The control of polycomb repressive complexes by long noncoding RNAs.

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.

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

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.

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

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.