A truncated and catalytically inactive isoform of KDM5B histone demethylase accumulates in breast cancer cells and regulates H3K4 tri-methylation and gene expression.

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 methylation and induces derepression of a gene cluster, including the tumor suppressor Cav1 and several genes involved in the interferon-alpha and -gamma response. We discuss the relevance of this finding considering the hypothesis that KDM5B may possess regulatory roles independent of its catalytic activity.

A gene expression atlas for different kinds of stress in the mouse brain.

Stressful experiences are part of everyday life and animals have evolved physiological and behavioral responses aimed at coping with stress and maintaining homeostasis. However, repeated or intense stress can induce maladaptive reactions leading to behavioral disorders. Adaptations in the brain, mediated by changes in gene expression, have a crucial role in the stress response. Recent years have seen a tremendous increase in studies on the transcriptional effects of stress. The input raw data are freely available from public repositories and represent a wealth of information for further global and integrative retrospective analyses. We downloaded from the Sequence Read Archive 751 samples (SRA-experiments), from 18 independent BioProjects studying the effects of different stressors on the brain transcriptome in mice. We performed a massive bioinformatics re-analysis applying a single, standardized pipeline for computing differential gene expression. This data mining allowed the identification of novel candidate stress-related genes and specific signatures associated with different stress conditions. The large amount of computational results produced was systematized in the interactive "Stress Mice Portal".

The Secret Garden of Neuronal circRNAs.

High-throughput transcriptomic profiling approaches have revealed that circular RNAs (circRNAs) are important transcriptional gene products, identified across a broad range of organisms throughout the eukaryotic tree of life. In the nervous system, they are particularly abundant, developmentally regulated, region-specific, and enriched in genes for neuronal proteins and synaptic factors. These features suggested that circRNAs are key components of an important layer of neuronal gene expression regulation, with known and anticipated functions. Here, we review major recognized aspects of circRNA biogenesis, metabolism and biological activities, examining potential new functions in the context of the nervous system.

Differential Expression of Hippocampal Circular RNAs in the BTBR Mouse Model for Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition with unknown etiology. Recent experimental evidences suggest the contribution of non-coding RNAs (ncRNAs) in the pathophysiology of ASD. In this work, we aimed to investigate the expression profile of the ncRNA class of circular RNAs (circRNAs) in the hippocampus of the BTBR T + tf/J (BTBR) mouse model and age-matched C57BL/6J (B6) mice. Alongside, we analyzed BTBR hippocampal gene expression profile to evaluate possible correlations between the differential abundance of circular and linear gene products. From RNA sequencing data, we identified circRNAs highly modulated in BTBR mice. Thirteen circRNAs and their corresponding linear isoforms were validated by RT-qPCR analysis. The BTBR-regulated circCdh9 was better characterized in terms of molecular structure and expression, highlighting altered levels not only in the hippocampus, but also in the cerebellum, prefrontal cortex, and amygdala. Finally, gene expression analysis of the BTBR hippocampus pinpointed altered biological and molecular pathways relevant for the ASD phenotype. By comparison of circRNA and gene expression profiles, we identified 6 genes significantly regulated at either circRNA or mRNA gene products, suggesting low overall correlation between circRNA and host gene expression. In conclusion, our results indicate a consistent deregulation of circRNA expression in the hippocampus of BTBR mice. ASD-related circRNAs should be considered in functional studies to identify their contribution to the etiology of the disorder. In addition, as abundant and highly stable molecules, circRNAs represent interesting potential biomarkers for autism.

Small Molecule Inhibitors of KDM5 Histone Demethylases Increase the Radiosensitivity of Breast Cancer Cells Overexpressing JARID1B.

Background: KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. Methods: In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). Results: we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. Conclusions: These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.

JARID1B expression and its function in DNA damage repair are tightly regulated by miRNAs in breast cancer.

