Unexpected Roles of a Shugoshin Protein at Subtelomeres

Home , Position effect, Subtelomere

Genes Genet. Syst. (2017) 92, p. 127–133 Roles of shugoshin at subtelomeres 127 Unexpected roles of a shugoshin protein at subtelomeres

Junko Kanoh* Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

(Received 3 April 2017, accepted 19 June 2017; J-STAGE Advance published date: 9 August 2017)

A chromosome is composed of structurally and functionally distinct domains. Telo- meres, which are located at the ends of linear chromosomes, play crucial roles in genome stability. Although substantial knowledge of telomeres has been accu- mulated, the regulation and function of subtelomeres, which are the domains adjacent to telomeres, remain largely unknown. In this review, I describe recent discoveries about the multiple roles of a shugoshin family protein, Sgo2, which is localized at centromeres in mitosis and contributes to precise chromosome segregation, in deﬁning chromatin structure and functions of the subtelomeres in ﬁssion

yeast. Sgo2 becomes enriched at the subtelomeres, particularly during G2 phase, and is essential for the formation of a highly condensed subtelomeric chromatin body called the knob. Furthermore, Sgo2 maintains the expression levels of subtelomeric genes and the timing of DNA replication at subtelomeric late origins.

Key words: subtelomere, shugoshin, heterochromatin, knob, replication timing

regulated remain outstanding issues. INTRODUCTION The telomere, a specialized chromatin structure at the Composition of subtelomeric DNA Telomeres are ends of chromosomes, plays important roles in genome composed of DNA with repeated sequence ([TTAGGG]n integrity, and the adjacent chromosomal region, the sub- in mammals) and its associated proteins. In contrast, telomere, has recently become a focus of attention in the subtelomeres do not possess telomere repeat sequences; medical field. Subtelomere deletion syndrome (STD) is however, they generally contain multiple segments that a congenital disease arising from deletion or rearrange- share high similarity with each other in a specific organ- ment in the subtelomeric region. STD patients exhibit ism, in addition to various genes. In budding yeast, mental retardation and multiple congenital anomalies, Saccharomyces cerevisiae, subtelomeres span approxi- which most likely develop from the haploinsufficiency of mately 25 kb and include X and Y’ elements with highly subtelomeric genes (de Vries et al., 2003). Furthermore, variable lengths. The X element contains a highly facioscapulohumeral muscular dystrophy is caused by an homologous X core region (475 bp) and an autonomously increase in the expression of the DUX4 gene, located at replicating sequence; the Y’ element contains an RNA the subtelomere, which is affected by telomere position helicase gene (Louis, 1995). Internal repeat sequences effects (Stadler et al., 2013). Therefore, maintenance of exist in some subtelomeres at the telomere-distal region subtelomeric gene expression appears to be critical for adjacent to the X’ and Y elements. The length of the human health. internal repeats is also variable; they sometimes contain Although substantial knowledge of telomeres has accu- genes encoding PAU (seripauperin multigene family), mulated, the regulation and function of subtelomeres MAL (multigene complex for maltose fermentation), MEL remain a long-term mystery: research on subtelomeres (α-galactosidase) or ERR (enolase) proteins (Louis, 1995) has been progressing much more slowly than research (Fig. 1A). In humans, subtelomeres possess a mosaic of on other chromosomal regions due to technical difficul- multiple common sequences (more than 40 types in total) ties resulting from the structural nature of subtelomeric containing various genes. The length of the homologous homologous sequences, which are very long and present regions of human subtelomeres varies from less than in large numbers of copies. In particular, how the con- 20 kb to more than 200 kb (Linardopoulou et al., 2005; densed and silenced chromatin structure at subtelomeres Riethman et al., 2005; Stong et al., 2014). is formed and how replication timing at subtelomeres is The fission yeast, Schizosaccharomyces pombe, possesses three chromosomes. Although genome sequencing is not yet complete, the subtelomeres of S. pombe also Edited by Kenji Ichiyanagi * Corresponding author. E-mail: [email protected] contain highly homologous sequences spanning 40–60 DOI: http://doi.org/10.1266/ggs.17-00016 kb in chromosomes 1 and 2 that harbor the RecQ-type Figure 1 Kanoh

