Natural antisense transcripts as versatile regulators of gene expression.

Long non-coding RNAs (lncRNAs) are emerging as a major class of gene products that have central roles in cell and developmental biology. Natural antisense transcripts (NATs) are an important subset of lncRNAs that are expressed from the opposite strand of protein-coding and non-coding genes and are a genome-wide phenomenon in both eukaryotes and prokaryotes. In eukaryotes, a myriad of NATs participate in regulatory pathways that affect expression of their cognate sense genes. Recent developments in the study of NATs and lncRNAs and large-scale sequencing and bioinformatics projects suggest that whether NATs regulate expression, splicing, stability or translation of the sense transcript is influenced by the pattern and degrees of overlap between the sense-antisense pair. Moreover, epigenetic gene regulatory mechanisms prevail in somatic cells whereas mechanisms dependent on the formation of double-stranded RNA intermediates are prevalent in germ cells. The modulating effects of NATs on sense transcript expression make NATs rational targets for therapeutic interventions.

Site-Directed Mutagenesis Protocol to Determine the Role of Amino Acid Residues in Polycomb Group (PcG) Protein Function.

Site-directed mutagenesis (SDM) is a technique in molecular biology and protein engineering that is widely used to determine the significance of specific residues involved in post-translational modifications (PTMs), protein structure, function, and stability. Here, we describe a simple and cost-effective polymerase chain reaction (PCR)-based SDM method. This method can be used to introduce point mutation, short addition, or deletions in protein sequences. Using polycomb repressive complex-2 (PRC2)-associated protein JARID2 as an example, we demonstrate how SDM can be used to study structural and consequently functional changes in a protein.

Antisense RNAs during early vertebrate development are divided in groups with distinct features.

Long noncoding RNAs or lncRNAs are a class of non-protein-coding RNAs that are >200 nt in length. Almost 50% of lncRNAs during zebrafish development are transcribed in an antisense direction to a protein-coding gene. However, the role of these natural antisense transcripts (NATs) during development remains enigmatic. To understand NATs in early vertebrate development, we took a computational biology approach and analyzed existing as well as novel data sets. Our analysis indicates that zebrafish NATs can be divided into two major classes based on their coexpression patterns with respect to the overlapping protein-coding genes. Group 1 NATs have characteristics similar to maternally deposited RNAs in that their levels decrease as development progresses. Group 1 NAT levels are negatively correlated with that of overlapping sense-strand protein-coding genes. Conversely, Group 2 NATs are coexpressed with overlapping protein-coding genes. In contrast to Group 1, which is enriched in genes involved in developmental pathways, Group 2 protein-coding genes are enriched in housekeeping functions. Group 1 NATs also show larger overlap and higher complementarity with the sense-strand mRNAs compared to other NATs. In addition, our transcriptomics data, quantifying RNA levels from cytoplasmic and nuclear compartments, indicates that Group 1 NATs are more abundant in the cytosol. Based on their expression pattern, cytosolic nature, and their higher complementarity to the overlapping developmental mRNAs, we speculate that Group 1 NATs function post-transcriptionally to silence spurious expression of developmental genes.

A long intergenic non-coding RNA regulates nuclear localization of DNA methyl transferase-1.

DNA methyl transferase-1 or DNMT1 maintains DNA methylation in the genome and is important for regulating gene expression in cells. Aberrant changes in DNMT1 activity and DNA methylation are commonly observed in cancers and many other diseases. Recently, a number of long intergenic non-protein-coding RNAs or lincRNAs have been shown to play a role in regulating DNMT1 activity. CCDC26 is a nuclear lincRNA that is frequently mutated in cancers and is a hotbed for disease-associated single nucleotide changes. However, the functional mechanism of CCDC26 is not understood. Here, we show that this lincRNA is concentrated on the nuclear periphery. Strikingly, in the absence of CCDC26 lincRNA, DNMT1 is mis-located in the cytoplasm, and the genomic DNA is significantly hypomethylated. This is accompanied by double-stranded DNA breaks and increased cell death. These results point to a previously unrecognized mechanism of lincRNA-mediated subcellular localization of DNMT1 and regulation of DNA methylation.

The Missing Link Between Cancer-Associated Variants and LncRNAs.

Despite studies indicating that long noncoding RNAs, or lncRNAs, can act as proto-oncogenes, the implications of large numbers of cancer-associated variants found within noncoding RNA loci remain largely unknown. Here, we draw upon emerging studies to speculate on how variants of lncRNAs might play a role in cancer development.

8q24.21 Locus: A Paradigm to Link Non-Coding RNAs, Genome Polymorphisms and Cancer.

