Transgenic mouse lines are invaluable tools for neuroscience but, as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, of either sex, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, although rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired‐end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories’. Some factories further specialize in transcribing responsive genes encoding micro‐RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.

Although it is widely assumed that active RNA polymerase tracks along its template, we find that DNA, not the polymerase, moves, suggesting that polymerase works by reeling in the template.

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Transgenic mouse lines are invaluable tools for neuroscience but as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically-encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, though rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.

We analyzed three human genes that were >200 kbp in length as they are switched on rapidly and synchronously by tumor necrosis factor alpha and obtained new insights into the transcription cycle that are difficult to obtain using continuously active, short, genes. First, a preexisting "whole-gene" loop in one gene disappears on stimulation; it is stabilized by CCCTC-binding factor and TFIIB and poises the gene for a prompt response. Second, "subgene" loops (detected using chromosome conformation capture) develop and enlarge, a result that is simply explained if elongating polymerases become immobilized in transcription factories, where they reel in their templates. Third, high-resolution localization confirms that relevant nascent transcripts (detected using RNA fluorescence in situ hybridization) lie close enough to be present on the surface of one factory. These dynamics underscore the complex transitions between the poised, initiating, and elongating transcriptional states. It is widely assumed that an RNA polymerase transcribes by first diffusing to a promoter wherever that promoter might be in the nucleus, binding, and then tracking down the template. However, an alternative sees the active form of the enzyme housed in a transcription factory; then, a promoter would diffuse to a factory, where it would bind a transiently immobilized polymerase, before that polymerase reeled in the template as it extruded the transcript (8, 32, 33). Nucleoplasmic factories are polymorphic (13,14), and in a HeLa cell one is typically associated with ϳ16 loops tethered through engaged polymerases and transcription factors to a ϳ90-nm core (10). Chromosome conformation capture (3C) and fluorescence in situ hybridization (FISH) provide strong support for this alternative; sequences lying far apart on the genetic map lie together in three-dimensional (3D) nuclear space, and the contacting sequences are often transcribed and/or associated with bound transcription factors (2,5,7,21,24,29,33,37,47).We present here a detailed analysis of the changing conformations of three human genes as they become active. Our approach depends on the use of a rapid and synchronous gene switch. Diploid human umbilical vein endothelial cells (HUVECs) are arrested in the G 0 phase of the cell cycle by serum starvation, and tumor necrosis factor alpha (TNF-␣) is added; this cytokine orchestrates the inflammatory response and induces a subset of genes to become active within minutes (44). We chose three rapidly responding genes of Ͼ200 kbp for analysis, since their great length provides ample temporal and spatial resolution. After switching them on, we used 3C to analyze their changing conformations over a period of 85 min. We found that "subgene" loops develop soon after initiation, and these then grow as pioneering polymerases elongate.We also analyzed an exceptional "whole-gene" loop seen in one gene before stimulation which, in contrast to the subgene loops, disappears on stimulation. Whole-gene loops have been detected in various organisms, includin...

Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-κB, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-κB binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-κΒ-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-κΒ and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-κΒ that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.[Supplemental material is available for this article.]Mammalian cells, embedded in a multicellular environment, require intricate mechanisms in order to interpret, integrate, and ultimately respond to extracellular stimuli. Inflammatory signaling constitutes a well-studied example (Bhatt and Ghosh 2014; Smale and Natoli 2014); tumor necrosis factor (TNF) rapidly remodels gene expression programs through its main effector, nuclear factor κB (NF-κB) (Hayden and Ghosh 2008;Bhatt et al. 2012). TNF activates the same genes across cell types (Moynagh 2005), but the cascade needs to unfold against the ongoing transcriptional program of the stimulated cell. Along these lines, recent work in adipocytes showed that NF-κB is involved in gene repression via redistribution of prebound factors . This is tightly linked to the choice of NF-κB binding in vivo, but the specific principles that guide this choice in three-dimensional (3D) nuclear space and over time are still not well understood, despite the wealth of data on the different phases of the inflammatory response. For example, we now know that signaling directs binding of its downstream effectors to already-active cis-regulatory ele-

An RNA polymerase has been thought to transcribe by seeking out a promoter, initiating and then tracking down the template. We add tumor necrosis factor α to primary human cells, switch on transcription of a 221-kb gene and monitor promoter position during the ensuing transcription cycle (using RNA fluorescence in situ hybridization coupled to super-resolution localization, chromosome conformation capture and Monte Carlo simulations). Results are consistent with a polymerase immobilized in a ‘factory’ capturing a promoter and reeling in the template, as the transcript and promoter are extruded. Initially, the extruded promoter is tethered close to the factory and so likely to re-initiate; later, the tether becomes long enough to allow re-initiation in another factory. We suggest close tethering underlies enhancer function and transcriptional ‘bursting’.