SWI1 Is Required for Meiotic Chromosome Remodeling Events

Molecular Plant

•

Volume 1

•

Number 4

•

Pages 620–633

•

July 2008

RESEARCH ARTICLE

SWI1 Is Required for Meiotic Chromosome Remodeling Events Kingsley A. Boatenga,2, Xiaohui Yanga,2, Fuqui Donga, Heather A. Owena,b and Christopher A. Makaroffa,1 a Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA b Present address: Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA

ABSTRACT The Arabidopsis dsy10 mutant was previously identified as being defective in the synapsis of meiotic chromosomes resulting in male and female sterility. We report here the molecular analysis of the mutation and show that it represents a T-DNA insertion in the third exon of the SWI1 gene. Four mutations have now been identified in SWI1, several of which exhibit different phenotypes. For example, the swi1-1 and dyad mutations only affect meiosis in megasporocytes, while the swi1-2 and dsy10 mutations block both male and female meiosis. Furthermore, as part of a detailed cytological characterization of dsy10 meiocytes, we identified several differences during male meiosis between the swi1-2 and dys10 mutants, including variations in the formation of axial elements, the distribution of cohesin proteins and the timing of the premature loss of sister chromatid cohesion. We demonstrate that dsy10 represents a complete loss-of-function mutation, while a truncated form of SWI1 is expressed during meiosis in swi1-2 plants. We further show that dys10 meiocytes exhibit alterations in modified histone patterns, including acetylated histone H3 and dimethylated histone H3-Lysine 4. Key words:

Arabidopsis; meiosis; sister chromatid cohesion; chromatin remodeling; histone.

INTRODUCTION Meiosis represents a highly conserved and coordinated series of events in which a single round of chromosome replication is followed by two rounds of chromosome segregation resulting in the production of haploid gametes (Zickler and Kleckner, 1999). Cytological and molecular studies in a number of organisms have defined numerous events that are crucial for the correct partitioning of genetic material; however, the most extensively studied system is yeast (reviewed in Zickler, 2006). The commitment to meiosis is thought to occur at premeiotic S-phase. During this time, a meiosis-specific cohesin complex is loaded onto the chromosomes and sister chromatid cohesion is established. The initiation of meiosis is first observed cytologically at leptotene, when chromosome condensation begins and the installation of axial elements along the chromosomes can be observed. During leptotene, homologs move towards each other in what is termed ‘long-range co-alignment’ (Tesse et al., 2003). By late leptotene, the homologs have moved closer together via a double strand break (DSB)-dependent process and are now referred to as being in presynaptic co-alignment. During this time, each chromosome develops a proteinaceous structure called an axial element (AE). In zygotene, the homologous chromosomes begin to synapse through the polymerization of a central element (CE) between the two AEs, which are then referred to as

lateral elements (LEs). This tripartite structure, called the synaptonemal complex (SC), is completed at pachytene and persists until diplotene, when the chromosomes are further condensed. Chiasmata, which are the cytological manifestation of homologous recombination events, maintain connections between homologous chromosomes. In order for homologs to separate during anaphase I, the chiasmata must be resolved and sister chromatid cohesion released along the chromosome arms, which is triggered at the metaphase to anaphase transition by separase, a cysteine protease (Ciosk et al., 1998; Uhlmann et al., 2000). Centromere cohesion is maintained by the Sgo proteins (Gregan et al., 2005; Kitajima et al., 2004), until it is released at anaphase II. Substantial progress has been made in elucidating factors associated with many meiotic events in a number of organisms, including plants reviewed in Hamant et al. (2006), Ma (2005), and Pawlowski and Cande (2005). However, some of the least 1 To whom correspondence should be addressed. E-mail: makaroca@ muohio.edu, fax 513–529–5715, tel. 513–529–2813. 2

These authors contributed equally to this work.

ª The Author 2008. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssn030, Advance Access publication 26 June 2008 Received 22 February 2008; accepted 7 April 2008

Boateng et al.

understood events involve the initiation of meiosis and the reorganization of the nucleus, which occur after DNA replication and are essential for the synapsis and recombination of homologous chromosomes. A number of studies have shown a close relationship between premeiotic S phase and the subsequent pairing and initiation of recombination between homologous chromosomes (Borde et al., 2000). For example, the absence of the B-type cyclins Clb5 and Clb6, which are required for premeiotic S phase in budding yeast, leads to defects in double-strand break (DSB) induction, recombination and synaptonemal complex (SC) formation (Smith et al., 2001; Stuart and Wittenberg, 1998). Deletion of the histone methyltransferase Set1 leads to a delay in premeiotic S-phase and subsequent alterations in centromere and telomere distribution (Trelles-Sticken et al., 2005). Likewise, defects in the establishment of sister chromatid cohesion have major effects on chromosome juxtapositioning and synapsis (Cai et al., 2003; Klein et al., 1999; Molnar et al., 2001). Therefore, chromatin configuration established during premeiotic S phase, and the subsequent nuclear reorganization events that occur in early prophase I, are critical for chromosome synapsis and recombination later in meiosis. We previously reported on the dsy10 mutation, which causes male and female sterility (Cai and Makaroff, 2001). Preliminary cytological characterization of male meiosis in dsy10 plants suggested that meiotic chromosomes aligned and paired during zygotene and pachytene, respectively. However, 10 univalents were observed at diakinesis, suggesting that the homologues either failed to undergo synapsis and recombination or underwent premature separation. Alterations in chromosome structure were observed beginning in pachytene and the premature release of sister chromatid cohesion was observed beginning at anaphase I. In this report, we describe the isolation and further characterization of the dsy10 mutation and show that it represents a T-DNA insertion in the fourth exon of the SWI1 gene. Four mutations have now been characterized in SWI1. Interestingly, different phenotypes are observed for several of the swi1 alleles (Agashe et al., 2002; Mercier et al., 2001, 2003; Motamayor et al., 2000; Siddiqi et al., 2000). For example, the swi1-1 and dyad mutations only affect meiosis in megasporocytes, while the swi1-2 and dsy10 mutations block both male and female meiosis. Furthermore, differences in chromosome behavior during male meiosis have been reported for dsy10 and swi1-2 plants (Cai and Makaroff, 2001; Mercier et al., 2001, 2003). The swi1-1 mutation is caused by a T-DNA insertion in the 5’ end of the gene. The swi1-2 allele is a nonsense mutation at amino acid 394, while the dyad allele represents a nonsense mutation at amino acid 509. We demonstrate here that dsy10 represents a complete loss-of-function mutation, while a truncated form of SWI1 is produced in swi1-2 plants. We provide evidence for several differences in between the swi1-2 and dys10 mutations and show that dys10 meiocytes exhibit alterations in modified histone H3 patterns. These results suggest that SWI1 is important for meiotic chromatin remodeling and may be a regulator of early meiotic events.

