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Review
. 2015 Jul 1;7(7):a018770.
doi: 10.1101/cshperspect.a018770.

Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

Robin C Allshire  1 , Karl Ekwall  2
Affiliations
Review

Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

Robin C Allshire et al. Cold Spring Harb Perspect Biol. .

Abstract

This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres.

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Figures

Figure 1.
Figure 1.
Life cycle of the fission yeast, S. pombe. Fission yeast has a short G1 taking less than 10% of the cell cycle (stippled area is expanded to aid representation). In rich medium, G1 cells proceed into S phase followed by a long G2 (∼70% of the cell cycle), mitosis, and cytokinesis. When starved of nitrogen, cells of opposite mating-type (+ and −) conjugate, after which nuclei fuse in a process known as karyogamy. Premeiotic replication and recombination allows meiosis I and II to proceed, resulting in four haploid nuclei that are separated into four spores in an ascus. Provision of rich medium allows germination of spores and resumption of the vegetative cell cycle.
Figure 2.
Figure 2.
Distinct outer repeat heterochromatin and central kinetochore domains at fission yeast centromeres. (A, top) Representation of a fission yeast centromere. The central domain (pink, kinetochore) is composed of imr and cnt elements, the outer repeats contain transcribed dg and dh repeats (green, heterochromatin). All three centromeres have a similar overall arrangement; however, the number of outer repeats differs: cen1 (40 kb) has two, cen2 (65 kb) has three, and cen3 (110 kb) has approximately 13. Clusters of transfer RNA (tRNA) genes (double arrowheads) occur in the imr region and at the extremities of all three centromeres. (Middle) Schematically shows transcription patterns of marker genes placed within the outer repeats, central domain, or beyond the centromere. (Bottom) Images showing the phenotype of S. pombe colonies of ade6+ transgenics inserted at various sites within the centromere. Cells expressing ade6+ from a transgene inserted in sequences outside the centromere form white colonies. When ade6+ is inserted at sites within the outer repeats, expression is silenced and red colonies are formed. Expression of ade6+ from the central domain is typically variegated, resulting in red, white, and sectored colonies. (B) A schematic representation of S. pombe chromosomes. The three chromosomes are depicted showing the four main regions of heterochromatin: centromere, telomere, mat2/3, and rDNA regions.
Figure 3.
Figure 3.
Centromeric chromatin domains in S. pombe. (A) A schematic representation of the symmetrical DNA arrangement in S. pombe centromeres. (B) Heterochromatin: outer repeats are packaged in nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me) by Clr4 as part of the CLRC complex. This allows the binding of chromodomain proteins Chp1 (a component of the RNAi RITS complex), Chp2, and Swi6. Collectively, these and other factors, including the SHREC complex-containing Clr3 histone deacetylase activity and the RDRC complex, act to assemble and propagate heterochromatin. Central “kinetochore” chromatin: CENP-A is found in the central domain where it probably replaces the majority of histone H3 to form specialized nucleosomes (coral colored). In addition to CENP-A, several proteins assemble at the central domain chromatin to form the inner and outer kinetochore multiprotein structures (coral arc). See Figure 6 for a description of the kinetochore.
Figure 4.
Figure 4.
Cell-cycle regulation of centromere heterochromatin assembly. (A) Heterochromatin located at chromosomal centromeres becomes differentially methylated and phosphorylated on histones throughout the cell cycle as indicated. These modifications control the binding of the heterochromatin protein Swi6. During mitosis Swi6 is displaced by H3S10 phosphorylation. Swi6 binding is reestablished during subsequent DNA replication (S phase) when a more accessible chromatin structure permits RNA Pol II to transcribe centromeric DNA. This, in turn, recruits the RNAi machinery to direct H3K9me methylation. (B) Replication-coupled RNAi model (Li et al. 2011). This figure illustrates an alternative model for how RNAi works at centromeres. Here, RNAi serves to release RNA Pol II from chromatin to avoid collision with DNA replication machinery during S phase. See text for further details. (A, Adapted from Djupedal and Ekwall 2008.)
Figure 5.
Figure 5.
Chromatin boundaries and the boundary mechanism involving Epe1 (for enhancement of position effect) in S. pombe. (A) A schematic representation of different types of boundary elements in S. pombe. (B) The mechanism of boundary function by Epe1. Epe1 associates with Swi6, however, when the antisilencing factor is ubiquitinated by the Cul4-Ddb1 ligase and degraded in the heterochromatin region to allow for heterochromatin assembly. However, at boundaries, Epe1 is somehow protected from degradation, thus restricting the spreading of heterochromatin. Phosphorylation of Swi6 contributes to the dissociation of Epe1 at heterochromatin, while promoting the association with the HDAC complex SHREC in maintaining histone hypoacetylation. (A, Adapted from Scott et al. 2007.)
