Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies

Introduction Placenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are conditions postulated to originate from initial failure of placentation, leading to clinical sequelae indicative of endothelial dysfunction. Vascular smooth muscle aberrations have also been implicated in the pathogenesis of both disorders via smooth muscle contractility and relaxation mediated by Myosin Light Chain Phosphatase (MLCP) and the oppositional contractile action of Myosin Light Chain Kinase. PPP1R12A is a constituent part of the MLCP complex responsible for dephosphorylation of myosin fibrils. We hypothesize that alternative splicing of micro-exons result in isoforms lacking the functional leucine zipper (LZ) domain which may give those cells expressing these alternative transcripts a tendency towards contraction and vasoconstriction. Methods Expression was determined by qRT-PCR. Epigenetic profiling consisted of bisulphite-based DNA methylation analysis and ChIP for underlying histone modifications. Results We identified several novel transcripts with alternative micro-exon inclusion that would produce LZ- PPP1R12A protein. qRT-PCR revealed some isoforms, including the PPP1R12A canonical transcript, are differentially expressed in placenta biopsies from PE and IUGR samples compared to uncomplicated pregnancies. Discussion We propose that upregulation of PPP1R12A expression in complicated pregnancies may be due to enhanced promoter activity leading to increased transcription as a response to physiological stress in the placenta, which we show is independent of promoter DNA methylation

DC Respond to Cognate T Cell Interaction in the Antigen-Challenged Lymph Node

Dendritic cells (DC) are unrivaled in their potential to prime naive T cells by presenting antigen and providing costimulation. DC are furthermore believed to decode antigen context by virtue of pattern recognition receptors and to polarize T cells through cytokine secretion toward distinct effector functions. Diverse polarized T helper (TH) cells have been explored in great detail. In contrast, studies of instructing DC have to date largely been restricted to in vitro settings or adoptively transferred DC. Here we report efforts to unravel the DC response to cognate T cell encounter in antigen-challenged lymph nodes (LN). Mice engrafted with antigen-specific T cells were immunized with nanoparticles (NP) entrapping adjuvants and absorbed with antigen to study the immediate DC response to T cell encounter using bulk and single cell RNA-seq profiling. NP induced robust antigen-specific TH1 cell responses with minimal bystander activation. Fluorescent-labeled NP allowed identification of antigen-carrying DC and focus on transcriptional changes in DC that encounter T cells. Our results support the existence of a bi-directional crosstalk between DC and T cells that promotes TH1 responses, including involvement of the ubiquitin-like molecule Isg15 that merits further study

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening.

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process

Engrafted parenchymal brain macrophages differ from microglia in transcriptome, chromatin landscape and response to challenge

Irradiation depletes brain microglia cells and induces replenishment of the pool by bone marrow (BM)-derived macrophage. Here the authors show, using mouse BM chimera, that BM-derived macrophages establish long-term residency in the brain, but remain distinct from resident microglia in their transcriptome and gene accessibility landscape

Dimerization of the ARF2A protein and its interaction.

<p>ARF2A was cloned downstream of the DNA-binding domain (DB-ARF2A) and co-transformed into yeast with either (A) ARF2A cloned downstream of the activation domain (AD-ARF2A); or (B) ASR1 cloned downstream of the activation domain (AD-ASR1), yeast growth on media lacking leucine, tryptophan, histidine and adenine indicated positive protein-protein interactions. (C) Relative expression levels of <i>ASR1</i> in WT cv. MicroTom fruit at five developmental stages (IG: immature green; MG: mature green; Br: breaker; Or: orange; and R: red), error bars represent SE; statistical significance was evaluated using an ANOVA test (JMP software, SAS) with three biological repeats based on the average of three technical replicates, values indicated by the same letter (a,b,c) are not statistically significant, p-value<0.05. (D) A Bimolecular Fluorescence Complementation assay (BiFC) was carried out by transient expression in tobacco leaves; ARF2A was cloned downstream of the amino-terminal region of YFP (yellow fluorescent protein; YN-ARF2A) and ASR1 was cloned downstream of the carboxy-terminal region of YFP (YC-ASR1); leaf regions were examined for fluorescent signal by light and confocal fluorescence microscopy. Inset zoom region shows that the ARF2A-ASR1 interaction is nuclear localized. Scale bars in the light and confocal fluorescence microscopy represent 50 μm and 10 μm, respectively.</p

Microarray analysis of <i>ARF2-OX</i> fruit.

<p>Gene expression was analyzed in WT at 42 dpa (mature green stage; WT-42G) and 53 dpa (red stage; WT-53R) and green (<i>ARF2-OX</i>-42G) and red (<i>ARF2-OX</i>-42R) patches from <i>ARF2-OX</i> fruit at 42 dpa by microarray analysis. Results are displayed as (A) a principal component analysis (PCA) and (B) a Venn diagram of the differentially expressed genes in the comparisons: <i>ARF2-OX</i>-42G to WT-42G; <i>ARF2-OX</i>-42R to WT-42G; and WT-53R to WT-42G. P-value<0.01 and FDR<0.05; dpa: days post anthesis.</p

Phenotype and expression levels of <i>ARF2</i> genes in <i>ARF2as</i> transgenic lines.

<p>Fruit of <i>ARF2as</i> lines were analysed for relative expression levels by qRT-PCR of (A) <i>ARF2A;</i> and (B) <i>ARF2B</i>. (C-D) The ripening of <i>ARF2as</i> fruit is delayed as compared to WT. (D-E) <i>ARF2as</i> fruit were parthenocarpic or nearly parthenocarpic with reduced number of seeds. (F) Principle component analysis (PCA) plot from untargeted analysis of metabolites. DPA: days post anthesis; error bars represent SE; Statistical significance was evaluated using a student’s t-test, **p-value<0.01.</p

<i>ARF2A</i> expression in tomato fruit.

<p>Relative expression levels of <i>ARF2A</i> analyzed by qRT-PCR, in (A) WT fruit at five developmental stages; (B) <i>rin</i> and <i>nor</i> mutants; (C) <i>TAGL1</i> over-expressing fruit (<i>35S</i>:<i>TAGL1</i>); and (D) <i>nr</i> mutant. Relative expression levels of <i>ARF2A</i> in fruit at three developmental stages, treated with (E) 1-MCP; (F) ethylene; (H) NAA; and (J) ABA. Relative expression levels of <i>ARF2A</i> in fruit at the MG stage, at 0, 2, 4 and 6 days post-treatment with (G) ethrel; (I) NAA; and (K) ABA. Error bars represent SE. Statistical significance was evaluated using a student’s t-test, *p-value<0.05, **p-value<0.01 and ***p-value<0.001; dpa: days post anthesis; IG: immature green; MG: mature green; Br: breaker; Or: orange; and R: red.</p

Metabolic analysis of <i>ARF2-OX</i> fruit.

<p>WT and <i>ARF2-OX</i> fruit were analyzed at 42 and 53 dpa by UPLC-qTOF-MS in positive mode, (A) results are visualized by a principle component analysis (PCA) plot; and displayed as histograms for (B) targeted flavonoids (upper row), and targeted glycoalkaloids (lower row). (C) Isoprenoids were analyzed in WT and <i>ARF2-OX</i> fruit at 42 and 53 dpa by HPLC. Grey bars represent WT, black bars <i>ARF2-OX</i> green/yellow patches and white bars <i>ARF2-OX</i> red patches. Error bars represent SE. Statistical significance was evaluated using a student’s t-test, *p-value<0.05; dpa: days post anthesis.</p