d

SWI1: An Early Meiotic Regulator

|

621

RESULTS Molecular Characterization of the dsy10 Mutation Preliminary genetic and molecular studies on dsy10 plants had shown that the line contains two T-DNA inserts (Peirson et al., 1996). Analysis of plant DNA at one of the T-DNA insertion sites (insertion site 1) revealed that the T-DNA is located 159 bp 5’ to the transcript terminus of GLX2-1. However, the T-DNA insertion did not alter the expression of GLX2-1 and the wild-type gene did not complement the mutation, indicating that it is not responsible for the dsy10 meiotic defects (Maiti et al., 1997). Cloning and sequence analysis of the second dsy10 insertion site revealed that the T-DNA is located within exon 4 of SWI1 (Figure 1A). The position of the T-DNA insertion within amino acid 139 of the 639 amino acid polypeptide suggested that it results in complete disruption of the gene. To investigate this possibility, the effect of the T-DNA insertion on SWI1 expression was analyzed by RT-PCR. Primer pairs 492 and 85 or 745 and 493 were used in amplification reactions of total bud cDNA produced from wild-type and dsy10 plants. Amplification products of ;1.0 kb were obtained from the wild-type

Figure 1. The SWI 1 Locus. (A) Map of the four SWI1 alleles. The positions of four swi1 alleles are shown above the map. Three transcripts have been identified for SWI1 that can encode proteins of 639, 635, and 578 amino acids. Primers used to amplify portions of SWI1 are shown below the map. N-Ab and C-Ab refer to the positions of the 19 aa and 153 aa SWI1 peptides used to raise amino-terminal and carboxy terminal antibodies, respectively. (B) Expression Analysis. Results from RT–PCR analysis of SWI1 expression using wild-type and dsy10 cDNA with primer pairs 492/ 85 or 745/493 followed by Southern blotting of the PCR products (primer pair 745/493). Actin served as an internal control.

622

|

Boateng et al.

d

SWI1: An Early Meiotic Regulator

cDNA with the two primer pairs, but none from dsy10 cDNA (Figure 1B). Control amplification reactions with primers corresponding to ACT8 produced products from both cDNA populations. Southern blot analysis on the RT–PCR products confirmed that SWI1 transcripts are absent in the dsy10 mutant. Therefore, the dsy10 T-DNA insertion reduces SWI1 transcript levels to below detectable levels.

Dsy10 Meiocytes Exhibit Alterations in Chromosome Pairing and the Premature Release of Cohesion Beginning at Anaphase I Preliminary cytological studies suggested that dsy10 plants exhibit a desynaptic phenotype (Cai and Makaroff, 2001). In contrast, the swi1-2 allele was reported to totally block homologous chromosome pairing (Mercier et al., 2001). In addition, swi1-2 plants exhibit a complete loss of sister chromatid cohesion by metaphase I, while 10 univalents were typically observed ay metaphase I in dsy10 meiocytes. Because of the apparent phenotypic differences between the dsy10 and swi1-2 alleles, we more carefully investigated the effect of the dsy10 mutation on male meiosis.

A detailed analysis of DAPI-stained chromosome spreads of meiotic chromosomes confirmed and clarified alterations during male meiosis in dsy10 meiocytes. Chromosomes begin to condense and up to 14 brightly stained heterochromatin regions (10 pericentromeric regions and four rDNAs) are typically observed at leptotene in wild-type male meiocytes (Figure 2A). Approximately six heterochromatin regions are observed as homologous chromosomes pair during zygotene (Figure 2B). Synapsis is complete at pachytene (Figure 2C). Chromosomes further condense and five bivalents are visible at diakinesis (Figure 2D). At metaphase I (Figure 2E), the bivalents congress and align on the spindle followed by separation of homologues at anaphase I (Figure 2F) and the sister chromatids at anaphase II (Figure 2G), resulting in four groups of five chromosomes at telophase II (Figure 2H). Meiocytes in dsy10 plants resembled wild-type meiocytes during leptotene and early zygotene (Figure 2I). Approximately six heterochromatin regions are observed and some long-range co-alignment of chromosomes appeared to occur in short stretches in some dsy10 meiocytes; however, typical zygotene chromosomes with partially paired homologues

Figure 2. Meiosis in Wild-Type and dsy10 Meiocytes. DAPI staining of chromosome spreads of selected stages during male meiosis in wild-type (A–H) and dsy10 meiocytes (I–P). (A, I) Leptotene, (B, J) Zygotene, (C) Pachytene, (D, L) Diakinesis, (E, M) Metaphase I, (F, N) Anaphase I, (G, O) Telophase I, (H, P) Telophase II, (K) Mid-prophase cell with unpaired homologs. Bar = 10 lm.

Boateng et al.

were not observed (Figure 2J). Normal examples of pachytene chromosomes were also not observed in dsy10 meiocytes (Figure 2K). Ten univalents were typically observed during diplotene and diakinesis (Figure 2L) that congressed and attached to the spindle at metaphase I (Figure 2M). Univalents as well as occasional separated sister chromatids were observed as the chromosomes segregated at anaphase I (Figure 2N). An uneven distribution of chromosomes was observed at telophase I (Figure 2O). By telophase II, multiple groups of chromosomes were observed (Figure 2P), which resulted in polyads with up to 10 microspores. These results indicate that homologous chromosomes in dsy10 plants fail to align and pair normally, but suggest that the presynaptic association of centromeres may occur. In addition, sister chromatid cohesion appears to be maintained until meiosis II in most dsy10 meiocytes. In order to investigate these questions further, FISH experiments were performed using a fluorescein-labeled centromere probe that localizes to the central domain of the pericentromeric heterochromatin

d

SWI1: An Early Meiotic Regulator

|

623

of Arabidopsis chromosomes (Fransz et al., 1998). In wild-type plants, eight to ten centromere signals were observed during meiotic interphase and leptotene (Figure 3A). During zygotene (Figure 3B), pachytene (Figure 3C), and diplotene (Figure 3D), four to five centromere signals were observed in most wildtype meiocytes. Beginning at diakinesis, the homologous centromeres began to separate, resulting in the presence of five pairs of signals (Figure 3E). Ten independent signals were typically observed from early anaphase I (Figure 3F and 3G), until anaphase II, when 20 signals were typically observed. Similar to the situation in wild-type plants, approximately 10 centromere signals were observed in dsy10 plants during meiotic interphase (average 9.5) and leptotene (average 9.3; Figure 3I). As the meiocytes progressed through prophase I, the number of centromere signals was significantly reduced (Figure 3J and 3K). The number of centromere signals ranged from one to eight in dsy10 meiocytes, with most cells exhibiting three to five signals (average 3.5, n = 60). This is similar to the situation observed in DAPI-stained dsy10 cells, and is

Figure 3. Centromere FISH in Wild-Type and dsy10 Meiocytes. FISH of a centromere probe to chromosomes in wild-type (A–H) and dsy10 (I–P) meiocytes. Merged photos of fluorescein-labeled 180-bp centromeric repeat (green) on chromosomes stained with DAPI (red). (A, I) Leptotene, (B, J) Early Zygotene, (C) Pachytene, (D, L) Diakinesis, (E, M) Metaphase I, (F, N) Anaphase I, (G, O) Early Anaphase II, (H, P) Telophase II, (K) Mid-prophase cell with clustered centromeres and unpaired homologs. Bar = 10 lm.

