17 research outputs found
Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast
The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin–euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin–euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains
H3K9me-Independent Gene Silencing in Fission Yeast Heterochromatin by Clr5 and Histone Deacetylases
Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci
Four chromo-domain proteins of <i>Schizosaccharomyces pombe</i> differentially repress transcription at various chromosomal locations
Expression-state boundaries in the mating-type region of fission yeast.
A transcriptionally silent chromosomal domain is found in the mating-type region of fission yeast. Here we show that this domain is delimited by 2-kb inverted repeats, IR-L and IR-R. IR-L and IR-R prevent the expansion of transcription-permissive chromatin into the silenced region and that of silenced chromatin into the expressed region. Their insulator activity is partially orientation dependent. The silencing defects that follow deletion or inversion of IR-R are suppressed by high dosage of the chromodomain protein Swi6. Combining chromosomal deletions and Swi6 overexpression shows that IR-L and IR-R provide firm borders in a region where competition between silencing and transcriptional competence occurs. IR-R possesses autonomously replicating sequence (ARS) activity, leading to a model where replication factors, or replication itself, participate in boundary formation
The Clr7 and Clr8 Directionality Factors and the Pcu4 Cullin Mediate Heterochromatin Formation in the Fission Yeast Schizosaccharomyces pombe
Fission yeast heterochromatin is formed at centromeres, telomeres, and in the mating-type region where it mediates the transcriptional silencing of the mat2-P and mat3-M donor loci and the directionality of mating-type switching. We conducted a genetic screen for directionality mutants. This screen revealed the essential role of two previously uncharacterized factors, Clr7 and Clr8, in heterochromatin formation. Clr7 and Clr8 are required for localization of the Swi6 chromodomain protein and for histone H3 lysine 9 methylation, thereby influencing not only mating-type switching but also transcriptional silencing in all previously characterized heterochromatic regions, chromosome segregation, and meiotic recombination in the mating-type region. We present evidence for physical interactions between Clr7 and the mating-type region and between Clr7 and the S. pombe cullin Pcu4, indicating that a complex containing these proteins mediates an early step in heterochromatin formation and implying a role for ubiquitination at this early stage prior to the action of the Clr4 histone methyl-transferase. Like Clr7 and Clr8, Pcu4 is required for histone H3 lysine 9 methylation, and bidirectional centromeric transcripts that are normally processed into siRNA by the RNAi machinery in wild-type cells are easily detected in cells lacking Clr7, Clr8, or Pcu4. Another physical interaction, between the nucleoporin Nup189 and Clr8, suggests that Clr8 might be involved in tethering heterochromatic regions to the nuclear envelope by association with the nuclear-pore complex
Two Portable Recombination Enhancers Direct Donor Choice in Fission Yeast Heterochromatin
<div><p>Mating-type switching in fission yeast results from gene conversions of the active <i>mat1</i> locus by heterochromatic donors. <i>mat1</i> is preferentially converted by <i>mat2-P</i> in M cells and by <i>mat3-M</i> in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the <i>mat2-mat3</i> heterochromatic domain. Cells whose silent cassettes are swapped to <i>mat2-M mat3-P</i> switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps occur through the recombination enhancers. Our observations lead to a model where heterochromatin biases competitions between the two recombination enhancers to achieve directionality.</p></div
The Clr7 and Clr8 Directionality Factors and the Pcu4 Cullin Mediate Heterochromatin Formation in the Fission Yeast <em>Schizosaccharomyces pombe</em>
Mating-type region and mating-type switching in <i>S. pombe</i>.
<p>(A) Schematic representation of the mating-type region showing the expressed, switchable, <i>mat1</i> locus and the silent <i>mat2-P</i> and <i>mat3-M</i> donor loci. Elements are described in the text. (B) Model for the replication-coupled gene conversions of <i>mat1</i> responsible for mating-type switching. (C) Pedigree of switching. (D) 2004 model for the directionality of switching (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003762#pgen.1003762-Jia1" target="_blank">[41]</a>). The model proposes that SRE3 attracts the Swi2/Swi5 recombination complex to the mating-type region. Swi2/Swi5 remains localized near <i>mat3-M</i> in P cells, facilitating the use of <i>mat3-M</i> as a donor, and spreads over the entire mating-type region in M cells, facilitating the use of <i>mat2-P</i>. Use of <i>mat2-P</i> is favored over <i>mat3-M</i> when Swi2/Swi5 is present at both <i>mat2-P</i> and <i>mat3-M</i> (as in wild-type M cells) or in the absence of a functional directionality mechanism (as in <i>SRE3Δ</i> or <i>swi2Δ</i> cells).</p
SRE2 and SRE3 can stimulate recombination in both cell types.
<p>(A) Iodine staining of strains with the indicated mating-type regions (M: yellow; P: light blue; SRE2: dark blue; SRE3: red) shows that SRE2 stimulates recombination at <i>mat3</i> when it is substituted for SRE3 (2×SRE2 strain) and that SRE3 stimulates recombination at <i>mat2</i> when it is substituted for SRE2 (2×SRE3 strain), albeit less efficiently. The strains were, from left to right: SP837, TP75, TP126, TP8, TP303. (B) Fluorescence microscopy and (C) quantification of <i>mat1</i> content by Southern blot confirm balanced donor use in 2×SRE2 cells showing that P cells use SRE2 (TP273 and TP126 strains). (D–E) Same analyses as (B–C) for 2×SRE3 (TP313 and TP303 strains) indicate that M cells are not fully proficient in the use of SRE3 and accumulate in 2×SRE3 populations.</p
SRE3-independent effects of Swi2 on donor choice.
<p>(A) Representation of the mating-type region showing the probe (<i>Dde</i>I-<i>Nsi</i>I fragment) and restriction sites used for the Southern blots in (C). (B) Iodine staining of sporulated colonies with indicated genotypes. Dark staining reflects efficient switching. Light staining indicates a switching defect that can be either asymmetric towards a preferred mating-type (no staining at colony junctions) or inefficient in both directions (staining at colony junctions). (C) Southern blots of genomic DNA digested with <i>Dde</i>I and hybridized to the <i>Dde</i>I-<i>Nsi</i>I probe shown in (A), for nine independent cultures of <i>h<sup>90</sup> swi2</i><sup>+</sup>: 968 (1–9); <i>h<sup>90</sup> swi2Δ</i>: TP133 (10–18); <i>SRE3Δ swi2</i><sup>+</sup>: TP75 (19–27); <i>SRE3Δ swi2Δ</i>: TP157 (28–36). (D) Quantification of <i>mat1</i> content estimated from the Southern blots shown in (C). P/(P+M) ratios are plotted as % P cells. (E) Independent measurement of cell type ratios by fluorescence microscopy using a dual reporter system, YFP under control of the M-specific <i>mfm3</i> promoter and CFP under control of the P-specific <i>map2</i> promoter. M cells are yellow and P cells cyan. <i>h<sup>90</sup> swi2</i><sup>+</sup>: TP220; <i>SRE3Δ swi2</i><sup>+</sup>: TP270. Cell counts from fluorescence microscopy are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003762#pgen.1003762.s006" target="_blank">Table S3</a>. Effects of Swi5 on donor choice are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003762#pgen.1003762.s001" target="_blank">Figure S1</a>. All Southern blot quantifications are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003762#pgen.1003762.s002" target="_blank">Figure S2</a>.</p