Reverse recruitment: the Nup84 nuclear pore subcomplex mediates Rap1/Gcr1/Gcr2 transcriptional activation - PubMed (original) (raw)

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

Balaraj B Menon et al. Proc Natl Acad Sci U S A. 2005.

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 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 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.

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Figures

Fig. 1.

Fig. 1.

Genome-wide genetic analysis of GCR1. A Δ_gcr1_ query strain (SD8) was crossed to an array of ≈4,700 deletion mutants to screen for synthetic growth defects in the resulting double mutants. Synthetic defects that result from combination with GCR1 deletion (arrows) fall into four categories based on Gene Ontology annotation (29) as follows: nuclear pore-associated genes (purple; large type), genes involved in cell growth and maintenance (light green), metabolic genes (blue), and uncharacterized ORFs (gray). Components of the Nup84 nuclear pore subcomplex and the related factor Nup100 are shown in bold. Deletion of NUP84 is also synthetically defective in combination with a GCR2 deletion, as indicated.

Fig. 2.

Fig. 2.

Physical association between the Rap1/Gcr1/Gcr2 activation complex and the nuclear periphery. Rap1 (A), Gcr1-myc (B), and Gcr2-myc (C) copurify with Pom152 (D) in nuclear envelope fractions; the nucleolar protein Nop1 (E) served as a negative control.

Fig. 3.

Fig. 3.

Epitope-tagged Gcr1 coimmunoprecipitates with three nuclear pore factors [Nup84 (A), Pom34 (B), and Pom152 (C)] and the NPC-associated β-importin Kap123 (D). Gcr1-myc was immunoprecipitated from whole cell extracts (input; lane 1); the last of four washes before elution (final wash; lane 2) was analyzed as a control. The corresponding protein eluates (pellet) were loaded in lane 3.

Fig. 4.

Fig. 4.

Transcriptional activation at the nuclear rim. The exclusively cytoplasmic nucleoporin Nup42 and the Seh1 component of the Nup84 subcomplex failed to activate transcription above background levels (vector) in WT cells. All other nucleoporins tested, including the human nucleoporin hNup98 (protooncogenic homolog of Nup145C), stimulated transcription of the reporter gene. The conventional activators Gcr1, Gcr2, Gcn4, and Swi4, as well as the mediator components Ssn8 and Sin4, are shown for comparison. Error bars represent standard error of the mean.

Fig. 5.

Fig. 5.

Activation and repression by the multifunctional regulator Rap1 at the nuclear periphery of S. cerevisiae. Known components of the Rap1 activation (Gcr1/Gcr2; refs. –, , , and 19) or silencing (Sir complex; refs. –, , , and 19) assemblages are shown. Essential nucleoporins are indicated with an asterisk; components of the GCR1 genetic network identified by SGA analysis (Fig. 1) are shown in bold. Note that combining deletion of GCR1 with deletion of each of several genes encoding components of the Rap1 silencing assemblage did not result in a synthetic growth defect (NUP2, NUP60, YKU70, YKU80, and SIR1 were tested by tetrad dissection). Dashed lines highlight presumptive perinuclear tethering interactions; NPC-associated factors shown to interact with Gcr1 (Fig. 3) are underlined. The representation shown here is not intended to rule out the existence of a unified complex that can switch between activation and repression of transcription (see text for further discussion).

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