624

|

Boateng et al.

d

SWI1: An Early Meiotic Regulator

slightly lower than the four to five centromere signals observed in wild-type cells during mid-prophase I (Figure 3B and 3C). Eight to ten centromere signals were observed as univalents condensed at diplotene and diakinesis (Figure 3L) and congressed at metaphase I (Figure 3N). The number of centromere signals gradually increased from 10 to approximately 20 as the meiocytes progressed from metaphase I to metaphase II (Figure 3N and 3O). The clustering of centromeres in dsy10 meiocytes could represent either the non-specific association of centromeres or the initial stages of homologous chromosome alignment. Therefore, we performed FISH using a telomere-derived fragment that strongly labels a centromere proximal region on chromosome 1 (Armstrong et al., 2001) in order to evaluate the association of homologous centromeres in dsy10 meiocytes. Two strong chromosome 1 signals, along with the numerous telomere signals, were observed during interphase, leptotene, and early zygotene in both wild-type and dsy10 meiocytes (Figure 4A and 4I). Beginning in zygotene

(Figure 4B) and extending through pachytene (Figure 4C), diplotene (Figure 4D) and diakinesis, one strong signal was observed in wild-type meiocytes. Two strong chromosome one signals, along with the weaker telomere-derived signals, were observed from diakinesis through prometaphase II (Figure 4E–4G). Four chromosome 1 signals were observed, beginning at anaphase II, when the sister chromatids separated. In contrast, two chromosome 1 signals were typically observed in meiocytes of dsy10 plants throughout prophase I (Figure 4I–4M). Two widely spaced chromosome 1 centromere signals were observed in approximately 65% of prophase I nuclei (n = 53). Approximately 20% of the nuclei contained two adjacent signals, and a single signal was observed in approximately 15% of the prophase I nuclei. Two centromere 1 signals were observed in all dsy10 metaphase I cells and most anaphase I cells (Figure 4M and 4N). Beginning at approximately telophase I, the average number of chromosome 1 signals increased to approximately 3.5 as sister chromatid cohesion was lost (Figure 4O and 4P).

Figure 4. Chromosome 1 FISH in Wild-Type and dsy10 Meiocytes. FISH of a chromosome 1 probe to chromosomes in wild-type (A–H) and dsy10 (I–P) meiocytes. Merged photos of a fluorescein-labeled telomere probe that labels sequences adjacent to the chromosome 1 centromere (green) and chromosomes stained with DAPI (red). (A, I) Leptotene, (B, J) Early Zygotene, (C) Pachytene, (D, L) Diakinesis, (E, M) Metaphase I, (F, N) Anaphase I, (G, O) Metaphase II, (H, P) Telophase II, (K) mid-prophase cell with clustered centromeres and unpaired homologs. Arrows show the two chromosome 1 centromere associated signals. The insets show a longer exposure of the telomere FISH signals. Bar = 10 lm.

Boateng et al.

d

SWI1: An Early Meiotic Regulator

|

625

Therefore, while we found no evidence of homologous chromosome alignment or pairing, the presynaptic clustering of centromeres does occur in dsy10 meiocytes, and is in fact more pronounced than in wild-type meiocytes. However, the centromere clustering observed in dsy10 meiocytes represents the non-specific association of centromeres and not the beginning of homologous chromosome pairing. Furthermore, while some dsy10 meiocytes maintain sister chromatid cohesion until meiosis II, the premature loss of sister chromatid cohesion can be detected as early as anaphase I.

dsy10 Meiocytes Exhibit Alterations in the Distribution of Axial Element and Cohesin Proteins Studies on swi1-2 plants had demonstrated that the axial element protein ASY1, the cohesin protein SYN1 and the recombination protein RAD51 all showed altered distribution patterns in the mutant (Mercier et al., 2003). Given the differences in when cohesion is lost between swi1-2 and dsy10 plants, we were interested in determining if the two mutants exhibited other differences. Therefore, we examined the distribution of ASY1, SYN1, SMC3, and RAD51 in dsy10 plants. ASY1 appeared during late interphase as numerous diffuse foci on the chromatin prior to chromosome condensation in wild-type meiocytes (Figure 5A). At leptotene and early zygotene, ASY1 co-localized with the developing univalent axes, forming thin threads (Figure 5B and 5C) and, during pachytene, it was associated with the axes of the synapsed chromosomes (Figure 5D). Most of the ASY1 signal disappeared from chromosomes beginning at diplotene. However, weak staining was observed on chromosomes up through tetrad stage (Figure 5E and 5F). ASY1 localization patterns were indistinguishable from those of wild-type meiocytes in dsy10 pre-leptotene and leptotene nuclei. ASY1 co-localized with chromatin and appeared as thin threads that represent AEs in wild-type meiocytes (Figure 5G and 5H). However, as prophase progressed, ASY1 labeling patterns became more diffuse (Figure 5I). In mid to late prophase nuclei, weak ASY1 signal was detected only in the nucleoplasm (Figure 4J); it did not associate with the chromosomes. ASY1 signal was not detected in meiocytes of dsy10 plants from approximately diakinesis to the polyad stage (Figure 5K and 5L). Immunolocalization experiments with antibodies to cohesin proteins also detected differences between dsy10, wild-type, and swi1-2 plants. SYN1 localized along the developing chromosome axes at leptotene and remained on the synapsed homologs at early (Figure 6A) and late pachytene (Figure 6B) in wild-type plants. Beginning at diplotene, the SYN1 signal gradually decreased, such that it was barely visible by metaphase I (Figure 6C). It was typically not detectable after the onset of anaphase I (Figure 6D and 6E). SYN1 distribution in the dsy10 mutant was similar to that of wild-type meiocytes during early meiosis, including leptotene, and early zygotene (Figure 6F). SYN1 labeling in nuclei of dsy10 plants later in prophase I also resembled wild-type (Figure 6G). By diakinesis,

Figure 5. dsy10 Meiocytes Form Axial Elements. ASY1 immunolocalization during meiosis in wild-type (A–F) and dys10 (G–L) meiocytes. Merged images of DAPI-stained chromosomes (red) and ASY1 immunolocalization (green) are shown. (A, G) Interphase, (B) Leptotene, (C) Early zygotene, (D) Pachytene, (E, K) Diakinesis, (F) Tetrad stage, (H) Early prophase; (I, J) Midprophase, (L) Polyad. Bar = 10 lm.

only isolated spots of SYN1 signal could be detected along the chromosomes, with the signal being weaker than in wild-type meiocytes (Figure 6H). SYN1 was barely detectable on the chromosomes at approximately pre-metaphase I, and was undetectable throughout the rest of meiosis (Figure 6I and 6J), similar to the situation in wild-type meiocytes. In order to further analyze the distribution of cohesin complexes and determine if the alterations in SYN1 labeling we observe actually reflect alterations in the binding of the cohesin complex, the distribution of a second cohesin protein, SMC3, was examined. Immunolocalization experiments on chromosome spreads of dsy10 plants using antibody to Arabidopsis SMC3 produced labeling patterns very similar to those observed with the SYN1 antibody (Supplemental Figure 1). SMC3 antibody strongly labeled the chromosomes during early and mid-prophase, became weaker during diplotene and diakinesis, and was undetectable on the chromosomes from approximately metaphase I in both wild-type and dsy10 plants. In contrast, but similar to the SYN1 localization previously reported, for swi1-2 plants, we found that SMC3 labeled the

