Reverse recruitment: The Nup84 nuclear pore subcomplex mediates Rap1/Gcr1/Gcr2 transcriptional activation

Abstract

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 Rap1͞Gcr1͞Gcr2 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 Rap1 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 Rap1 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.chromatin boundaries ͉ leukemia ͉ silencing ͉ synthetic genetic array ͉ gene regulation A n underlying assumption of both the stepwise and preassembly alternatives (1) of the recruitment model of in vivo gene activation (2-6) is that activators work by bringing the transcriptional machinery to the DNA, i.e., that the machinery itself diffuses relatively freely within the nuclear compartment. We have been studying the repressor͞activator protein Rap1 of Saccharomyces cerevisiae, which recognizes identical motifs in mediating either transcriptional activation (of glycolytic genes and ribosomal protein genes; refs. 7-9) or repression (of silent mating type loci and telomeres; refs. 10-15) and with its coactivators Gcr1 and Gcr2 participates in coordination of growth with cell-cycle progression (16,17). Numerous aspects of Rap1 activation have conformed poorly with the ''free diffusion'' aspect of the recruitment model for transcriptional activation. One such aspect is the presence of an unusually large activation domain that is easily inactivated by means of mutations throughout the N-terminal 280 residues of Gcr1, spanning four distinct hypomutable regions (8,17,18); two of these hypomutable regions overlap with putative transmembrane domains.We report here independent approaches demonstrating that the Rap1͞Gcr1͞Gcr2 activation assemblage (7-9, 19), like its silencing counterpart, is anchored at the nuclear periphery. For example, synthetic genetic array (SGA) analysis identified a robust genetic network that connects the Ra...

A Point Mutation in theAspergillus nidulans sonBNup98 Nuclear Pore Complex Gene Causes Conditional DNA Damage Sensitivity

“…For example, in Saccharomyces cerevisiae the NPC has been demonstrated to play roles in tethering telomeres to the nuclear periphery, which helps to facilitate transcriptional silencing of subtelomeric genes (Galy et al 2000;Feuerbach et al 2002;Therizols et al 2006). Somewhat paradoxically, certain nucleoporins have been demonstrated to preferentially associate with transcriptionally active genes (Ishii et al 2002;Casolari et al 2004Casolari et al , 2005Dilworth et al 2005;Menon et al 2005;Schmid et al 2006). Interestingly, budding yeast nucleoporin null alleles that display sensitivity to DNA-damaging agents have been identified (Galy et al 2000;Bennett et al 2001;Chang et al 2002;Loeillet et al 2005;Therizols et al 2006).…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…It has also been proposed that gene localization within the nucleus is sometimes regulated in response to regulatory signals (Menon et al 2005;Sarma et al 2007). According to this model, genes and their associated activators move to the nuclear periphery where they encounter the transcriptional machinery at nuclear pore complexes.…”

A role for DNA sequence in controlling the spatial organization of the genome

“…38 Likewise, tethering nucleoporins of the Nup84 subcomplex to reporter genes leads to constitutive activation, suggesting that localization at the NPC is sufficient to promote transcription. 39 The hsp70 gene cluster localizes at the nuclear periphery, interacts with the NPC and expression of these genes depends on proteins that interact with the NPC. 23 The localization of the male X chromosome in Drosophila to the nuclear periphery involves interaction with the NPC and this is important for interaction with MSL dosage compensation factors, suggesting a correlation between peripheral localization and increased transcription.…”