Elavl1 Impacts Osteogenic Differentiation and mRNA Levels of Genes Involved in ECM Organization.

Posttranscriptional gene regulation by Adenylate Uridylate (AU) rich element RNA binding protein, Elavl1 has been implicated in embryonic development as well as progenitor cell differentiation. Elavl1 binds to hundreds of cellular messenger RNAs predominantly through interactions with AU-rich elements (AREs) found in the untranslated regions (UTRs) and functions by regulating their stability. Biological functions of Elavl1 during osteogenic differentiation of bone marrow derived mesenchymal stem cells is not well-understood. Here we report that specific knockdown of nuclear localized Elavl1 by RNA interference in multipotent BMSCs led to increased osteogenic differentiation. Differential gene expression analysis following unbiased total RNA sequencing upon Elavl1 depletion during osteogenic differentiation of BMSCs showed increased levels of multiple mRNAs that are involved in extracellular matrix organization. We further show that many of these mRNAs contain Elavl1 binding consensus motifs that are preserved in their 3' UTRs. RNA stability analyses indicated that depletion of Elavl1 prolongs the steady state RNA levels of several of these mRNAs. Together, our data points to Elavl1 mediated negative regulation of multiple genes involved in ECM organization that play a functional role in MSC osteogenic differentiation.

Endogenous antisense RNA curbs CD39 expression in Crohn's disease.

CD39 is an ectonucleotidase that initiates conversion of extracellular nucleotides into immunosuppressive adenosine. CD39 is expressed by regulatory T (Treg)-cells, where it mediates immunosuppression, and by a subset of T-helper (Th) 17-cells, where it limits pathogenicity. CD39 is regulated via single-nucleotide-polymorphisms and upon activation of aryl-hydrocarbon-receptor and oxygen-mediated pathways. Here we report a mechanism of CD39 regulation that relies on the presence of an endogenous antisense RNA, transcribed from the 3'-end of the human CD39/ENTPD1 gene. CD39-specific antisense is increased in Treg and Th17-cells of Crohn's disease patients over controls. It largely localizes in the cell nucleus and regulates CD39 by interacting with nucleolin and heterogeneous-nuclear-ribonucleoprotein-A1. Antisense silencing results in CD39 upregulation in vitro and amelioration of disease activity in a trinitro-benzene-sulfonic-acid model of colitis in humanized NOD/scid/gamma mice. Inhibition/blockade of antisense might represent a therapeutic strategy to restore CD39 along with immunohomeostasis in Crohn's disease.

PRMT5 inhibition promotes osteogenic differentiation of mesenchymal stromal cells and represses basal interferon stimulated gene expression.

Protein arginine methyltransferases (PRMTs) catalyze symmetric and asymmetric methylation on arginine residues of multiple protein targets including histones and have essential roles in organismal development and disease. PRMT5 mediates symmetric di-methylation (sDMA) of arginine 2 (H3R2me2s) and arginine 8 on histone 3 (H3R8me2s), arginine 3 on histones 2A and 4 (H2A/H4R3me2s) as well as several non-histone substrates like Sm proteins. Here, we found that selective inhibition of PRMT5 in mesenchymal stromal cells (MSCs) led to a reduction in colony forming units (CFUs) and increased osteoblast differentiation. PRMT5 inhibition blocked global symmetric dimethylation of H3R8 and H4R3 but not on H3R2. Genome-wide expression analysis by total RNA sequencing of mesenchymal stromal cells undergoing osteogenic differentiation revealed significant reduction in the intrinsic expression of several interferon-stimulated genes (ISGs) upon PRMT5 inhibition. Effects of PRMT5 inhibition on basal ISG expression and osteogenic differentiation was effectively blocked by exogenous activation of type I IFN signaling. Together, these results indicate important functions for PRMT5 in the regulation of basal interferon gene expression in MSCs and in the control of differentiation potential of MSCs during osteogenic differentiation.

BPI Fold-Containing Family A Member 2/Parotid Secretory Protein Is an Early Biomarker of AKI.

AKI is a major cause of morbidity and mortality and an important contributor to the development and progression of CKD. Molecular biomarkers that improve the detection and prognostication of AKI are therefore required. We assessed the utility as such of BPI fold-containing family A member 2 (BPIFA2), also known as parotid secretory protein, which we identified via a multiplex quantitative proteomics screen of acutely injured murine kidneys. In physiologic conditions, BPIFA2 is expressed specifically in the parotid glands and is abundant in salivary secretions. In our study, AKI induced Bpifa2 expression in the kidneys of mice within 3 hours. Furthermore, we detected BPIFA2 protein in plasma and urine in these models as early as 6 hours after injury. However, renal injury did not induce the expression of Bpifa2 in mice lacking Nur77, an immediate early gene expressed in the kidneys during AKI. Notably, patients with AKI had higher blood and urine levels of BPIFA2 than did healthy individuals. Together, our results reveal that BPIFA2 is a potential early biomarker of AKI.

Noncoding RNA and epigenetic gene regulation in renal diseases.

