Complex small-world regulatory networks emerge from the 3D organisation of the human genome.

The discovery that overexpressing one or a few critical transcription factors can switch cell state suggests that gene regulatory networks are relatively simple. In contrast, genome-wide association studies (GWAS) point to complex phenotypes being determined by hundreds of loci that rarely encode transcription factors and which individually have small effects. Here, we use computer simulations and a simple fitting-free polymer model of chromosomes to show that spatial correlations arising from 3D genome organisation naturally lead to stochastic and bursty transcription as well as complex small-world regulatory networks (where the transcriptional activity of each genomic region subtly affects almost all others). These effects require factors to be present at sub-saturating levels; increasing levels dramatically simplifies networks as more transcription units are pressed into use. Consequently, results from GWAS can be reconciled with those involving overexpression. We apply this pan-genomic model to predict patterns of transcriptional activity in whole human chromosomes, and, as an example, the effects of the deletion causing the diGeorge syndrome.

Extrusion without a motor: a new take on the loop extrusion model of genome organization.

Chromatin loop extrusion is a popular model for the formation of CTCF loops and topological domains. Recent HiC data have revealed a strong bias in favour of a particular arrangement of the CTCF binding motifs that stabilize loops, and extrusion is the only model to date which can explain this. However, the model requires a motor to generate the loops, and although cohesin is a strong candidate for the extruding factor, a suitable motor protein (or a motor activity in cohesin itself) has yet to be found. Here we explore a new hypothesis: that there is no motor, and thermal motion within the nucleus drives extrusion. Using theoretical modelling and computer simulations we ask whether such diffusive extrusion could feasibly generate loops. Our simulations uncover an interesting ratchet effect (where an osmotic pressure promotes loop growth), and suggest, by comparison to recent in vitro and in vivo measurements, that diffusive extrusion can in principle generate loops of the size observed in the data. Extra View on : C. A. Brackley, J. Johnson, D. Michieletto, A. N. Morozov, M. Nicodemi, P. R. Cook, and D. Marenduzzo "Non-equilibrium chromosome looping via molecular slip-links", Physical Review Letters 119 138101 (2017).

Nonequilibrium Chromosome Looping via Molecular Slip Links.

We propose a model for the formation of chromatin loops based on the diffusive sliding of molecular slip links. These mimic the behavior of molecules like cohesin, which, along with the CTCF protein, stabilize loops which contribute to organizing the genome. By combining 3D Brownian dynamics simulations and 1D exactly solvable nonequilibrium models, we show that diffusive sliding is sufficient to account for the strong bias in favor of convergent CTCF-mediated chromosome loops observed experimentally. We also find that the diffusive motion of multiple slip links along chromatin is rectified by an intriguing ratchet effect that arises if slip links bind to the chromatin at a preferred "loading site." This emergent collective behavior favors the extrusion of loops which are much larger than the ones formed by single slip links.

Simulating topological domains in human chromosomes with a fitting-free model.

We discuss a polymer model for the 3D organization of human chromosomes. A chromosome is represented by a string of beads, with each bead being "colored" according to 1D bioinformatic data (e.g., chromatin state, histone modification, GC content). Individual spheres (representing bi- and multi-valent transcription factors) can bind reversibly and selectively to beads with the appropriate color. During molecular dynamics simulations, the factors bind, and the string spontaneously folds into loops, rosettes, and topologically-associating domains (TADs). This organization occurs in the absence of any specified interactions between distant DNA segments, or between transcription factors. A comparison with Hi-C data shows that simulations predict the location of most boundaries between TADs correctly. The model is "fitting-free" in the sense that it does not use Hi-C data as an input; consequently, one of its strengths is that it can - in principle - be used to predict the 3D organization of any region of interest, or whole chromosome, in a given organism, or cell line, in the absence of existing Hi-C data. We discuss how this simple model might be refined to include more transcription factors and binding sites, and to correctly predict contacts between convergent CTCF binding sites.

Merging drops in a Teflon tube, and transferring fluid between them, illustrated by protein crystallization and drug screening.

