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The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accu-mulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A), followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in doublestrand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following doublestrand breaks and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing. We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection. [ABSTRACT FROM AUTHOR]

Protein arginine methyltransferase 5 (PRMT5) is a member of the arginine methyltransferase protein family that critically mediates the symmetric dimethylation of Arg-3 at histone H4 (H4R3me2s) and is involved in many key cellular processes, including hematopoiesis. However, the post-translational modifications (PTMs) of PRMT5 that may affect its biological functions remain less well-understood. In this study, using MS analyses, we found that PRMT5 itself is methylated in human erythroleukemia Lys-562 cells. Biochemical assays revealed that coactivator-associated arginine methyltransferase 1 (CARM1) interacts directly with and methylates PRMT5 at Arg-505 both in vivo and in vitro. Substitutions at Arg-505 significantly reduced PRMT5's methyltransferase activity, decreased H4R3me2s enrichment at the γ-globin gene promoter, and increased the expression of the γ-globin gene in Lys-562 cells. Moreover, CARM1 knockdown consistently reduced PRMT5 activity and activated γ-globin gene expression. Importantly, we show that CARM1-mediated methylation of PRMT5 is essential for the intracellular homodimerization ofPRMT5to its active form. These results thus reveal a critical PTM of PRMT5 that represses human γ-globin gene expression.Weconclude that CARM1-mediated asymmetric methylation of PRMT5 is critical for its dimerization and methyltransferase activity leading to the repression of γ-globin expression. Given PRMT5's crucial role in diverse cellular processes, these findings may inform strategies for manipulating its methyltransferase activity for managing hemoglobinopathy or cancer. [ABSTRACT FROM AUTHOR]

Production of hemoglobin during development is tightly regulated. For example, expression from the human β-globin gene locus, comprising β-, δ-, ∊-, and γ-globin genes, switches from ∊-globin to γ-globin during embryonic development and then from γ-globin to β-globin after birth. Expression of human ∊-globin in mice has been shown to ameliorate anemia caused by β-globin mutations, including those causing β-thalassemia and sickle cell disease, raising the prospect that reactivation of ∊-globin expression could be used in managing these conditions in humans. Although the human globin genes are known to be regulated by a variety of multiprotein complexes containing enzymes that catalyze epigenetic modifications, the exact mechanisms controlling ∊-globin gene silencing remain elusive. Here we found that the heterochromatin protein HP1γ, a multifunctional chromatin- and DNA-binding protein with roles in transcriptional activation and elongation, represses ∊-globin expression by interacting with a histone-modifying enzyme, lysine methyltransferase SUV4-20h2. Silencing of HP1γ expression markedly decreased repressive histone marks and the multimethylation of histone H3 lysine 9 and H4 lysine 20, leading to a significant decrease in DNA methylation at the proximal promoter of the ∊-globin gene and greatly increased ∊-globin expression. In addition, using chromatin immunoprecipitation, we showed that SUV4-20h2 facilitates the deposition of HP1γ on the ∊-globin-proximal promoter. Thus, these data indicate that HP1γ is a novel epigenetic repressor of ∊-globin gene expression and provide a potential strategy for targeted therapies for β-thalassemia and sickle cell disease. [ABSTRACT FROM AUTHOR]

The T-synthase (Core 1 β3-galactosyltransferase) and its molecular chaperone Cosmc regulate the biosynthesis of mucin type O-glycans on glycoproteins, and evidence suggests that both T-synthase and Cosmc are transcriptionally suppressed in several human diseases, although the transcriptional regulation of these two genes is not understood. Here we characterized the promoters essential for human Cosmc and T-synthase transcription. The upstream regions of the genes lack a conventional TATA box but contain CpG-islands: cCpG-I and cCpG-II for Cosmc and tCpG for T-synthase. Using luciferase reporter assays, site-directed mutagenesis, ChIP assays, and Mithramycin A treatment, we identified the core promoters within cCpG-II and tCpG, which contain two binding sites for Kruppel-like transcription factors (KLF), including SP1/SP3, respectively. Methylome analysis of Tn4 B cells, which harbor a silenced Cosmc, confirmed the hypermethylation of Cosmc core promoter, but not for that of T-synthase. These results demonstrate that Cosmc and T-synthase are transcriptionally regulated at a basal level by SP/KLF family transcription factors, which explains their ubiquitous and coordinated expression, and also indicates that they are differentially epigenetically regulated beyond X-chromosome imprinting. These results are important in understanding the regulation of these genes that have roles in human diseases, such as IgA Nephropathy and cancer. [ABSTRACT FROM AUTHOR]