Figure 6.
Figure 6.
Centromeric chromatin and kinetochores in S. pombe. The central domain of S. pombe centromeres is composed of CENP-A-containing chromatin and a multiprotein network that makes up the kinetochore. The inner constitutive centromere-associated network (CCAN) and outer KMN kinetochore protein networks are depicted and the different protein components listed below. Kinetochore assembly within the central domain mediates attachment to microtubules on spindle formation and chromosome segregation.
Figure 7.
Figure 7.
CENP-A chromatin establishment and propagation through the cell cycle. (A) Central domain DNA alone is unable to establish a functional centromere; outer repeats are required. Loss of heterochromatin from established centromeres does not affect CENP-Acnp1 or kinetochore maintenance in the central domain. This suggests that heterochromatin may, in some way, initially direct the site of CENP-Acnp1 chromatin and thus kinetochore assembly. (B) Cell-cycle dependency of CENP-ACnp1 recruitment in S. pombe. In S. pombe, centromeric DNA is replicated and existing CENP-ACnp1 is diluted by nucleosome segregation to sister chromatids during S phase. Recruitment of new CENP-A occurs during the G2 phase, indicated by the pink nucleosomes. The Sim3 histone chaperone interacts with new CENP-ACnp1 and delivers it to the centromere, where it is received by Scm3 and assembled into nucleosomes by unknown factors and mechanisms. Nucleosome gaps could be filled or H3 nucleosomes could be replaced. Scm3 is shown as a dimer interacting directly with Mis18. Scm3 recruitment at centromeres requires the Sim4/Mis6 and Mis16/Mis18 complexes. Mis16/Mis18 and Scm3 are removed from centromeres during mitosis and reassociate in G1. (B, Adapted from Mellone et al. 2009.)
Figure 8.
Figure 8.
Defective heterochromatin leads to abnormal centromere structures. (A) Cells lacking RNAi or heterochromatin components display elevated rates of chromosome loss and lagging chromosomes (indicated by yellow arrows) on late anaphase spindles. Chromosomal DNA is stained by DAPI (blue) and mitotic spindle microtubules are labeled for immunofluorescence (IF) (red). (B) A schematic three-dimensional figure of a normal centromere illustrates the outer heterochromatin regions (green circles) decorated with Swi6 (black circles), which recruits cohesin to ensure sister-chromatid cohesion. The central domain consists of CENP-A-containing chromatin (red circles), associated with opposing kinetochores on each sister chromatid. Lagging chromosomes in cells with defective heterochromatin may be the result of disorganized kinetochores such that one centromere may attach to microtubules from opposite poles. Such merotelic orientation could persist into anaphase, in which the breakage of attachment with one pole or the other would lead to random segregation and result in chromosome loss/gain events.
Figure 9.
Figure 9.
Nuclear organization in S. pombe. (A) Electron microscopy analysis of an S. pombe nucleus. (Top) Micrograph of a cross-section through a high-pressure fixed and Lowicryl-embedded interphase S. pombe cell. The cellular structures are indicated: cell wall, nuclear envelope, nucleolus, heterochromatin region, and SPB. (Bottom) A higher magnification of the same nucleus. The nuclear structures indicated are SPB, γ-tubulin region, anchor structure, and the centromeric heterochromatin. (B) Two interphase nuclei with heterochromatin (centromeres, telomeres, and the silent mat2-mat3 loci) decorated by red fluorescent immunolocalization of Swi6, and kinetochore chromatin (centromeres only) decorated by green fluorescent immunolocalization of CENP-ACnp1. The red signals, not in close proximity to green, represent telomeres or the mat2-mat3 loci. All centromeres are clustered at the nuclear periphery adjacent to the SPB. (C) A model for chromatin organization at the fission yeast nuclear periphery. (Top) Genes with low expression levels tend to associate with the nuclear periphery, whereas highly expressed genes tend to reside in the nuclear interior. (Top) Localization of divergent intergenic regions and H2A.Z at the nuclear envelope may present a mechanism for anchoring the promoters of convergent gene pairs at the periphery. (Bottom left) Differential localization of Ima1, nuclear pores, and Man1. The inner membrane proteins Ima1 and Man1 are not equally distributed at the nuclear periphery, but rather occupy distinct areas that interact with different chromosomal regions. The subtelomeric chromatin is associated with Man1-rich peripheral regions in which Swi6 is also located. Ima1 is colocalized with Dcr1 and Rdp1 at nuclear pores. (Bottom right) Organization of centromeric DNA at the SPB. Central domain cnt and imr regions are localized closer to the SPB than the heterochromatic dg and dh repeats. The two centromeric domains are shaded in colors symbolizing the different IF localization of Swi6 (dg-dh repeats) and CENP-ACnp1 (imr/cnt regions). (A, Reprinted from Kniola et al. 2001; C, bottom right, Adapted from Takahashi et al. 1992.)
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