The majority of the human genome is comprised of non-protein-coding genes, but the relevance of non-coding RNAs in complex diseases has yet to be fully elucidated. One class of non-coding RNAs is long non-coding RNAs or lncRNAs, many of which have been identified to play a range of roles in transcription and translation. While the clinical importance of the majority of lncRNAs have yet to be identified, it is puzzling that a large number of disease-associated genetic variations are seen in lncRNA genes. The 8q24.21 locus is rich in lncRNAs and very few protein-coding genes are located in this region. Interestingly, the 8q24.21 region is also a hot spot for genetic variants associated with an increased risk of cancer. Research focusing on the lncRNAs in this area of the genome has indicated clinical relevance of lncRNAs in different cancers. In this review, we summarise the lncRNAs in the 8q24.21 region with respect to their role in cancer and discuss the potential impact of cancer-associated genetic polymorphisms on the function of lncRNAs in initiation and progression of cancer.

Combined transcriptomic and phosphoproteomic analysis of BMP4 signaling in human embryonic stem cells.

Human embryonic stem cells (hESCs) are an invaluable tool in the fields of embryology and regenerative medicine. Activin A and BMP4 are well-characterised growth factors implicated in pluripotency and differentiation. In the current study, hESCs are cultured in a modified version of mTeSR1, where low concentrations of ActivinA substitute for TGFß. This culture system is further used to investigate the changes induced by BMP4 on hESCs by employing a combination of transcriptomic and phosphoproteomic approaches. Results indicate that in a pluripotent state, hESCs maintain WNT signaling under negative regulation by expressing pathway inhibitors. Initial stages of differentiation are characterized by upregulation of WNT pathway ligands, TGFß pathway inhibitors which have been shown in Xenopus to expand the BMP signaling range essential for embryonic patterning, and mesendodermal transcripts. Moreover, BMP4 enhances the phosphorylation of proteins associated with migration and transcriptional regulation. Results further indicate the vital regulatory role of Activin A and BMP4 in crucial fate decisions in hESCs.

Autoregulation of JARID2 through PRC2 interaction with its antisense ncRNA.

OBJECTIVE: JARID2 is a member of chromatin-modifying Polycomb Repressive Complex-2 or PRC2. It plays a role in recruiting PRC2 to developmental genes and regulating its activity. JARID2 along with PRC2 is indispensable for normal development. However, it remains unclear how JARID2 expression itself is regulated. Recently a number of non-protein-coding RNAs or ncRNAs are shown to regulate transcription. An antisense ncRNA, JARID2-AS1, is expressed from the first intron of JARID2 isoform-1 but its role in regulation of JARID2 expression has not been investigated. The objective of this study was to explore the role of JARID2-AS1 in regulating JARID2 and consequently PRC2. RESULTS: We found that JARID2-AS1 is localised in the nucleus and shows anti-correlated expression pattern to that of JARID2 isoform-1 mRNA. More interestingly, data mining approach strongly indicates that JARID2-AS1 binds to PRC2. These are important observations that provide insights into transcriptional regulation of JARID2, especially because they indicate that JARID2-AS1 by interacting and probably recruiting PRC2 participates in an auto-regulatory loop that controls levels of JARID2. This holds importance in regulation of developmental and differentiation processes. However, to support this hypothesis, further in-depth studies are needed which can verify JARID2-AS1-PRC2 interactions.

Functional annotation of human long noncoding RNAs via molecular phenotyping.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

A novel form of JARID2 is required for differentiation in lineage-committed cells.

Polycomb repressive complex-2 (PRC2) is a group of proteins that play an important role during development and in cell differentiation. PRC2 is a histone-modifying complex that catalyses methylation of lysine 27 of histone H3 (H3K27me3) at differentiation genes leading to their transcriptional repression. JARID2 is a co-factor of PRC2 and is important for targeting PRC2 to chromatin. Here, we show that, unlike in embryonic stem cells, in lineage-committed human cells, including human epidermal keratinocytes, JARID2 predominantly exists as a novel low molecular weight form, which lacks the N-terminal PRC2-interacting domain (ΔN-JARID2). We show that ΔN-JARID2 is a cleaved product of full-length JARID2 spanning the C-terminal conserved jumonji domains. JARID2 knockout in keratinocytes results in up-regulation of cell cycle genes and repression of many epidermal differentiation genes. Surprisingly, repression of epidermal differentiation genes in JARID2-null keratinocytes can be rescued by expression of ΔN-JARID2 suggesting that, in contrast to PRC2, ΔN-JARID2 promotes activation of differentiation genes. We propose that a switch from expression of full-length JARID2 to ΔN-JARID2 is important for the up-regulation differentiation genes.

