Evidence accumulated over the past decade shows that long non-coding RNAs (lncRNAs) are widely expressed and have key roles in gene regulation. Recent studies have begun to unravel how the biogenesis of lncRNAs is distinct from that of mRNAs and is linked with their specific subcellular localizations and functions. Depending on their localization and their specific interactions with DNA, RNA and proteins, lncRNAs can modulate chromatin function, regulate the assembly and function of membraneless nuclear bodies, alter the stability and translation of cytoplasmic mRNAs and interfere with signalling pathways. Many of these functions ultimately affect gene expression in diverse biological and physiopathological contexts, such as in neuronal disorders, immune responses and cancer. Tissue-specific and condition-specific expression patterns suggest that lncRNAs are potential biomarkers and provide a rationale to target them clinically. In this Review, we discuss the mechanisms of lncRNA biogenesis, localization and functions in transcriptional, post-transcriptional and other modes of gene regulation, and their potential therapeutic applications.

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Gene regulation by long non-coding RNAs and its biological functions.

The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure.

Control of Cell Identity Genes Occurs in Insulated Neighborhoods in Mammalian Chromosomes

Long non-coding RNAs (lncRNAs) are diverse transcription products emanating from thousands of loci in mammalian genomes. Cis-acting lncRNAs, which constitute a substantial fraction of lncRNAs with an attributed function, regulate gene expression in a manner dependent on the location of their own sites of transcription, at varying distances from their targets in the linear genome. Through various mechanisms, cis-acting lncRNAs have been demonstrated to activate, repress or otherwise modulate the expression of target genes. We discuss the activities that have been ascribed to cis-acting lncRNAs, the evidence and hypotheses regarding their modes of action, and the methodological advances that enable their identification and characterization. The emerging principles highlight lncRNAs as transcriptional units highly adept at contributing to gene regulatory networks and to the generation of fine-tuned spatial and temporal gene expression programmes.

Regulation of gene expression by cis-acting long non-coding RNAs.

Precise control of gene expression is fundamental to cell function and development. Although ultimately gene expression relies on DNA-binding transcription factors to guide the activity of the transcription machinery to genes, it has also become clear that chromatin and histone post-translational modification have fundamental roles in gene regulation. Polycomb repressive complexes represent a paradigm of chromatin-based gene regulation in animals. The Polycomb repressive system comprises two central protein complexes, Polycomb repressive complex 1 (PRC1) and PRC2, which are essential for normal gene regulation and development. Our early understanding of Polycomb function relied on studies in simple model organisms, but more recently it has become apparent that this system has expanded and diverged in mammals. Detailed studies are now uncovering the molecular mechanisms that enable mammalian PRC1 and PRC2 to identify their target sites in the genome, communicate through feedback mechanisms to create Polycomb chromatin domains and control transcription to regulate gene expression. In this Review, we discuss and contextualize the emerging principles that define how this fascinating chromatin-based system regulates gene expression in mammals.

The molecular principles of gene regulation by Polycomb repressive complexes.

