Microfluidic chambers using fluid walls for cell biology.

Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.

Transcription-driven genome organization: a model for chromosome structure and the regulation of gene expression tested through simulations.

Current models for the folding of the human genome see a hierarchy stretching down from chromosome territories, through A/B compartments and topologically-associating domains (TADs), to contact domains stabilized by cohesin and CTCF. However, molecular mechanisms underlying this folding, and the way folding affects transcriptional activity, remain obscure. Here we review physical principles driving proteins bound to long polymers into clusters surrounded by loops, and present a parsimonious yet comprehensive model for the way the organization determines function. We argue that clusters of active RNA polymerases and their transcription factors are major architectural features; then, contact domains, TADs and compartments just reflect one or more loops and clusters. We suggest tethering a gene close to a cluster containing appropriate factors-a transcription factory-increases the firing frequency, and offer solutions to many current puzzles concerning the actions of enhancers, super-enhancers, boundaries and eQTLs (expression quantitative trait loci). As a result, the activity of any gene is directly influenced by the activity of other transcription units around it in 3D space, and this is supported by Brownian-dynamics simulations of transcription factors binding to cognate sites on long polymers.

Shaping epigenetic memory via genomic bookmarking.

Reconciling the stability of epigenetic patterns with the rapid turnover of histone modifications and their adaptability to external stimuli is an outstanding challenge. Here, we propose a new biophysical mechanism that can establish and maintain robust yet plastic epigenetic domains via genomic bookmarking (GBM). We model chromatin as a recolourable polymer whose segments bear non-permanent histone marks (or colours) which can be modified by 'writer' proteins. The three-dimensional chromatin organisation is mediated by protein bridges, or 'readers', such as Polycomb Repressive Complexes and Transcription Factors. The coupling between readers and writers drives spreading of biochemical marks and sustains the memory of local chromatin states across replication and mitosis. In contrast, GBM-targeted perturbations destabilise the epigenetic patterns. Strikingly, we demonstrate that GBM alone can explain the full distribution of Polycomb marks in a whole Drosophila chromosome. We finally suggest that our model provides a starting point for an understanding of the biophysics of cellular differentiation and reprogramming.

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.

Why the activity of a gene depends on its neighbors.

Sixty years ago, the position of a gene on a chromosome was seen to be a major determinant of gene activity; however, position effects are rarely central to current discussions of gene expression. We describe a comprehensive and simplifying view of how position in 1D sequence and 3D nuclear space underlies expression. We suggest that apparently-different regulatory motifs including enhancers, silencers, insulators, barriers, and boundaries act similarly - they are active promoters that tether target genes close to, or distant from, appropriate transcription sites or 'factories'. We also suggest that any active transcription unit regulates the firing of its neighbors - and thus can be categorized as one or other type of motif; this is consistent with expression quantitative trait loci (eQTLs) being widely dispersed.

Simulated binding of transcription factors to active and inactive regions folds human chromosomes into loops, rosettes and topological domains.

Biophysicists are modeling conformations of interphase chromosomes, often basing the strengths of interactions between segments distant on the genetic map on contact frequencies determined experimentally. Here, instead, we develop a fitting-free, minimal model: bivalent or multivalent red and green 'transcription factors' bind to cognate sites in strings of beads ('chromatin') to form molecular bridges stabilizing loops. In the absence of additional explicit forces, molecular dynamic simulations reveal that bound factors spontaneously cluster-red with red, green with green, but rarely red with green-to give structures reminiscent of transcription factories. Binding of just two transcription factors (or proteins) to active and inactive regions of human chromosomes yields rosettes, topological domains and contact maps much like those seen experimentally. This emergent 'bridging-induced attraction' proves to be a robust, simple and generic force able to organize interphase chromosomes at all scales.

Ephemeral Protein Binding to DNA Shapes Stable Nuclear Bodies and Chromatin Domains.

Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear bodies exchange rapidly with the soluble pool while the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins able to switch between an "on" (binding) and an "off" (nonbinding) state. This system provides a model for any DNA-binding protein that can be posttranslationally modified to change its affinity for DNA (e.g., through phosphorylation). Protein switching is a nonequilibrium process, and it leads to the formation of clusters of self-limiting size, where individual proteins in a cluster exchange with the soluble pool with kinetics similar to those seen in photobleaching experiments. This behavior contrasts sharply with that exhibited by nonswitching proteins, which are permanently in the on-state; when these bind to DNA nonspecifically, they form clusters that grow indefinitely in size. To explain these findings, we propose a mean-field theory from which we obtain a scaling relation between the typical cluster size and the protein switching rate. Protein switching also reshapes intrachromatin contacts to give networks resembling those seen in topologically associating domains, as switching markedly favors local (short-range) contacts over distant ones. Our results point to posttranslational modification of chromatin-bridging proteins as a generic mechanism driving the self-assembly of highly dynamic, nonequilibrium, protein clusters with the properties of nuclear bodies.

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.

Dissecting the nascent human transcriptome by analysing the RNA content of transcription factories.

While mapping total and poly-adenylated human transcriptomes has now become routine, characterizing nascent transcripts remains challenging, largely because nascent RNAs have such short half-lives. Here, we describe a simple, fast and cost-effective method to isolate RNA associated with transcription factories, the sites responsible for the majority of nuclear transcription. Following stimulation of human endothelial cells with the pro-inflammatory cytokine TNFα, we isolate and analyse the RNA content of factories by sequencing. Comparison with total, poly(A)(+) and chromatin RNA fractions reveals that sequencing of purified factory RNA maps the complete nascent transcriptome; it is rich in intronic unprocessed transcript, as well as long intergenic non-coding (lincRNAs) and enhancer-associated RNAs (eRNAs), micro-RNA precursors and repeat-derived RNAs. Hence, we verify that transcription factories produce most nascent RNA and confer a regulatory role via their association with a set of specifically-retained non-coding transcripts.

