Replication Pausing and Imprinting while bands corresponding to the full-length mat1 fragment and the fragments generated by the DSB are given to the left. (C) 2D-gel analysis of the strains shown in panel A. Genomic DNA from log-phase cells was digested with NdeI and subjected to 2D-gel electrophoresis. A line drawing of the analyzed fragment and the positions of the restriction enzyme sites and the probe is shown below. The site of the imprint is indicated with a circle. The obtained blots were probed with a mat1M-specific probe. The strain names are given above each 2D gel. doi:10.1371/journal.pgen.1001328.g002 Identification of a region required for imprinting that acts as a spacer To characterize the region defined by the deletion in strain SS13 in more detail, we constructed two sets of nested deletions, starting from both ends of the region (Figure 3). Deletion of 31 bp, 111 bp or 150 bp starting from the centromere-proximal side (strains SS6, SS5 and SS12, respectively) led to a gradual reduction in levels of imprinting, from 87.7% to 70.7% and ending with 46.6% of the wild-type level, suggesting that each deleted segment of this region partly contributes to the effectiveness of the imprinting process (Figure 3A, 3B). Similarly, introducing nested deletions of 55 bp, 94 bp of 174 bp in length starting from the centromere-distal side (strains SS22, SS23 and SS24, respectively), led to reduced imprinting to 85.5%, 55.5% and 16.9% of the wild-type level (Figure 3A, 3B). Again, this suggested that each of the deleted segments of this region makes a contribution to the imprinting process. All the deletions had a similar effect on replication pausing at MPS1, leading to an approximately 50% reduction in the intensity of the pause signal (Figure 3A, 3C). There are two possible explanations for the observed effect on imprinting; either specific sequences within this region contribute separately to the efficiency of the imprinting process or the 205-bp region acts as a spacer to separate regions of importance on either side. In the latter case, the observed gradual reduction in imprinting would be due to the gradual decrease in the distance between these flanking regions. If this region indeed acts as a spacer, reinstating the distance with an unrelated DNA sequence should restore imprinting. When we performed this experiment, the level of imprinting was indeed restored to 72.3% of the wild-type level and the level of pausing was restored to 80% (Figure 3, strain SS25). This result confirms the main role of this 205-bp region as a spacer separating flanking regions and the importance of this function for imprinting. The requirement of this 205-bp region for the imprinting process was also confirmed by deleting the equivalent sequences from the mat3M donor locus of a wild-type strain (Figure 3D). Imprinting in this strain was undetectable (Figure 3F) and colonies only displayed 1% sporulation efficiency compared to 72% observed for the wild-type colonies (Figure 3E). These results are consistent with the transposition of the mutant cassette sequence from the mat3M donor locus to the expressed mat1 locus, where the cis- acting deletion prevents subsequent imprinting events, and therefore any further mating-type switching. Transcription initiated from the Mc gene does not interfere with imprinting One possible function of the spacer region is that it separates the transcriptional unit Mc, which is encoded by the gene cassette, from the region of imprinting. Mc transcription is initiated from a promoter located within the cassette and terminates just prior to the spacer region where transcription termination and poly- adenylation signals are located. (Figure 4B). Thus, deletion of the spacer region brings these signals closer to the site of the imprint, where they could interfere with the imprinting process. It has been shown that insertion of the repressible nmt1 promoter at a position centromere-distal to the H1 domain such that it transcribes into the imprinting region leads to transcription-dependent inhibition of replication pausing as well as imprinting [11]. To test if Mc transcription interferes with imprinting as well when the spacer region is deleted, we used two approaches. Mc transcription has been shown to be repressed in rich media [8], therefore the levels of imprinting in the spacer region deletion strains were investigated under different growth conditions (Figure 4A). Quantification of the imprinting levels showed only minor differences between strains grown in poor (PMA+) and rich (YEA) media, suggesting that Mc transcription did not interfere with the imprinting process. To exclude the possibility that repressed levels of Mc transcription were still sufficient to inhibit imprinting, we deleted the promoter region of the transcriptional unit (Figure 4B–4D, strain SS66). Abolishment of the Mc transcript was confirmed by northern analysis, and this deletion produced only a slight reduction in the level of imprinting (90.6% of the wild-type). Importantly, the combination of the promoter deletion with the spacer deletion (strain SS63) did not lead to a restoration of imprinting (Figure 4B–4D). Thus, the reduction in imprinting observed when the spacer region is deleted is not due to inhibitory effects of Mc transcription. The region just centromere-proximal to the spacer region contains the strong priming site that we determined previously by RIP mapping [12], in addition to the putative Mc transcription termination and poly-adenylation signals. Therefore, the spacer deletions could be causing an inhibitory effect by bringing this region closer to the site of imprinting. To investigate this possibility, a 271-bp deletion was introduced that covered most of the spacer region and the potentially inhibitory region containing the priming site and the putative transcription signals (Figure 4E, strain SV20). This deletion had no effect on replication pausing, however, imprinting was still abolished in this strain. Therefore, the reduced levels of imprinting when the spacer region is shortened or fully removed are not due to a flanking inhibitory region brought closer to the site of the imprint. The spacer region influences the position of lagging- strand priming sites The spacer deletion could affect the replication process by changing where RNA primers are synthesized relative to the imprint site during lagging-strand replication. Therefore, high- resolution Southern blot analysis of replication intermediates was performed, using a single-stranded probe specific to the nascent lagging-strand (Figure 5A–5D). This method is the most direct way to analyze replication intermediates and avoids the problem of numerous unspecific bands observed when using the RIP mapping analysis. Two strong bands were detected in the imprinting- proficient wild-type strain (WT) and strain SS25, where the spacer region has been replaced by a scrambled sequence, the migrations of which are consistent with that predicted for fragments generated by hydrolysis of the imprinted parental upper strand (Figure 5B). Additionally, two weaker signals approximately 30 bp and 350 bp into the cassette were detected in these strains, which could be putative priming sites (indicated by arrowheads in Figure 5B). The signal 350 bp into the cassette corresponds to the position of the strong priming site that we had detected previously using RIP mapping [12]. Interestingly, both of these signals were absent in the lane containing replication intermediates from strain SS13, which carries the spacer deletion, suggesting that the deletion PLoS Genetics | www.plosgenetics.org 5 March 2011 | Volume 7 | Issue 3 | e1001328