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

Abstract

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy (Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach-Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. (2006) Nature 441, 770 -773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.

Connecting the transcription site to the nuclear pore: a multi-tether process that regulates gene expression

“…In light of these findings and because of the earlier observation that the interaction of Nup2 with GAL gene promoters does not require SAGA components (Schmid et al, 2006), it is conceivable that gene repositioning to the NPC occurs mainly in two steps: the interaction of specific promoter-bound transcription activators with nucleoporins mediates the initial binding to the NPC, whereas the subsequent recruitment of SAGA might stabilize gene-NPC anchoring by interacting with TREX2 at a minimum. Moreover, chromatin immunoprecipitation (ChIP) experiments revealed that the NPCassociated protein Mlp1 binds to the UAS of active genes through direct interactions with SAGA components (Luthra et al, 2007). These data, and the fact that Mlp1 is required for efficient gene-NPC anchoring (Dieppois et al, 2006;Tan-Wong et al, 2009), suggest that -in addition to TREX2 -the interaction between Mlp1 and SAGA contributes to stabilizing gene-NPC associations during and following the early transcription activation process (Fig.…”

“…In addition to the proposed tethering mediated by Nup2, genes are tethered to the NPC through interaction of the SAGA coactivator complex with the NPC-bound TREX2 complex (consisting of Sac3, Thp1, Sus1 and Cdc31) (Cabal et al, 2006;Fischer et al, 2004;Fischer et al, 2002;Rodriguez-Navarro et al, 2004). The nuclear basket Mlp proteins might also contribute to gene anchoring, as they bind the promoter of active genes through interactions with SAGA subunits (Dieppois et al, 2006;Luthra et al, 2007). The nucleoporins Nup1 and Nup60 are the docking sites of TREX2 and Mlp1, respectively (Feuerbach et al, 2002;Fischer et al, 2002).…”

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

“…[42][43][44] Previous work has shown that components of the SAGA histone acetyltransferase complex are required for the peripheral localization of the INO1 and GAL1 genes. 21,[45][46][47] The SAGA complex has been suggested to physically associate with the nuclear pore complex through a bridging interaction between the Sac3 and Sus1 proteins. 48 Acetylation by the catalytic subunit of SAGA, the Gcn5 protein, promotes transcription initiation of the INO1 and GAL1 genes, both of which are targeted to the nuclear periphery upon activation.…”

Gene positioning is regulated by phosphorylation of the nuclear pore complex by Cdk1

“…5,6 Localization at the nuclear periphery correlates with an interaction of the promoters of these genes with the nuclear pore complex (NPC) 5,11 and requires nucleoporins. [12][13][14] We have focused on the mechanism by which the INO1 gene is targeted to the NPC. We find that INO1 relocalizes to the nuclear periphery rapidly, independent of mRNA production 12 and requires two DNA "zip codes" in the promoter called Gene Recruitment Sequences (GRSs 15 ) and several components of the NPC (Nup1, Nup2, Nup60, Nup157, Nup42, Gle2) and associated proteins (Mlp2, Gcn5, Spt7, Spt20, Sus1, Sac3 and Thp1).…”