Splicing of many human genes involves sites embedded within introns.

The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3' end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon-exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome.

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.

Biocompatibility of fluids for multiphase drops-in-drops microfluidics.

This paper addresses the biocompatibility of fluids and surfactants in the context of microfluidics and more specifically in a drops-in-drops system for mammalian cell based drug screening. In the drops-in-drops approach, three immiscible fluids are used to manipulate the flow of aqueous microliter-sized drops; it enables merging of drops containing cells with drops containing drugs within a Teflon tube. Preliminary tests showed that a commonly-used fluid and surfactant combination resulted in significant variability in gene expression levels in Jurkat cells after exposure to a drug for four hours. This result led to further investigations of potential fluid and surfactant combinations that can be used in microfluidic systems for medium to long-term drug screening. Results herein identify a fluid combination, HFE-7500 and 5-cSt silicone oil + 0.25% Abil EM180, which enabled the drops-in-drops approach; this combination also allowed gene expression at normal levels comparable with the conventional drug screening in both magnitude and variability.

Nonspecific bridging-induced attraction drives clustering of DNA-binding proteins and genome organization.

Molecular dynamics simulations are used to model proteins that diffuse to DNA, bind, and dissociate; in the absence of any explicit interaction between proteins, or between templates, binding spontaneously induces local DNA compaction and protein aggregation. Small bivalent proteins form into rows [as on binding of the bacterial histone-like nucleoid-structuring protein (H-NS)], large proteins into quasi-spherical aggregates (as on nanoparticle binding), and cylinders with eight binding sites (representing octameric nucleosomal cores) into irregularly folded clusters (like those seen in nucleosomal strings). Binding of RNA polymerase II and a transcription factor (NFκB) to the appropriate sites on four human chromosomes generates protein clusters analogous to transcription factories, multiscale loops, and intrachromosomal contacts that mimic those found in vivo. We suggest that this emergent behavior of clustering is driven by an entropic bridging-induced attraction that minimizes bending and looping penalties in the template.

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'.

Transcription factories, chromatin loops, and the dysregulation of gene expression in malignancy.

Pathologists recognize and classify cancers according to nuclear morphology, but there remains little scientific explanation of why malignant nuclei possess their characteristic features, or how those features are related to dysregulated function. This essay will discuss a basic structure-function axis that connects one central architectural motif in the nucleus-the chromatin loop-to the vital nuclear function of transcription. The loop is attached to a "transcription factory" through components of the transcription machinery (either polymerases or transcriptional activators/repressors), and the position of a gene within a loop determines how often that gene is transcribed. Then, dysregulated transcription is tightly coupled to alterations in structure, and vice versa. We also speculate on how the experimental approaches being used to analyze loops and factories might be applied to study the problems of tumour initiation and progression.

The proteomes of transcription factories containing RNA polymerases I, II or III.

Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.

Stable diffusion gradients in microfluidic conduits bounded by fluid walls.

Assays mimicking in vitro the concentration gradients triggering biological responses like those involved in fighting infections and blood clotting are essential for biomedical research. Microfluidic assays prove especially attractive as they allow precise control of gradient shape allied to a reduction in scale. Conventional microfluidic devices are fabricated using solid plastics that prevent direct access to responding cells. Fluid-walled microfluidics allows the manufacture of circuits on standard Petri dishes in seconds, coupled to simple operating methods; cell-culture medium sitting in a standard dish is confined to circuits by fluid walls made of an immiscible fluorocarbon. We develop and experimentally validate an analytical model of diffusion between two or more aqueous streams flowing at different rates into a fluid-walled conduit with the cross-section of a circular segment. Unlike solid walls, fluid walls morph during flows as pressures fall, with wall shape changing down the conduit. The model is validated experimentally for Fourier numbers < 0.1 using fluorescein diffusing between laminar streams. It enables a priori prediction of concentration gradients throughout a conduit, so allowing rapid circuit design as well as providing bio-scientists with an accurate way of predicting local concentrations of bioactive molecules around responsive and non-responsive cells.

A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy.

In our brains, different neurons make appropriate connections; however, there remain few in vitro models of such circuits. We use an open microfluidic approach to build and study neuronal circuits in vitro in ways that fit easily into existing bio-medical workflows. Dumbbell-shaped circuits are built in minutes in standard Petri dishes; the aqueous phase is confined by fluid walls - interfaces between cell-growth medium and an immiscible fluorocarbon, FC40. Conditions are established that ensure post-mitotic neurons derived from human induced pluripotent stem cells (iPSCs) plated in one chamber of a dumbbell remain where deposited. After seeding cortical neurons on one side, axons grow through the connecting conduit to ramify amongst striatal neurons on the other - an arrangement mimicking unidirectional cortico-striatal connectivity. We also develop a moderate-throughput non-contact axotomy assay. Cortical axons in conduits are severed by a media jet; then, brain-derived neurotrophic factor and striatal neurons in distal chambers promote axon regeneration. As additional conduits and chambers are easily added, this opens up the possibility of mimicking complex neuronal networks, and screening drugs for their effects on connectivity.

Bridging condensins mediate compaction of mitotic chromosomes.

Eukaryotic chromosomes compact during mitosis into elongated cylinders-and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamic simulations involving two types of such proteins with different activities. One, which we refer to as looping condensins, anchors long-lived chromatin loops to create bottlebrush structures. The second, referred to as bridging condensins, forms multivalent bridges between distant parts of these loops. We show that binding of bridging condensins leads to the formation of shorter and stiffer mitotic-like cylinders without requiring any additional energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes seen experimentally and points to a mechanistic basis for the structure of common fragile sites in mitotic chromosomes.