The ability to manipulate drops with small volumes has many practical applications. Current microfluidic devices generally exploit channel geometry and/or active external equipment to control drops. Here we use a Teflon tube attached to a syringe pump and exploit the properties of interfaces between three immiscible liquids to create particular fluidic architectures. We then go on to merge any number of drops (with volumes of micro- to nano-liters) at predefined points in time and space in the tube; for example, 51 drops were merged in a defined order to yield one large drop. Using a different architecture, specified amounts of fluid were transferred between 2 nl drops at specified rates; for example, 2.5 pl aliquots were transferred (at rates of ~500 fl s(-1)) between two drops through inter-connecting nano-channels (width ~40 nm). One proof-of-principle experiment involved screening conditions required to crystallize a protein (using a concentration gradient created using such nano-channels). Another demonstrated biocompatibility; drugs were mixed with human cells grown in suspension or on surfaces, and the treated cells responded like those grown conventionally. Although most experiments were performed manually, moderate high-throughput potential was demonstrated by mixing ~1000 different pairs of 50 nl drops in ~15 min using a robot. We suggest this reusable, low-cost, and versatile methodology could facilitate the introduction of microfluidics into workflows of many experimental laboratories.

A simple model for DNA bridging proteins and bacterial or human genomes: bridging-induced attraction and genome compaction.

We present computer simulations of the phase behaviour of an ensemble of proteins interacting with a polymer, mimicking non-specific binding to a piece of bacterial DNA or eukaryotic chromatin. The proteins can simultaneously bind to the polymer in two or more places to create protein bridges. Despite the lack of any explicit interaction between the proteins or between DNA segments, our simulations confirm previous results showing that when the protein-polymer interaction is sufficiently strong, the proteins come together to form clusters. Furthermore, a sufficiently large concentration of bridging proteins leads to the compaction of the swollen polymer into a globular phase. Here we characterise both the formation of protein clusters and the polymer collapse as a function of protein concentration, protein-polymer affinity and fibre flexibility.

Depletion effects and loop formation in self-avoiding polymers.

Langevin dynamics is employed to study the looping kinetics of self-avoiding polymers both in ideal and crowded solutions. A rich kinetics results from the competition of two crowding-induced effects: the depletion attraction and the enhanced viscous friction. For short chains, the enhanced friction slows down looping, while for longer chains, the depletion attraction renders it more frequent and persistent. We discuss the possible relevance of the findings for chromatin looping in living cells.

Transcription factories: structures conserved during differentiation and evolution.

Many cellular functions take place in discrete compartments, but our textbooks make little reference to any compartments involved in transcription. We review the evidence that active RNA polymerases and associated factors cluster into 'factories' that carry out many (perhaps all) of the functions required to generate mature transcripts. Clustering ensures high local concentrations and efficient interaction. Then, a gene must associate with the appropriate factory before it can be transcribed. Recent results show that the density and diameter of nucleoplasmic factories remain roughly constant as cells differentiate, despite large changes in the numbers of active polymerases and nucleoplasmic volumes.

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux.

Exposure to light can destroy the ability of a molecule to fluoresce. Such photobleaching limits the use of fluorescence and confocal microscopy in biological studies. Loss of fluorescence decreases the signal-to-noise ratio and so image resolution; it also prevents the acquisition of meaningful data late during repeated scanning (e.g. when collecting three-dimensional images). The aim of this work was to investigate the role of oxygen in the photobleaching of fluorophores bound to DNA in fixed cells, and to explore whether anoxia could minimize such bleaching. Anoxia significantly reduced bleaching rates and changed the order of reaction of both propidium iodide (an intercalator) and chromomycin A3 (a minor-groove binder) bound to DNA; it afforded the greatest protection at low photon fluxes. However, it had no effect on the bleaching of the green fluorescent protein (GFP) covalently attached to a histone and so bound to DNA, probably because the protein shielded the chromophore from oxygen. Bleaching of all three fluorophores depended on photon flux. Practical ways of minimizing bleaching were examined, and examples of three-dimensional images of DNA marked by propidium and GFP (collected under standard and optimized conditions) are presented.

Quantifying the syncytialisation of human placental trophoblast BeWo cells grown in vitro.

We have generated lines of BeWo cells that constitutively and stably express either histone H2B tagged with the green fluorescent protein (GFP), or the mitochondrial targeting sequence of subunit VIII of cytochrome c oxidase fused with a red fluorescent protein; one line has nuclei that fluoresce green, the other mitochondria that fluoresce red. Expression of these tagged proteins has no effect on the rates of DNA, RNA and protein synthesis, or on the amounts of human chorionic gonadotropin (hCG) secreted after treatment with forskolin. We used fluorescence-activated cell sorting (FACS) to monitor the extent of cell fusion (syncytialisation) between these two lines; fused cells are readily and accurately detected by their green/red fluorescence. This assay should prove useful in the investigation of the molecular mechanisms involved in trophoblast syncytialisation.

