SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope

Abstract

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis.

Actively Transcribed GAL Genes Can Be Physically Linked to the Nuclear Pore by the SAGA Chromatin Modifying Complex

“…This finding, in combination with published work showing that the Ada2 component of the SAGA complex is required for localization of the active GAL genes at nuclear pores (9), raises the possibility that this interaction between the Mlp proteins and the SAGA complex could contribute to the recruitment of transcriptionally active SAGA-dependent GAL genes to the nuclear periphery. If SAGA does mediate the interaction of Mlp proteins with chromatin, we would hypothesize that the Mlp proteins should interact with the same regions of the chromatin as the SAGA complex.…”

“…Transcription factors, chromatin modifying complexes, and the transcription machinery itself have each been independently implicated in directing active loci to the NPC (8 -12), perhaps suggesting a recruitment mechanism dependent upon transcription initiation. In addition, some interactions between components of the NPC and active loci are RNA-dependent (7), and mRNA processing and export factors have also been implicated in tethering loci to the NPC (9,13,14), suggesting that the interaction between active genes and the NPC may instead be dependent upon ongoing transcription and mRNA maturation. Recent, independent studies of GAL and HXK1 recruitment have yielded different results regarding the role of the transcription machinery and transcriptional co-activators in this process (8 -10), raising the possibility that distinct mechanisms of recruitment may operate for individual loci.…”

“…These transcribed genes interact with components of the nuclear pore complex (NPC) 4 (5,(7)(8)(9)(10), perhaps facilitating efficient mRNA processing and export. Although this phenomenon of locus association with the nuclear periphery has been reproducibly observed in yeast (5)(6)(7)(8)(9)(10), critical questions remain unanswered regarding the mechanism of locus recruitment to the NPC. Transcription factors, chromatin modifying complexes, and the transcription machinery itself have each been independently implicated in directing active loci to the NPC (8 -12), perhaps suggesting a recruitment mechanism dependent upon transcription initiation.…”

“…These components of the NPC include the Mlp proteins, Nic96, Nup1, Nup2, Nup60, and Nup116 (5,7,9). We focused on the two Mlp proteins as they are localized to the nuclear basket of the NPC (15), have roles in mRNA processing and export (16 -18), and have been implicated in the recruitment of multiple and diverse genes including the GAL genes, ␣ factor-responsive genes, and many other genes that are highly expressed under normal growth conditions (5,7).…”

Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

“…36 Furthermore, the cluster has been shown to produce antisense lncRNAs that are eliminated exclusively by the Dcp2p and Rat1p pathway instead of the 3 0 -5 0 Rrp6p-dependent pathway which is common to many lncRNAs. 22 Thus, it is tempting to speculate that the elimination of GAL lncRNAs may mirror the outcome of Rhoinduced aberrant HXK1 mRNPs in using the same degradation pathway.…”