JARID1B/KDM5B histone demethylase's mRNA is markedly overexpressed in breast cancer tissues and cell lines and the protein has been shown to have a prominent role in cancer cell proliferation and DNA repair. However, the mechanism of its post-transcriptional regulation in cancer cells remains elusive. We performed a computational analysis of transcriptomic data from a set of 103 breast cancer patients, which, along with JARID1B upregulation, showed a strong downregulation of 2 microRNAs (miRNAs), mir-381 and mir-486, potentially targeting its mRNA. We showed that both miRNAs can target JARID1B 3'UTR and reduce luciferase's activity in a complementarity-driven repression assay. Moreover, MCF7 breast cancer cells overexpressing JARID1B showed a strong protein reduction when transfected with mir-486. This protein's decrease is accompanied by accumulation of DNA damage, enhanced radiosensitivity and increase of BRCA1 mRNA, 3 features previously correlated with JARID1B silencing. These results enlighten an important role of a miRNA's circuit in regulating JARID1B's activity and suggest new perspectives for epigenetic therapies.

miR-135a Regulates Synaptic Transmission and Anxiety-Like Behavior in Amygdala.

MicroRNAs are a class of non-coding RNAs with a growing relevance in the regulation of gene expression related to brain function and plasticity. They have the potential to orchestrate complex phenomena, such as the neuronal response to homeostatic challenges. We previously demonstrated the involvement of miR-135a in the regulation of early stress response. In the present study, we examine the role of miR-135a in stress-related behavior. We show that the knockdown (KD) of miR-135a in the mouse amygdala induces an increase in anxiety-like behavior. Consistently with behavioral studies, electrophysiological experiments in acute brain slices indicate an increase of amygdala spontaneous excitatory postsynaptic currents, as a result of miR-135a KD. Furthermore, we presented direct evidences, by in vitro assays and in vivo miRNA overexpression in the amygdala, that two key regulators of synaptic vesicle fusion, complexin-1 and complexin-2, are direct targets of miR-135a. In vitro analysis of miniature excitatory postsynaptic currents on miR-135a KD primary neurons indicates unpaired quantal excitatory neurotransmission. Finally, increased levels of complexin-1 and complexin-2 proteins were detected in the mouse amygdala after acute stress, accordingly to the previously observed stress-induced miR-135a downregulation. Overall, our results unravel a previously unknown miRNA-dependent mechanism in the amygdala for regulating anxiety-like behavior, providing evidences of a physiological role of miR-135a in the modulation of presynaptic mechanisms of glutamatergic neurotransmission.

Leptin induction following irradiation is a conserved feature in mammalian epithelial cells and tissues.

PURPOSE: Leptin (LEP) is a peptide hormone with multiple physiological functions. Besides its systemic actions, it has important peripheral roles such as a mitogen action on keratinocytes following skin lesions. We previously showed that LEP mRNA is significantly induced in response to neutron irradiation in mouse skin and that the protein increases in the irradiated epidermis and in the related subcutaneous adipose tissue. In this work, we investigated the post-transcriptional regulation of LEP by miRNAs and the conservation of LEP's role in radiation response in human cells. METHODS: We used microarray analysis and real-time polymerase chain reaction (RT-PCR) to analyze modulation of miRNAs potentially targeting LEP in mouse skin following irradiation and bioinformatic analysis of transcriptome of irradiated human cell lines and cancer tissues from radiotherapy-treated patients to evaluate LEP expression. RESULTS AND CONCLUSIONS: We show that a network of miRNAs potentially targeting LEP mRNA is modulated in irradiated mouse skin and that LEP itself is significantly modulated by irradiation in human epithelial cell lines and in breast cancer tissues from radiotherapy-treated patients. These results confirm and extend the previous evidence that LEP has a general and important role in the response of mammalian cells to irradiation.

RNA-binding protein HuR and the members of the miR-200 family play an unconventional role in the regulation of c-Jun mRNA.

Post-transcriptional gene regulation is a fundamental step for coordinating cellular response in a variety of processes. RNA-binding proteins (RBPs) and microRNAs (miRNAs) are the most important factors responsible for this regulation. Here we report that different components of the miR-200 family are involved in c-Jun mRNA regulation with the opposite effect. While miR-200b inhibits c-Jun protein production, miR-200a tends to increase the JUN amount through a stabilization of its mRNA. This action is dependent on the presence of the RBP HuR that binds the 3'UTR of c-Jun mRNA in a region including the mir-200a binding site. The position of the binding site is fundamental; by mutating this site, we demonstrate that the effect is not micro-RNA specific. These results indicate that miR-200a triggers a microRNA-mediated stabilization of c-Jun mRNA, promoting the binding of HuR with c-Jun mRNA. This is the first example of a positive regulation exerted by a microRNA on an important oncogene in proliferating cells.

An high-throughput in vivo screening system to select H3K4-specific histone demethylase inhibitors.

BACKGROUND: Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes. METHODOLOGY/PRINCIPAL FINDINGS: In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity. CONCLUSIONS/SIGNIFICANCE: Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.

Acute stress alters amygdala microRNA miR-135a and miR-124 expression: inferences for corticosteroid dependent stress response.

The amygdala is a brain structure considered a key node for the regulation of neuroendocrine stress response. Stress-induced response in amygdala is accomplished through neurotransmitter activation and an alteration of gene expression. MicroRNAs (miRNAs) are important regulators of gene expression in the nervous system and are very well suited effectors of stress response for their ability to reversibly silence specific mRNAs. In order to study how acute stress affects miRNAs expression in amygdala we analyzed the miRNA profile after two hours of mouse restraint, by microarray analysis and reverse transcription real time PCR. We found that miR-135a and miR-124 were negatively regulated. Among in silico predicted targets we identified the mineralocorticoid receptor (MR) as a target of both miR-135a and miR-124. Luciferase experiments and endogenous protein expression analysis upon miRNA upregulation and inhibition allowed us to demonstrate that mir-135a and mir-124 are able to negatively affect the expression of the MR. The increased levels of the amygdala MR protein after two hours of restraint, that we analyzed by western blot, negatively correlate with miR-135a and miR-124 expression. These findings point to a role of miR-135a and miR-124 in acute stress as regulators of the MR, an important effector of early stress response.

Exposure of Toll-like receptors 4 to bacterial lipopolysaccharide (LPS) impairs human colonic smooth muscle cell function.

Endotoxemia by bacterial lipopolysaccharide (LPS) has been reported to affect gut motility specifically depending on Toll-like receptor 4 activation (TLR4). However, the direct impact of LPS ligation to TLR4 on human smooth muscle cells (HSMC) activity still remains to be elucidated. The present study shows that TLR4, its associated molecule MD2, and TLR2 are constitutively expressed on cultured HSMC and that, once activated, they impair HSMC function. The stimulation of TLR4 by LPS induced a time- and dose-dependent contractile dysfunction, which was associated with a decrease of TLR2 messenger, a rearrangement of microfilament cytoskeleton and an oxidative imbalance, i.e., the formation of reactive oxygen species (ROS) together with the depletion of GSH content. An alteration of mitochondria, namely a hyperpolarization of their membrane potential, was also detected. Most of these effects were partially prevented by the NADPH oxidase inhibitor apocynin or the NFkappaB inhibitor MG132. Finally, a 24 h washout in LPS-free medium almost completely restored morphofunctional and biochemical HSMC resting parameters, even if GSH levels remained significantly lower and no recovery was observed in TLR2 expression. Thus, the exposure to bacterial endotoxin directly and persistently impaired gastrointestinal smooth muscle activity indicating that HSMC actively participate to dysmotility during infective burst. The knowledge of these interactions might provide novel information on the pathogenesis of infection-associated gut dysmotility and further clues for the development of new therapeutic strategies.