128 J. KANOH

A S. cerevisiae

Internal repeats X

B S. pombe

Highly homologous Mosaic of Unique sequence common segments

Subtelomeric homologous region Fig. 1. Composition of subtelomeres. (A) Representative structure of the subtelomeres in S. cerevisiae. (B) Structure of the subtelomeres of chromosomes 1 and 2 in S. pombe. See main text for details. helicase genes (tlh1/2 +) (http://www.pombase.org/status/ In contrast, S. cerevisiae does not possess H3K9me or sequencing-status). The telomere-proximal regions are HP1 homologs; instead, the telomere-binding protein Rap1 composed of a mosaic of common segments exhibiting associates with Sir3-Sir4 (silencing factors), which then variations like those in humans (Sugawara, 1988). The recruits Sir2, a histone deacetylase, to deacetylate his- telomere-distal regions of approximately 50 kb do not tones H3 and H4. After this, Sir3-Sir4 is again recruited contain sequences that are homologous between subtelo- to the deacetylated chromatin and again recruits Sir2, to meres. However, they share a common feature of chro- expand the heterochromatin (Huang, 2002) (Fig. 3B). matin structure: the levels of histone modifications, such Heterochromatin generally brings about a transcription- as methylation and acetylation, are very low compared ally silent state. Thus, the expression of genes located with the adjacent euchromatin and subtelomeric hetero- within subtelomeric heterochromatin is repressed; how- chromatin regions (see below) (Buchanan et al., 2009) ever, the physiological functions of these gene products (Fig. 1B and 2). The number of subtelomeres varies from remain largely unknown. Moreover, the roles of subtelo- four to six per S. pombe haploid genome, depending on meric heterochromatin itself have not yet been clarified. the presence or absence of a short subtelomereric homologous sequence (approximately 15 kb) and a chromosome Sgo2 is localized at subtelomeres during inter- 3-specific sequence (1.1 kb) between the telomeres and phase Shugoshin is a well conserved centromere pro- the rDNA repeats at the ends of chromosome 3 (Ohno et tein and plays crucial roles in centromeric cohesion al., 2016). and/or proper chromosome segregation in mitosis and meiosis (Kerrebrock et al., 1995; Kitajima et al., 2004; Subtelomeric heterochromatin structure Subtelo- Marston et al., 2004; Rabitsch et al., 2004; Salic et al., meres generally include heterochromatin, where histone 2004; Kawashima et al., 2007; Vanoosthuyse et al., 2007). H3 methylated at K9 (H3K9me) is highly enriched and Schizosaccharomyces pombe possesses two shugoshin heterochromatin protein 1 (HP1) homologs are associ- paralogs, Sgo1 and Sgo2. Sgo1 is expressed only dur- ated (Elgin and Grewal, 2003). At least two factors can ing meiosis and plays a critical role in centromeric cohe- independently establish subtelomeric heterochromatin in sion of sister chromatids in meiosis I (Kitajima et al., S. pombe. One is the telomere-binding protein complex 2004). In contrast, Sgo2 is continuously expressed and called shelterin; the other is the RNAi machinery, which contributes to precise chromosome segregation during acts at the centromere-homologous (cenH) sequence (the M phase (Kawashima et al., 2007). Intriguingly, it was template of siRNA) within the tlh1/2 + genes in the sub- reported that Sgo2 changes its localization from the cen- telomeres. After a methyltransferase, Clr4, methylates tromeres to the vicinity of the telomeres upon entry into H3K9 and an HP1 homolog, Swi6, is recruited to the interphase (Kawashima et al., 2010); however, detailed telomere-proximal sites, the heterochromatin spreads out information about Sgo2 localization and the physiologi- around the subtelomeric homologous regions, presumably cal roles of Sgo2 near telomeres remained unknown. In via the association of Swi6 with itself and Clr4 (Hall et the following sections, I describe our recent discovery of al., 2002; Cam et al., 2005; Kanoh et al., 2005; Wang et unexpected roles of Sgo2 at the subtelomeres in S. pombe al., 2016) (Fig. 3A). (Tashiro et al., 2016). Figure 2 Kanoh