626

|

Boateng et al.

d

SWI1: An Early Meiotic Regulator

Figure 6. Cohesin Localization Patterns in Wild-Type and dsy10 Meiocytes. SYN1 immunolocalization in wild-type (A–E) and dsy10 (F–J). Each panel shows a merged image of DAPI-stained chromosomes (red) and SYN1 immunolocalization (green). (A) Early pachytene, (B) Late pachytene, (C) Metaphase I, (D) Anaphase I, (E) Tetrad, (F, G) Mid-prophase, (H) Diakinesis, (I) Anaphase I, (J) polyad. Normal (K, O) and detergent (0.1% Tween-20)-washed (L–N, P–R) immunolocalization of SYN1 and SMC3 antibodies during meiosis in wild-type (K–N) and dsy10 (O–R) meiocytes. Each panel shows a merged image of DAPI-stained chromosome (red) and SYN1 (K–M, O–P) or SMC3 (N, R) (green), (K–N) Pachytene, (O–R) Prophase.

chromosomes of swi1-2 meiocytes from interphase to the polyad stage (Supplemental Figure 1). While the signal was reduced at diplotene and diakinesis, it was still detectable during these stages and quite strong during meiosis II. During the course of this study, we observed that washing conditions can affect the cohesin immunolocalization results in dsy10 plants and that including detergent (0.1% Tween20) in the wash buffer resulted in pronounced differences in cohesin labeling patterns between wild-type and dsy10 meiocytes. SYN1 antibodies strongly labeled the lateral elements along the lengths of the chromosomes throughout pachytene in wild-type meiocytes in the presence or absence of 0.1% Tween-20 (Figure 6K–6M). In contrast, SYN1 signals were dramatically reduced in prophase I nuclei of dsy10 meiocytes when 0.1% Tween-20 was included in the wash buffer

(Figure 6O–6Q). Furthermore, the labeling that was observed was punctate and did not appear to label the lateral elements in detergent washed slides of dsy10 meiocytes. Similar to the SYN1 immunolocalization patterns, SMC3 was also prematurely released from the detergent-washed mid-prophase dsy10 chromosomes (Figure 6R) in contrast to wild-type pachytene nuclei, which retained strong SMC3 labeling (Figure 6N). Therefore, cohesin proteins are not properly bound to the chromosomes in dsy10 meiocytes. We also investigated the behavior of RAD51, an essential component of the recombination machinery, to study the progression of recombination in dsy10 meiocytes. During meiosis in wild-type plants, numerous AtRAD51 foci appeared on leptotene chromosomes, presumably marking the sites of early recombination events. During leptotene and zygotene, there

Boateng et al.

d

SWI1: An Early Meiotic Regulator

|

627

was a progressive reduction in the number of signals, such that by early pachytene, only a few RAD51 foci remained (Supplemental Figure 2A–2C). In contrast, AtRAD51 foci were never detected in dsy10 meiocytes (Supplemental Figure 2D–2F). Therefore, in contrast to our results for ASY1, SYN1, and SMC3, the RAD51 localization patterns were similar between dsy10 and swi1-2 plants.

A Truncated Form of SWI1 Is Expressed in swi1-2 Plants The absence of axial element formation and the total loss of sister chromatid cohesion at metaphase I in swi1-2 plants appears to represent a more severe phenotype than that observed for dsy10 plants. It has been suggested that swi1-2 is a null mutation and that phenotypic differences observed for different swi1 alleles may be the result of leaky SWI1 expression in other mutants (Mercier et al., 2003). However, SWI1 transcripts are present in buds of swi1-2 plants (Mercier et al., 2001), raising the possibility that a truncated SWI1 protein may actually be produced in swi1-2 plants. In order to investigate this possibility, SWI1 immunolocalization studies were conducted on wild-type, dsy10, and swi1-2 meiocytes. When an antibody to the SWI1 C-terminus (C-Ab; Figure 1) was used, SWI1 signal was detected during premeiotic interphase of wild-type, but not dsy10 or swi1-2, meiocytes (Supplemental Figure 3). However, this antibody recognizes a region of SWI1 after the premature stop codon in the swi1-2 mutant. To confirm that SWI1 protein is in fact absent in dsy10 plants and test if a truncated version of SWI1 is produced in swi1-2 plants, we obtained a SWI1 peptide antibody to amino acids 89–107 of the protein (N-Ab; Figure 1). Immunolocalization experiments on chromosome spreads of wild-type plants using the amino terminal antibody produced localization patterns identical to those obtained with the C-terminal antibody. SWI1 signal was detected during meiotic interphase (Figure 7A and 7B) and early leptotene (Figure 7C), and was absent from chromosomes from approximately late leptotene onwards in wild-type plants (Figure 7D). Contrary to the situation in wild-type meiocytes and consistent with our data obtained using the C-terminal antibody, SWI1 protein was never detected in dsy10 meiocytes (Figure 7E–7H). In contrast, SWI1 signal was detected in meiocytes of swi1-2 plants. A strong signal was detected during meiotic interphase, with the strongest labeling in the nucleolus (Figure 7I). In nuclei at late interphase and early leptotene, the labeling was more uniform (Figure 7J and 7K). SWI1 labeling decreased during leptotene (Figure 7L) and was undetectable by late leptotene. These results demonstrate that dsy10 represents a complete loss-of-function mutation, while a truncated form of SWI1 is produced in swi1-2 plants.

The dsy10 Mutation Leads to Defects in Histone H3 Patterns Meiotic chromosomes in dsy10 plants typically appear larger and less condensed than chromosomes in wild-type plants. This suggested that the mutation affects chromatin structure.

Figure 7. SWI1 Protein Is Not Detected in dsy10 Meiocytes. SWI1 immunolocalization during meiosis in wild-type (A–D), dsy10 (E–H), and swi1-2 (I–L) meiocytes using an amino-terminal SWI1 antibody (N-Ab). In each panel, the upper image shows SWI1 (green), and the lower image shows a DAPI-stained nucleus (red). (A, B, E) Interphase I, (F, I, J) Early leptotene, (C, G, K) Late leptotene, (D, H, L) Early zygotene. Bar = 10 lm.

Therefore, we performed immunolocalization studies with antibodies to acetylated histone H3 (AH3) and dimethylated histone H3-Lys 4 (H3-K4me2) to examine their distribution in meiocytes of wild-type and dsy10 plants. In wild-type plants, the AH3 antibody labeled chromosomes in both somatic and meiotic cells. The labeling patterns were generally uniform, with the exception that the nucleolus and chromocenters were not labeled. In wild-type meiocytes, acetylated histone H3 signal was strongest during interphase (Figure 8A). The signal gradually decreased during prophase, becoming very weak by early zygotene (Figure 8B) and undetectable during zygotene and pachytene (Figure 8C). A weak, diffuse AH3 signal was detected again in late diplotene (Figure 8D), and persisted through to telophase I (data not shown). The AH3 labeling pattern of dsy10 plants was comparable to wild-type plants during interphase (Figure 8E). However, in contrast to wild-type meiocytes, the signal remained relatively constant throughout prophase in dsy10 plants (Figure 8F–8H).