A mutation in the largest (catalytic) subunit of RNA polymerase II and its relation to the arrest of the cell cycle in G(1) phase.

Transcriptional activity of RNA polymerase II is modulated during the cell cycle. We previously identified a temperature-sensitive mutation in the largest (catalytic) subunit of RNA polymerase II (RPB1) that causes cell cycle arrest and genome instability. We now characterize a different cell line that has a temperature-sensitive defect in cell cycle progression, and find that it also has a mutation in RPB1. The temperature-sensitive mutant, tsAF8, of the Syrian hamster cell line, BHK21, arrests at the non-permissive temperature in the mid-G(1) phase. We show that RPB1 in tsAF8--which is found exclusively in the nucleus at the permissive temperature--is also found in the cytoplasm at the non-permissive temperature. Comparison of the DNA sequences of the RPB1 gene in the wild-type and mutant shows the mutant phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one RPB1 allele; this gives rise to an ala-to-asp substitution at residue 315 in the protein. Aligning the amino acid sequences from various species reveals that ala(315) is highly conserved in eukaryotes.

Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B.

Histones H2A and H2B form part of the same nucleosomal structure as H3 and H4. Stable HeLa cell lines expressing histones H2B, H3, and H4 tagged with green fluorescent protein (GFP) were established; the tagged molecules were assembled into nucleosomes. Although H2B-GFP was distributed like DNA, H3-GFP and H4-GFP were concentrated in euchromatin during interphase and in R-bands in mitotic chromosomes. These differences probably result from an unregulated production of tagged histones and differences in exchange. In both single cells and heterokaryons, photobleaching revealed that H2B-GFP exchanged more rapidly than H3-GFP and H4-GFP. About 3% of H2B exchanged within minutes, whereas approximately 40% did so slowly (t(1/2) approximately 130 min). The rapidly exchanging fraction disappeared in 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and so may represent H2B in transcriptionally active chromatin. The slowly exchanging fraction was probably associated with chromatin domains surrounding active units. H3-GFP and H4-GFP were assembled into chromatin when DNA was replicated, and then >80% remained bound permanently. These results reveal that the inner core of the nucleosome is very stable, whereas H2B on the surface of active nucleosomes exchanges continually.

Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap.

Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant on this approach. (J Histochem Cytochem 49:803-808, 2001)

Coupled transcription and translation within nuclei of mammalian cells.

It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes and that no translation occurs in nuclei. We localized translation sites by incubating permeabilized mammalian cells with [3H]lysine or lysyl-transfer RNA tagged with biotin or BODIPY; although most nascent polypeptides were cytoplasmic, some were found in discrete nuclear sites known as transcription "factories." Some of this nuclear translation also depends on concurrent transcription by RNA polymerase II. This coupling is simply explained if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria.

General anaesthesia in a man with mitochondrial myopathy undergoing eye surgery.

Mitochondrial myopathies are rare complex multisystem disorders, which can present several potential anaesthetic problems. A 49-year-old man with mitochondrial myopathy was anaesthetized successfully for cataract extraction using propofol and alfentanil infusions. Vecuronium sensitivity was apparent, with a prolonged duration of action, despite dosage reduction.

Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene.

Persistent expression of a transgene at therapeutic levels is required for successful gene therapy, but many small vectors with heterologous promoters are prone to vector loss and transcriptional silencing. The delivery of genomic DNA would enable genes to be transferred as complete loci, including regulatory sequences, introns, and native promoter elements. These elements may be critical to ensure prolonged, regulated, and tissue-specific transgene expression. Many studies point to considerable advantages to be gained by using complete genomic loci in gene expression. Large-insert vectors incorporating elements of the bacterial artificial chromosome (BAC) cloning system, and the episomal maintenance mechanisms of Epstein-Barr virus (EBV), can shuttle between bacteria and mammalian cells, allowing large genomic loci to be manipulated conveniently. We now demonstrate the potential utility of such vectors by stably correcting a human genetic deficiency in vitro. When the complete hypoxanthine phosphoribosyltransferase (HPRT) locus of 115 kilobases (kb) was introduced into deficient human cells, the transgene was both maintained as an episome and expressed stably for six months in rapidly dividing cell cultures. The results demonstrate for the first time that gene expression from an episomal genomic transgene can correct a cell culture disease phenotype for a prolonged period.