Roles of shugoshin at subtelomeres 129

Sgo2 Swi6 Swi6 Swi6 Knob Heterochromatin

Low levels of Euchromatin High H3K9 methylation Telomere histone modifications

Subtelomere (~100 kb) Fig. 2. Schematic illustration of the chromatin structure of S. pombe subtelomeres. Heterochromatin containing Figurehighly 3 methylated Kanoh H3K9 and Swi6 is formed around the subtelomeric homologous region (approximately 50 kb). Sgo2 associates with the whole subtelomere (approximately 100 kb), and is required for the formation of a knob (approximately 50 kb) next to the heterochromatin, where various histone modiﬁcations are maintained at low levels. A euchromatin region, where gene transcription is active, is adjacent to the subtelomere.

Swi6 Establishment A S. pombe of heterochromatin Swi6 Spreading of heterochromatin RNA Clr4 Clr4

Swi6 RNAi Shelterin

Subtelomere siRNA template Telomere (cenH in tlh1/2)

B Establishment S. cerevisiae of heterochromatin Sir2

Spreading of heterochromatin Sir3-4

Sir3-Sir4 Rap1

Subtelomere Telomere Fig. 3. Formation of subtelomeric heterochromatin. (A) In S. pombe, the shelterin proteins (Taz1 or Ccq1) associated with the telomeres and with the telomere-proximal region of the subtelomere and RNAi machinery acting at the cenH sequence independently recruit Clr4 to methylate H3K9. H3K9me recruits Swi6 (HP1), and then the heterochromatin spreads out to the chromosomal inner region. (B) In S. cerevisiae, the telomere-binding protein Rap1 recruits Sir3-Sir4, which in turn recruits Sir2 to deacetylate histones H3 and H4. Sir3-Sir4 then associates with the deacetylated histones and again recruits Sir2 to expand the heterochromatin region.

