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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00819

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Nongenic transcription, gene regulation and action at a distance

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

View larger version (23K): [in a new window]   Fig. 1. Transcribed regulatory regions in four loci. (A) Drosophila 87A7 heat-shock locus. Two hsp70 genes (promoters and direction of transcription indicated) are protected from the spread of heterochromatin (grey arrowheads) by scs and scs' (which both contain promoters). (B) Drosophila bithorax complex. Ubx, abd-A and Abd-B are regulated by many silencers and enhancers found in the regions shown below (grey arrows indicate target genes) and various cellular memory modules (CMMs); various barriers (e.g. Mcp, Fab7, Fab8) separate these regions. Not all ORFs, regulators, barriers or transcription units are shown. (C) Yeast HMR locus. A silent domain containing mating type (MAT) loci (a2, a1) is maintained by barriers flanking E and I. Rap1/Abf1/ORC bind to ARS317 (at E) and ARS318 (at I), followed by recruitment of SIR1-4, spread of SIR2-4 to flanking barriers and nucleosomal deacetylation by SIR2. The barrier next to I contains a long terminal repeat (LTR) and a tRNAThr gene; the one next to E probably contains one (or two) LTRs, and so also contains promoters. ORF YCR097W-A (downstream of a1) is not shown. (D) Human ß-globin locus. A (transcribed) LCR regulates (grey arrows) the activity of the locus that contains five genes and one pseudogene (ß; not shown). The vertical arrows mark hypersensitive sites (HS).

View larger version (22K): [in a new window]   Fig. 2. Different loop ties. (A) Structural. DNA repeats (green) in two cells bind to the same protein complexes (yellow ovals, red diamonds), looping the fibre. After cutting with a nuclease and removing detached fragments, the same set of repeats from each cell remain bound. When 10% DNA remains, repeats are enriched tenfold. (B) Functional. The fibre is looped by attachment to a protein complex, but both attachments and proteins in the complex change from moment to moment. After cutting and removing detached fragments, a different set of fragments remains attached in the two cells. When 10% DNA remains, no fragment is enriched tenfold. This result is obtained if cutting and removal are carried out in isotonic buffers; essentially all active transcription complexes also remain attached.

View larger version (24K): [in a new window]   Fig. 3. Two methods used to illustrate looping. (A) 3C. A loop is shown, tethered by transcription units a and c to a protein cluster (small pink circle). The 3C method involves fixation to covalently cross-link DNA sequences lying next to each other (usually through DNA:protein:DNA links shown as the red line), before removing unlinked proteins, cutting DNA, dilution and ligation. Dilution favours intramolecular ligation, so the ends of two different DNA molecules in one DNA:protein:DNA complex are joined together more frequently than those in different molecules/complexes. Then, two DNA sequences initially lying together in 3D space are often ligated together (i.e. a with c, but not b), and (after reversing crosslinks) their juxtaposition is detected using PCR. (B) RNA TRAP. Only the region around transcription units a and c is illustrated. This method also involves fixation to preserve molecular contacts, in situ hybridization of a tagged intron probe to specific primary transcripts at a target transcription site (i.e. a), immunolabelling to bring peroxidase activity to the tagged probe, and reaction with biotintyramide to generate free-radicals that biotinylate proteins in the vicinity. Then DNA sequences initially lying near the target site in 3D space (c, but not b) co-purify with biotinylated chromatin and can be detected by PCR.

View larger version (25K): [in a new window]   Fig. 4. Model for the organization of the chromatin fibre in a HeLa nucleus. DNA is wound into a nucleosome, and then a zig-zagging string of nucleosomes is tied to a factory through a cluster of transcription factors (diamond) or an active polymerase (oval). Components of the factory exchange with the soluble pool, and attachments to the factory are made and broken as factors dissociate and transcription terminates. 10-20 loops (only three are shown) of 5-200 kbp form a cloud around the transcription factory; long, static, loops are likely to become heterochromatic and attached to the lamina. 50-100 clouds then form a chromosome territory. Modified, with permission, from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. (Cook, 2001).

View larger version (26K): [in a new window]   Fig. 5. A transcription cycle. A chromatin loop is attached through transcription factors to a factory; these attachments are made and broken continuously as components in the factory exchange with the soluble pool. On initiation, the promoter (white circle) binds to a polymerase in the factory, and the transcript is extruded as the template slides (white arrows) through the polymerase; on termination, the template detaches. Between initiation and termination the template is bound to the factory both stably (the bond is one of the stablest noncovalent ones known) and persistently (it takes 5 minutes to transcribe a typical human gene) (Kimura et al., 2002). Therefore, such attachments are much more stable and persistent than those mediated by transcription factors, and this may underlie the difference in `strength' between transcribed and nontranscribed enhancers.

View larger version (36K): [in a new window]   Fig. 6. General (left) and specific views (right) of how transcriptional activity is regulated by local (A,B) and distant motifs (C-F). Left: thick and thin blue lines represent transcription units (with promoters as white circles) and intervening DNA, respectively. Right: half a loop is shown with unit t temporarily attached to, and transcribed by, a polymerase in a factory (pink circle); as a transcript is extruded (wavy red line), chromatin between s and t is reeled in and `opened'. (A,B) Left: a repressor (or activator) binds close to the transcription start site to reduce (increase) the chances that a polymerase will be recruited. Right: repressor (or activator) binding to the promoter of s reduces (increases) the chances that s binds to the factory. In both cases, the repressor (or activator) has the same effect by binding to the polymerase (rather than the promoter). (C) Left: inserting a nontranscribed motif (e.g. some enhancers) increases the activity of a distant unit. Right: the motif has an affinity for transcription factors in the factory, and – once it binds – promoter s becomes tethered close to the factory, increasing its chances of attaching and initiating. A nontranscribed silencer (not shown) could have the opposite effect and reduce the chances that promoter s could bind (perhaps by directly blocking access to polymerases in the factory, or indirectly because DNA between the motif and promoter s was too short/rigid to loop back). (D) Left: inserting a transcribed motif (e.g. some enhancers, LCRs) increases the activity of a distant unit. Right: unit s attaches to the factory and initiates; this brings promoter r closer to the factory, increasing its chances of attaching and initiating. If the orientation of s were reversed, its transcription would progressively tether r ever closer to the factory and this might explain why orientation affects the activity of the ß-globin LCR (Tanimoto et al., 1999). (E) Left: inserting an active unit near another, silences one of the two. Right: the attachment of unit t prevents s from attaching, perhaps because all polymerases are now occupied, DNA between t and s is too short/rigid to loop back, or transcription of t leads to recruitment of inappropriate factors to the factory; alternatively, r might attach to another (blue) factory lacking the polymerase and/or factors required by s, so distancing s from the (pink) factory with the appropriate polymerase/factors (tRNAThr in HMR probably silences URA3 like this). (F) Left: Heterochromatin spreads leftwards (grey arrow) down the fibre, but inserting a barrier (yellow) restricts the spread and allows the unit to be expressed. Right: unit t acts as a barrier preventing heterochromatic spread to inactivate s.