In Saugetieren konnen genetische und epigenetische Unterschiede zwischen den elterlichen Allelen zu allele-spezifischer Genexpression (AGE) fuhren. RNA-seq wurde seit der Entwicklung von Hochdurchsatzsequenzierung verwendet um AGE in Mensch- und Mausgeweben zu detektieren. Da man die DNA-Sequenz der Eltern benotigt, erweist sich ein genomweiter Nachweis von allele-spezifischer Genexpression im Menschen als schwierig. Ein geeigneteres Modell um AGE nachzuweisen stellen Kreuzungen von genetisch unterschiedlichen Stammen von Labormause dar, da die genetischen Unterschiede zwischen vielen Stammen bereits bekannt sind. Obwohl AGE Detektion mittels RNA-seq eine anerkannte Methode ist, gab es zu Beginn dieser Studie keine bioinformatische Software, die prazise und mit einer niedrigen Fehlerquote, AGE detektiert. Deshalb entwickelten wir im ersten Teil dieser Studie die benutzerfreundliche Software Allelome.PRO, welche mit hoher Prazision allele-spezifische Genexpression oder Histonmodifikationen erfasst. Zusatzlich klassifiziert die Software jedes Gen in einem Zelltyp in eine der folgenden Kategorien: biallelisch, Mausstamm-spezifisch, gepragt oder nicht-informativ. Dadurch wird das gesamte allele-spezifische Bild aller aktiven Gene – „Allelome“ genannt - abgebildet. Im nachsten Schritt verwendeten wir Allelome.PRO um AGE fur Protein und nicht-Protein-kodierenden (nk) Gene in 23 Geweben, in jeweils verschiedenen Entwicklungsstadien der Maus, zu identifizieren um das Maus Allelome zu erhalten. Diese Entwicklungsstadien beinhalten pluripotente, embryonale, extra-embryonale, neugeborene und adulte Gewebe. Diese an der Maus neuartige Analyse fuhrte zu folgenden drei Erkenntnissen. Erstens wurden viele Gene mit gewebespezifischer Mausstamm-spezifischer Expression identifiziert und dass dieses Expressionsmuster von naheliegenden Mausstamm-spezifischen Expressions-Aktivatoren reguliert wird. Diese Aktivatoren werden von Histonen mit der H3K27ac Modifikation markiert. Zweitens wurden in 19 weiblichen Geweben eine unerwartet grose Zahl an Genen, die dem Prozess der X-Chromosom-Inaktivierung entkommen, genannt „Escaper“, gefunden. Im Gegensatz zu den meisten Studien, die von niedrigen Escaper-Prozenten in der Maus berichten, fanden wir einen dem Menschen ahnlichen Prozentsatz von 15 Prozent. Bemerkenswert ist der hohe Escaper-Anteil von 50 Prozent im adulten Beinmuskel. Drittens konnten wir zeigen das gepragte Gengruppen viel groser sind als bisher angenommen und die Grose dramatisch zwischen Geweben und Entwicklungsstadien variiert. Insbesondere zeigen wir anhand von genetischen Mausmodellen, dass sich die gepragte Igf2r Gengruppe in der Plazenta uber zehn Megabasen erstreckt und somit die groste, gepragte Region in der Maus reprasentiert. Zusammenfassend zeigen unsere Ergebnisse, dass AGE welche durch genetische Unterschiede zwischen den Allelen oder durch epigenetische Prozesse, wie X-Chromosom-Inaktivierung oder genomischer Pragung entstehen, uberraschend oft gewebespezifisch ist.

Mapping the mouse Allelome reveals tissue-specific regulation

lncRNA-Induced Spread of Polycomb Controlled by Genome Architecture, RNA Abundance, and CpG Island DNA.

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is cotransfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. We show that CRISPR-Bac can be used to knock down proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.

/pdf/a-piggybac-based-toolkit-for-inducible-genome-editing-in-17y16hfeco.pdf

A piggyBac-based toolkit for inducible genome editing in mammalian cells.

The polycomb repressive complexes 1 and 2 (PRCs; PRC1 and PRC2) are conserved histone-modifying enzymes that often function cooperatively to repress gene expression. The PRCs are regulated by long noncoding RNAs (lncRNAs) in complex ways. On the one hand, specific lncRNAs cause the PRCs to engage with chromatin and repress gene expression over genomic regions that can span megabases. On the other hand, the PRCs bind RNA with seemingly little sequence specificity, and at least in the case of PRC2, direct RNA-binding has the effect of inhibiting the enzyme. Thus, some RNAs appear to promote PRC activity, while others may inhibit it. The reasons behind this apparent dichotomy are unclear. The most potent PRC-activating lncRNAs associate with chromatin and are predominantly unspliced or harbor unusually long exons. Emerging data imply that these lncRNAs promote PRC activity through internal RNA sequence elements that arise and disappear rapidly in evolutionary time. These sequence elements may function by interacting with common subsets of RNA-binding proteins that recruit or stabilize PRCs on chromatin. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

The control of polycomb repressive complexes by long noncoding RNAs.

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically-dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is co-transfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. Using Mus musculus-derived embryonic and trophoblast stem cells, we show that CRISPR-Bac can be used to knockdown proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.

/pdf/a-piggybac-based-toolkit-for-inducible-genome-editing-in-329ya68to3.pdf

Keean C.A. Braceros

Papers

lncRNA-Induced Spread of Polycomb Controlled by Genome Architecture, RNA Abundance, and CpG Island DNA.

A piggyBac-based toolkit for inducible genome editing in mammalian cells.

The control of polycomb repressive complexes by long noncoding RNAs.

A piggyBac-based toolkit for inducible genome editing in mammalian cells