Chromatin immunoprecipitation (ChIP)–chip analyses Phosphorylation of histone H2A is important for revealed that Sgo2 is enriched not only at the centro- Sgo2 localization What is the requirement for localiza- meres but also throughout the subtelomeres of both chro- tion of Sgo2 at the subtelomeres? Sgo2 remains attached mosomes 1 and 2, extending more than 100 kb from each to the subtelomeres in cells lacking telomeric DNA, indi- telomere (Fig. 2). However, Sgo2 was barely detectable cating that the telomeric structure is dispensable for near the telomeres of chromosome 3 in a strain that pos- the association of Sgo2 with the subtelomeres. In con- sesses no subtelomeric sequence in chromosome 3. ChIP trast, deletion of bub1 +, which encodes the sole protein analyses using synchronous cell culture showed that Sgo2 kinase that phosphorylates serine 121 of histone H2A associates with the subtelomeres preferentially during (H2A-S121) for the activation of the spindle assembly interphase, with a peak in the middle of G2 phase. checkpoint (Kawashima et al., 2010), abolishes the subtelomeric localization of Sgo2 almost completely. Sub- 130 J. KANOH stituting H2A-S121 with a non-phosphorylatable alanine of an exogenous marker gene inserted at a telomere- (h2a-S121A) or the bub1-KD (kinase-dead) mutation proximal site within a subtelomere demonstrated that results in almost the same level of removal of Sgo2 from Swi6-heterochromatin is dominant to repress gene expres- the subtelomeres as observed in bub1∆. Therefore, the sion in the telomere-proximal region. In contrast, there phosphorylation of H2A-S121 by Bub1 is important for was a marked increase in the expression of the marker the association of Sgo2 with subtelomeres. gene when it was placed at a telomere-distal site within a subtelomere in sgo2∆ and bub1∆ cells, whereas only a Sgo2 is crucial for the formation of a highly con- small increase was observed in swi6∆ cells, indicating densed chromatin body, the knob What are the func- that the subtelomeric localization of Sgo2 is required for tions of Sgo2 at the subtelomeres in interphase? Knobs this repressive effect (Tashiro et al., 2016). These results are highly condensed chromatin bodies (more condensed indicate that the association of Sgo2 with subtelomeres than heterochromatin) in S. pombe that are intensely forms a new type of repressive chromatin domain that is stained with the DNA-labeling dye DAPI (4’,6-diamidino- distinct from H3K9me-Swi6-mediated heterochromatin. 2-phenylindole). They are located in the vicinity of the telomeres, adjacent to the subtelomeric heterochroma- Sgo2 maintains replication timing at subtelo- tin (marked by H3K9me), specifically during interphase meres Does the subtelomeric localization of Sgo2 have (Matsuda et al., 2015). Interestingly, the subnuclear other effects on cellular functions? Schizosaccharomyces localization of Sgo2 always overlaps with the knobs, pombe subtelomeres contain many DNA replication which are completely lost in sgo2Δ cells. Consistent with origins that fire during late S phase (Hayashi et al., this, bub1Δ and h2a-S121A mutant cells, in which Sgo2 2007). The replication timing of some subtelomeric late is dissociated from the subtelomeres, also completely lack origins is regulated by the telomere-binding proteins knobs. Thus, the association of Sgo2 with subtelomeres Taz1 and Rif1 (Hayano et al., 2012; Tazumi et al., 2012) plays a crucial role in knob formation. (see Hisao Masai et al.’s review in this issue); however, the overall regulatory mechanism of subtelomeric replica- Sgo2 represses transcription of subtelomeric tion timing remains elusive. Intriguingly, the clusters of genes What is the consequence of Sgo2-mediated late origins at the subtelomeres largely overlap with Sgo2 knob formation? Condensed chromatin is generally localization. Indeed, in sgo2Δ, bub1-KD and h2a-S121A expected to be inhibitory to transcription (Politz et al., cells, the subtelomeric late origins replicate earlier than 2013). Genome-wide microarray analyses showed in wild-type cells, whereas the replication timing in other that the transcript levels of genes at subtelomeres are chromosomal regions does not change (Tashiro et al., increased in sgo2Δ cells, whereas gene expression is not 2016). Thus, the association of Sgo2 with subtelomeres strongly affected by sgo2Δ in other chromosomal regions has a repressive effect on replication to maintain proper including centromeres, where Sgo2 is highly enriched in replication timing at the subtelomeric late origins. M phase. Notably, the locations of the genes affected by sgo2Δ correlate with the subtelomeric distribution of Sgo2 regulates loading of Sld3 onto subtelomeric Sgo2. Transcription of subtelomeric genes is also dere- replication origins How does Sgo2 regulate replica- pressed in the bub1Δ and h2a-S121A mutants, and RNA tion timing? A replication factor, Sld3, is recruited to polymerase II (RNAPII) occupancy at subtelomeric gene origins during the earliest step of the activation of pre- loci is considerably higher in the sgo2∆, bub1∆ and h2a- replicative complex upon S phase entry (Yabuuchi et al., S121A mutants than in the wild-type strain. Further- 2006). Sld3 is enriched at the early origins but not at more, the levels of histone modifications associated with the late origins in wild-type cells arrested immediately transcriptionally active chromatin, such as dimethylation before the stage of replication initiation. In contrast, of lysine 4 and trimethylation of lysine 36 in histone H3 in sgo2Δ cells, recruitment of Sld3 is accelerated at the (Noma et al., 2001; Kizer et al., 2005; Morris et al., 2005), subtelomeric late origins, but not at the internal late are increased at subtelomeric gene loci in sgo2∆, bub1∆ origins. Therefore, Sgo2 represses the initiation step of and h2a-S121A cells. Collectively, these studies indicate replication by limiting Sld3 loading at the subtelomeric that Sgo2 maintains gene expression at the transcrip- late origins, which is required for proper replication tim- tional level, accompanied by histone modifications, spe- ing. cifically at the subtelomeres. Conclusion and future prospects Sgo2 is localized Sgo2 forms a new type of repressive chromatin at the centromeres and contributes to precise chromo- domain As described above, heterochromatin generally some segregation in M phase. On the other hand, dur- inhibits the expression of nearby genes. Do Sgo2 and ing interphase, Sgo2 is recruited to the subtelomeres and subtelomeric heterochromatin regulate gene expression plays important roles in the formation of knobs, in the independently of each other? Monitoring the expression repression of gene transcription, and in the repression of Roles of shugoshin at subtelomeres 131