Kidneys have a major role in normal physiology and metabolic homeostasis. Loss or impairment of kidney function is a common occurrence in several metabolic disorders, including hypertension and diabetes. Chronic kidney disease (CKD) affect nearly 10% of the population worldwide; ranks 18th in the list of causes of death; and contributes to a significant proportion of healthcare costs. The tissue repair and regenerative potential of kidneys are limited and they decline during aging. Recent studies have demonstrated a key role for epigenetic processes and players, such as DNA methylation, histone modifications, noncoding (nc)RNA, and so on, in both kidney development and disease. In this review, we highlight these recent findings with an emphasis on aberrant epigenetic changes that accompany renal diseases, key targets, and their therapeutic value.

In Vitro Corticogenesis from Embryonic Stem Cells Recapitulates the In Vivo Epigenetic Control of Imprinted Gene Expression.

In vitro corticogenesis from embryonic stem cells (ESCs) is an attractive model of cortical development and a promising tool for cortical therapy. It is unknown to which extent epigenetic mechanisms crucial for cortex development and function, such as parental genomic imprinting, are recapitulated by in vitro corticogenesis. Here, using genome-wide transcriptomic and methylation analyses on hybrid mouse tissues and cells, we find a high concordance of imprinting status between in vivo and ESC-derived cortices. Notably, in vitro corticogenesis strictly reproduced the in vivo parent-of-origin-dependent expression of 41 imprinted genes (IGs), including Mest and Cdkn1c known to control corticogenesis. Parent-of-origin-dependent DNA methylation was also conserved at 14 of 18 imprinted differentially methylated regions. The least concordant imprinted locus was Gpr1-Zdbf2, where the aberrant bi-allelic expression of Zdbf2 and Adam23 was concomitant with a gain of methylation on the maternal allele in vitro. Combined, our data argue for a broad conservation of the epigenetic mechanisms at imprinted loci in cortical cells derived from ESCs. We propose that in vitro corticogenesis helps to define the still poorly understood mechanisms that regulate imprinting in the brain and the roles of IGs in cortical development.

Imprinting control regions (ICRs) are marked by mono-allelic bivalent chromatin when transcriptionally inactive.

Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire life-span. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover, they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters, regardless of the developmental stage, thereby contributing to protect cell identity.

ICR noncoding RNA expression controls imprinting and DNA replication at the Dlk1-Dio3 domain.

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells.

PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome.

Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

Transcription and histone methylation changes correlate with imprint acquisition in male germ cells.

Genomic imprinting in mammals is controlled by DNA methylation imprints that are acquired in the gametes, at essential sequence elements called 'imprinting control regions' (ICRs). What signals paternal imprint acquisition in male germ cells remains unknown. To address this question, we explored histone methylation at ICRs in mouse primordial germ cells (PGCs). By 13.5 days post coitum (d.p.c.), H3 lysine-9 and H4 lysine-20 trimethylation are depleted from ICRs in male (and female) PGCs, indicating that these modifications do not signal subsequent imprint acquisition, which initiates at ∼15.5 d.p.c. Furthermore, during male PGC development, H3 lysine-4 trimethylation becomes biallelically enriched at 'maternal' ICRs, which are protected against DNA methylation, and whose promoters are active in the male germ cells. Remarkably, high transcriptional read-through is detected at the paternal ICRs H19-DMR and Ig-DMR at the time of imprint establishment, from one of the strands predominantly. Combined, our data evoke a model in which differential histone modification states linked to transcriptional events may signal the specificity of imprint acquisition during spermatogenesis.

Epigenetic transitions in germ cell development and meiosis.

Germ cell development is controlled by unique gene expression programs and involves epigenetic reprogramming of histone modifications and DNA methylation. The central event is meiosis, during which homologous chromosomes pair and recombine, processes that involve histone alterations. At unpaired regions, chromatin is repressed by meiotic silencing. After meiosis, male germ cells undergo chromatin remodeling, including histone-to-protamine replacement. Male and female germ cells are also differentially marked by parental imprints, which contribute to sex determination in insects and mediate genomic imprinting in mammals. Here, we review epigenetic transitions during gametogenesis and discuss novel insights from animal and human studies.

Cancer therapy via modulation of micro RNA levels: a promising future.

Micro RNAs (miRNAs) are a class of naturally occurring ∼ 22 nt long non-coding small RNA molecules that regulate the expression of a wide range of genes involved in development, growth, proliferation and apoptosis. miRNAs are evolutionarily conserved from plants to animals, and they regulate and fine-tune a diverse array of biological processes. Recently, they have been shown to act as either oncogenes or tumor suppressors in a wide variety of tumors. Here, we review the studies that document the role of miRNAs as key players in human cancer and the potential therapeutic modality of exploiting miRNAs for cancer prognosis and treatment.

Genome-wide DNA demethylation in mammals.

The cytidine deaminase AID and elongator-complex proteins contribute to the extensive removal of DNA methylation in mammalian primordial germ cells and in the paternal pronucleus of the zygote.

RNAi in X inactivation: contrasting findings on the role of interference.

X inactivation is the process that brings about the dosage equivalence of X-linked genes in females to that of males. This complex process initiated at a very early stage of female embryonic development is orchestrated by long non-coding RNAs transcribed in both sense and antisense orientation. Recent studies present contradicting evidence for the role of small RNAs and RNase III enzyme Dicer in the X inactivation process. In this review, I discuss these results in the overall perspective of X inactivation and gene silencing.