell lines. RESULTS: We identified significant up-regulation of CTCF in the ND samples. Importantly, the expression of CTCF returned to normal levels after CR but rebounded in the RE samples. In the pre-B ALL cell line Nalm-6, siRNA-mediated silencing of CTCF expression promoted cell apoptosis and reduced cell proliferation; accordingly, over-expression of a cDNA encoding full-length CTCF protected cells from apoptosis and enhanced cell proliferation. Furthermore, inhibition or activation of the nuclear factor-kappa B (NF-κB) pathway resulted in marked variations in the levels of CTCF mRNA and protein in leukemic cells, indicating that CTCF may be involved downstream of the NF-κB pathway. Moreover, inhibition of the NF-κB pathway increased cell apoptosis, which was partially rescued by ectopic over-expression of CTCF, suggesting that CTCF may play a significant role in the anti-apoptotic pathway mediated by NF-κB. CONCLUSIONS: Our results indicate that CTCF serves as both an anti-apoptotic factor and a proliferative factor in leukemic cells. It potentially contributes to leukemogenesis through the NF-κB pathway in pediatric ALL patients.

Arabidopsis floral initiator SKB1 confers high salt tolerance by regulating transcription and pre-mRNA splicing through altering histone H4R3 and small nuclear ribonucleoprotein LSM4 methylation.

Plants adapt their growth and development in response to perceived salt stress. Although DELLA-dependent growth restraint is thought to be an integration of the plant's response to salt stress, little is known about how histone modification confers salt stress and, in turn, affects development. Here, we report that floral initiator Shk1 kinase binding protein1 (SKB1) and histone4 arginine3 (H4R3) symmetric dimethylation (H4R3sme2) integrate responses to plant developmental progress and salt stress. Mutation of SKB1 results in salt hypersensitivity, late flowering, and growth retardation. SKB1 associates with chromatin and thereby increases the H4R3sme2 level to suppress the transcription of FLOWERING LOCUS C (FLC) and a number of stress-responsive genes. During salt stress, the H4R3sme2 level is reduced, as a consequence of SKB1 disassociating from chromatin to induce the expression of FLC and the stress-responsive genes but increasing the methylation of small nuclear ribonucleoprotein Sm-like4 (LSM4). Splicing defects are observed in the skb1 and lsm4 mutants, which are sensitive to salt. We propose that SKB1 mediates plant development and the salt response by altering the methylation status of H4R3sme2 and LSM4 and linking transcription to pre-mRNA splicing.

Structural insights into protein arginine symmetric dimethylation by PRMT5.

Symmetric and asymmetric dimethylation of arginine are isomeric protein posttranslational modifications with distinct biological effects, evidenced by the methylation of arginine 3 of histone H4 (H4R3): symmetric dimethylation of H4R3 leads to repression of gene expression, while asymmetric dimethylation of H4R3 is associated with gene activation. The enzymes catalyzing these modifications share identifiable sequence similarities, but the relationship between their catalytic mechanisms is unknown. Here we analyzed the structure of a prototypic symmetric arginine dimethylase, PRMT5, and discovered that a conserved phenylalanine in the active site is critical for specifying symmetric addition of methyl groups. Changing it to a methionine significantly elevates the overall methylase activity, but also converts PRMT5 to an enzyme that catalyzes both symmetric and asymmetric dimethylation of arginine. Our results demonstrate a common catalytic mechanism intrinsic to both symmetric and asymmetric arginine dimethylases, and show that steric constrains in the active sites play an essential role in determining the product specificity of arginine methylases. This discovery also implies a potentially regulatable outcome of arginine dimethylation that may provide versatile control of eukaryotic gene expression.

Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

INO80 chromatin remodeling complex promotes the removal of UV lesions by the nucleotide excision repair pathway.

The creation of accessible DNA in the context of chromatin is a key step in many DNA functions. To reveal how ATP-dependent chromatin remodeling activities impact DNA repair, we constructed mammalian genetic models for the INO80 chromatin remodeling complex and investigated the impact of loss of INO80 function on the repair of UV-induced photo lesions. We showed that deletion of two core components of the INO80 complex, INO80 and ARP5, significantly hampered cellular removal of UV-induced photo lesions but had no significant impact on the transcription of nucleotide excision repair (NER) factors. Loss of INO80 abolished the assembly of NER factors, suggesting that prior chromatin relaxation is important for the NER incision process. Ino80 and Arp5 are enriched to UV-damaged DNA in an NER-incision-independent fashion, suggesting that recruitment of the remodeling activity likely takes place during the initial stage of damage recognition. These results demonstrate a critical role of INO80 in creating DNA accessibility for the NER pathway and provide direct evidence that repair of UV lesions and perhaps most bulky adduct lesions requires chromatin reconfiguration.

Generation of GM130 Conditional Knockout Mouse.

The Golgi apparatus is a common and highly dynamic organelle in eukaryotic cells. It plays an important role in secretory trafficking and cargo modifications. Increasing evidence suggests that structural changes and functional disorders of the Golgi apparatus are involved in many human diseases, but whether Golgi dysfunction is a causal factor in regard to the progression of these diseases remains unknown. GM130 has been postulated to play roles in Golgi stack formation and vesicular transport based on studies on cultured cells and in vitro reconstitutions. To define the role of GM130 in animal, a GM130 knockout mouse has recently been created. Based on the principle of homologous recombination, the GM130 conditional knockout mouse model was established through gene targeting, stem cell screening, and blastocyst injection. Such model has been successfully applied for studies of physiological functions of GM130 and Golgi apparatus at the cellular and animal levels.

PRMT5 determines the pattern of polyploidization and prevents liver from cirrhosis and carcinogenesis.

Human hepatocellular carcinoma (HCC) occurs almost exclusively in cirrhotic livers. Here, we report that hepatic loss of protein arginine methyltransferase 5 (PRMT5) in mice is sufficient to cause cirrhosis and HCC in a clinically relevant way. Furthermore, pathological polyploidization induced by hepatic loss of PRMT5 promotes liver cirrhosis and hepatic tumorigenesis in aged liver. The loss of PRMT5 leads to hyper-accumulation of P21 and endoreplication-dependent formation of pathological mono-nuclear polyploid hepatocytes. PRMT5 and symmetric dimethylation at histone H4 arginine 3 (H4R3me2s) directly associate with chromatin of P21 to suppress its transcription. More importantly, loss of P21 rescues the pathological mono-nuclear polyploidy and prevents PRMT5-deficiency-induced liver cirrhosis and HCC. Thus, our results indicate that PRMT5-mediated symmetric dimethylation at histone H4 arginine 3 (H4R3me2s) is crucial for preventing pathological polyploidization, liver cirrhosis and tumorigenesis in mouse liver.