Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation

Abstract Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

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TET-mediated 5-methylcytosine oxidation in tRNA promotes translation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014226 ◽

2020 ◽

pp. jbc.RA120.014226

Author(s):

Hui Shen ◽

Robert Jordan Ontiveros ◽

Michael C Owens ◽

Monica Yun Liu ◽

Uday Ghanty ◽

...

Keyword(s):

Messenger Rna ◽

Biological Function ◽

Catalytic Domain ◽

Embryonic Stem ◽

Wild Type ◽

Oxidation Activity ◽

Tet Enzymes ◽

Mediated Oxidation

Oxidation of 5-methylcytosine (5mC) in DNA by the Ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNAs). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison to wild type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

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Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression

Scientific Reports ◽

10.1038/s41598-021-86834-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sara Busacca ◽

Qi Zhang ◽

Annabel Sharkey ◽

Alan G. Dawson ◽

David A. Moore ◽

...

Keyword(s):

Small Molecule ◽

Selective Vulnerability ◽

Dependent Manner ◽

Histone H4 ◽

Wild Type ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Methylthioadenosine Phosphorylase ◽

Protein Arginine Methyltransferase 5

AbstractWe hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

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Developmental Neurotoxicity of Fipronil and Rotenone on a Human Neuronal In Vitro Test System

Neurotoxicity Research ◽

10.1007/s12640-021-00364-8 ◽

2021 ◽

Author(s):

Anne Schmitz ◽

Silke Dempewolf ◽

Saime Tan ◽

Gerd Bicker ◽

Michael Stern

Keyword(s):

Cell Migration ◽

Neurite Outgrowth ◽

Neuronal Differentiation ◽

Precursor Cell ◽

Developmental Neurotoxicity ◽

In Vitro Studies ◽

Human Model ◽

In Vitro Test ◽

Dependent Manner

AbstractPesticide exposure during in utero and early postnatal development can cause a wide range of neurological defects. However, relatively few insecticides have been recognized as developmental neurotoxicants, so far. Recently, discovery of the insecticide, fipronil, in chicken eggs has raised public concern. The status of fipronil as a potential developmental neurotoxicant is still under debate. Whereas several in vivo and in vitro studies suggest specific toxicity, other in vitro studies could not confirm this concern. Here, we tested fipronil and its main metabolic product, fipronil sulfone both at concentrations between 1.98 and 62.5 µM, alongside with the established developmental neurotoxicant, rotenone (0.004–10 µM) in vitro on the human neuronal precursor cell line NT2. We found that rotenone impaired all three tested DNT endpoints, neurite outgrowth, neuronal differentiation, and precursor cell migration in a dose-dependent manner and clearly separable from general cytotoxicity in the nanomolar range. Fipronil and fipronil sulfone specifically inhibited cell migration and neuronal differentiation, but not neurite outgrowth in the micromolar range. The rho-kinase inhibitor Y-27632 counteracted inhibition of migration for all three compounds (EC50 between 12 and 50 µM). The antioxidant, n-acetyl cysteine, could ameliorate the inhibitory effects of fipronil on all three tested endpoints (EC 50 between 84 and 164 µM), indicating the involvement of oxidative stress. Fipronil sulfone had a stronger effect than fipronil, confirming the importance to test metabolic products alongside original pesticides. We conclude that in vitro fipronil and fipronil sulfone display specific developmental neurotoxicity on developing human model neurons.

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RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2

Nature Communications ◽

10.1038/s41467-021-21828-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xia Wang ◽

Jin Wang ◽

Yu-Man Tsui ◽

Chaoran Shi ◽

Ying Wang ◽

...

Keyword(s):

Stem Cells ◽

Mrna Stability ◽

Rna Binding ◽

Embryonic Stem ◽

Molecular Characteristics ◽

Specific Gene ◽

Functional Studies ◽

Poor Differentiation ◽

Development In Vitro

AbstractGrowing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-β2 mRNA stability by decreasing N6-methyladenosine (m6A) modification. TGF-β signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-β2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

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The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽

10.7554/elife.03573 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 21

Author(s):

Yick W Fong ◽

Jaclyn J Ho ◽

Carla Inouye ◽

Robert Tjian

Keyword(s):

Stem Cell ◽

Es Cells ◽

Embryonic Stem ◽

In Vitro Transcription ◽

Specific Gene ◽

Transcriptional Level ◽

Ips Cell ◽

Ribonucleoprotein Complex ◽

Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

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Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽

10.1210/en.2004-0061 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5525-5531 ◽

Cited By ~ 50

Author(s):

Gary M. Leong ◽

Sofia Moverare ◽

Jesena Brce ◽

Nathan Doyle ◽

Klara Sjögren ◽

...