Fig. 4. Sgo2 plays various roles at the centromeres and subtelomeres in S. pombe. Sgo2 is localized at the centromeres in M phase for precise chromosome segregation. On the other hand, during interphase, Sgo2 changes its localization to the subtelomeres and regulates knob formation, the expression of subtelomeric genes, and the replication timing of subtelomeric late origins.

replication to ensure proper gene expression and replica- pombe Sgo2 may also interact with a protein phosphatase tion timing at the subtelomeres. Thus, Sgo2 is a mul- to counteract the activity of DDK and thereby regulate tifunctional protein shuttling between centromeres and Sld3 loading. subtelomeres during the cell cycle (Tashiro et al., 2016) Third, how is the highly condensed knob structure (Fig. 4). formed? Intriguingly, knobs are not formed at the cen- Many questions have been raised from the study of tromeres (Matsuda et al., 2015), despite the high accu- Sgo2. First, how is Sgo2 targeted to the subtelomeres mulation of Sgo2 at that location in M phase, suggesting during interphase? Phosphorylation of H2A-S121 is that Sgo2 by itself is not sufficient and requires other observed outside the subtelomeres in interphase (Tashiro factors to form knobs. The mechanism underlying the et al., 2016), indicating that H2A-S121 phosphoryla- subtelomere-specific formation of knobs remains unclear; tion alone is not sufficient and that another factor is however, one possibility is that a unique chromatin required to confine Sgo2 to the subtelomeres. Because structure with low levels of acetylated and methylated Sgo2 dynamically changes its location during the cell histones (Buchanan et al., 2009) contributes to knob for- cycle, some cell-cycle regulators and/or modifications of mation. The low level of H3K9 methylation in the telo- Sgo2 (such as phosphorylation) may be involved in Sgo2 mere-distal region of the subtelomeres (adjacent to the recruitment to the subtelomeres. subtelomeric heterochromatin region) is in sharp con- Second, how does Sgo2 regulate knob formation, gene trast to the high level of H3K9 methylation in the Sgo2- expression and replication timing? Is it related to the associated outer regions of the centromeres (Kawashima functions of Sgo2 at centromeres? One possibility is et al., 2007; Buchanan et al., 2009). In fact, knobs are that Sgo2 regulates subtelomeric gene expression and observed preferentially in the telomere-distal regions of replication timing through the knobs. The highly con- the subtelomeres (Matsuda et al., 2015) (Fig. 2). densed knob structure may inhibit the recruitment of Fourth, are the shugoshin family proteins in other RNAPII, histone modification enzymes and replication organisms also involved in subtelomere functions? Sub- factors. The other possibility is that Sgo2 acts at the telomeric localization of shugoshin in other organisms subtelomeres independently of the knobs. As described has not yet been reported. In humans, however, main- in Masai et al.’s review, the telomere-binding protein tenance of the expression levels of subtelomeric genes Rif1 regulates the replication timing of late origins by is important for health. Thus, one can speculate that interacting with PP1 phosphatase to counteract the there exist some regulatory mechanisms, possibly involv- action of Dbf4-dependent kinase (DDK) at the initia- ing shugoshin, for the local organization of a repressive tion step of replication (Davé et al., 2014; Hiraga et al., chromatin domain at subtelomeres in other eukaryotes, 2014; Mattarocci et al., 2014). Interestingly, Sgo1 forms as in S. pombe. a complex with PP2A phosphatase to protect cohesins in S. pombe, S. cerevisiae and mammals (Kitajima et al., REFERENCES 2006; Riedel et al., 2006; Tang et al., 2006; Ishiguro et Buchanan, L., Durand-Dubief, M., Roguev, A., Sakalar, C., al., 2010), and human Sgo2 also interacts with PP2A for Wilhelm, B., Strålfors, A., Shevchenko, A., Aasland, the protection of cohesion (Tanno et al., 2010). Thus, S. R., Shevchenko, A., Ekwall, K., et al. (2009) The 132 J. KANOH

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