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Identification of a novel type of spacer element required for imprinting in fission yeast

PLoS Genetics (2011) 7(3)
DOI: 10.1371/journal.pgen.1001328
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Abstract

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging-strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers. © 2011 Sayrac et al.

Figures

  • Figure 1. Mating-type switching in S. pombe. (A) Switching pedigree. The cells of minus (M) and plus (P) mating type are represented with light and dark brown colours, respectively. Division of an unswitchable (u) cell leads to the formation of an unswitchable and a switchable (s) cell of the same mating type. Division of the switchable cell gives rise to a switchable cell of the same mating type and an unswitchable cell of the opposite mating type. (B) The mating-type region on chromosome II. The transcriptionally active mat1 locus and the silenced donor loci, mat2P and mat3M, are shown. The M and P information is indicated with light and dark brown colours, respectively. The homology domains H1, H2 and H3 flanking the loci are represented by grey boxes. The centromere (cen2) and the origin of replication located centromere-distal to mat1 are shown with a hollow box and a circle, respectively. The replication termination element RTS1 is indicated with grey triangles, and direction of fork movement through mat1 is shown above with grey arrows, representing unidirectional replication. The imprint is indicated by a yellow star. (C) The replication of the mat1 locus in unswitchable and switchable cells. The nascent leading and lagging strands are represented by red and blue lines, respectively. In the second generation, the inherited template strands are also shown in red or blue, depending on which strand they were replicated as during the S phase of the parent. The polarities of DNA strands are indicated with 39 and 59 symbols. The S phases of both cell types involves pausing of the replication fork at mat1 and the introduction of the imprint in the newly synthesized upper strand. In addition, during the S phase of a switchable cell, the imprint on the upper template strand acts as a lesion to block leading-strand synthesis. The stalled leading-strand replication complex induces homologous recombination between mat1 and the donor loci leading to mating-type switching. doi:10.1371/journal.pgen.1001328.g001
  • Figure 2. Identification of two regions required for imprinting and pausing within the mat1M cassette. (A) Line drawing on the left, a simple representation of the intact mat1M cassette flanked with the homology domains is shown. The site of the imprint is indicated by a vertical arrow. The nucleotide length of the region is given. The positions of the two encoded transcripts, Mi and Mc, are shown by horizontal arrows. The fulllength wild-type DNA is represented by an intact line, whereas deletions are shown as gapped lines and the nucleotide positions of the start and the end of each deletion are given below the lines. The position of the specific lagging-strand priming site described in 12] is marked by a vertical line. The text box on right shows the names of the strains used, followed by quantification of the double-strand break (DSB) products (including standard error) and of the replication pause signal observed on 2D gels. The results are given as percentages of the wild-type values (100%). For DSB products, the values lower or equal to the negative control threshold are indicated as ‘‘below detection’’ (B.D.), whilst the absence of a pause signal is indicated by a dash. (B) Southern analysis of the strains shown in panel A. Genomic DNA was digested with HindIII and probed with the mat1P HindIII fragment. During DNA isolation, the imprint is hydrolyzed to a DSB and the resulting break products migrate as two bands. The strain names are given on top,
  • Figure 4. Transcription of the mating-type-specific gene Mc does not affect imprinting. (A) The names of the strains and quantification of the DSB products generated during growth in either low-nitrogen sporulation (PMA+) or rich (YEA) media are given. The levels of imprinting, as assessed by DSB products, are similar for both growth conditions. (B) The effect of abolishing Mc transcription on the spacer deletion phenotype. Left, schematic representation of the mat1M cassette and the strains used is given. The positions of the Mi and Mc transcripts are shown by horizontal arrows. Deletion of the promoter region between the two transcripts (in strains SS66 and SS63) was performed, thus abolishing transcription. Right, the names of the strains and quantification of the DSB products (including standard error) are given. (C) Southern analysis of the strains shown in panel B. See Figure 2 legend for method. (D) Northern analysis of total RNA from the strains shown in panel B. The blot was probed for Mc using a double-stranded fragment generated by PCR. (E) Characterization of the strain (SV20) carrying a deletion of most of the spacer region and the region containing the previously mapped priming site. See Figure 2 legend for method. The position of the deletion is shown as a line drawing. Please note that fork-regression at MPS1 is observed in the 2D-gel displayed, in the absence of the imprint. This observation is fully analyzed in a related study (Vengrova and Dalgaard, in preparation). doi:10.1371/journal.pgen.1001328.g004
