Glucose Regulates m6A Methylation of RNA in Pancreatic Islets

Type 2 diabetes is characterized by chronic hyperglycemia associated with impaired insulin action and secretion. Although the heritability of type 2 diabetes is high, the environment, including blood components, could play a major role in the development of the disease. Amongst environmental effects, epitranscriptomic modifications have been recently shown to affect gene expression and glucose homeostasis. The epitranscriptome is characterized by reversible chemical changes in RNA, with one of the most prevalent being the m6A methylation of RNA. Since pancreatic β cells fine tune glucose levels and play a major role in type 2 diabetes physiopathology, we hypothesized that the environment, through variations in blood glucose or blood free fatty acid concentrations, could induce changes in m6A methylation of RNAs in pancreatic β cells. Here we observe a significant decrease in m6A methylation upon high glucose concentration, both in mice and human islets, associated with altered expression levels of m6A demethylases. In addition, the use of siRNA and/or specific inhibitors against selected m6A enzymes demonstrate that these enzymes modulate the expression of genes involved in pancreatic β-cell identity and glucose-stimulated insulin secretion. Our data suggest that environmental variations, such as glucose, control m6A methylation in pancreatic β cells, playing a key role in the control of gene expression and pancreatic β-cell functions. Our results highlight novel causes and new mechanisms potentially involved in type 2 diabetes physiopathology and may contribute to a better understanding of the etiology of this disease.

Core promoter mutation contributes to abnormal gene expression in bladder cancer

Background Bladder cancer is one of the most mortal cancers. Bladder cancer has distinct gene expression signature, highlighting altered gene expression plays important roles in bladder cancer etiology. However, the mechanism for how the regulatory disorder causes the altered expression in bladder cancer remains elusive. Core promoter controls transcriptional initiation. We hypothesized that mutation in core promoter abnormality could cause abnormal transcriptional initiation thereby the altered gene expression in bladder cancer. Methods In this study, we performed a genome-wide characterization of core promoter mutation in 77 Spanish bladder cancer cases. Results We identified 69 recurrent somatic mutations in 61 core promoters of 62 genes and 28 recurrent germline mutations in 20 core promoters of 21 genes, including TERT, the only gene known with core promoter mutation in bladder cancer, and many oncogenes and tumor suppressors. From the RNA-seq data from bladder cancer, we observed altered expression of the core promoter-mutated genes. We further validated the effects of core promoter mutation on gene expression by using luciferase reporter gene assay. We also identified potential drugs targeting the core promoter-mutated genes. Conclusions Data from our study highlights that core promoter mutation contributes to bladder cancer development through altering gene expression.

Unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating Ethylene and Abscisic acid pathway

Submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates Galactosyltransferase (GALT9) and their expression is inversely affected at 24 hours of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild type and MIM775 Arabidopsis shoot. A similar recovery phenotype of galt9 mutant indicates the role of miR775-GALT9 module in post-submergence recovery. We predicted Golgi-localized GALT9 to be potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29, and ORE1), ethylene signalling (EIN2 and EIN3) and ABA biosynthesis (NCED3) pathway genes in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role of miR775-GALT9 module in post-submergence recovery through a crosstalk with ethylene and ABA pathway.

Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.

Transcriptome Analysis of an Aedes albopictus Cell Line Single- and Dual-Infected with Lammi Virus and WNV

Understanding the flavivirus infection process in mosquito hosts is important and fundamental in the search for novel control strategies that target the mosquitoes’ ability to carry and transmit pathogenic arboviruses. A group of viruses known as insect-specific viruses (ISVs) has been shown to interfere with the infection and replication of a secondary arbovirus infection in mosquitoes and mosquito-derived cell lines. However, the molecular mechanisms behind this interference are unknown. Therefore, in the present study, we infected the Aedes albopictus cell line U4.4 with either the West Nile virus (WNV), the insect-specific Lammi virus (LamV) or an infection scheme whereby cells were pre-infected with LamV 24 h prior to WNV challenge. The qPCR analysis showed that the dual-infected U4.4 cells had a reduced number of WNV RNA copies compared to WNV-only infected cells. The transcriptome profiles of the different infection groups showed a variety of genes with altered expression. WNV-infected cells had an up-regulation of a broad range of immune-related genes, while in LamV-infected cells, many genes related to stress, such as different heat-shock proteins, were up-regulated. The transcriptome profile of the dual-infected cells was a mix of up- and down-regulated genes triggered by both viruses. Furthermore, we observed an up-regulation of signal peptidase complex (SPC) proteins in all infection groups. These SPC proteins have shown importance for flavivirus assembly and secretion and could be potential targets for gene modification in strategies for the interruption of flavivirus transmission by mosquitoes.