Regulation of Leukocytes by TspanC8 Tetraspanins and the "Molecular Scissor" ADAM10.

A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane protein that functions as a "molecular scissor" to cleave the extracellular regions from its transmembrane target proteins. ADAM10 is well characterized as the ligand-dependent activator of Notch proteins, which control cell fate decisions. Indeed, conditional knockouts of ADAM10 in mice reveal impaired B-, T-, and myeloid cell development and/or function. ADAM10 cleaves many other leukocyte-expressed substrates. On B-cells, ADAM10 cleavage of the low-affinity IgE receptor CD23 promotes allergy and asthma, cleavage of ICOS ligand impairs antibody responses, and cleavage of the BAFF-APRIL receptor transmembrane activator and CAML interactor, and BAFF receptor, reduce B-cell survival. On microglia, increased ADAM10 cleavage of a rare variant of the scavenger receptor triggering receptor expressed on myeloid cells 2 may increase susceptibility to Alzheimer's disease. We and others recently showed that ADAM10 interacts with one of six different regulatory tetraspanin membrane proteins, which we termed the TspanC8 subgroup, comprising Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. The TspanC8s are required for ADAM10 exit from the endoplasmic reticulum, and emerging evidence suggests that they dictate ADAM10 subcellular localization and substrate specificity. Therefore, we propose that ADAM10 should not be regarded as a single scissor, but as six different scissors with distinct substrate specificities, depending on the associated TspanC8. In this review, we collate recent transcriptomic data to present the TspanC8 repertoires of leukocytes, and we discuss the potential role of the six TspanC8/ADAM10 scissors in leukocyte development and function.

Exercise and high-fat feeding remodel transcript-metabolite interactive networks in mouse skeletal muscle.

Enhanced coverage and sensitivity of next-generation 'omic' platforms has allowed the characterization of gene, metabolite and protein responses in highly metabolic tissues, such as, skeletal muscle. A limitation, however, is the capability to determine interaction between dynamic biological networks. To address this limitation, we applied Weighted Analyte Correlation Network Analysis (WACNA) to RNA-seq and metabolomic datasets to identify correlated subnetworks of transcripts and metabolites in response to a high-fat diet (HFD)-induced obesity and/or exercise. HFD altered skeletal muscle lipid profiles and up-regulated genes involved in lipid catabolism, while decreasing 241 exercise-responsive genes related to skeletal muscle plasticity. WACNA identified the interplay between transcript and metabolite subnetworks linked to lipid metabolism, inflammation and glycerophospholipid metabolism that were associated with IL6, AMPK and PPAR signal pathways. Collectively, this novel experimental approach provides an integrative resource to study transcriptional and metabolic networks in skeletal muscle in the context of health and disease.

Exon junction complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster.

Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, we found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosomes. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. We also show that eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, whereas Y14 and MAGO fractionate separately. Cumulatively, our data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood.

Identification of RNA-Protein Interactions Through In Vitro RNA Pull-Down Assays.

Recent advances in next-generation sequencing have revealed that majority of the human genome is transcribed into long and short RNA (ncRNA) transcripts. Many ncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore identifying proteins that interact with ncRNAs is central to deciphering ncRNA functions. Here we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example we describe pull-down of proteins interacting with ncRNA XIST, which assists in the recruitment of the polycomb-repressive complex-2 (PRC2) and drives X-chromosomal inactivation.

Remote Axial Tuning in Microscopy Utilizing Hydrogel-Driven Tunable Liquid Lens.

This work presents the use of a tunable-focus thermo-responsive hydrogel based liquid lens in combination with an objective lens to achieve remote axial focusing in a conventional microscopy. The goal of this design is to eliminate image distortion due to sample vibrations caused by mechanical stage scanning. This approach reduces the mechanical complexity and power consumption due to the use of electrically tunable lenses while achieving a two-fold increase in the axial scanning range. The merits of the proposed design were demonstrated by characterizing a customized microscope system over a scanning range of 1700 µm. A lateral resolution of 2 µm was obtained consistently throughout the scanning range. Healthy Spodoptera frugiperda Sf21 insect cells imaging was used to verify the depth scanning ability and the resolution of our remote focusing microscope system.

Micro-Fresnel-Zone-Plate Array on Flexible Substrate for Large Field-of-View and Focus Scanning.