  • Figure 5. Southern analysis of mat1 replication intermediates. (A) Line drawing of the fragments generated for different strains for denaturing polyacrylamide gel electrophoresis (PAGE) analysis of replicating mat1M DNA shown in panels B and C. The positions of the restriction enzyme sites and the mapped priming site, and the lengths of the fragments are given. The site of the imprint is indicated with a circle. (B) Denaturing PAGE analysis of the mat1M lagging strand. Replicating DNA from log-phase cells was isolated as for 2D-gel analysis, digested with restriction enzymes given in panel A, then was subjected to denaturing PAGE analysis. After blotting, the membrane was probed with a strandspecific probe that hybridizes to the nascent lagging strand and the parental upper strand. Denatured and labelled DNA ladder is given to the left with size markers. The strains that exhibit imprinting (WT and SS25) produced two strong bands that correspond to the centromere-distal and proximal DSB products created by hydrolysis of the imprint (1d and 1p, respectively). Specific priming products at the site of the imprint (2) migrate to the same position as the distal DSB product observed for the WT and SS25 strains (1d). See panel D. Two fainter putative priming products that are shared between WT and SS25 are marked by arrowheads. (C) Denaturing PAGE analysis of the mat1M leading strand. The nascent leading strand and the parental lower strand were detected by the strand-specific probe. The co-migrating signals from the leading strand intermediates stalled at the imprint in WT and SS25 strains (3) and at MPS1 in the strain SS13 (4) are indicated. See panel D. Denatured and labelled DNA ladder is given to the left with size markers. (D) Line drawings of the replication intermediates observed in panel B and C are given. Fragments that produce bands in PAGE
  • Figure 6. Characterization of a cis-acting region named abc that is required for replication pausing and imprinting. (A) Left, line drawing representation of the strains used. A gap represents a deletion and a gray line segment represents a substitution of a DNA sequence. The defined abc region is shown and the borders of the element are indicated by vertical lines. Right, names of the strains and quantification of DSB products and pause signals on 2D gels (including standard error) are given. (B) Southern analysis of the strains shown in panel A. See Figure 2 legend for method. (C) 2D-gel analysis of the strains shown in panel A. See Figure 2 legend for method. doi:10.1371/journal.pgen.1001328.g006
  • Figure 7. Characterization of the cis-acting region required for replication pausing. (A) DNA sequence of the abc region, aligned to the corresponding sequence in the P cassette, is given. The borders of a, b, and c regions are indicated by double arrows above the sequence. The sequence was divided into 10 segments and each segment was substituted by a random DNA sequence and denominated as substitution 1 to 10 (Sub1-10). (B) 2D-gel analysis of plasmids that carry different substitutions within the abc region. Top left, line drawing of the parent plasmid. The
  • Figure 8. Out-competition of the putative trans-acting factor(s) that interacts with the M abc and P abc regions. (A) The effect of plasmids that carry arrays of the abc region on sporulation efficiency. Two different plasmids (constructed using pREP3X or pREP4X parental vectors) that each carries an array of approximately 10 copies of either the M abc or P abc region were introduced in the h90 wild-type background (strain JZ1), such that two plasmid-borne arrays of either M or P abc were present. The sporulation efficiency relative to that observed for a strain carrying empty vectors (100%) are given as a graph. The deviation is shown as error bars. (B) Quantification of mat1 imprinting in strains used in panel A. HindIII fragments were run and probed with the mat1P HindIII fragment. The intensity of the signal from the P abc array is higher due to greater sequence homology. Quantification of intensities of the DSB products relative to the strain that carries the empty vectors (100%) is given below. (C) Neither P abc nor M abc induces replication pausing at the transcriptionally silenced donor loci. Left, 2D-gel analyses of mat2P and mat3M replication are shown. Right, line drawings of the fragments analyzed are given. The direction of replication through each locus is indicated with grey arrows. The positions of the restriction enzyme sites and the probes used are shown. The sizes of the fragments are given. doi:10.1371/journal.pgen.1001328.g008
  • Figure 9. Substitutions in the region around and of the imprinted nucleotides. (A) The wild-type sequence is given on top. The H1 domain sequences are highlighted with a grey shadow. The position of the inverted repeat that flank the imprint is shown with arrows. Below, the mutant sequences introduced are given. Substituted nucleotides are shown in italics and are underlined. (B) Southern analysis of the strains given in panel A. See Figure 2 legend for method. The position of the DSB products is shown. (C) High-resolution Southern blot of the wild-type and mutant strains carrying substitutions at the imprinted nucleotides. A strand-specific probe that hybridizes to the imprinted strand was used. For detailed explanation of the method, see the Materials and Methods section. The positions of the fragments generated by hydrolysis of the imprint are shown. doi:10.1371/journal.pgen.1001328.g009

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CITATION STYLE

APA

Sayrac, S., Vengrova, S., Godfrey, E. L., & Dalgaard, J. Z. (2011). Identification of a novel type of spacer element required for imprinting in fission yeast. PLoS Genetics, 7(3). https://doi.org/10.1371/journal.pgen.1001328

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