PRMT1 and PRMT8 regulate retinoic acid-dependent neuronal differentiation with implications to neuropathology.

Retinoids are morphogens and have been implicated in cell fate commitment of embryonic stem cells (ESCs) to neurons. Their effects are mediated by RAR and RXR nuclear receptors. However, transcriptional cofactors required for cell and gene-specific retinoid signaling are not known. Here we show that protein arginine methyl transferase (PRMT) 1 and 8 have key roles in determining retinoid regulated gene expression and cellular specification in a multistage neuronal differentiation model of murine ESCs. PRMT1 acts as a selective modulator, providing the cells with a mechanism to reduce the potency of retinoid signals on regulatory "hotspots." PRMT8 is a retinoid receptor target gene itself and acts as a cell type specific transcriptional coactivator of retinoid signaling at later stages of differentiation. Lack of either of them leads to reduced nuclear arginine methylation, dysregulated neuronal gene expression, and altered neuronal activity. Importantly, depletion of PRMT8 results in altered expression of a distinct set of genes, including markers of gliomagenesis. PRMT8 is almost entirely absent in human glioblastoma tissues. We propose that PRMT1 and PRMT8 serve as a rheostat of retinoid signaling to determine neuronal cell specification in a context-dependent manner and might also be relevant in the development of human brain malignancy

Arginine Methyltransferase Prmt8 Provides Cellular Stress Tolerance In Aging Motoneurons

Aging contributes to cellular stress and neurodegeneration. Our understanding is limited regarding the tissue-restricted mechanisms providing protection in postmitotic cells throughout life. Here, we show that spinal cord motoneurons exhibit a high abundance of asymmetric dimethyl arginines (ADMAs) and the presence of this posttranslational modification provides protection against environmental stress. We identify protein arginine methyltransferase 8 (PRMT8) as a tissue-restricted enzyme responsible for proper ADMA level in postmitotic neurons. Male PRMT8 knock-out mice display decreased muscle strength with aging due to premature destabilization of neuromuscular junctions. Mechanistically, inhibition of methyltransferase activity or loss of PRMT8 results in accumulation of unrepaired DNA double-stranded breaks and decrease in the cAMP response-element-binding protein 1 (CREB1) level. As a consequence, the expression of CREB1-mediated prosurvival and regeneration-associated immediate early genes is dysregulated in aging PRMT8 knock-out mice. The uncovered role of PRMT8 represents a novel mechanism of stress tolerance in long-lived postmitotic neurons and identifies PRMT8 as a tissue-specific therapeutic target in the prevention of motoneuron degeneration

A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules.

Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s

Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid

Adipocyte differentiation and function have become the major research targets due to the increasing interest in obesity and related metabolic conditions. Although, late stages of adipogenesis have been extensively studied, the early phases remain poorly understood. Here we present that supplementing ascorbic acid (AsA) to the adipogenic differentiation cocktail enables the robust and efficient differentiation of mouse embryonic stem cells (mESCs) to mature adipocytes. Such ESC-derived adipocytes mimic the gene-expression profile of subcutaneous isolated adipocytes in vivo remarkably well, much closer than 3T3-L1 derived ones. Moreover, the differentiated cells are in a monolayer, allowing a broad range of genome-wide studies in early and late stages of adipocyte differentiation to be performed

Labelled regulatory elements are pervasive features of the macrophage genome and are dynamically utilized by classical and alternative polarization signals

The concept of tissue-specific gene expression posits that lineage-determining transcription factors (LDTFs) determine the open chromatin profile of a cell via collaborative binding, providing molecular beacons to signal-dependent transcription factors (SDTFs). However, the guiding principles of LDTF binding, chromatin accessibility and enhancer activity have not yet been systematically evaluated. We sought to study these features of the macrophage genome by the combination of experimental (ChIP-seq, ATAC-seq and GRO-seq) and computational approaches. We show that Random Forest and Support Vector Regression machine learning methods can accurately predict chromatin accessibility using the binding patterns of the LDTF PU.1 and four other key TFs of macrophages (IRF8, JUNB, CEBPA and RUNX1). Any of these TFs alone were not sufficient to predict open chromatin, indicating that TF binding is widespread at closed or weakly opened chromatin regions. Analysis of the PU.1 cistrome revealed that two-thirds of PU.1 binding occurs at low accessible chromatin. We termed these sites labelled regulatory elements (LREs), which may represent a dormant state of a future enhancer and contribute to macrophage cellular plasticity. Collectively, our work demonstrates the existence of LREs occupied by various key TFs, regulating specific gene expression programs triggered by divergent macrophage polarizing stimuli.This work has been supported by Hungarian Scientific Research Fund [NKFIH K116855, K124298, KH126885 to L.N.]; GINOP-2.3.2-15-2016-0006; GINOP-2.1.7-15-2016-01487; NIH [R01DK115924]; Campus Hungary Scholarship at Centro Nacional de Análisis Genómico (to A.H.); American Heart Association (AHA) [17POST33660450 to B.D.]. Funding for open access charge: Hungarian Scientific Research Fund [NKFIH K116855, K124298, KH126885]; NIH [R01DK115924]; GINOP 2.1.7-15-2016-01487; GINOP 2.3.2-15-2016-000

Measurement of siRNA expression with small RNA specific UPL-based quantitative PCR assays.

<p>Sequence specificity of PRMT1 specific siRNA for exon 3 of PRMT1 (A). PRMT1 specific siRNA levels as detected by qPCR and the corresponding mPRMT1 mRNA as well as protein levels detected by qPCR and Western blot analysis (B).</p

Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.

<p>Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.</p

Sensitivity and specificity of miRNA specific UPL-based quantitative PCR system.

<p>Amplification plot of mmu-mir-1 in range from 10 ng to 10^–3 ng input mouse heart total RNA (A). Sequence similarities and differences between mir-181a, b, and c (B). Amplification plot of synthetic mir-181a miRNA ranging from 10 pM to 10^–4 pM input mir-181a amplicon (C). Standard curve of synthetic mir-181a miRNA (D). Specificity and relative detection capacity of mir-181 specific UPL-based qPCR assays. Numbers represent the percentage of the signals measured on the synthetic amplicons. 100% is always the signal measured by an assay on its specific synthetic amplicon, like mir-180a assay on the mir-181a synthetic amplicon. In brackets the corresponding Cp values are shown.(E).</p