Field of view and accommodative focus are two fundamental attributes of many imaging systems, ranging from human eyes to microscopes. Here, we present arrays of Fresnel zone plates fabricated on a flexible substrate, which allows for the adjustment of both the field of view and optical focus. Such zone plates function as compact and lightweight microlenses and are fabricated using silicon nanowires. Inspired by compound eyes in nature, these microlenses are designed to point along various angles in order to capture images, offering an exceptionally wide field of view. Moreover, by flexing the substrate, the lens position can be adjusted, thus achieving axial focus scanning. An array of microlenses on a flexible substrate was incorporated into an optical system to demonstrate high resolution imaging of objects located at different axial and angular positions. These silicon based microlenses could be integrated with electronics and have a wide range of potential applications, from medical imaging to surveillance.

Genome-wide regulatory analysis reveals that T-bet controls Th17 lineage differentiation through direct suppression of IRF4.

The complex relationship between Th1 and Th17 cells is incompletely understood. The transcription factor T-bet is best known as the master regulator of Th1 lineage commitment. However, attention is now focused on the repression of alternate T cell subsets mediated by T-bet, particularly the Th17 lineage. It has recently been suggested that pathogenic Th17 cells express T-bet and are dependent on IL-23. However, T-bet has previously been shown to be a negative regulator of Th17 cells. We have taken an unbiased approach to determine the functional impact of T-bet on Th17 lineage commitment. Genome-wide analysis of functional T-bet binding sites provides an improved understanding of the transcriptional regulation mediated by T-bet, and suggests novel mechanisms by which T-bet regulates Th cell differentiation. Specifically, we show that T-bet negatively regulates Th17 lineage commitment via direct repression of the transcription factor IFN regulatory factor-4 (IRF4). An in vivo analysis of the pathogenicity of T-bet-deficient T cells demonstrated that mucosal Th17 responses were augmented in the absence of T-bet, and we have demonstrated that the roles of T-bet in enforcing Th1 responses and suppressing Th17 responses are separable. The interplay of the two key transcription factors T-bet and IRF4 during the determination of T cell fate choice significantly advances our understanding of the mechanisms underlying the development of pathogenic T cells.

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming.

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.

T-bet and GATA3 orchestrate Th1 and Th2 differentiation through lineage-specific targeting of distal regulatory elements.

T-bet and GATA3 regulate the CD4+ T cell Th1/Th2 cell fate decision but little is known about the interplay between these factors outside of the murine Ifng and Il4/Il5/Il13 loci. Here we show that T-bet and GATA3 bind to multiple distal sites at immune regulatory genes in human effector T cells. These sites display markers of functional elements, act as enhancers in reporter assays and are associated with a requirement for T-bet and GATA3. Furthermore, we demonstrate that both factors bind distal sites at Tbx21 and that T-bet directly activates its own expression. We also show that in Th1 cells, GATA3 is distributed away from Th2 genes, instead occupying T-bet binding sites at Th1 genes, and that T-bet is sufficient to induce GATA3 binding at these sites. We propose these aspects of T-bet and GATA3 function are important for Th1/Th2 differentiation and for understanding transcription factor interactions in other T cell lineage decisions.

Genome-wide analyses of Zta binding to the Epstein-Barr virus genome reveals interactions in both early and late lytic cycles and an epigenetic switch leading to an altered binding profile.

The Epstein-Barr virus (EBV) genome sustains substantial epigenetic modification involving chromatin remodelling and DNA methylation during lytic replication. Zta (ZEBRA, BZLF1), a key regulator of the EBV lytic cycle, is a transcription and replication factor, binding to Zta response elements (ZREs) in target promoters and EBV lytic origins of replication. In vitro, Zta binding is modulated by DNA methylation; a subset of CpG-containing Zta binding sites (CpG ZREs) is bound only in a DNA methylation-dependent manner. The question of how the dynamic epigenetic environment impacts Zta interaction during the EBV lytic cycle is unknown. To address this, we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) to identify Zta binding sites across the EBV genome before and after viral DNA replication. Replication did not alter the association of Zta across many regions of the EBV genome, but a striking reduction in Zta binding occurred at some loci that contain CpG ZREs. Separating Zta-bound DNA into methylated and nonmethylated fractions, we found that promoters that contain CpG ZREs were enriched in the methylated fraction but that Zta binding to promoters lacking CpG ZREs was not reduced. We hypothesize that the loss of DNA methylation on the EBV genome during the lytic cycle causes the reduced binding to CpG ZREs; this may act as a lytic cycle epigenetic switch. However, the epigenetic changes associated with the replicated EBV genome do not affect the interaction of Zta with many loci that are rich in non-CpG ZREs; this leads to sustained binding at these regions.