Poised Transcription Factories Prime Silent uPA Gene Prior to Activation

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Abstract
The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs) and is thought to require de novo association with transcription factories. We identify two types of factory: “poised transcription factories,” containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from “active factories” associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA) gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p+S2p−) factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p+S2p+) factories and loops out from its CT. However, the levels of gene association with poised or active transcription factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised transcription factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction. The spatial organization of the genome inside the cell nucleus is important in regulating gene expression and in the response to external stimuli. Examples of changing spatial organization are the repositioning of genes outside chromosome territories during the induction of gene expression, and the gathering of active genes at transcription factories (discrete foci enriched in active RNA polymerase). Recent genome-wide mapping of RNA polymerase II has identified its presence at many genes poised for activation, raising the possibility that such genes might associate with poised transcription factories. Using an inducible mammalian gene, urokinase-type plasminogen activator (uPA), and a system in which this gene is poised for expression, we show that uPA associates with poised transcription factories prior to activation. Gene activation induces two independent events: repositioning towards the exterior of its chromosome territory and association with active transcription factories. Surprisingly, genes inside the interior of the chromosome territory prior to activation are less likely to be actively transcribed, suggesting that positioning at the territory interior has a role in gene silencing.