Role of GDNF, GFRα1 and GFAP in a Bifidobacterium-Intervention Induced Mouse Model of Intestinal Neuronal Dysplasia

ObjectiveTo investigate the effects of glial cell-derived neurotrophic factor (GDNF), GDNF family receptor alpha 1 (GFRα1), and glial fibrillary acidic protein (GFAP) on colonic motility in a mouse model of intestinal neuronal dysplasia by intervention with Bifidobacterium and to explore the influence of Bifidobacterium on enteric glial cells (EGCs).MethodsWestern blotting and qRT-PCR were employed to detect the expression of GFRα1 and GFAP in colonic tissues of mice with or without Tlx2 mutations, and ELISA was used to detect the expression of GDNF in serum. IHC was used to detect the appearance of the ganglion cells. Subsequently, Tlx2 homozygous mutant (Tlx2−/−) mice were treated with Bifidobacterium. Colonic motility was measured before and after intervention by measuring the glass bead expelling time. The variations in abdominal circumference and GDNF, GFRα1, and GFAP expression were measured. In addition, 16SrRNA gene sequencing was performed to detect the abundance of the intestinal microbiota.ResultsThe mRNA and protein expression of GFRα1 and GFAP was decreased in the colonic tissues of Tlx2−/− mice and GDNF expression was decreased in serum compared with Tlx2+/− and WT mice. After confirming the colonization of Bifidobacterium by 16S rRNA gene sequencing, the expelling time and abdominal distension were ameliorated, and the expression of GFAP, GDNF, and GFRα1 was increased.ConclusionsThe expression of GDNF, GFRα1, and GFAP is associated with colonic motility. The altered expression of EGC-related factors suggested that Bifidobacterium may be involved in the EGC activation process. The amelioration of IND symptoms after intervention with Bifidobacterium prompted the elicitation of adjuvant therapy.

MicroRNAs as Modulators of the Immune Response in T-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukaemia (ALL) is an aggressive haematological tumour driven by the malignant transformation and expansion of B-cell (B-ALL) or T-cell (T-ALL) progenitors. The evolution of T-ALL pathogenesis encompasses different master developmental pathways, including the main role played by Notch in cell fate choices during tissue differentiation. Recently, a growing body of evidence has highlighted epigenetic changes, particularly the altered expression of microRNAs (miRNAs), as a critical molecular mechanism to sustain T-ALL. The immune response is emerging as key factor in the complex multistep process of cancer but the role of miRNAs in anti-leukaemia response remains elusive. In this review we analyse the available literature on miRNAs as tuners of the immune response in T-ALL, focusing on their role in Natural Killer, T, T-regulatory and Myeloid-derived suppressor cells. A better understanding of this molecular crosstalk may provide the basis for the development of potential immunotherapeutic strategies in the leukemia field.

Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis

FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analogue approved for multiple sclerosis (MS) therapy, which can functionally antagonize the S1P receptor, S1P1. Vitamin B12 (B12) deficiency produces neurological manifestations resembling MS. Here, we report a new mechanism where FTY720 suppresses neuroinflammation by regulating B12 metabolic pathways. Nuclear RNA-seq of c-Fos-activated astrocytes (called ieAstrocytes) from experimental autoimmune encephalomyelitis (EAE) spinal cords identified up-regulation of CD320, a transcobalamin 2 (TCN2)-B12 receptor, by S1P1 inhibition. CD320 was reduced in MS plaques. Deficiency of CD320 or dietary B12 worsened EAE and eliminated FTY720 efficacy, while concomitantly down-regulating type I interferon signaling. TCN2 functioned as a chaperone for FTY720 and sphingosine, which induced astrocytic CD320 internalization. An accompanying paper identified a requirement for astrocyte sphingosine kinases in FTY720 efficacy and its altered expression in MS brains, molecularly linking MS and B12 deficiency that can be accessed by sphingolipid/fingolimod metabolic pathways.

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.