628

|

Boateng et al.

d

SWI1: An Early Meiotic Regulator

during mid-prophase (Figure 8O). Weak H3-K4me2 signal was again observed in the dsy10 meiocytes during diplotene and later stages of meiosis (Figure 8P). Therefore, dys10 meiocytes exhibit alterations in the distribution of modified histone H3 during mid-prophase.

DISCUSSION Alleles of SWI1 Exhibit Different Phenotypes

Figure 8. Histone H3 Acetylation and Dimethylation Patterns Are Altered in the dsy10 Meiocytes. Immunodetection of acetylated histone H3 (AH3) and anti-dimethylated Histone H3-Lys 4 (H3-K4me2) in wild-type (A–D, I–L) and dsy10 meiocytes (E–H, M–P). In each panel, the upper image shows a DAPI-stained nucleus (red) and the lower image shows immunolocalization (green) for AH3 (A–H) and H3-K4me2 (I–P). (A, E, I, M) Interphase, (B,F, J, N) Zygotene, (C,K) Pachytene, (D,H, L, P) Diplotene, (G, O) Prophase I. Bar = 10 lm.

Alterations were also detected with the H3-K4me2 antibody. The H3-K4me2 antibody produced a diffuse labeling pattern during interphase in wild-type plants (Figure 8I). The signal was found on the condensing chromosomes at leptotene, zygotene, pachytene, and at later stages of meiosis in wild-type plants (Figure 8I–8L). The labeling was mainly in the euchromatin and absent from the nucleolus and chromocenters. While similar labeling patterns were observed during interphase and leptotene in dsy10 nuclei (Figure 8M and 8N), H3-K4me2 labeling was not detectable in dsy10 meiocytes

SWI1 is complex in several regards. Three different transcription start-sites, which can produce proteins with slightly different N-termini, have been identified. The longest transcript can encode a 639 amino acid protein, while the shortest transcript has the potential to encode a 579 amino acid protein. Furthermore, four alleles of Arabidopsis SWI1 (swi1-1, swi1-2 dyad, and dsy10) have now been characterized, several of which exhibit phenotypic differences (Agashe et al., 2002; Mercier et al., 2001, 2003; Motamayor et al., 2000; Siddiqi et al., 2000). The swi1-1 mutation is caused by a T-DNA insertion in the 5’ untranslated region of the second longest transcript. The swi1-2 allele is a nonsense mutation at amino acid 394, while the dyad allele represents a nonsense mutation at position 509. We demonstrate here that the dsy10 mutation results from a T-DNA insertion at position 139 in the 639 amino acid protein. All four alleles result in female sterility. Analysis of female meiosis in swi1-1 and dyad plants showed that chromosomes fail to undergo synapsis and formed univalents at metaphase I that segregated evenly into two daughter cells, resembling a mitotic division (Agashe et al., 2002; Mercier et al., 2001; Motamayor et al., 2000). The two daughter nuclei then underwent a second division that was sometimes mitotic-like, but more often aberrant with uneven chromosome segregation. Furthermore, it was recently shown that dyad plants can in fact form seeds that are triploid if an unreduced dyad female gamete is fertilized by a haploid male gametophyte (Ravi et al., 2008). Male meiosis appears to occur normally in swi1-1 and dyad plants (Motamayor et al., 2000; Siddiqi et al., 2000), while swi1-2 and dsy10 plants are both male and female sterile (Cai and Makaroff, 2001; Mercier et al., 2001). Finally, while the swi1-2 and dsy10 mutations exhibit many of the same alterations during male meiosis, they also show several differences. Both swi1-2 and dsy10 block homologue pairing, synapsis and recombination, and cause the premature loss of sister chromatid cohesion. A difference between the two mutants is the observation that swi1-2 plants are unable to assemble normal axes (Mercier et al., 2003), whereas early axial element formation appears relatively normal in dsy10 plants. ASY1 is a meiosis-specific protein that associates with the chromosome axes during prophase I (Armstrong et al., 2002). While ASY1 colocalizes with the condensing chromosomes in swi1-2 meiocytes, it does not localize to the threads that typify leptotene AEs in wild-type plants (Mercier et al., 2003). In contrast, ASY1 localization patterns in dsy10 meiocytes were similar to those

Boateng et al.

of wild-type plants from interphase through early zygotene; it appeared as thin threads that represent AEs. The presence of relatively normal ASY1 staining patterns during leptotene and early zygotene indicates that the initial formation of axial elements may be relatively normal in dsy10 plants. This is similar to the situation in Sordaria spo11 and ski8 mutants. In the absence of SPO11 or SKI8, axial elements are built along the chromosomes, but homologues show only rare signs of recognition and do not form SCs (Storlazzi et al., 2003; Tesse et al., 2003). Differences in the distribution of cohesin proteins were also observed between dsy10 and swi1-2 plants. Cohesin labeling patterns are indistinguishable between wild-type, dys10, and swi1-2 plants during early prophase. The cohesins localized along the developing chromosome axes at leptotene and strongly labeled the chromosomes at early zygotene in all three lines. Wild-type plants exhibited strong stable labeling of pachytene chromosomes, which gradually decreased beginning in diplotene, and, by metaphase I, cohesin signals were barely visible on the chromosomes. Although the cohesin signals appeared relatively normal in mid-prophase cells of dsy10 plants, the proteins were not tightly bound to the chromosomes and could be removed by washing with 0.1% Tween-20. In contrast, the cohesins are not released from the chromosomes of swi1-2 plants under normal washing conditions and instead were detected on the 10 univalents, the 20 individual chromatids and in polyads (Supplemental Figure 1; Mercier et al., 2003). Cohesins are released in three steps during meiosis in plants. The bulk of the cohesin is released during late prophase I in an ESP1-independent process. Residual arm cohesin and centromere cohesin are released in ESP1-dependent processes at anaphase I and anaphase II, respectively (Liu and Makaroff, 2006). Swi1-2 plants appear to be defective in the prophase I removal of cohesin. Interestingly, cohesion between the sister chromatids is lost earlier in swi1-2 meiocytes than it is in dsy10 plants. At this time, the reason for this apparent discrepancy is not clear. However, there seem to be differences in the way the cohesins interact with chromosomes in the two mutants. It was previously suggested that the swi1-2 mutation represents a null mutation and the reason that the swi1-1 and dyad mutations represent weaker alleles is because of leaky expression of or the production of a partially functional protein in swi1-1 and dyad plants, respectively (Mercier et al., 2003). We have shown that dsy10 represents a complete loss-offunction mutation, while an altered form of SWI1 is produced in swi1-2 plants. The observation that the swi1-2 mutation appears to have a somewhat more severe effect on male meiosis than dsy10 raises the possibility that expression of a truncated form of SWI1 has a negative effect on chromatin structure. It is possible that the protein could exhibit altered activity or specificity that results in a chromatin structure different from that caused by the complete absence of SWI1. These differences could then affect cohesin binding and ultimately the formation of axial elements. Cohesins are important for the localization of LE-associated proteins, acting

d

SWI1: An Early Meiotic Regulator

|

629

together with SC proteins to generate the characteristic axis-loop structure of meiotic chromosomes (Page and Hawley, 2004; Revenkova and Jessberger, 2006). SWI1 exhibits little to no similarity with known proteins and structure/function relationships have not yet been established for the different regions of SWI1. However, the observation that dyad plants, which presumably express a SWI1 protein missing the Cterminal 135 amino acids, are male fertile, while swi1-2 plants, which express a protein missing the C-terminal 245 amino acids, are male sterile suggests that the region from 390 to 505 is essential for SWI1 function in male meiocytes.

