Locus folding mechanisms determine modes of antigen receptor gene assembly.

The dynamic folding of genomes regulates numerous biological processes, including antigen receptor (AgR) gene assembly. We show that, unlike other AgR loci, homotypic chromatin interactions and bidirectional chromosome looping both contribute to structuring Tcrb for efficient long-range V(D)J recombination. Inactivation of the CTCF binding element (CBE) or promoter at the most 5'Vß segment (Trbv1) impaired loop extrusion originating locally and extending to DßJß CBEs at the opposite end of Tcrb. Promoter or CBE mutation nearly eliminated Trbv1 contacts and decreased RAG endonuclease-mediated Trbv1 recombination. Importantly, Trbv1 rearrangement can proceed independent of substrate orientation, ruling out scanning by DßJß-bound RAG as the sole mechanism of Vß recombination, distinguishing it from Igh. Our data indicate that CBE-dependent generation of loops cooperates with promoter-mediated activation of chromatin to juxtapose Vß and DßJß segments for recombination through diffusion-based synapsis. Thus, the mechanisms that fold a genomic region can influence molecular processes occurring in that space, which may include recombination, repair, and transcriptional programming.

SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation-Phosphorylation Cross-talk.

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. SIGNIFICANCE: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007.

Aberrant RNA methylation triggers recruitment of an alkylation repair complex.

Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.

CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism.

The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.

Ceramide in apoptosis and oxidative stress in allergic inflammation and asthma.

BACKGROUND: Nothing is known about the mechanisms by which increased ceramide levels in the lung contribute to allergic responses and asthma severity. OBJECTIVE: We sought to investigate the functional role of ceramide in mouse models of allergic airway disease that recapitulate the cardinal clinical features of human allergic asthma. METHODS: Allergic airway disease was induced in mice by repeated intranasal administration of house dust mite or the fungal allergen Alternaria alternata. Processes that can be regulated by ceramide and are important for severity of allergic asthma were correlated with ceramide levels measured by mass spectrometry. RESULTS: Both allergens induced massive pulmonary apoptosis and also significantly increased reactive oxygen species in the lung. Prevention of increases in lung ceramide levels mitigated allergen-induced apoptosis, reactive oxygen species, and neutrophil infiltration. In contrast, dietary supplementation of the antioxidant α-tocopherol decreased reactive oxygen species but had no significant effects on elevation of ceramide level or apoptosis, indicating that the increases in lung ceramide levels in allergen-challenged mice are not mediated by oxidative stress. Moreover, specific ceramide species were altered in bronchoalveolar lavage fluid from patients with severe asthma compared with in bronchoalveolar lavage fluid from individuals without asthma. CONCLUSION: Our data suggest that elevation of ceramide level after allergen challenge contributes to the apoptosis, reactive oxygen species generation, and neutrophilic infiltrate that characterize the severe asthmatic phenotype. Ceramide might be the trigger of formation of Creola bodies found in the sputum of patients with severe asthma and could be a biomarker to optimize diagnosis and to monitor and improve clinical outcomes in this disease.

RNA Modifications: Reversal Mechanisms and Cancer.

An emerging molecular understanding of RNA alkylation and its removal is transforming our knowledge of RNA biology and its interplay with cancer chemotherapy responses. DNA modifications are known to perform critical functions depending on the genome template, including gene expression, DNA replication timing, and DNA damage protection, yet current results suggest that the chemical diversity of DNA modifications pales in comparison to those on RNA. More than 150 RNA modifications have been identified to date, and their complete functional implications are still being unveiled. These include intrinsic roles such as proper processing and RNA maturation; emerging evidence has furthermore uncovered RNA modification "readers", seemingly analogous to those identified for histone modifications. These modification recognition factors may regulate mRNA stability, localization, and interaction with translation machinery, affecting gene expression. Not surprisingly, tumors differentially modulate factors involved in expressing these marks, contributing to both tumorigenesis and responses to alkylating chemotherapy. Here we describe the current understanding of RNA modifications and their removal, with a focus primarily on methylation and alkylation as functionally relevant changes to the transcriptome. Intriguingly, some of the same RNA modifications elicited by physiological processes are also produced by alkylating agents, thus blurring the lines between what is a physiological mark and a damage-induced modification. Furthermore, we find that a high level of gene expression of enzymes with RNA dealkylation activity is a sensitive readout for poor survival in four different cancer types, underscoring the likely importance of examining RNA dealkylation mechanisms to cancer biology and for cancer treatment and prognosis.

A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.

ORMDL proteins regulate ceramide levels during sterile inflammation.

