INO1 transcriptional memory leads to DNA zip code-dependent interchromosomal clustering

Abstract

Many genes localize at the nuclear periphery through physical interaction with the nuclear pore complex (NPC). We have found that the yeast INO1 gene is targeted to the NPC both upon activation and for several generations after repression, a phenomenon called epigenetic transcriptional memory. Targeting of INO1 to the NPC requires distinct cis-acting promoter DNA zip codes under activating conditions and under memory conditions. When at the nuclear periphery, active INO1 clusters with itself and with other genes that share the GRS I zip code. Here, we show that during memory, the two alleles of INO1 cluster in diploids and endogenous INO1 clusters with an ectopic INO1 in haploids. After repression, INO1 does not cluster with GRS I - containing genes. Furthermore, clustering during memory requires Nup100 and two sets of DNA zip codes, those that target INO1 to the periphery when active and those that target it to the periphery after repression. Therefore, the interchromosomal clustering of INO1 that occurs during transcriptional memory is dependent upon, but mechanistically distinct from, the clustering of active INO1. Finally, while localization to the nuclear periphery is not regulated through the cell cycle during memory, clustering of INO1 during memory is regulated through the cell cycle.

Keywords: DNA zip code; epigenetic inheritance; interchromosomal clustering; nuclear pore; transcriptional memory.

Figure 1. FIGURE 1: Experimental system.

(A) Schematic of the INO1 promoter, with the relevant regulatory elements and DNA zip codes highlighted. GRS: Gene Recruitment Sequence ; MRS: Memory Recruitment Sequence ; UAS_INO: Upstream Activating Sequence regulated by inositol. (B and C) Experimental setups for studying interchromosomal clustering using two different repressor arrays (B) or two identical arrays (C). (D) Representative confocal micrographs of cells having two GFP-marked arrays. Scale bar = 1µm.

Figure 2. FIGURE 2: INO1 transcriptional memory leads to interchromosomal clustering.

(A) Haploid cells having the endogenous INO1 gene marked with the TetO and URA3:INO1 marked with the LacO, expressing GFP-TetR and mRFP-LacI were grown under INO1 repressing (+ inositol) or memory (- inositol → + inositol, 3h) conditions, fixed and processed for immunofluorescence against GFP and mRFP. Left: The distribution of distances between the two loci in ~100 cells, binned into 0.2 µm bins. P values were calculated using a Wilcoxon Rank Sum Test. Right: the fraction of cells in which the two loci were ≤ 0.55 µm. P values were calculated using a Fisher Exact Test. Note: the distribution of the repressed condition has been previously published and is shown only for comparison to the distribution under the experimental (memory) condition. (B-D) Subsampling analysis. Full datasets (n = 100) or randomly generated subsamples of 50 or 40 measurements (r50 or r40, respectively) were compared pairwise using a Wilcoxon Rank Sum test. The numbers in each cell are the P values, color-coded as described in the legend. (B) A biological replicate compared with itself. (C) Two biological replicates compared with each other. (D) Distributions from repressing and memory conditions compared with each other.

Figure 3. FIGURE 3: INO1 interchromosomal clustering during memory is specific and MRS-dependent.

(A-C) Haploid cells having the endogenous INO1 gene marked with the TetO and either URA3 (A-C) or GAL1 (A) marked with the LacO, expressing GFP-TetR and mRFP-LacI were grown under INO1 memory (activating → repressing, 3h) conditions, fixed and processed for immunofluorescence against GFP and mRFP. Left: The distribution of distances between the two loci in ~ 100 cells, binned into 0.2 µm bins. P values were calculated using a Wilcoxon Rank Sum Test. Right: the fraction of cells in which the two loci were ≤ 0.55 µm. P values were calculated using a Fisher Exact Test. Note that the data used to generate the distribution for the control of INO1-TetO vs. URA3:INO1-LacO is the same in all three panels. The combinations tested were INO1-TetO vs. URA3:INO1-LacO (A-C), or INO1-TetO vs. GAL1-LacO (A), INO1-TetO vs. URA3:mrsINO1-LacO (B) and INO1-TetO vs. URA3:MRS50-LacO (C).

Figure 4. FIGURE 4: INO1 interchromosomal clustering during transcriptional memory requires clustering of active INO1.

(A and B) Haploid cells having the LacO array integrated at URA3 and the TetO array integrated at INO1, and expressing GFP-TetR and mRFP-LacI were fixed and processed for immunofluorescence against GFP and mRFP. Left: The distribution of distances between the two loci in ~ 100 cells, binned into 0.2 µm bins. P values were calculated using a Wilcoxon Rank Sum Test. Right: the fraction of cells in which the two loci were ≤ 0.55 µm. P values were calculated using a Fisher Exact Test. (A) INO1-TetO vs. URA3:INO1-LacO or URA3:grs1,2 INO1-LacO grown under memory conditions. (B) INO1-TetO vs. URA3:GRS I-LacO under either activating or memory conditions. (C) INO1-TetO vs URA3:GRS II-LacO under activating or memory conditions. (D) INO1-TetO vs. URA3:INO1-LacO put3∆ cells under activating or memory conditions.

Figure 5. FIGURE 5: Nup100 is specifically required for INO1 clustering during memory.

Diploid cells having both alleles of INO1 marked with the LacO array and expressing GFP-LacI were grown under repressing, activating or memory conditions. Left: The distribution of distances between the two loci in ~ 100 cells, binned into 0.2 µm bins. P values were calculated using a Wilcoxon Rank Sum Test. Right: the fraction of cells in which the two loci were ≤ 0.55 µm. P values were calculated using a Fisher Exact Test. (A and B) NUP100/NUP100 diploids. (C and D) nup100∆/nup100∆ diploids.

Figure 6. FIGURE 6: Interchromosomal clustering of recently repressed INO1 is regulated through the cell cycle.

(A and B) An asynchronous population of cells having INO1:TetO and INO1:LacO, expressing GFP-TetR, GFP-LacI and Pho88-mCherry (ER/nuclear envelope membrane protein) were grown under repressing, activating or memory conditions. Left: The distribution of distances between the two loci in ~ 100 cells, binned into 0.2 µm bins. P values were calculated using a Wilcoxon Rank Sum Test. Right: the fraction of cells in which the two loci were ≤ 0.55 µm. P values were calculated using a Fisher Exact Test. (C) Bright field (top) and green fluorescence (bottom) channels of typical cells used to measure distances in cells with different bud morphologies. The outline of the cell above is overlaid on the green channel (hatched line). (D) Cells were scored for both their bud morphology and the distance between the two loci and the fraction of each class of cells that was ≤ 0.55 µm was determined. P values were calculated using the Fisher Exact Test.

Figure 7. FIGURE 7: Model for zip code-dependent INO1 targeting to the NPC and interchromosomal clustering.

(A) The long-term repressed gene is positioned in the nucleoplasm and both the active and recently repressed memory state of the gene are positioned at the nuclear periphery through interaction with the NPC. The GRS elements control targeting to the NPC under activating conditions. The Put3 transcription factor binds the GRS I zip code and is required for GRS I-mediated peripheral targeting and interchromosomal clustering . The MRS element controls targeting to the NPC under memory conditions and requires Nup100 . (B) The INO1 gene clusters with other GRS I-containing loci under activating conditions (top) and this is a prerequisite for clustering with itself (and potentially other loci) in an MRS-dependent cluster for several generations after repression, during transcriptional memory.