What Is the Role of SWI1? Our data, together with results from others, suggest that Arabidopsis SWI1 is required for meiotic chromatin remodeling, the establishment of sister chromatid cohesion, and the pairing, synapsis, and recombination between homologues during meiosis. SWI1 is first detected in G1, strongly labels meiotic S-phase cells and gradually disappears during G2 (Mercier et al., 2003), indicating that it plays a very early role in meiosis. Based on the effect of SWI1 mutations on female meiosis and the finding that SWI1 has partial sequence similarity to maize AMEIOTIC1 (am1), it was suggested that SWI1 could function as a switch that controls the transition from mitosis to meiosis (Hamant et al., 2006; Motamayor et al., 2000). However, studies have shown that both SWI1 megasporocytes and meiocytes actually enter meiosis (Agashe et al., 2002; Mercier et al., 2001). It has also been postulated (Mercier et al., 2003) that SWI1 may function in a manner analogous to yeast Eco1/Ctf7, which facilitates the establishment of cohesion after cohesin complexes have loaded onto the chromosomes (Kenna and Skibbens, 2003; Toth et al., 1999). Based primarily on studies in yeast, several related models have been proposed for the establishment of cohesion (Lengronne et al., 2006; Milutinovich et al., 2007). It is generally accepted that Scc2p and Scc4p facilitate the binding of cohesin complexes to the chromosomes prior to DNA replication (Ciosk et al., 2000; Gillespie and Hirano, 2004). Sister chromatid cohesion is then established in a Ctf7-dependent step that occurs either during or soon after DNA replication fork passage. Pds5, which physically associates with yeast Ctf7 and the cohesin complex, facilitates the binding of Ctf7 to the chromosomes (Noble et al., 2006). It is therefore possible that SWI1 could play a role similar to Ctf7 or Pds5 in yeast. The dsy10 mutation affects the patterns of several modified forms of histone H3, including the failure to deacetylate histone H3 during leptotene, zygotene, and pachytene, and the absence of H3-K4me2 labeling during zygotene and pachytene. While changes in modified histones have been observed during meiosis in a number of organisms, less is known about how the changes in chromatin structure specifically affect meiosis or how these changes are controlled in any organism (Ivanovska and Orr-Weaver, 2006). Alterations in chromatin structure, including histone methylation patterns, have been associated with several meiotic defects. In C. elegans, altered

630

|

Boateng et al.

d

SWI1: An Early Meiotic Regulator

meiotic recombination and chromosome segregation have been linked with the reduced and delayed accumulation of histone H3-K9 methylation in HIM-17 depleted cells (Reddy and Villeneuve, 2004). In budding yeast, mutations in Set1, which is responsible for the methylation of H3 lysine 4, block bouquet formation (Trelles-Sticken et al., 2005), while telomere clustering at the spindle pole body during meiosis in S. pombe requires the methylation of H3 on lysine 9 (Tuzon et al., 2004). Furthermore, mice containing a mutation in Meisetz, which encodes a histone methyltransferase responsible for trimethylation of histone H3 lysine 4, exhibit defects in homologous chromosome pairing, DSB repair and impaired sex body formation (Hayashi et al., 2005). Therefore, some of the later meiotic alterations observed in dsy10 plants could result from the reduced levels of H3-K4me2 during prophase; however, the H3-K4me2 alterations are likely not the primary defect in dsy10 plants. Likewise, the deacetylation of histone H3 is important for the proper condensation of meiotic chromosomes in Xenopus egg cells (Magnaghi-Jaulin and Jaulin, 2006). However, because the alterations we observe in AH3 patterns occur later in prophase, we again believe that these represent secondary alterations and they result from defects earlier in meiosis. The presence of alterations in modified histone patterns, along with the apparent alterations in cohesin complex binding and cohesion formation does, however, raise the possibility that SWI1 could be either directly or indirectly involved in early chromatin remodeling events that are required for the proper binding of cohesin complexes and the subsequent establishment of cohesion. Studies are underway to more specifically analyze early meiotic chromosome structure in dsy10 plants and determine if SWI1 directly controls chromatin structural modifications during meiosis.

METHODS Plant Material Arabidopsis thaliana L. Heynh, ecotype Wassilewskija (WS) was the source of both wild-type and mutant plants. The dsy10 mutant was selected during a large-scale screen of T-DNA transformants at the Du Pont Company, Wilmington, DE, and was previously described (Peirson et al., 1996). Drs Christine Me´zard and Christine Horlow kindly provided seeds for the swi1-2 mutant. Plants were grown in a commercial potting mix at 22
C with 16 h of light and 8 h of darkness.

Cloning of the dsy10 Mutation Genomic DNA was isolated from sterile individuals in a segregating population of plants containing the dsy10 mutation using the CTAB method (Doyle and Doyle, 1990). The dsy10 T-DNA insertion was monitored in sterile plants over several generations and in F2 plants after backcrossing with wild-type WS using Southern blotting and PCR analyses. The dsy10 insertion segregated with the sterility phenotype in 100% (.200) of

the plants examined. Sequences flanking the dsy10 T-DNA insertion site were isolated from a genomic library constructed with DNA isolated from homozygous dsy10 plants as previously described (Maiti et al., 1997). Wild-type sequences corresponding to the dsy10 locus were isolated from a wild Arabidopsis genomic lambda library. Total RNA was isolated from unopened flower buds. Poly (A) RNA was isolated using oligo d(T) cellulose and used for cDNA synthesis and cloning. Genomic and cDNA clones were sub-cloned and sequenced using the dideoxynucleotide chain termination method.

Expression Analysis SWI1 transcripts in buds of WT and dsy10 plants were investigated using RT–PCR. Equal amounts (4 lg) of total RNA was used to synthesize cDNA with a Thermoscript RT–PCR system (Invitrogen). Primers for ACTIN8 (ACT8) were used as a control to standardize the cDNA (An et al., 1996). SWI1 transcript levels were determined by PCR with the primers 494 and 492 or 745 and 493 (Figure 1). PCR products were analyzed by Southern blot analysis with a 32P-labeled SWI1-specific probe generated by PCR with primers 745 and 493. Radioactivity was detected using a Molecular Dynamics Phosphorimager (Sunnyvale, CA, USA).

