Interaction of a DNA zip code with the nuclear pore complex promotes H2A.Z incorporation and INO1 transcriptional memory

Abstract

DNA "zip codes" in the promoters of yeast genes confer interaction with the NPC and localization at the nuclear periphery upon activation. Some of these genes exhibit transcriptional memory: after being repressed, they remain at the nuclear periphery for several generations, primed for reactivation. Transcriptional memory requires the histone variant H2A.Z. We find that targeting of active INO1 and recently repressed INO1 to the nuclear periphery is controlled by two distinct and independent mechanisms involving different zip codes and different interactions with the NPC. An 11 base pair memory recruitment sequence (MRS) in the INO1 promoter controls both peripheral targeting and H2A.Z incorporation after repression. In cells lacking either the MRS or the NPC protein Nup100, INO1 transcriptional memory is lost, leading to nucleoplasmic localization after repression and slower reactivation of the gene. Thus, interaction of recently repressed INO1 with the NPC alters its chromatin structure and rate of reactivation.

Figure 1. Different cis acting DNA elements control peripheral localization of active and recently repressed INO1

A. Schematic of the lac operator array plasmid, with or without INO1, integrated at URA3. B. Quantitative localization assay of URA3, URA3:INO1 or grs I mutant URA3:INO1. The hatched blue line indicates the baseline for this assay. Cells were grown in the presence or absence of 100μM inositol or switched from medium lacking inositol to medium containing inositol for one hour (−ino → +ino). C. Time course of peripheral localization after repression. The indicated strains were shifted from activating to repressing conditions and cells were harvested and fixed for immunofluorescence and chromatin localization at the indicated times. D. Top panel: left, schematic of INO1 promoter mutants; right, deleted sequences. Bottom panel: peripheral localization from strains having grs I mutant INO1 (data same as in panel B) or grs I mutant INO1 with each of the deletion mutations at URA3.

Figure 2. The MRS is a sequence-specific DNA zip code

A. The MRS functions as a DNA zip code. Top panel: sequences of inserts tested for peripheral localization when integrated at URA3 using the strategy shown in Figure 3A. Bottom panel: peripheral localization of URA3 (data same as in Figure 1B) or URA3 having each of the indicated DNA fragments integrated nearby. B. Top, schematic of INO1 promoter, indicating relative positions of the grs I mutation (red bar) and the MRS. Bottom panel: peripheral localization of either the grs I mutant (labeled WT; same data as shown in Figure 1B) or the indicated single base pair substitutions within the MRS integrated beside URA3. C. & D. Peripheral localization of mrs mutant INO1 or grs I mrs mutant INO1 integrated at URA3 (C) or at the endogenous INO1 locus (D).

Figure 3. The MRS and the histone variant H2A.Z control peripheral targeting of recently repressed INO1

A. Scheme for integrating DNA elements for localization experiments (Ahmed et al., 2010). B. Peripheral localization of URA3, URA3:MRS and URA3:MRS in the htz1Δ strain. C. Chromatin immunoprecipitation of HA-H2A.Z (Meneghini et al., 2003) from wild type and mrs mutant INO1strains and quantified using primers to amplify −197 to −284 relative to the INO1 ORF or primers to amplify the BUD3 promoter. D. Top panel: map of nucleosomes in the INO1 promoter. Shown are the positions of GRS I (red box), the MRS (yellow box), the TATA box (grey box), two UAS_INO elements (green boxes) and the PCR products associated with each nucleosome. Bottom panel: ChIP of HA-H2A.Z from either a wild type or mrs mutant INO1 strain, quantified using primers corresponding to the locations in the top panel or the BUD3 promoter.

Figure 4. The MRS is sufficient to induce H2A.Z incorporation

A. Integration scheme for inserting DNA elements for ChIP experiments (as in Ahmed et al., 2010). B. ChIP of HA-H2A.Z at URA3. The MRS or a control insert were integrated at URA3. Immunoprecipitations were performed with or without 12CA5 mAb against the HA tag. C. ChIP of HA-H2A.Z from URA3:MRS, mrsmut:URA3 or URA3:MRS swr1Δ strains. D. & E. ChIP of HA-H2A.Z (D) or peripheral localization (E) from strains having the MRS (with or without Swr1), the mrs C2A or the Reb1bs integrated at URA3. For panels B–D, immunoprecipitated DNA was quantified relative to input DNA by using real time PCR with primers for both the INO1 promoter and BUD3 promoter.

Figure 5. The MRS is required for transcriptional memory

A. INO1 activation. At time = 0, cells were shifted from repressing medium containing 100μM inositol (red arrow in schematic) to medium without inositol (green arrow in schematic). Cells were harvested at indicated time points and INO1 mRNA levels were quantified relative to ACT1 mRNA levels by RT-qPCR. B. INO1 reactivation. Cells were shifted from activating medium to repressing medium containing 100μM inositol for 3h. At time = 0, cells were harvested and returned to medium without inositol. Cells were harvested at indicated time points and INO1 mRNA levels were quantified relative to ACT1 mRNA levels by RT-qPCR. C. ChIP with anti-Rbp1 antibody at the indicated time points during activation (same scheme as in A). D. ChIP with anti-Rbp1 antibody at the indicated time points during reactivation (same scheme as in B). E. ChIP with anti-Rbp1 antibody (8WG16) from wild type or mrs mut INO1 strains after repression. F. ChIP using anti-Rpb1, anti-phospho Ser2 CTD or anti-phospho Ser5 CTD antibodies from wild type or mrs mut INO1 strains under activating conditions or after 3h of repression.

Figure 6. NPC proteins required for targeting of active and recently repressed INO1

A and B. Peripheral localization of recently repressed INO1 in NPC mutant strains. The wild type and mutant strains were grown overnight in medium lacking inositol, inositol was added for 1h and cells were fixed form immunofluorescence. Red bars highlight strains that targeted both active and recently repressed INO1 to the nuclear periphery. Blue bars highlight strains that failed to target both active and recently repressed INO1 to the nuclear periphery. Purple bars highlight strains that targeted active INO1 to the nuclear periphery but failed to target recently repressed INO1 to the nuclear periphery. C. ChIP of TAP-tagged Nup2 or Nup100. Immunoprecipitated DNA was quantified relative to input DNA using real time PCR and primers specific for the INO1 promoter and RPA34 intergenic region (negative control).

Figure 7. Nup100 is essential for INO1 transcriptional memory

A and B. ChIP of HA-H2A.Z in wild type, nup100Δ and swr1Δ strains grown without inositol or shifted from −inositol to +inositol for one hour before cross-linking and processing for ChIP. Immunoprecipitated DNA was quantified relative to input DNA using primers against the INO1 promoter and the BUD3 promoter. C. Wild type, nup100Δ or mrs mutant INO1 cells were harvested at indicated times after shifting from repressing to activating conditions. INO1 mRNA levels were quantified relative to ACT1 mRNA levels by RT-qPCR. D. After 3h of repression, at time = 0 the strains were shifted back to activating medium and harvested at the indicated times. INO1 mRNA levels were quantified relative to ACT1 mRNA levels by RT-qPCR. E. ChIP with anti-Rbp1 after repression from wild type and htz1Δ strains. F. ChIP with anti-Rbp1 after repression from wild