Keyword(s):

Promoter Activity ◽

Hepatoma Cells ◽

Cytokine Signaling ◽

Mrna Levels ◽

Dependent Manner ◽

Wild Type ◽

Hepatic Expression ◽

Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

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Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽

10.1242/dev.116.supplement.157 ◽

1992 ◽

Vol 116 (Supplement) ◽

pp. 157-165 ◽

Cited By ~ 20

Author(s):

R. S. P. Beddington ◽

P. Rashbass ◽

V. Wilson

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Primitive Streak ◽

Coat Colour ◽

Es Cell ◽

Wild Type ◽

Mesoderm Cell ◽

Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

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Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

10.1101/2021.04.05.438547 ◽

2021 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Wen-Hsin Lee ◽

Sandhya Bangaru ◽

Andrew B Ward ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Dependent Manner ◽

Receptor Binding Domain ◽

Wild Type ◽

Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

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Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽

10.1161/circ.118.suppl_18.s_576-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Bin Ren ◽

Arpita Mukhopadhyay* ◽

Anthony A Lanahan ◽

Zhen W Zhuang ◽

Karen L Moodie ◽

...

Keyword(s):

Endothelial Cells ◽

Branching Morphogenesis ◽

Erk Signaling ◽

Dependent Manner ◽

Wild Type ◽

Erk Activation ◽

Arterial Development ◽

Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

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Using a Multi-Stage hESC Model to Characterize BDE-47 Toxicity During Neurogenesis

Toxicological Sciences ◽

10.1093/toxsci/kfz136 ◽

2019 ◽

Vol 171 (1) ◽

pp. 221-234 ◽

Cited By ~ 3

Author(s):

Hao Chen ◽

Helia Seifikar ◽

Nicholas Larocque ◽

Yvonne Kim ◽

Ibrahim Khatib ◽

...

Keyword(s):

Neural Development ◽

Polybrominated Diphenyl Ethers ◽

Neuronal Development ◽

Embryonic Stem ◽

Cellular Level ◽

Environmental Chemicals ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Neurodevelopmental Toxicity

Abstract Although the ramifications associated with polybrominated diphenyl ethers (PBDEs) exposures during human pregnancy have yet to be determined, increasing evidence in humans and animal models suggests that these compounds cause neurodevelopmental toxicity. Human embryonic stem cells (hESCs) models can be used to study the effects of environmental chemicals throughout the successive stages of neuronal development. Here, using a hESC differentiation model, we investigated the effects of common PBDE congeners (BDE-47 or -99) on the successive stages of early neuronal development. First, we determined the points of vulnerability to PBDEs across 4 stages of in vitro neural development by using assays to assess for cytotoxicity. Differentiated neural progenitors were identified to be more sensitive to PBDEs than their less differentiated counterparts. In follow-up investigations, we observed BDE-47 to inhibit functional processes critical for neurogenesis (eg, proliferation, expansion) in hESC-derived neural precursor cells (NPCs) at sub-lethal concentrations. Finally, to determine the mechanism(s) underlying PBDE-toxicity, we conducted global transcriptomic and methylomic analyses of BDE-47. We identified 589 genes to be differentially expressed due to BDE-47 exposure, including molecules involved in oxidative stress mediation, cell cycle, hormone signaling, steroid metabolism, and neurodevelopmental pathways. In parallel analyses, we identified a broad significant increase in CpG methylation. In summary our results suggest, on a cellular level, PBDEs induce human neurodevelopmental toxicity in a concentration-dependent manner and sensitivity to these compounds is dependent on the developmental stage of exposure. Proposed mRNA and methylomic perturbations may underlie toxicity in early embryonic neuronal populations.

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