Antibodies The C-terminal 153 amino acids of SWI1 were cloned into pET21b (Novagen) to generate a C-terminal HIS-tag fusion protein, which was overexpressed in E. coli BL21RIL cells (Novagen). The protein was purified using nickel-affinity chromatography, followed by SDS polyacrylamide gel electrophoresis and used for polyclonal antiserum production in rabbits. A peptide to amino acids from 85 to 105 (HFDYSRMNRNKPMKKRSGG) of SWI1 was synthesized, coupled to KLH, and also used to produce a rabbit polyclonal antiserum (Genemed Synthesis Inc.). The SWI1 antibodies were used at a working dilution of 1:500. ASY1 and RAD51 polyclonal antibodies, kindly provided by Chris Franklin, were used at working dilutions of 1:1000 and 1:500, respectively (Armstrong et al., 2002; Mercier et al., 2003). SYN1 and SMC3 antibodies were used at a working dilution of 1:500 (Cai et al., 2003; Lam et al., 2005). The dimethylated histone H3-Lys 4 (H3-K4me2) and acetylated histone H3 (AH3, acetylated on Lys-9 and 14) antibodies were obtained from Upstate Group (LLC) and used at 1:500 dilutions.

Immunolocalization and Cytological Procedures The analysis of male meiosis was performed essentially as described (Ross et al., 1997) with the following modification. Digested anthers were washed twice with cold 10 mM sodium citrate for 5 min each, cleared in 60% acetic acid for 2 min, covered with a cover glass and squashed by gentle tapping. The slides were frozen on dry ice, the cover glass quickly removed and dried overnight. The slides were washed in phosphate-buffered saline (PBS) for 15 min and stained with DAPI (4,6-diamino-2-phenylindole dihydrochloride; Vector Laboratories, Inc., Burlingame, CA, 1.5 lg ml 1).

Boateng et al.

Immunolocalization studies were conducted on buds of wild-type and dsy10 plants that were fixed for 2 h at room temperature in Buffer A containing 4% paraformaldehyde (Cai et al., 2003). Anthers were squashed between two perpendicular poly-L-lysine-coated slides and dried overnight. The meiocytes were then soaked in PBS for 20 min and treated with 1.4% w/v b-glucuronidase, 0.3% w/v cytohelicase, 0.3% w/v pectolyase, 0.3% w/v cellulase in 10 mM sodium citrate buffer. After washing in PBS, the slides were blocked (PBS, 5% w/v BSA) and incubated overnight with primary antibody. The slides were washed in PBS, 1 mM EDTA, with or without 0.1% Tween-20, and treated with Alexa-488-labeled secondary antibody (1:300). After washing, the samples were stained with DAPI in 1,4-diazabicyclo-[2,2,2]-octane (DABCO) and observed with an Olympus 1X81 fluorescence deconvolution microscope system. Data were analyzed with Image Pro Plus (Media Cybernetics, Silver Spring, MD) and organized with Adobe Photoshop. The images shown reflect the typical cell observed at each stage. At least 20 examples of each stage were observed for each of the antibodies used. Fluorescence in-situ hybridization (FISH) was conducted on inflorescences that were fixed in acetic alcohol (ethanol:glacial acetic acid, 3:1). Staged buds were subjected to FISH using previously published procedures (Caryl et al., 2000; Fransz et al., 1996). The pAL1 clone containing a pericentromeric 180-bp repeat was used to detect centromere sequences (MartinezZapater et al., 1986). Primary PCR amplification of pAL1 was conducted using the M13 forward and reverse primers followed by random primer labeling in the presence of Biotin-labeled dUTP (Roche, Indianapolis, IN 46250). The biotin-labeled probe was used in hybridization solution at 5 lg ml 1 and detected with 10 lg ml 1 fluorescein-labeled streptavidin. Chromosome 1, centromere-associated telomere sequences were detected by hybridization with the 5’ end FITC-labeled oligonucleotide probe, FITC-(CCCTAAA)6 at 5 lg ml 1. Slides were counterstained with DAPI, mounted and viewed as above.

SUPPLEMENTARY DATA Supplementary Data are available at www.mplant.oxford journals.org.

SWI1: An Early Meiotic Regulator

|

631

Makaroff Lab, and the reviewers for helpful comments and suggestions on the manuscript. No conflict of interest declared.

REFERENCES Agashe, B., Prasad, C.K., and Siddiqi, I. (2002). Identification and analysis of DYAD: a gene required for meiotic chromosome organization and female meiotic progression in Arabidopsis. Development 129, 3935–3943. An, Y., McDowell, J., Huang, S., McKinney, E., Chambliss, S., and Meagher, R. (1996). Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J. 10, 107–121. Armstrong, S., Caryl, A., Jones, G., and Franklin, F. (2002). Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica. J. Cell Sci. 115, 3645–3655. Armstrong, S., Franklin, F., and Jones, G. (2001). Nucleolusassociated telomere clustering and pairing preceed meiotic chromosome synapsis in Arabidopsis thaliana. J. Cell Sci. 114, 4207–04217. Borde, V., Goldman, A.S.H., and Lichten, M. (2000). Direct coupling between meiotic DNA replication and recombination initiation. Science. 290, 806–809. Cai, X., and Makaroff, C. (2001). The dsy10 mutation of Arabidopsis results desynapsis and a general breakdown in meiosis. Sex. Plant Repro. 14, 63–67. Cai, X., Dong, F.G., Edelmann, R.E., and Makaroff, C.A. (2003). The Arabidopsis SYN1 cohesin protein is required for sister chromatid arm cohesion and homologous chromosome pairing. J. Cell Sci. 116, 2999–3007. Caryl, A.P., Armstrong, S.J., Jones, G.H., and Franklin, F.C.H. (2000). A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1. Chromosoma. 109, 62–71. Ciosk, R., Shirayama, M., Shevchenko, A., Tanaka, T., Toth, A., and Nasmyth, K. (2000). Cohesin’s binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol. Cell. 5, 243–254. Ciosk, R., Zachariae, W., Michaelis, C., Shevchenko, A., Mann, M., and Nasmyth, K. (1998). An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell. 93, 1067–1076. Doyle, J., and Doyle, J. (1990). Isolation of plant DNA from fresh tissue. BRL Focus. 12, 13–15.

FUNDING National Science Foundation MCB0718191 to C.A.M.).

d

(MCB0322171

to

C.A.M.,

ACKNOWLEDGMENTS We are grateful to Chris Franklin for providing the ASY1 and RAD51 antibodies, Christine Horlow and Christine Mezard for the swi1-2 seeds, and Richard Edelmann and Matthew L. Duley for technical support on the microscope. Thanks go to Meldomi Asante, the

Fransz, P., Alonso-Blanco, C., Liharska, T.B., Peeters, A.J.M., Zabel, P., and Hans de Jung, J. (1996). High resolution physcial mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibers. Plant J. 9, 421–430. Fransz, P., Armstrong, S., AlonsoBlanco, C., Fischer, T.C., TorresRuiz, R.A., and Jones, G. (1998). Cytogenetics for the model system Arabidopsis thaliana. Plant J. 13, 867–876. Gillespie, P.J., and Hirano, T. (2004). Scc2 couples replication licensing to sister chromatid chromatid in Xenopus egg extracts. Curr. Biol. 14, 1598–1603.

