A ROS-Ca2+ signalling pathway identified from a chemical screen for modifiers of sugar-activated circadian gene expression.

Sugars are essential metabolites for energy and anabolism that can also act as signals to regulate plant physiology and development. Experimental tools to disrupt major sugar signalling pathways are limited. We have performed a chemical screen for modifiers of activation of circadian gene expression by sugars to discover pharmacological tools to investigate and manipulate plant sugar signalling. Using a library of commercially available bioactive compounds, we identified 75 confident hits that modified the response of a circadian luciferase reporter to sucrose in dark-adapted seedlings. We validated the transcriptional effect on a subset of the hits and measured their effects on a range of sugar-dependent phenotypes for 13 of these chemicals. Chemicals were identified that appear to influence known and unknown sugar signalling pathways. Pentamidine isethionate (PI) was identified as a modifier of a sugar-activated Ca2+ signal that acts downstream of superoxide in a metabolic signalling pathway affecting circadian rhythms, primary metabolism and plant growth. Our data provide a resource of new experimental tools to manipulate plant sugar signalling and identify novel components of these pathways.

LncRNA PVT1 Promotes Bladder Cancer Progression by Forming a Positive Feedback Loop With STAT5B

Background: Plasmacytoma Variant Translocation 1 (LncRNA PVT1) and signal transducer and activator of transcription 5B (STAT5B) have been reported to play important roles in various cancers, but their interaction in bladder cancer (BC) remains unclear. Purpose: In this study, we aimed to explore the interaction between lncRNA PVT1 and STAT5B in BC tumorigenesis. Methods: The association of the expression of the lncRNA PVT1 and STAT5B to the prognosis of patient with BC was evaluated via bioinformatic analysis. Loss- and gain-of-function assays were performed to determine the biological functions of lncRNA PVT1 and STAT5B in BC cells. Quantitative real time polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence were used to detect lncRNA PVT1 and STAT5B expression. Fluorescence in situ hybridization, RNA pull-down and RNA immunoprecipitation assays were conducted to determine the regulatory effect of lncRNA PVT1 on STAT5B. The transcriptional effect of STAT5B on lncRNA PVT1 gene was determined using luciferase reporter assay, chromatin immunoprecipitation and DNA-affinity precipitation assays.Results: We found that lncRNA PVT1 and STAT5B enhance the expression of each other and promote the malignant phenotypes in BC, including cell viability and invasion. lncRNA PVT1 stabilizes STAT5B by decreasing ubiquitination, enhances STAT5B phosphorylation, and promotes the translocation to the nucleus of STAT5B to trigger further carcinogenesis activities. In the nucleus, STAT5B activates the transcription of lncRNA PVT1 by binding directly to its promoter region, leading to a positive feedback.Conclusions: We first identified the lncRNA PVT1/STAT5B positive feedback loop for bladder carcinogenesis, which may provide new molecular targets for interventions of BC.

PARP7 mono-ADP-ribosylates the Agonist Conformation of the Androgen Receptor in the Nucleus

We recently described a signal transduction pathway that contributes to AR regulation based on site-specific ADP-ribosylation by PARP7, a mono-ADP-ribosyltransferase implicated in several human cancers. ADP-ribosylated AR is specifically recognized by PARP9/DTX3L, a heterodimeric complex that contains an ADP-ribose reader (PARP9) and a ubiquitin E3 ligase (DTX3L). Here, we have characterized the cellular and biochemical requirements for AR ADP-ribosylation by PARP7. We found that the reaction requires nuclear localization of PARP7 and an agonist-induced conformation of AR. PARP7 contains a Cys3His1-type zinc finger (ZF), which we found is critical for AR ADP-ribosylation. The Parp7 ZF is required for efficient nuclear localization by the nuclear localization signal (NLS) encoded in PARP7, but rescue experiments indicate the ZF makes a contribution to AR ADP-ribosylation that is transport-independent. ZF structure appears to be dispensable for PARP7 catalytic activity and for PARP7 binding to AR. Androgen induction of the MYBPC1 gene is regulated by AR and PARP7, and we determined that the ZF is required for the PARP7 transcriptional effect on MYBPC1. Our data indicate the PARP7 ZF plays an important role in modulating the subcellular localization of PARP7 and its capacity to ADP-ribosylate and promote AR-dependent transcription.

Effect of circular RNA, mmu_circ_0000296, on neuronal apoptosis in chronic cerebral ischaemia via the miR-194-5p/Runx3/Sirt1 axis

Chronic cerebral ischaemia (CCI) is a common pathological disorder, which is associated with various diseases, such as cerebral arteriosclerosis and vascular dementia, resulting in neurological dysfunction. As a type of non-coding RNA, circular RNA is involved in regulating the occurrence and development of diseases, such as ischaemic brain injury. Here, we found that HT22 cells and hippocampus treated with CCI had low expression of circ_0000296, Runx3, Sirt1, but high expression of miR-194-5p. Overexpression of circ_0000296, Runx3, Sirt1, and silenced miR-194-5p significantly inhibited neuronal apoptosis induced by CCI. This study demonstrated that circ_0000296 specifically bound to miR-194-5p; miR-194-5p bound to the 3′UTR region of Runx3 mRNA; Runx3 directly bound to the promoter region of Sirt1, enhancing its transcriptional activity. Overexpression of circ_0000296 by miR-194-5p reduced the negative regulatory effect of miR-194-5p on Runx3, promoted the transcriptional effect of Runx3 on Sirt1, and inhibited neuronal apoptosis induced by CCI. mmu_circ_0000296 plays an important role in regulating neuronal apoptosis induced by CCI through miR-194-5p/Runx3/Sirt1 pathway.

Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

Background: Differences in gene expression, which arises from divergence in cis-regulatory elements or alterations in transcription factors (TFs) binding specificity, are one of the most important causes of phenotypic diversity during evolution. On one hand, changes in the cis-elements located in the vicinity of target genes affect TF binding and/or local chromatin environment, thereby modulating gene expression in one-to-one manner. On the other hand, alterations in trans-factors influence the expression of their target genes in a more pleiotropic fashion. Although evolution of amino acid sequences is much slower than that of non-coding regulatory elements, particularly for the TF DNA binding domains (DBD)， it is still possible that changes in TF-DBD might have the potential to drive large phenotypic changes if the resulting effects have a net positive effect on the organism’s fitness. If so, species-specific changes in TF-DBD might be positively selected. So far, however, this possibility has been largely unexplored.Results: By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.Conclusions: There were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Moreover, Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.

Acute Transcriptional Effects of Dexamethasone on Mouse Adrenal Gland Transcriptome

Researchers have long known that dexamethasone causes cellular and functional changes in the adrenal gland. For example, long-term dexamethasone treatment leads to reversible adrenal cortex atrophy. In the adrenal medulla, dexamethasone treatment alters the maturation and function of the neural crest-derived chromaffin cells. Here we aim to study the acute transcriptional effect of dexamethasone on mouse adrenal gland at the transcriptome level. Our data suggested that a one-hour dexamethasone treatment had a cell type-specific effect on the adrenal transcriptome. There were 922 dexamethasone-induced genes and 853 dexamethasone-suppressed genes. GO analysis showed that the upregulated genes were primarily linked to neuronal cell function. Clustered heatmaps further showed that many genes involved in the catecholamine synthesis were upregulated by dexamethasone treatment, whereas most genes involved in the steroidogenesis pathway were downregulated. Interestingly, steroidogenic factor 1 (SF1, encoded by Nr5a1), the critical transcription factor that regulates steroidogenesis, had a >2-fold decrease under the one-hour dexamethasone treatment, suggesting a possible mechanism of the acute suppression of steroidogenic activity. Our findings indicate that the acute effects of dexamethasone stimulate catecholamine synthesis in the medulla, whereas steroidogenesis in the cortex is suppressed by dexamethasone.

Nonlinear control of transcription through enhancer-promoter interactions

Chromosome structure in mammals is thought to regulate transcription by modulating the three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated interactions and topologically associating domains (TADs)1–4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. To address this question we use a novel assay to position an enhancer at a large number of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. Quantitative analysis of hundreds of cell lines reveal that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a non-linear relationship. Mathematical modeling and validation against experimental data further provide evidence that nonlinearity arises from transient enhancer-promoter interactions being memorized into longer-lived promoter states in individual cells, thus uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism for how enhancers control transcription across large genomic distances despite rarely meeting their target promoters, and for how TAD boundaries can block distal enhancers. We finally show that enhancer strength additionally determines not only absolute transcription levels, but also the sensitivity of a promoter to CTCF-mediated functional insulation. Our unbiased, systematic and quantitative measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

TuMV infection alters miR168/AGO1 and miR403/AGO2 systems regulation in Arabidopsis

Viral infections trigger a strong response of the plant RNA silencing machinery. The cargo protein AGO1 has a well-established role in siRNA loading. Several plant viruses have convergently evolved a molecular counter-attack reliant on inactivation of the AGO1-based plant defense by altering the regulatory loop that comprises miR168 and AGO1. Upon viral infections, another AGO protein, AGO2, is induced. AGO2 is in turn post-transcriptionally regulated by miR403. The simultaneous study of both regulatory systems working along an infection time-frame is lacking. Here it is shown the molecular response of Arabidopsis Turnip Mosaic Virus (TuMV) infection from early to late stages post-infection. Molecular analyses confirmed that TuMV induced mature miR168 accumulation in Arabidopsis, as reported also for other plant-virus interactions. However, differently from reported plant-virus interactions, miRNA precursors pre-miR168a and b were down-regulated and no significant transcriptional effect was detected on miR168a promoter. AGO1 mRNA was induced, but AGO1 protein levels were unchanged. Analyses of the miR403/AGO2 system showed a similar pattern, except for an induction of AGO2 protein levels. Additionally, low molecular weight forms of AGO1 and 2 proteins were detected and relatively overaccumulated under TuMV infections. Our results suggest that TuMV alters the biogenesis of miR168 and miR403 at the processing step. Whilst TuMV induces both miR168 and miR403 overaccumulation, it fails to prevent overaccumulation of the antiviral AGO2 at both mRNA and protein levels.

Genome-wide role of codon usage on transcription and identification of potential regulators

Codon usage bias is a fundamental feature of all genomes and plays an important role in determining gene expression levels. The codon usage was thought to influence gene expression mainly due to its impact on translation. Recently, however, codon usage was shown to affect transcription of fungal and mammalian genes, indicating the existence of a gene regulatory phenomenon with unknown mechanism. In Neurospora, codon usage biases strongly correlate with mRNA levels genome-wide, and here we show that the correlation between codon usage and RNA levels is maintained in the nucleus. In addition, codon optimality is tightly correlated with both total and nuclear RNA levels, suggesting that codon usage broadly influences mRNA levels through transcription in a translation-independent manner. A large-scale RNA sequencing-based genetic screen in Neurospora identified 18 candidate factors that when deleted decreased the genome-wide correlation between codon usage and RNA levels and reduced the codon usage effect on gene expression. Most of these factors, such as the H3K36 methyltransferase, are chromatin regulators or transcription factors. Together, our results suggest that the transcriptional effect of codon usage is mediated by multiple transcriptional regulatory mechanisms.