Chromosomal contact permits transcription between coregulated genes.

Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and interchromosomal contact. This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.

A simple recipe for the non-expert bioinformaticist for building experimentally-testable hypotheses for proteins with no known homologs.

The study of the protein-protein interactions (PPIs) of unique ORFs is a strategy for deciphering the biological roles of unique ORFs of interest. For uniform reference, we define unique ORFs as those for which no matching protein is found after PDB-BLAST search with default parameters. The uniqueness of the ORFs generally precludes the straightforward use of structure-based approaches in the design of experiments to explore PPIs. Many open-source bioinformatics tools, from the commonly-used to the relatively esoteric, have been built and validated to perform analyses and/or predictions of sorts on proteins. How can these available tools be combined into a protocol that helps the non-expert bioinformaticist researcher to design experiments to explore the PPIs of their unique ORF? Here we define a pragmatic protocol based on accessibility of software to achieve this and we make it concrete by applying it on two proteins-the ImuB and ImuA' proteins from Mycobacterium tuberculosis. The protocol is pragmatic in that decisions are made largely based on the availability of easy-to-use freeware. We define the following basic and user-friendly software pathway to build testable PPI hypotheses for a query protein sequence: PSI-PRED â†’ MUSTER â†’ metaPPISP â†’ ASAView and ConSurf. Where possible, other analytical and/or predictive tools may be included. Our protocol combines the software predictions and analyses with general bioinformatics principles to arrive at consensus, prioritised and testable PPI hypotheses.

Antisense-oligonucleotide-mediated perturbation of long non-coding RNA reveals functional features in stem cells and across cell types.

Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.

Functional analysis of a small cryptic plasmid pYS1 from Nocardia.

Bacteria of the genus Nocardia cause opportunistic infections of lung, brain and central nervous system, and cutaneous tissue. They are also producers of antibiotics and industrially important enzymes. As studies describing plasmids in this genus are limited, we have characterized a 4326bp cryptic plasmid pYS1 from Nocardia aobensis IFM 10795. Three open reading frames (ORFs) were predicted. Both sequence analyses and detection of single-stranded intermediates suggested a rolling-circle mechanism as the mode of replication of pYS1. Mutageneses and deletion analyses revealed both the predicted double- and single-stranded origins to be indispensable in replication, suggesting a lack of secondary signals for leading and lagging strand synthesis. The replicon of pYS1 is broad-host-range and compatible to that of pAL5000 of mycobacteria, making it potentially useful in genetic manipulation of various actinomycetes. Insertion analyses showed orf1, despite its sequence similarity to plasmid transfer genes, is involved in plasmid stability rather than conjugation and is lethal in the absence of a functional orf3. This situation is somewhat analogous to the kil/kor system of pIJ101 of Streptomyces, except that orf3 was unrelated to korA and was shown by promoter-probe assays to encode a novel transcriptional repressor negatively regulating orf1 expression.

Publisher Correction: Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments.

In the version of this article initially published, '+' and '-' labels were missing from the graph keys at the bottom of Fig. 8d. The error has been corrected in the HTML and PDF versions of the article.

Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments.

Accumulation of trimethylation of histone H3 at lysine 4 (H3K4me3) on immune-related gene promoters underlies robust transcription during trained immunity. However, the molecular basis for this remains unknown. Here we show three-dimensional chromatin topology enables immune genes to engage in chromosomal contacts with a subset of long noncoding RNAs (lncRNAs) we have defined as immune gene-priming lncRNAs (IPLs). We show that the prototypical IPL, UMLILO, acts in cis to direct the WD repeat-containing protein 5 (WDR5)-mixed lineage leukemia protein 1 (MLL1) complex across the chemokine promoters, facilitating their H3K4me3 epigenetic priming. This mechanism is shared amongst several trained immune genes. Training mediated by ß-glucan epigenetically reprograms immune genes by upregulating IPLs in manner dependent on nuclear factor of activated T cells. The murine chemokine topologically associating domain lacks an IPL, and the Cxcl genes are not trained. Strikingly, the insertion of UMLILO into the chemokine topologically associating domain in mouse macrophages resulted in training of Cxcl genes. This provides strong evidence that lncRNA-mediated regulation is central to the establishment of trained immunity.

C1 CAGE detects transcription start sites and enhancer activity at single-cell resolution.

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3'-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5'-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-ß of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization.

Visualization of Enhancer-Derived Noncoding RNA.

Enhancers are principal regulators that allow spatiotemporal tissue-specific control of gene expression. While mounting evidence suggests that enhancer-derived long noncoding RNAs (long ncRNAs), including enhancer RNAs (eRNAs), are an important component of enhancer function, their expression has not been broadly analyzed at a single cell level via imaging techniques. This protocol describes a method to image eRNA in single cells by in situ hybridization followed by tyramide signal amplification (TSA). The procedure can be multiplexed to simultaneously visualize both eRNA and protein-coding transcript at the site of transcriptional elongation, thereby permitting analysis of dynamics between the two transcript species in single cells. Our approach is not limited to eRNAs, but can be implemented on other transcripts.

Are genes switched on when they kiss?

Chromatin loops are pervasive and permit the tight compaction of DNA within the confined space of the nucleus. Looping enables distal genes and DNA elements to engage in chromosomal contact, to form multigene complexes. Advances in biochemical and imaging techniques reveal that loop-mediated contact is strongly correlated with transcription of interacting DNA. However, these approaches only provide a snapshot of events and therefore are unable to reveal the dynamics of multigene complex assembly. This highlights the necessity to develop single cell-based assays that provide single molecule resolution, and are able to functionally interrogate the role of chromosomal contact on gene regulation. To this end, high-resolution single cell imaging regimes, combined with genome editing approaches, are proving to be pivotal to advancing our understanding of loop-mediated dynamics.

lncRNA and gene looping: what's the connection?

Recent functional studies have unveiled the significant role chromatin topology plays in gene regulation. Several lines of evidence suggest genes access necessary factors for transcription by forming chromatin loops. A clearer picture of the players involved in chromatin organization, including lncRNA, is emerging.

Phage as a source of antibacterial genes: Multiple inhibitory products encoded by Rhodococcus phage YF1.

Bacteriophage-encoded proteins which inhibit or modify cellular components may contribute to antibacterial drug discovery by allowing the identification of novel targets. Given their abundance and diversity, phages may have various strategies in host inhibition and therefore may possess a variety of such proteins. Using Rhodococcus equi and phage YF1, we show that a single phage possesses numerous genes that inhibit the host when introduced into the host on a plasmid. These genes mostly encode proteins of unknown function, confirming the potential that this approach may have in providing new antibacterial targets.