Summary How is chromatin architecture established and what role does it play in activation of transcription? We show that a regulatory locus in yeast (the UASg) bears, in addition to binding sites for the activator Gal4, sites bound by the protein RSC. RSC tightly positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that suffices to impose characteristic features of chromatin architecture. Removal of RSC allows ordinary nucleosomes to form more broadly over the UASg, and these nucleosomes compete with (but do not exclude) Gal4 binding to its sites. Taken with our previous work, the results show that both prior to and following induction specific DNA binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are found scattered throughout the yeast genome. We surmise, also, that Gal4 works in higher eukaryotes despite whatever obstacle broadly positioned nucleosomes present.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially-regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long-range to predominantly short-range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome “remodeler” rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4′s action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription—one that requires the action of SWI/SNF recruited by the activator, and a slower one that does not—clarifies our understanding of the early events of gene activation, and in particular corrects earlier reports that SWI/SNF plays no role in GAL gene induction. Our finding that chromatin structure is irrelevant for repression as studied here—that is, repression sets in as efficiently whether or not promoter nucleosomes are allowed to reform—contradicts the widely held, but little tested, idea that nucleosomes are required for repression. These findings were made possible by our nucleosome occupancy assay. The assay, we believe, will prove useful in studying other outstanding issues in the field.

We previously showed that RanGTP forms a 1:1 complex with karyopherin ␤ that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel, G. (1996) J. Biol. Chem. 271, 5313-5316) and karyopherin ␤ functionally inactive (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). Recycling of both factors for another round of function requires dissociation of the RanGTP-karyopherin ␤ complex. Here we show using BIAcore™, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that karyopherin ␤ and RanGTP are recycled efficiently in a reaction that involves karyopherin ␣, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that karyopherin ␣ first releases RanGTP from karyopherin ␤ in a reaction that does not require GTP hydrolysis. The released RanGTP is then sequestered by RanBP1, and the newly formed karyopherin ␣␤ binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind karyopherin ␣␤ stimulate recycling of karyopherin ␤ and Ran in a manner similar to the C terminus of Nup1.Transport of proteins that contain a nuclear localization signal (NLS) 1 into the nucleus of the cell requires energy, mobile transport factors, and nuclear pore complexes (NPC) in the nuclear envelope. Karyopherin ␣␤ heterodimer (also termed importin ␣␤, NLS receptor-p97 complex, PTAC, or Kap60/95) binds proteins that contain an NLS similar to that of the SV40 large T-antigen or nucleoplasmin (NLS protein) and brings them to the NPC (3-12). Karyopherin ␣ binds the NLS protein (2, 3, 7, 13-15), whereas karyopherin ␤ increases the affinity of karyopherin ␣ for the NLS (2, 16) and docks the karyopherin ␣-NLS-protein complex to a subfamily of NPC proteins (nucleoporins) that contain XXFG-peptide repeats (2,14,(17)(18)(19)(20). The subsequent translocation across the NPC requires Ran/TC4 (21, 22) and p10/NTF2 (23, 24). p10 is a dimer (24, 25) that binds RanGDP (26, 27) and karyopherin ␤ (26, 28) and functions to tether RanGDP to karyopherin ␣␤ heterodimers that are docked to nucleoporins (26). When Ran is in its GTP-bound form it disrupts the interaction of karyopherin ␤ with karyopherin ␣ and with FXFG regions of nucleoporins by forming a complex with karyopherin ␤ (2). The repetitive interaction of transport factors, substrates, and nucleoporins at the NPC may facilitate the transport of substrates across the NPC (2, 17).Accessory factors regulate nuclear transport by modulating Ran. The GTPase-activating protein for Ran, RanGAP (termed RanGAP1, or Rna1 in yeast) (29 -32), and the nucleotide exchange factor for Ran, RanGEF (termed RCC1, or Prp20 in yeast) (33)(34)(35), are required to sustain efficient transport of substrates across the NPC (36 -39). The Ran binding protein 1, RanBP1 (40), is also involved in nuclear transport (41, 42). As the RanGTP-karyopher...