Binding of nuclear factor κB to noncanonical consensus sites reveals its multimodal role during the early inflammatory response.

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.

Prefrontal Dopamine D1 and D2 Receptors Regulate Dissociable Aspects of Decision Making via Distinct Ventral Striatal and Amygdalar Circuits.

Mesocortical dopamine (DA) regulates a variety of cognitive functions via actions on D1 and/or D2 receptors. For example, risk/reward decision making is modulated differentially by these two receptors within the prefrontal cortex (PFC), with D2 receptors enabling flexible decision making and D1 receptors promoting persistence in choice biases. However, it is unclear how DA mediates opposing patterns of behavior by acting on different receptors within the same terminal region. We explored the possibility that DA may act on separate networks of PFC neurons that are modulated by D1 or D2 receptors and in turn interface with divergent downstream structures such as the basolateral amygdala (BLA) or nucleus accumbens (NAc). Decision making was assessed using a probabilistic discounting task in which well trained male rats chose between small/certain or large/risky rewards, with the odds of obtaining the larger reward changing systematically within a session. Selective disruption of D1 or D2 modulation of separate PFC output pathways was achieved using unilateral intra-PFC infusions of DA antagonists combined with contralateral inactivation of the BLA or NAc. Disrupting D2 (but not D1) modulation of PFC→BLA circuitry impaired adjustments in decision biases in response to changes in reward probabilities. In contrast, disrupting D1 modulation of PFC→NAc networks reduced risky choice, attenuating reward sensitivity and increasing sensitivity to reward omissions. These findings reveal that mesocortical DA can facilitate dissociable components of reward seeking and action selection by acting on different functional networks of PFC neurons that can be distinguished by the subcortical projection targets with which they interface.SIGNIFICANCE STATEMENT Prefrontal cortical dopamine regulates a variety of executive functions governed by the frontal lobes via actions on D1 and D2 receptors. These receptors can in some instances mediate different patterns of behavior, but the mechanisms underlying these dissociable actions are unclear. Using a selective disconnection approach, we reveal that D1 and D2 receptors can facilitate diverse aspects of decision making by acting on separate networks of prefrontal neurons that interface with distinct striatal or amygdalar targets. These findings reveal an additional level of complexity in how mesocortical DA regulates different forms of cognition via actions on different receptors, highlighting how it may act upon distinct cortical microcircuits to drive different patterns of behavior.

Super-resolution measurement of distance between transcription sites using RNA FISH with intronic probes.

Nascent transcripts being copied from specific human genes can be detected using RNA FISH (fluorescence in situ hybridization) with intronic probes, and the distance between two different nascent transcripts is often measured when studying structure-function relationships. Such distance measurements are limited by the resolution of the light microscope. Here we describe methods for measuring these distances in cultured cells with a precision of a few tens of nanometers, using equipment found in most laboratories (i.e., a wide-field fluorescence microscope equipped with a charged-coupled-device camera). Using images of pairs of transcripts that are often co-transcribed, we discuss how selection of cell type, design of FISH probes, image acquisition, and image processing affect the precision that can be achieved.

TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed.

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.

Promoter type influences transcriptional topography by targeting genes to distinct nucleoplasmic sites.

Both the sequence of a promoter and the position of a gene in 3D nuclear space play crucial roles in gene regulation, but few studies address their inter-relationship. Using human and viral promoters on mini-chromosomes and RNA fluorescence in situ hybridization coupled to 'high-precision' localization, we show that promoters binding the same transcription factors and responding to the same signaling pathways tend to be co-transcribed in the same transcription factories. We go on to suggest how such spatial co-association might drive co-regulation of genes under the control of similar cis-elements.

Space exploration by the promoter of a long human gene during one transcription cycle.

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'.

Active RNA polymerases: mobile or immobile molecular machines?

It is widely assumed that active RNA polymerases track along their templates to produce a transcript. We test this using chromosome conformation capture and human genes switched on rapidly and synchronously by tumour necrosis factor alpha (TNFalpha); one is 221 kbp SAMD4A, which a polymerase takes more than 1 h to transcribe. Ten minutes after stimulation, the SAMD4A promoter comes together with other TNFalpha-responsive promoters. Subsequently, these contacts are lost as new downstream ones appear; contacts are invariably between sequences being transcribed. Super-resolution microscopy confirms that nascent transcripts (detected by RNA fluorescence in situ hybridization) co-localize at relevant times. Results are consistent with an alternative view of transcription: polymerases fixed in factories reel in their respective templates, so different parts of the templates transiently lie together.

Maximum precision closed-form solution for localizing diffraction-limited spots in noisy images.

Super-resolution techniques like PALM and STORM require accurate localization of single fluorophores detected using a CCD. Popular localization algorithms inefficiently assume each photon registered by a pixel can only come from an area in the specimen corresponding to that pixel (not from neighboring areas), before iteratively (slowly) fitting a Gaussian to pixel intensity; they fail with noisy images. We present an alternative; a probability distribution extending over many pixels is assigned to each photon, and independent distributions are joined to describe emitter location. We compare algorithms, and recommend which serves best under different conditions. At low signal-to-noise ratios, ours is 2-fold more precise than others, and 2 orders of magnitude faster; at high ratios, it closely approximates the maximum likelihood estimate.

Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines.

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.

Modulation of risk/reward decision making by dopaminergic transmission within the basolateral amygdala.

RATIONALE: Dopamine (DA) transmission within cortico-limbic-striatal circuitry is integral in modulating decisions involving reward uncertainty. The basolateral amygdala (BLA) also plays a role in these processes, yet how DA transmission within this nucleus regulates cost/benefit decision making is unknown. OBJECTIVES: We investigated the contribution of DA transmission within the BLA to risk/reward decision making assessed with a probabilistic discounting task. METHODS: Rats were well-trained to choose between a small/certain reward and a large/risky reward, with the probability of obtaining the larger reward decreasing (100-12.5 %) or increasing (12.5-100 %) over a session. We examined the effects of antagonizing BLA D1 (SCH 23390, 0.1-1 µg) or D2 (eticlopride, 0.1-1 µg) receptors, as well as intra-BLA infusions of agonists for D1 (SKF 81297, 0.1-1 µg) and D2 (quinpirole, 1-10 µg) receptors. We also assessed how DA receptor stimulation may induce differential effects related to baseline levels of risky choice. RESULTS: BLA D1 receptor antagonism reduced risky choice by decreasing reward sensitivity, whereas D2 antagonism did not affect overall choice patterns. Stimulation of BLA D1 receptors optimized decision making in a baseline-dependent manner: in risk-averse rats, infusions of a lower dose of SKF81297 increased risky choice when reward probabilities were high (50 %), whereas in risk-prone rats, this drug reduced risky choice when probabilities were low (12.5 %). Quinpirole reduced risky choice in risk-prone rats, enhancing lose-shift behavior. CONCLUSIONS: These data highlight previously uncharacterized roles for BLA DA D1 and D2 receptors in biasing choice during risk/reward decision making through mediation of reward/negative feedback sensitivity.

Dynamic reconfiguration of long human genes during one transcription cycle.

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.