The bioactive sphingolipid metabolite, ceramide, regulates physiological processes important for inflammation and elevated levels of ceramide have been implicated in IL-1-mediated events. Although much has been learned about ceramide generation by activation of sphingomyelinases in response to IL-1, the contribution of the de novo pathway is not completely understood. Because yeast ORM1 and ORM2 proteins negatively regulate ceramide levels through inhibition of serine palmitoyltransferase, the first committed step in ceramide biosynthesis, we examined the functions of individual mammalian ORM orthologs, ORM (yeast)-like (ORMDL)1-3, in regulation of ceramide levels. In HepG2 liver cells, downregulation of ORMDL3 markedly increased the ceramide precursors, dihydrosphingosine and dihydroceramide, primarily from de novo biosynthesis based on [U-(13)C]palmitate incorporation into base-labeled and dual-labeled dihydroceramides, whereas downregulation of each isoform increased dihydroceramides [(13)C]labeled in only the amide-linked fatty acid. IL-1 and the IL-6 family cytokine, oncostatin M, increased dihydroceramide and ceramide levels in HepG2 cells and concomitantly decreased ORMDL proteins. Moreover, during irritant-induced sterile inflammation in mice leading to induction of the acute-phase response, which is dependent on IL-1, expression of ORMDL proteins in the liver was strongly downregulated and accompanied by increased ceramide levels in the liver and accumulation in the blood. Together, our results suggest that ORMDLs may be involved in regulation of ceramides during IL-1-mediated sterile inflammation.

Aberrant ORM (yeast)-like protein isoform 3 (ORMDL3) expression dysregulates ceramide homeostasis in cells and ceramide exacerbates allergic asthma in mice.

BACKGROUND: Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known. OBJECTIVES: We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis. METHODS: We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice. RESULTS: Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite-induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite-challenged mice. CONCLUSIONS: Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma.

K63-linked polyubiquitination of transcription factor IRF1 is essential for IL-1-induced production of chemokines CXCL10 and CCL5.

Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.

MAPK14 cooperates with MAPK3/1 to regulate endothelin-1-mediated prostaglandin synthase 2 induction and survival in leiomyoma but not in normal myometrial cells.

We recently reported that in ELT3 uterine leiomyoma cells, but not in normal myometrial cells, endothelin (ET)-1 exerts a survival effect insensitive to MAPK3/1(ERK1/2) inhibition. In the present work, we investigated the potential role of MAPK14 (p38) in this ET-1-mediated effect. We demonstrated that, in ELT3, but not in normal myometrial cells, ET-1 activated MAPK14. Data based on pharmacological and siRNA approaches indicate that ETA and ETB receptors contributed to the activation of MAPK14 by ET-1 through a mechanism involving Gi protein, but not PI3-kinase. The inhibition of MAPK3/1 by U0126 did not affect the activation of MAPK14 by ET-1. Conversely, the inhibition of MAPK14 by SB203580 and the down-regulation of MAP2K3/MAP2K6 (kinases upstream of MAPK14) by specific siRNA did not alter the activation of MAPK3/1. These data indicate that MAPK14 was activated by ET-1 independently from MAPK3/1. Furthermore, ET-1 increased protein expression of prostaglandin synthase 2 (PTGS2 or COX2), prostaglandin E2 (PGE2) production, and subsequent ELT3 cell survival. The inhibition of PTGS2 induction and subsequent survival induced by ET-1 required the coinhibition of MAPK14 and MAPK3/1. Our findings provide evidence that ET-1 activated MAPK14 only in ELT3 cells, but not in normal myometrial cells. This MAPK14 activation was required, in addition to MAPK3/1 in ET-1-mediated survival through the COX2/prostaglandin axis, and may explain the absence of ET-1 antiapoptotic effect in normal myometrial cells. Our data reinforce the role of ET-1 and associated signaling pathways in leiomyoma pathology.

Endothelin-1: physiological and pathological roles in myometrium.

Endothelin-1 (ET-1), a member of endothelin peptide family is released by many different tissues including uterine smooth muscle. ET-1 acts through ETA and ETB receptors and is implicated in a wide range of biological and pathological functions that explain the great attention of the pharmacological industry for ET-1 receptors as potential therapeutic targets in vascular pathologies and cancers. It is now well established that ET-1 is also able to regulate myometrial functions. In the present review, we focused on ET axis and related signaling pathways involved in the regulation of myometrial contraction, as well as cell proliferation and survival. Such ET-1-mediated cellular functions play a critical role in normal pregnancy, preterm birth and uterine leiomyoma.