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'

A dynamical model reveals gene co-localizations in nucleus

Co-localization of networks of genes in the nucleus is thought to play an important role in determining gene expression patterns. Based upon experimental data, we built a dynamical model to test whether pure diffusion could account for the observed co-localization of genes within a defined subnuclear region. A simple standard Brownian motion model in two and three dimensions shows that preferential co-localization is possible for co-regulated genes without any direct interaction, and suggests the occurrence may be due to a limitation in the number of available transcription factors. Experimental data of chromatin movements demonstrates that fractional rather than standard Brownian motion is more appropriate to model gene mobilizations, and we tested our dynamical model against recent static experimental data, using a sub-diffusion process by which the genes tend to colocalize more easily. Moreover, in order to compare our model with recently obtained experimental data, we studied the association level between genes and factors, and presented data supporting the validation of this dynamic model. As further applications of our model, we applied it to test against more biological observations. We found that increasing transcription factor number, rather than factory number and nucleus size, might be the reason for decreasing gene co-localization. In the scenario of frequency-or amplitude-modulation of transcription factors, our model predicted that frequency-modulation may increase the co-localization between its targeted genes

Active RNA Polymerases: Mobile or Immobile Molecular Machines?

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

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

Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

The authors perform a cryo-EM study of the 1.9 MDa human Mediator-RNA polymerase II-TFIIF assembly, which reveals the structural organization of the human transcription initiation apparatus

TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed: Specialized transcription factories

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

DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift

T7 RNA Polymerase Functions In Vitro without Clustering

Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using ‘pulldowns’ and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a Kd<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein

Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells

Gene expression states influence the three-dimensional conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features, which define promoter activation states or CTCF binding sites. Single cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fibre fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions