The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4

Myogenic microRNAs are important regulators of muscle development and differentiation. To better understand the roles of chromatin-modifying and remodeling enzymes in the activation of myogenic microRNA expression, we have functionally analyzed two different protein arginine methyltransferases, Prmt5 and Prmt4, both of which have previously been implicated in the regulation of myogenic mRNA expression. Both Prmts are required for myogenic microRNA induction during differentiation. Prmt5 is indirectly required due to the necessity of Prmt5 for expression of the transcriptional regulator, myogenin, as ectopic expression of myogenin eliminates Prmt5 dependency. By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences. Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression

Outer Membrane Vesicles Derived From Escherichia coli Regulate Neutrophil Migration by Induction of Endothelial IL-8

Outer membrane vesicles (OMVs) are spherical, proteolipid nanostructures that are constitutively released by Gram-negative bacteria including Escherichia coli. Although it has been shown that administration of E. coli OMVs stimulates a strong pulmonary inflammatory response with infiltration of neutrophils into the lungs in vivo, the mechanism of E. coli OMV-mediated neutrophil recruitment is poorly characterized. In this study, we observed significant infiltration of neutrophils into the mouse lung tissues in vivo, with increased expression of the neutrophil chemoattractant CXCL1, a murine functional homolog of human IL-8, on intraperitoneal administration of E. coli OMVs. In addition, OMVs and CD31-positive endothelial cells colocalized in the mouse lungs. Moreover, in vitro results showed that E. coli OMVs significantly increased IL-8 release from human microvascular endothelial cells and toll-like receptor (TLR)4 was found to be the main component for recognizing E. coli OMVs among human endothelial cell-associated TLRs. Furthermore, the transmigration of neutrophils was suppressed in the lung tissues obtained from TLR4 knockout mice treated with E. coli OMVs. Taken together, our data demonstrated that E. coli OMVs potently recruit neutrophils into the lung via the release of IL-8/CXCL1 from endothelial cells in TLR4- and NF-κB-dependent manners

Bromodomain protein 7 interacts with PRMT5 and PRC2, and is involved in transcriptional repression of their target genes

Histone modification regulates gene expression, and one major regulatory step in this process is the ability of proteins that recognize epigenetic marks to recruit enzymes required to specify transcriptional outcome. Here we show that BRD7 is a component of hSWI–SNF complexes that interacts with PRMT5 and PRC2. Recruitment studies revealed that BRD7 co-localizes with PRMT5 and PRC2 on ‘suppressor of tumorigenecity 7’ (ST7) and retinoblastoma-like protein 2 (RBL2) promoters in patient-derived B cell lines, and that its association with these target genes correlates with hypermethylation of H3R8, H4R3 and H3K27. Furthermore, inhibition of BRD7 expression reduces PRMT5 and PRC2 recruitment to ST7 and RBL2 promoters; however, only ST7 becomes transcriptionally derepressed. Evaluation of the PRMT5- and PRC2-induced epigenetic marks revealed that while H3(Me2)R8, H4(Me2)R3 and H3(Me3)K27 marks are erased from the ST7 promoter, demethylation of RBL2 promoter histones is incomplete. We also show that the arginine demethylase (RDM) JMJD6, which can erase PRMT5-induced H4R3 methylation, and the H3K27-lysine-specific demethylases, KDM6A/UTX and KDM6B/JMJD3, are differentially recruited to ST7 and RBL2. These findings highlight the role played by BRD7 in PRMT5- and PRC2-induced transcriptional silencing, and indicate that recruitment of specific RDMs and KDMs is required for efficient transcriptional derepression

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase delta Catalytic Subunit Gene, POLD1

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage

hnRNP K Supports High-Amplitude D Site-Binding Protein mRNA (Dbp mRNA) Oscillation To Sustain Circadian Rhythms

Circadian gene expression is defined by the gene-specific phase and amplitude of daily oscillations in mRNA and protein levels. D site-binding protein mRNA (Dbp mRNA) shows high-amplitude oscillation; however, the underlying mechanism remains elusive. Here, we demonstrate that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator that activates Dbp transcription via the poly(C) motif within its proximal promoter. Biochemical analyses identified hnRNP K as a specific protein that directly associates with the poly(C) motif in vitro. Interestingly, we further confirmed the rhythmic binding of endogenous hnRNP K within the Dbp promoter through chromatin immunoprecipitation as well as the cycling expression of hnRNP K. Finally, knockdown of hnRNP K decreased mRNA oscillation in both Dbp and Dbp-dependent clock genes. Taken together, our results show rhythmic protein expression of hnRNP K and provide new insights into its function as a transcriptional amplifier of Dbp

PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27^Kip1

<div><p>Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27^Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27^Kip1 expression in human melanoma cells.</p></div

PRMT5 associates with Mep50, but not cyclin D1 or STAT3 in a subset of melanoma cell lines.

<p>A, HEM and representative human melanoma cell lines were harvested and subjected to total cell lysis followed by immunoprecipitation. 10% of input protein, as well as protein from IgG control and IP directed against Mep50 and cyclin D1, underwent immunoblot analysis for IP target proteins as well as PRMT5. B, IP against STAT3 was performed on the same cell line panel, and subjected to immunoblot analysis using antibodies against STAT3 or PRMT5.</p

PRMT5 is upregulated in human malignant melanoma tumors compared to normal epidermis, and subcellular localization of PRMT5 is heterogeneous amongst patient tumors.

<p>A, Brightfield image of PRMT5 staining in a Stage 2/3 tumor and its adjacent normal epidermis. PRMT5 protein was visualized with Vulcan FastRed label (pink); samples were counterstained with hematoxylin to delineate nuclei (arrows) and connective tissue (blue). B, malignant skin tumor of pathologic stage 4, showing predominantly nuclear PRMT5. C, malignant skin tumor of pathologic stage 4, showing predominantly cytoplasmic PRMT5 (arrows indicate hematoxylin-stained nuclei). D, representative images of PRMT5 staining in patient tumors of pathologic stages I–IV.</p