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Regulation of gene transcription is a key feature of developmental, homeostatic, and oncogenic processes, The reverse recruitment model of transcriptional control postulates that eukaryotic genes become active by moving to contact transcription factories at nuclear substructures; our previous work showed that at least some of these factories are tethered to nuclear pores. We demonstrate here that the nuclear periphery is the site of key events in the regulation of glucose-repressed genes, which together compose one-sixth of the Saccharomyces cerevisiae genome. We also show that the canonical glucose-repressed gene SUC2 associates tightly with the nuclear periphery when transcriptionally active but is highly mobile when repressed. Strikingly, SUC2 is both derepressed and confined to the nuclear rim in mutant cells where the Mig1 repressor is nuclear but not perinuclear. Upon derepression all three subunits ([alpha], [beta], and [gamma]) of the positively acting Snf1 kinase complex localize to the nuclear periphery, resulting in phosphorylation of Mig1 and its export to the cytoplasm. Reverse recruitment therefore appears to explain a fundamental pathway of eukaryotic gene regulation.

The budding yeast Saccharomyces cerevisiae has proven to be a fertile model system with which to identify glucose signaling factors, determine the relevant functional and physical interrelationships, and characterize the corresponding metabolic, transcriptomic, and proteomic readouts. The early events in glucose signaling appear to require both extracellular sensing by transmembrane proteins and intracellular sensing by G proteins.

Growth of Saccharomyces cerevisiae requires coordination of cell cycle events (e.g., new cell wall deposition) with constitutive functions like energy generation and duplication of protein mass. The latter processes are stimulated by the phosphoprotein Gcr1p, a transcriptional activator that operates through two different Rap1p-mediated mechanisms to boost expression of glycolytic and ribosomal protein genes, respectively. Simultaneous disruption of both mechanisms results in a loss of glucose responsiveness and a dramatic drop in translation rate. Since a critical rate of protein synthesis (CRPS) is known to mediate passage through Start and determine cell size by modulating levels of Cln3p, we hypothesized that GCR1 regulates cell cycle progression by coordinating it with growth. We therefore constructed and analyzed gcr1[DELTA] cln3[DELTA] and gcr1[DELTA] cln1[DELTA] cln2[DELTA] strains. Both strains are temperature and cold sensitive; interestingly, they exhibit different arrest phenotypes. The gcr1[DELTA] cln3[DELTA] strain becomes predominantly unbudded with 1N DNA content ([G.sub.1] arrest), whereas gcr1[DELTA] cln1[DELTA] cln2[DELTA] cells exhibit severe elongation and apparent M phase arrest. Further analysis demonstrated that the Rap1p/Gcr1p complex mediates rapid growth in glucose by stimulating both cellular metabolism and CLN transcription.

The multifunctional regulatory factor Rap1p of Saccharomyces cerevisiae accomplishes one of its tasks, transcriptional activation, by complexing with Gcr1p. An unusual feature of this heteromeric complex is its apparent capacity to contact simultaneously two adjacent DNA elements ([UAS.sub.RPG] and the CT box, bound specifically by Rap1p and Gcr1p, respectively). The complex can activate transcription through isolated [UAS.sub.RPG] but not CT elements. In promoters that contain both DNA signals its activity is enhanced, provided the helical spacing between the two elements is appropriate; this suggests that at least transient DNA loop formation is involved. We show here that this CT box-dependent augmentation of Rap1p/Gcr1p activation requires the presence of a third protein Gcr2p; the Gcr2 growth defect appears to result from a genome-wide loss of the CT box effect. Interestingly, a hyperphosphorylated form of Gcr1p disappears in [Delta]gcr2 cells but reappears if they harbor a doubly point-mutated GCR1 allele that bypasses the [Gcr2.sup.-] growth defect. Gcr2p therefore appears to induce a conformation change in Gcr1p and/or stimulate its hyperphosphorylation; one or both of these effects can be mimicked in the absence of GCR2 by mutation of GCR1. This improved view of Rap1p/Gcr1p/Gcr2p function reveals a new aspect of eukaryotic gene regulation: modification of an upstream activator, accompanied by at least transient DNA loop formation, mediates its improved capacity to activate transcription.

A method called unigenic evolution, a statistical analysis of mutability in evolutionary variants of a single gene, is described. It was applied to map the functionally important domains in Gcr1p of Saccharomyces cerevisiae. Multiple rounds of error-prone PCR mutagenesis on the variants of Gcr1p generated in vitro and in vivo identified four hypomutable regions of GCR1. Analysis of the regions by alternating insertion, deletion and point mutation showed that some of these regions may correspond to functionally important surface domains that are important for Gcr1p homodimerization. In particular, a putative leucine zipper was identified in region D. A diagram of the unigenic evolution of GCR1 is presented.

Efficient transcription of ribosomal protein (RP) and glycolytic genes requires the Rap1p/Gcr1p regulatory complex. A third factor, Gcr2p, is required for only the glycolytic (specialized) mode of transcriptional activation. It is recruited to the complex by Gcr1p and likely mediates a change in the phosphorylation state and/or conformation of the latter. We show here that leucine zipper motifs in Gcr1p and Gcr2p (1LZ and 2LZ) are each specific to one of the two activation mechanisms--mutations in 1LZ and 2LZ impair transcription of RP and glycolytic genes, respectively. Although neither class of mutations causes more than a mild growth defect, simultaneous impairment of 1LZ and 2LZ results in a severe synthetic defect and a reduction in the expression of both sets of genes. Intracistronic complementation by point mutations in the charged e and g positions confirmed that Gcr1p/Gcr1p and Gcr2p/Gcr2p homodimers are the forms required for the different roles of the activator complex. Direct heterodimerization between 1LZ and 2LZ apparently does not occur. Dichotomous Rap1p activation and its striking requirement for distinct homodimeric subunits give cells the capacity to switch between coordinated and uncoupled RP and glycolytic gene regulation.

The recruitment model for gene activation presumes that DNA is a platform on which the requisite components of the transcriptional machinery are assembled. In contrast to this idea, we show here that Rap1/Gcr1/Gcr2 transcriptional activation in yeast cells occurs through a large anchored protein platform, the Nup84 nuclear pore subcomplex. Surprisingly, Nup84 and associated subcomplex components activate transcription themselves in vivo when fused to a heterologous DNA-binding domain. The Rapi coactivators Gcr1 and Gcr2 form an important bridge between the yeast nuclear pore complex and the transcriptional machinery. Nucleoporin activation may be a widespread eukaryotic phenomenon, because it was first detected as a consequence of oncogenic rearrangements in acute myeloid leukemia and related syndromes in humans. These chromosomal translocations fuse a homeobox DNA-binding domain to the human homolog (hNup98) of a transcriptionally active component of the yeast Nup84 subcomplex. We conclude that Rapi target genes are activated by moving to contact compartmentalized nuclear assemblages, rather than through recruitment of the requisite factors to chromatin by means of diffusion. We term this previously undescribed mechanism "reverse recruitment" and discuss the possibility that it is a central feature of eukaryotic gene regulation. Reverse recruitment stipulates that activators work by bringing the DNA to an nuclear pore complex-tethered platform of assembled transcriptional machine components. [ABSTRACT FROM AUTHOR]