632

|

Boateng et al.

d

SWI1: An Early Meiotic Regulator

Gregan, J., Rabitsch, P.K., Sakem, B., Csutak, O., Latypov, V., Lehmann, E., Kohli, J., and Nasmyth, K. (2005). Novel genes required for segregation are identified meiotic chromosome by a high-throughput knockout screen in fission yeast. Curr. Biol. 15, 1663–1669. Hamant, O., Ma, H., and Cande, W.Z. (2006). Genetics of meiotic prophase I in plants. Annu. Rev. Plant Biol. 57, 267–302. Hayashi, K., Yoshida, K., and Matsui, Y. (2005). A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature. 438, 374–378. Ivanovska, I., and Orr-Weaver, T.L. (2006). Histone modifications and the chromatin scaffold for meiotic chromosome architecture. Cell Cycle. 5, 2064–2071. Kenna, M.A., and Skibbens, R.V. (2003). Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p, a cohesion establishment factor, associates with three different replication factor C complexes. Mol. Cell Biol. 23, 2999–3007. Kitajima, T.S., Kawashima, S.A., and Watanabe, Y. (2004). The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis. Nature. 427, 510–517.

Milutinovich, M., Unal, E., Ward, C., Skibbens, R.V., and Koshland, D. (2007). A multi-step pathway for the establishment of sister chromatid cohesion. Plos Genetics. 3. Molnar, M., Parisi, S., Kakihara, Y., Nojima, H., Yamamoto, A., Hiraoka, Y., Bozsik, A., Sipiczki, M., and Kohli, J. (2001). Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe. Genetics. 157, 519–532. Motamayor, J.C., Vezon, D., Bajon, C., Sauvanet, A., Grandjean, O., Marchand, M., Bechtold, N., Pelletier, G., and Horlow, C. (2000). Switch (Swi1), an Arabidopsis thaliana mutant affected in the female meiotic switch. Sex. Plant Reprod. 12, 209–218. Noble, D., Kenna, M.A., Dix, M., Skibbens, R.V., Unal, E., and Guacci, V. (2006). Intersection between the regulators of sister chromatid cohesion establishment and maintenance in budding yeast indicates a multi-step mechanism. Cell Cycle. 5, 2528–2536. Page, S.L., and Hawley, R.S. (2004). The genetics and molecular biology of the synaptonemal complex. Annu. Rev. Cell Dev. Biol. 20, 525–558. Pawlowski, W.P., and Cande, W.Z. (2005). Coordinating the events of the meiotic prophase. Trends Cell Biol. 15, 674–681.

Klein, F., Mahr, P., Galova, M., Buonomo, S.B.C., Michaelis, C., Nairz, K., and Nasmyth, K. (1999). A central role for cohesions in sister chromatid cohesion, formation of axial elements and recombination during yeast meiosis. Cell. 98, 91–103.

Peirson, B.N., Owen, H.A., Feldmann, K.A., and Makaroff, C.A. (1996). Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis. Sex. Plant Repro 9, 1–16.

Lam, W.S., Yang, X.H., and Makaroff, C.A. (2005). Characterization of Arabidopsis thaliana SMC1 and SMC3: evidence that ASMC3 may function beyond chromosome cohesion. J. Cell Sci. 118, 3037–3048.

Ravi, M., Marimuthu, M.P.A., and Siddiqi, I. (2008). Gamete formation without meiosis in Arabidopsis. Nature 451, 1121–1125.

Lengronne, A., McIntyre, J., Katou, Y., Kanoh, Y., Hopfner, K.P., Shirahige, K., and Uhlmann, F. (2006). Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol. Cell. 23, 787–799. Liu, Z., and Makaroff, C.A. (2006). Arabidopsis separase AESP is essential for embryo development and the release of cohesin during meiosis. Plant Cell. 18, 1213–1225. Ma, H. (2005). Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants. Annu. Rev. Plant Biol. 56, 393–434. Magnaghi-Jaulin, L., and Jaulin, C. (2006). Histone deacetylase activity is necessary for chromosome condensation during meiotic maturation in Xenopus laevis. Chromosome Res. 14, 319–332. Maiti, M.K., Krishnasamy, S., Owen, H.A., and Makaroff, C.A. (1997). Molecular characterization of glyoxalase II from Arabidopsis thaliana. Plant Mol. Biol. 35, 471–481. Martinez-Zapater, J.M., Estelle, M.A., and Somerville, C.R. (1986). A highly repeated DNA sequence in Arabidopsis thaliana. Mol. Gen. Genet. 204, 417–423. Mercier, R., Armstrong, S.J., Horlow, C., Jackson, N.P., Makaroff, C.A., Vezon, D., Pelletier, G., Jones, G.H., and Franklin, F.C.H. (2003). The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis. Development. 130, 3309–3318. Mercier, R., Vezon, D., Bullier, E., Motamayor, J.C., Sellier, A., Lefevre, F., Pelletier, G., and Horlow, C. (2001). SWITCH1 (SWI1): a novel protein required for the establishment of sister chromatid cohesion and for bivalent formation at meiosis. Genes Dev. 15, 1859–1871.

Reddy, K.C., and Villeneuve, A.M. (2004). C-elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination. Cell. 118, 439–452. Revenkova, E., and Jessberger, R. (2006). Shaping meiotic prophase chromosomes: cohesins and synaptonemal complex proteins. Chromosoma. 115, 235–240. Ross, K.J., Fransz, P., Armstrong, S.J., Vizir, I., Mulligan, B., Franklin, F.C.H., and Jones, G.H. (1997). Cytological characterization of four meiotic mutants of Arabidopsis isolated from T-DNAtransformed lines. Chromosome Res. 5, 551–559. Siddiqi, I., Ganesh, G., Grossniklaus, U., and Subbiah, V. (2000). The dyad gene is required for progression through female meiosis in Arabidopsis. Development. 127, 197–207. Smith, K.N., Penkner, A., Ohta, K., Klein, F., and Nicolas, A. (2001). B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis. Curr. Biol. 11, 88–97. Storlazzi, A., Tesse, S., Gargano, S., James, F., Kleckner, N., and Zickler, D. (2003). Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division. Genes Dev. 17, 2675–2687. Stuart, D., and Wittenberg, C. (1998). CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint. Genes Dev. 12, 2698–2710. Tesse, S., Storlazzi, A., Kleckner, N., Gargano, S., and Zickler, D. (2003). Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition. Proc. Natl Acad. Sci. U S A 100, 12865–12870. Toth, A., Ciosk, R., Uhlmann, F., Galova, M., Schleiffer, A., and Nasmyth, K. (1999). Yeast Cohesin complex requires a conserved

Boateng et al.

protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev. 13, 320–333. Trelles-Sticken, E., Adelfalk, C., Loidl, J., and Scherthan, H. (2005). Meiotic telomere clustering requires actin for its formation and cohesin for its resolution. J. Cell Biol. 170, 213–223. Tuzon, C.T., Borgstrom, B., Weilguny, D., Egel, R., Cooper, J.P., and Nielsen, O. (2004). The fission yeast heterochromatin protein Rik1 is required for telomere clustering during meiosis. J. Cell Biol. 165, 759–765.

d

SWI1: An Early Meiotic Regulator

|

633

Uhlmann, F., Wernic, D., Poupart, M.A., Koonin, E.V., and Nasmyth, K. (2000). Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell. 103, 375– 386. Zickler, D. (2006). From early homologue recognition to synaptonemal complex formation. Chromosoma. 115, 158–174. Zickler, D., and Kleckner, N. (1999). Meiotic chromosomes: Integrating structure and function. Annu. Rev. Genet. 33, 603–754.