Nup-PI: The Nucleopore-Promoter Interaction of Genes in Yeast Manfred Schmid, Ghislaine Arib, Caroline Laemmli, Junichi Nishikawa, Thérèse Durussel, Ulrich K. Laemmli  Molecular Cell  Volume 21, Issue 3, Pages 379-391 (February 2006) DOI: 10.1016/j.molcel.2005.12.012 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 ChEC Approach and Nup-PI at GAL Locus (A) The Nup2p receptor of the NPC basket was MN tagged (Nup2-MN) to determine by ChEC experiments in vivo the sites of interaction of the basket with the highly mobile genome. (B) Mapping of in vivo ChEC cleavage sites of Nup2-MN (strain TD320) by indirect end-labeling Southern blots at the GAL locus. Cells were either grown under repressive (glucose, left blot) or inducting conditions (galactose, right blot). These blots demonstrate the strong, galactose-dependent cleavage (dots) by Nup2-MN near the TATA boxes of the GAL1 and GAL10 genes that we termed Nup-PI (nucleopore-promoter interaction). The genomic map is aligned on the right. Digestion times were 0, 5, and 10 min. The full-length 6 kb fragment is indicated. Weak cleavage bands at HS sites (arrowheads) were marked. (C) Galactose-dependent anchoring of the GAL locus at the NPC basket. Strain YGA133 bearing a LacO array near the GAL locus was grown in glucose (row 1) or galactose (row 2). LacO arrays are detected by the fluorescence of GFP-LacI (green), and the NPC was revealed by immunostaining (red). DNA was counterstained with DAPI (gray). The bottom rows show control cells stained identically that express Gbd-Nup2 to force tether the GAL locus to NPC basket irrespective of growth conditions (glucose, galactose). Molecular Cell 2006 21, 379-391DOI: (10.1016/j.molcel.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 High-Resolution Analysis of ChEC Cleavage Patterns at GAL10 The ChEC patterns for several MN-tagged strains (indicated) were analyzed by high resolution with the primer extension method. The patterns shown for the Gal4-MN strain (MSY12) were from undigested (0′, lane 1) or from 1 min digested samples (1′, lane 2) obtained from galactose-grown cells. The H2b-MN (TD304), TBP-MN (TD348), and Nup2-MN (TD 320) patterns were obtained from glucose (−) or galactose (+) grown cells. Digestion conditions were for H2b-MN 1 min on ice, for TBP-MN 10 min on ice, and for Nup2-MN 5 min at 30°C. Shown is the autoradiograph of a sequencing gel at the GAL10 promoter (map). Bands that are most significantly enhanced in the Nup2-MN galactose pattern as compared to the H2b-MN galactose pattern are marked by dots. The lane scans of the galactose samples of H2b-MN (lane 4, gray line) and Nup2-MN (lane 8, black line) are shown on the right. Note that Nup-PI is principally limited to the 14–68 region upstream of the transcription initiation sites (position 1 in the map). Molecular Cell 2006 21, 379-391DOI: (10.1016/j.molcel.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Nup-PI at GAL Requires Gal4p but Not Intact SAGA and Transcription In vivo ChEC cleavage pattern of Nup2-MN (top, strains TD320, YGA107, YGA101, YGA95) and TBP-MN (bottom, strains TD327, YGA115, YGA121) in wt, gal4Δ, spt20Δ, and sus1Δ mutant backgrounds at the GAL1/10 locus as described in Figure 1. Specific Nup2-MN cleavage bands are marked by dots. Asterisks mark TBP-MN cleavage bands. Molecular Cell 2006 21, 379-391DOI: (10.1016/j.molcel.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Nup-PI Requires the UASg and TATA Box DNA Elements In vivo ChEC cleavage patterns of Nup2-MN (top) and TBP-MN (bottom) in strains (listed in Table S1) with modified GAL1 promoters. Glucose- and galactose-grown cells are compared and digestion carried out for 0, 5, and 10 min either on ice (TBP-MN) or at 30°C (Nup2-MN). The pGAL1 constructs are depicted on the bottom, aligned to a genomic map, and positions are shown relative to the endogenous GAL1 start codon. Regions deleted in the constructs are shown as dashed lines. All constructs lack the GAL10 proximal promoter elements. For indirect end labelling, Southern DNA was digested with PvuII/NotI to analyze specifically the GAL1 promoter region of these strains. Main Nup2-MN and TBP-MN cleavage sites were marked by dots and asterisks, respectively. Molecular Cell 2006 21, 379-391DOI: (10.1016/j.molcel.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Nup-PI at Several Genes of Yeast Chromosome VI In vivo ChEC cleavage patterns of Nup2-MN (strain TD320) and of H2b-MN (strain TD304) at several genomic loci on chromosome VI. Genomic maps are shown on the right of the blots. Significantly enhanced Nup2-MN cleavage bands that coincide with HS sites (visible in H2b-MN pattern) were marked by arrowheads, while those that do not map to HS sites were labeled by dots. Digestion conditions were for Nup2-MN 0, 5, and 10 min at 30°C and, for H2b-MN 0, 1, and 2 min on ice (indicated). (A) ChEC patterns at HXK1 from glucose and galactose-grown cells for Nup2-MN. (B) Same as (A) for H2b-MN. (C) ChEC patterns at CEN6 of Nup2-MN and H2b-MN from galactose-grown cells. (D) Same as (C) probed at FRS2. (E) Same as (C) probed at ACT1. An open arrowhead marks the strong H2b-MN cleavage band (strong HS site) at the 3′ end of ACT1. Molecular Cell 2006 21, 379-391DOI: (10.1016/j.molcel.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Tiling Microarray Cleavage Pattern for Nup2-MN throughout Chromosome VI (A) ChEC pattern of Nup2-MN throughout chromosome VI corrected for hypersensitive sites with the help of the H2b-MN data (Experimental Procedures, Figure S5). The vertical bars are the log2 ratios of the Nup2-MN to H2b-MN hybridization signals. Error bars are standard deviations of three independent experiments. Hence, positive values represent genomic regions, where Nup2-MN interacts with genuine preference. (B) Schematic outline for the preparation of gw probes that are enriched for DNA flanking MN cuts and are delimited by adjoining MboI sites. For details, refer to Figure S2. Molecular Cell 2006 21, 379-391DOI: (10.1016/j.molcel.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Speculations about Nup-PI (A) Depicted is the boundary assay described previously (Ishii et al., 2002), where the ADE2 gene is epigenetically silenced (hatched) by insertion in the HML locus (top). Nup-PI (nucleopore-to-gene-promoter interaction) was not observed with silenced or repressed genes. The bottom cartoons the situation, where ADE2 is force tethered to the Nup2p receptor of the NPC basket with the Gbd-Cse1 (gray) fusion protein that is bound to the UASg (red) element. We propose that this synthetic tether forces Nup-PI (yellow) despite the silenced chromatin state. In turn, this may facilitate the assembly of a productive transcription complex and perhaps of mRNA export (see text). The upstream activation sequence (UASa, green) and TATA box (blue) elements of the ADE2 promoter are depicted. (B) The physiological situations of GAL promoters with the NPC basket are shown. The top shows the glucose-repressed state, where the locus remains mobile. The bottom shows the galactose-activated situation, where the promoter is bound to the NPC basket. The mediating proteins are unknown. We observed that Nup-PI is an early step of gene activation and propose that the interaction of the promoter with the NPC basket facilitates the formation of a productive expression complex and perhaps the export of mRNA. Molecular Cell 2006 21, 379-391DOI: (10.1016/j.molcel.2005.12.012) Copyright © 2006 Elsevier Inc. Terms and Conditions